Complete Genome Sequence and Pathogenicity of Two

Swine Parainfluenzaviruses Isolated from Pigs in the

United States

Dan Qiao

Thesis submitted to the faculty of the Virginia Polytechnic Institute and State

University in partial fulfillment of the requirements for the degree of

Master of Science

In

Biomedical and Veterinary Sciences

Elankumaran Subbiah, Chair

Xiang-Jin Meng, Member

Chris Roberts, Member

Lijuan Yuan, Member

Kevin Myles, Member

May 21st , 2009

Blacksburg, VA

Keywords: Paramyxovirus, Bovine Parainfluenzavirus 3, Cross-species infection, Pigs

Complete Genome Sequence and Pathogenicity of Two Swine

Parainfluenzaviruses Isolated from Pigs in the United States

Dan Qiao

Abstract

Members of the family Paramyxoviridae are non-segmented, negative-strand RNA

viruses. A large and diverse host species are infected by paramyxoviruses, including avian,

porcine, canine, bovine, equine, ovine, reptiles, aquatic species and humans. In the last few

decades, many novel paramyxoviruses have emerged causing catastrophic illnesses in different

aquatic and terrestrial species of animals and some of them also made the species jump to

humans. Two novel paramyxoviruses 81-19252 (Texas81) and 92-7783 (ISU92) were isolated in

the 1980s and 1990s from the brain of pigs that experienced respiratory and central nervous

system disease from South and North Central United States. To understand their importance as

swine pathogens, molecular characterization and pathogenicity studies were undertaken. The

complete genome of Texas81 virus was 15456 nucleotides (nt) and ISU92 was 15480 nt in

length consisting of six non-overlapping genes coding for the nucloeo- (N), phospho- (P), matrix

(M), fusion (F), hemagglutinin-neuraminidase (HN) and large polymerase (L) proteins in the

order 3'-N-P/C/V-M-F-HN-L-5'. The features related to virus replication and found to be

conserved in most members of Paramyxoviridae were also found in swine viruses. These

include: conserved and complementary 3’ leader and 5’ trailer regions, trinucleotide intergenic

sequences, highly conserved gene start and gene stop signal sequences. The length of each gene

of these two viruses was similar except for the F gene, in which ISU92 had an additional 24 nt

iii

“U” rich 3’ untranslated region (UTR). The P gene of these viruses were predicted to express the

P protein from the primary transcript and edit a portion of its mRNA to encode V and D proteins

and the C protein was expected to be expressed from alternate translation initiation from the P

gene as in Respiroviruses. Sequence specific features related to virus replication and host

specific amino acid signatures in P, F, HN and L proteins indicated that these viruses probably

originated from bovine parainfluenzavirus 3. Pairwise comparisons of deduced amino acid

sequences of swine viral proteins with members of Paramyxoviridae and phylogenetic analysis

based on individual genes as well as predicted amino acid sequences suggested that these viruses

were novel members of the genus Respirovirus of the Paramyxovirinae subfamily and genotype

A of bovine parainfluenzavirus type 3. The mild clinical signs and undetectable gross and

microscopic lesions observed in swine parainfluenzavirus (sPIV3)-infected pigs indicate the

inapparent nature of these viruses in pigs. Limited seroprevalence studies in serum samples

collected from pig farms in Minnesota and Iowa in 2007-2008 by indirect ELISA revealed that

sPIV3 are not circulating in these farms. The mild pathogenicity of sPIV3 can facilitate its

development as a vaccine vector. The screening ELISA developed by us could be used to detect

seroprevalence of sPIV3 in animal and human populations.

iv

This thesis is dedicated to my family and friends. For the

love, support and encouragement from my parents, Yonge

Guo and Wei-min Qiao, and many friends.

v

Acknowledgements

This thesis arose in part out of years of research that has been done since I came to Dr.

Subbiah’s group. During that time, I have worked with a great number of people whose

contribution in assorted ways helped in the research and made me obtain a great and

unforgettable research experience.

In the first place I would like to record my gratitude to Dr. Elankumaran Subbiah for his

supervision, advice, and guidance from the very early stage of my research as well as giving me

an extraordinary experience throughout the work. Above all and the most needed, he provided

me with patience, kindness, unflinching encouragement and support to keep me going through

when I had hard times. His scientific passion and ideas to research always inspired me to be a

good scientist in the future. I would also express my gratefulness to my committee members, Dr.

X.J.Meng, Dr.Lijuan Yuan, Dr. Chris Roberts and Dr. Kevin Myles, who always gave valuable

advice, critical comments on my project and helped me improve my work. I especially want to

acknowledge Dr. Meng and Dr. Yuan who are more like mentors to me, giving me guidance,

believing in me even when I didn’t believe in myself and always gave me their precious time

when I turn to them for extra help in any aspects.

I gratefully thank Dr. Laure Deflube for her help. I learnt not only a lot of techniques but

also an earnest, industrious attitude towards research and her constructive comments on my

research are always useful.

Collective and individual acknowledgments are also owed to my colleagues in

Dr.Subbiah’s lab, Shobana Raghunath, Vrushali Pramod Chavan, Thomas Rogers-Cotrone,

vi

Gopakumar Moorkanat, as well as all in CMMID, whose presence perpetually refreshed and

helped me and made the experience memorable. Many thanks to them for giving me such a

pleasant time when working together.

vii

Table of Contents

Abstract………………………………………………………………………………………...….ii

Dedication…………………………………………………………………………………...….iv

Acknowledgements…………………………………………………………………………...…v

Table of Contents………………………………………………………………………...............vii

List of Abbreviations…………………………………………………………………………….xii

General Introduction…………………………………………………………………………….xvi

Chapter 1. Literature Review………………………………………………….....1

1.1 Introduction …………………………………………………………………………………...1

1.2 Classification of Paramyxoviruses………………………………………………………….....2

1.3 Emerging Paramyxoviruses……………………………………………………………….......3

1.4 Clinical Signs and Lesions……………………………………………………………….........6

1.5 Molecular Biology of Paramyxovirus………………………………………………………..11

1.5.1 Virion Structure and Genome Organization………………………………………...…11

1.5.1.1 Nucleocapsid Protein………………………………………………………......13

1.5.1.2 The P gene and its encoded proteins………………………………………...…13

1.5.1.3 Matrix Protein……………………………………………………………..…...17

1.5.1.4 L Protein………………………………………………………………………..17

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1.5.1.5 Viral Glycoproteins………………………………………………………….…18

1.5.2 Strategy of replication………………………………………………………….......…..23

1.6 Parainfluenzaviruses……………………………………………………………………...…24

1.6.1 History of parainfluenzaviruses…………………………………………………….....24

1.6.2 Bovine parainfluenzavirus type 3……………………………………………………...25

1.6.2.1 bPIV3 as a pathogen………………………………………………………......25

1.6.2.2 Cross-species infection of bPIV3 ………………………………………….......26

1.6.2.3 Virion Structure and Genome organization……………………………………27

1.6.2.4 Antigenic relatedness with other parainfluenzaviruses………………………28

1.6.2.5 Diagnostic methods for bPIV3 ………………………………………………...29

1.7 Swine Paramyxoviruses…………………………………………………………………….30

1.7.1 History………………………………………………………………………………….30

1.7.2 Laboratory Examination……………………………………………………………….31

1.7.3 Pathogenicity studies………………………………………………………………...32

1.7.4 Prevalence studies…………………………………………………………………......32

1.7.5 Texas81 history ……………………………………………………………………….33

1.8 Paramyxoviruses as vaccine vector………………………………………………………….33

1.9 Objectives of thesis…………………………………………………………………………..35

1.10 References…………………………………………………………………………………..36

1.11 Tables……………………………………………………………………………………….61

1.12 Figures………………………………………………………………………………………62

ix

Chapter 2. Molecular Characterization of Glycoprotein Genes and

Phylogenetic Analysis of Two Swine Paramyxoviruses Isolated from the

United States………………………………….………………………………..65

Abstract……………………………………………………………………………………...…...66

2.1 Introduction…………………………………………………………………………………..67

2.2 Materials and Methods……………………………………………………………………….69

2.3 Results and Discussion…………………………………………………………………...….78

2.3.1 Morphological and Biological Properties……………………………………………...78

2.3.2 Antigenic analysis………………………………………………………………...……78

2.3.3 Transcriptional start and stop sequences……………………………………………….79

2.3.4 F gene…………………………………………………………………………………80

2.3.5 HN gene………………………………………………………………………………..82

2.3.6 Phylogenetic analysis…………………………………………………………………..84

2.4 Acknowledgments……………………………………………………………………………85

2.5 References…………………………………………………………………………...……….86

2.6 Tables………………………………………………………………………………………...92

2.7 Figures………………………………………………………………………………………..96

Chapter 3. Complete Genome Sequence and Pathogenicity of Two Swine

Parainfluenzaviruses Isolated from Pigs in the United States …….…...……103

Abstract………………………………………………………………………………………....104

3.1 Introduction…………………………………………………………………………………106

x

3.2 Materials and Methods……………………………………………………………………...108

3.2.1 Viruses and Cells……………………………………………………………………..108

3.2.2 Reverse Transcription (RT)-Polymerase Chain Reaction (PCR) and Sequencing…...109

3.2.3 Data Analysis ………………………………………………………………………...110

3.2.4 Accession Numbers for Sequence Analysis…………………………………….…....110

3.2.5 Pathogenicity Studies in Pigs…………………………………………………………112

3.2.6 Development of indirect Enzyme-linked immunosorbent assay (ELISA) …………113

3.3 Results………………………………………………………………………………………114

3.3.1 Genome Features……………………………………………………………...……...114

3.3.2 Genome organization………………………………………………………...……….115

3.3.2.1 The Nucleoprotein (N) Gene………………………………………...…….…116

3.3.2.2 The Phosphoprotein (P) Gene and P/C/V Editing…………………...…...…..117

3.3.2.3 The Matrix protein (M) Gene………………………………………...………118

3.3.2.4 The Fusion Protein (F) Gene and Hemagglutinin-Neuraminidase

Protein (HN) Gene……………………….……………………....................119

3.3.2.5 The Large Polymerase Protein (L) Gene…………………………………......119

3.3.3 Phylogenetic analysis…………………………………………………………………120

3.3.4 Pathogenicity………………………………………………………………………….121

3.3.5 Serum antibody responses to sPIV3 in pigs detected by indirect ELISA ….………...121

3.4 Discussion…………………………………………………………………………………..122

3.5 Acknowledgments…………………………………………………………………………..128

3.6 References…………………………………………………………………………………..129

3.7 Tables……………………………………………………………………………………….158

xi

3.8 Figures………………………………………………………………………………………163

General Conclusion…………………………………………………………….171

xii

Abbreviations

aa Amino acid

aMPV Avian metapneumovirus

APHIS Animal and Plant Health Inspection Service

aPMV2/6 Avian paramyxovirus type 2/6

ASPV Atlantic salmon paramyxovirus

BeV Beilong virus

bPIV3 Bovine parainfluenza virus type 3

bRSV Bovine respiratory syncytial virus

CA Croup associated

cDNA Complementary DNA

CDV Canine distemper virus

CNS Central nervous system

CPE Cytopathic effect

DMV Dolphin morbillivirus

DNA Deoxyribonucleic acid

dpi Day(s) post inoculation

dsRNA Double strand RNA

ELISA Enzyme-linked immunosorbent assay

F Fusion protein

FDLV Fer-de-lance virus

GE Gene-end

GS Gene-start

HA Hemagglutination

HeV Hendra virus

hMPV Human metapneumovirus

HN Hemagglutinin-neuraminidase

hpi Hour(s) post inoculation

hPIV1/2/3 Human parainfluenza virus type 1/2/3

HRP Horseradish peroxidase

xiii

hRSV Human respiratory syncytial virus

ICTV International Committee on the Taxonomy of Viruses

IFA Indirect fluorescent antibody

IFN Interferon

IGS Intergenic sequence

J-V J-virus

kDA Kilo dalton

L Large polymerase protein

LPMV La Piedad Michoacán paramyxovirus

M Matrix protein

Mabs Monoclonal antibodies

MenV Menangle virus

MeV Measles virus

MOI Multiplicity of infection

MoV Mossman virus

MPRV Mapuera virus

MuV Mumps virus

N Nucleocapsid protein

NA Neuraminidase

NDV Newcastle disease virus

NiV Nipah virus

nm Nanometer

NNSV Nonsegmented negative-strand RNA viruses

nt Nucleotides

NVSL National Veterinary Services Laboratory

ORF Open reading frame

P Phosphoprotein

PBS Phosphate buffered saline

PCR Polymerase chain reaction

PCV Porcine circovirus

xiv

PEG Polyethylene glycol

pfu Plaque forming unit

PGI Proliferative gill inflammation

PIV Parainfluenza virus

PK15 Porcine kidney cell line

PoRV/LPMV Porcine rubulavirus

PPRV Peste-des-petits ruminants virus

PRCV Porcine respiratory coronavirus

PRRSV Porcine reproductive and respiratory syndrome virus

PRV Pseudorabies

RACE Rapid amplification of cDNA ends

RBCs Red blood cells

RDRP RNA dependent RNA polymerase

RNA Ribonucleic acid

RPV Rinderpest virus

RT Reverse transcription

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SeV Sendai virus

SF Shipping fever

SIV Swine influenza virus

SPF Specific pathogen free

sPIV Swine parainfluenza virus

sPMV Swine paramyxovirus

SV5 Simian parainfluenza virus 5

TCID50 50% Tissue culture infective dose

TioV Tioman virus

TM Transmembrane

TPMV Tupaia paramyxovirus

TtPIV-1 T. truncates parainfluenza virus type 1

µl Microliter

xv

USDA United States Department of Agriculture

UTR Untranslated region

VLP Virus-like particle

VN virus neutralization

VSV Vesicular stomatitis virus

xvi

General Introduction

Members of the family Paramyxoviridae are non-segmented, negative-strand RNA

viruses. Members of Paramyxoviridae have been isolated from many host species and this family

includes viruses that are found associated with central nervous and respiratory system diseases.

There are many important disease-causing viruses in this family, including animal pathogens that

are economically important in poultry and livestock industry and human pathogens. Members in

Paramyxoviridae family are not only important in economic and public health aspects, some of

them are also extensively studied to be developed as a vaccine vector or for use as anticancer

agents.

Paramyxoviruses are classified in the order Mononegavirales. Based on virus particle

morphology, genome organization, and structure and sequence relatedness, family

Paramyxoviridae can be divided into two subfamilies; Paramyxovirinae and Pneumovirinae.

Currently, there are five genera within the subfamily Paramyxovirinae: Rubulavirus, Avulavirus,

Respirovirus, Morbillivirus, and Henipavirus, and two genera within the subfamily

Pneumovirinae: Pneumovirus and Metapneumovirus.

A large and diverse host species are infected by paramyxoviruses including avian,

porcine, canine, bovine, equine, ovine, reptiles, aquatic species and humans. There are also

extensive records of zoonotic paramyxoviruses derived from sick pigs in the recent decades all

over the world.

The family Paramyxoviridae has a typical negative-stranded RNA virus structural

pattern. The enveloped virus particle contains a left-handed, helically symmetrical

xvii

ribonucleoprotein core, called nucleocapsid, which comprises the non-segmented single-

negative-stranded RNA genome surrounded by the nucleocapsid proteins, coated within a bilayer

viral envelope. The genome length of family Paramyxoviridae ranges from 15,000 to 19,000

nucleotides (nt), encoding six to ten tandemly linked genes in the order 3'-N-P/C/V-M-F-HN-L-

5'. Interestingly, the genome length of most members of Paramyxoviridae is divisible by six,

known as “rule of six”.

Two swine paramyxoviruses (sPMV) (81-19252 (Texas81), and 92-7783 (ISU92)) were

isolated from the pigs that experienced respiratory and central nervous system disease in the

1980s and 1990s from South and North Central United States, respectively. Texas81 virus was

isolated from the brain of pigs that exhibited respiratory and neurological disease in Texas State,

in 1981. Further information on the history of this virus is not available. ISU92 virus was

isolated from a swine operation in the1990s in north central United States. Affected pigs showed

high fever and mild cough. Encephalitic signs were observed in several pigs with persistent

squealing, head pressing, whole body tremors, and hind-limb ataxia leading to excessive

mortality. Antigenic analysis by indirect fluorescent antibody assay at the National Veterinary

Services Laboratory (NVSL), Ames, Iowa indicated that these two viruses were closely related

to human parainfluenza virus (hPIV) type 1 and 3 and bovine parainfluenza virus type 3 (bPIV3).

As new viruses emerging that causes severe diseases in pigs and have the potential to

cross species barrier and infect other hosts including humans, it is important that these new

viruses to be studied further. The objectives of my thesis is to perform molecular characterization

and pathogenicity studies to understand the significance of these two strains of sPMV for swine

health and to determine their taxonomic status and to examine the pathogenicity of the two

xviii

viruses in conventionally reared pigs. This will establish a foundation for a reverse genetics

system to study pathogenesis and develop them into vaccine vectors.

1

Chapter 1

Literature Review

1.1 Introduction

In the last few decades, many novel paramyxoviruses have emerged causing catastrophic

illnesses in different aquatic and terrestrial species of animals and some of them also made the

species jump to humans. Members of Paramyxoviridae have been isolated from many host

species and this family includes viruses which are found associated with central nervous and

respiratory system diseases (Lamb and Parks, 2007). There are many important disease-causing

viruses in this family, including animal pathogens that are economically important in poultry and

livestock industry, such as Newcastle disease virus (NDV), Rinderpest virus (RPV) and Peste-

des-Petits ruminants virus (PPRV), and human pathogens such as measles virus (MeV), mumps

virus (MuV), respiratory syncytial virus (RSV) and parainfluenza viruses (PIV) (Lamb and

Parks, 2007). Members in Paramyxoviridae family are not only important in economic and

public health aspects, some of them such as MeV and NDV are also extensively studied to be

developed as a vaccine vector or for use as anticancer agents (Cassel and Garrett, 1965;

Elankumaran et al., 2006; Russell and Peng, 2009; Vigil et al., 2008).

Members of the family Paramyxoviridae are non-segmented, negative-strand RNA

viruses. The paramyxovirus contains a lipid bilayer envelope that is derived from plasma

membrane of the host cell during budding. Viral glycoprotein spikes are inserted into the

envelope and can be visualized by electron microscopy. Inside the viral membrane is the helical

filamentous nucleocapsid core formed by large polymerase, nucleocapsid, and phosphoproteins

2

bound to the genomic RNA. They have a close relationship with two other negative strand RNA

viruses: Rhabdoviruses (for their unique nonsegmented genomes organization and its expression)

and Orthomyxoviruses (for the biological properties of the envelope glycoproteins). The

genomic RNA of Paramyxoviruses functions as a (i) template for synthesis of mRNA as well as

(ii) the template for the antigenome strand for making further copies of the genomic RNA. This

virion structural pattern and replication strategy are common in all negative-stranded RNA

viruses (Lamb and Parks, 2007).

1.2 Classification of Paramyxoviruses

Paramyxoviruses are enveloped RNA viruses possessing non-segmented negative-strand

genomes in the order Mononegavirales (Lamb et al., 2005b). Based on virion morphology,

genome organization, and structure and sequence relatedness, family Paramyxoviridae can be

divided into two subfamilies, Paramyxovirinae and Pneumovirinae. Currently, there are five

genera within the subfamily Paramyxovirinae: Rubulavirus, Avulavirus, Respirovirus,

Morbillivirus, and Henipavirus, and two genera within the subfamily Pneumovirinae:

Pneumovirus and Metapneumovirus (Lamb and Parks, 2007) (Table 1). Respiroviruses and

rubulaviruses demonstrate both hemagglutinating (HA) and neuraminidase (NA) activity.

Morbilliviruses and henipaviruses exhibit only HA but lack NA activity. Pneumoviruses do not

cause detectable HA in mammalian and avian erythrocytes and their narrower nucleocapsids also

morphologically distinguish them from Paramyxovirinae. In addition, the differential coding

potential of the P genes, and the presence of an extra gene (SH) are also considered for the

classification (Lamb and Parks, 2007).

3

Some recently identified paramyxoviruses: Menangle virus (MenV) (Philbey et al.,

1998), Tupaia paramyxovirus (Tidona et al, 1999), Tioman virus (TioV) (Chua et al., 2001),

Mossman virus (Miller et al, 2003), J-virus (J-V) (Jack et al., 2005; Jun et al., 1977), Beilong

virus (BeV) (Li et al., 2006b), Mapuera virus (MPRV) (Karabatsos, 1985), Salem virus (Glaser

et al., 2002) and Fer-de-lance virus (Clark et al., 1979) are also officially classified into

Paramyxoviridae by International Committee on the Taxonomy of Viruses (ICTV); however, the

subfamily and genera are still need to be assigned (Lamb et al., 2005a; Lamb and Parks, 2007).

Two more prospective members are, T. truncates parainfluenza virus type 1 (TtPIV-1) isolated

from bottlenose dolphins (Nollens et al., 2008), and Atlantic salmon paramyxovirus (ASPV)

(Nylund et al., 2008).

1.3 Emerging Paramyxoviruses

A large and diverse host species are infected by paramyxoviruses, including avian,

porcine, canine, bovine, equine, ovine, reptiles, aquatic species and humans (Franke et al., 2001;

Horwood et al., 2008; Lamb and Parks, 2007; Nollens et al., 2008; Nylund et al., 2007; Nylund

et al., 2008). Many bat-associated paramyxoviruses have emerged and some of them caused

diseases in animals and humans (Wang et al., 2001). There are also extensive records of

paramyxoviruses derived from sick pigs. Mapuera virus (MPRV) was isolated in 1979 from an

apparently healthy tropical fruit bat (Sturnira lilium) in Brazil (Karabatsos, 1985). Genomic

studies showed that MPRV was closely related to La Piedad Michoacán paramyxovirus (LPMV)

(Wang et al., 2007), also known as porcine rubulavirus (PoRV), the only well studied

neurotropic paramyxovirus isolated from pigs prior to the 1990s (Moreno-Lopez et al., 1986).

4

The host range and disease-causing potential of MPRV is still unknown. There was also

concurrent infection of porcine reproductive and respiratory syndrome virus (PRRSV) and a

paramyxovirus in Germany in the 1990s that had been subsequently named “SER” virus (Heinen

et al., 1998; Tong et al., 2002).

In 1972, a disease outbreak characterized by respiratory distress, lethargy, and death

occurred on a snake farm in Zurich, Switzerland(Clark et al., 1979). A virus subsequently was

isolated from lung tissue of one of the dead snakes. This virus initially was classified as a

paramyxo-like virus and designated Fer-de-Lance Virus (Clark et al., 1979). Since the original

disease outbreak and identification of a viral etiology in 1972, ophidian paramyxoviruses

(OPMV) have emerged as important pathogens of viperid snakes. The first report of OPMV

infection in the United States occurred in 1980 in a private collection of snakes in Florida

(Jacobson et al., 1980). Similar viruses also have been isolated from non-viperid snakes

including a black mamba, corn snakes, beauty snakes, and Moellendorff’s rat snakes (Jacobson

et al., 1980). Clinical signs of OPMV include a sudden gaping of the mouth, followed by violent

convulsions, regurgitation, and expulsion of a brownish fluid from the glottis. Death usually

follows within hours of the first convulsion. A variety of lesions have been described in snakes

dying with OPMV infections, including proliferative pneumonia, encephalitis, and pancreatic

hyperplasia (Jacobson et al., 1981).

In late 1994 in Australia, Hendra virus (HeV) caused an outbreak of severe respiratory

disease resulting in the death of 13 horses and their trainer (Murray et al., 1995), followed by

sporadic HeV outbreaks in horses and humans (Field et al., 2007a). Subsequent serosurveillance

of wild-caught wildlife showed evidence that flying foxes (genus Pteropus, order Chiroptera)

5

were the natural hosts of HeV (Field et al., 2007b). A closely related virus, Nipah virus (NiV)

from Malaysia caused severe febrile encephalitis and death in pigs and humans (Chua et al.,

2000), which spread to Bangladesh and India (Chadha et al., 2006; Hsu et al., 2004). HeV and

NiV were assigned as a genus, Henipavirus, in Paramyxovirinae (Eaton et al., 2006).

In 1997, another paramyxovirus, named Menangle virus (MenV) has been isolated in

Australia from still-born pigs with deformities (Philbey et al., 1998), and associated human

illness (Chant et al., 1998). Neutralizing antibodies against this virus could be detected in several

fruit bat species (Philbey et al., 1998). In 2000, Tioman virus (TioV) was isolated from urine

collected beneath a fruit bat colony on Tioman Island, Malaysia (Chua et al., 2001). Molecular

characterization revealed that MenV and TioV are closely related novel members of the genus

Rubulavirus (Bowden and Boyle, 2005; Chua et al., 2001; Chua et al., 2002). J-virus (JV),

isolated from wild mice in Australia, and Beilong virus (BeV), originally isolated from human

mesangial cells in China and subsequently detected in rat mesangial cells, may represent a new

group of paramyxoviruses (Basler et al., 2005; Jack et al., 2005; Jun et al., 1977; Li et al., 2006b;

Schomacker et al., 2004).

Recently, T. truncates parainfluenza virus type 1 (TtPIV-1) isolated from bottlenose

dolphins with severe respiratory illness (Nollens et al., 2008), and Atlantic salmon

paramyxovirus (ASPV) suffering from proliferative gill inflammation (Kvellestad et al., 2003;

Nylund et al., 2008) are also suggested to be classified in Paramyxovirinae. However, most of

these recently identified paramyxoviruses need to be further classified by the ICTV. For many of

the new and emerging viruses, the host range, pathogenicity and geographic distribution are

unknown.

6

1.4 Clinical Signs and Lesions

The clinical signs associated with paramyxovirus infections are essentially similar across

species. Many members of the family Paramyxoviridae were recognized as important respiratory

tract pathogens in many species of animals and humans, while some can also lead to neurological

symptoms. Parainfluenza virus 1 to 3 are major causes of croup, known as severe acute

laryngotracheobronchitis, and hPIV3 can also cause pneumonia and bronchiolitis, in infants and

children (Chanock, 1956; Chanock et al., 1958). hPIV4 is less severe and can cause mild upper

respiratory tract illness in young adults (Ruth A. Karron, 2007). hPIV1, 2, or 3 can produce

sensitive and permissive infections in hamsters experimentally. In addition, guinea pigs, cotton

rats, and ferrets are also semipermissive, however mice are poorly permissive (Ruth A. Karron,

2007). Chimpanzees and monkeys can also be infected with hPIV1-3 but only hPIV3 causes

symptomatic illness in chimpanzees and African green monkeys. Infection usually remains

subclinical and pulmonary pathology is minimal or undetectable in most experimental animals

due to the limited virus replication (Lamb and Parks, 2007).

Canine parainfluenza virus 2 (known as PIV5) is one of several etiologic agents of

kennel-cough (Appel and Percy, 1970). Infected dogs exhibit clinical signs ranging from dry

cough to pneumonia with fever, malaise, coughing with copious amounts of nasal discharge

(Appel and Percy, 1970; Rosenberg et al., 1971). A canine PIV was isolated from the

cerebrospinal fluid of a dog that experienced incoordination and posterior paresis (Evermann et

al., 1980), and this isolate induced hydrocephalus with central nervous system (CNS) depression

and severe inspiratory-expiratory dyspnea in dogs after experimental inoculation (Baumgartner

et al., 1982a; Baumgartner et al., 1982b).

7

Canine distemper is caused by the canine distemper virus (CDV) which is a contagious,

incurable, often fatal, multisystemic viral disease that affects the respiratory, gastrointestinal and

central nervous systems, and was the leading cause of death in unvaccinated dog of all ages

worldwide (Appel, 1969). CDV occurs among domestic dogs and many other carnivores,

including raccoons, skunks, foxes (Appel, 1969), and large cats such as lions, leopards, cheetahs,

and tigers (Myers et al., 1997a), but does not cause disease in domestic cats (Bart et al., 2000).

The development of a vaccine in the early 1960s led to a dramatic reduction in the number of

infected domestic dogs, and canine distemper occurs now only as sporadic outbreaks (Kapil et

al., 2008; Norris et al., 2006). Young puppies between 3 and 6 months old are most susceptible

to infection and disease, and are more likely to die than infected adults. Nonimmunized older

dogs are also highly susceptible to infection and disease (Appel, 1970).

bPIV3 was first isolated from nasal mucus of calves showing clinical signs of shipping

fever including rapid fever, respiratory symptoms, nasal discharge, cough, lacrimation,

conjunctivitis and inappetence (Andrewes et al., 1959; Reisinger et al., 1959). bPIV3 can also be

isolated from clinically normal cattle (Rosenberg et al., 1971). It is generally accepted that co-

infection of bPIV3 along with other viruses and Mannheimia/Pasturella species could result in a

clinical disease known as shipping fever (Battrell, 1995; Woods, 1968). A variety of factors such

as environmental temperature, transportation, hygiene, stocking density, co-mingling, host

immune status can contribute to increased susceptibility to secondary bacterial infection and

severity of clinical disease (Kahn and Line, 2005).

Mumps was a worldwide common childhood disease, characterized by painful salivary

gland swelling (95% of symptomatic cases), and orchitis in infected teenage and adult males

8

before introduction of the mumps vaccine (Philip et al., 1959). One third of MuV infections are

subclinical (Center for Disease Control (CDC) Mumps- United States, 1984-1986). Multiple

organs can be symptomatically involved during infection including testes, CNS, epididymis,

prostate, ovary, liver, pancreas, spleen, thyroid, kidneys, labyrinth, eyes, thymus, heart,

mammary glands, lungs, bone marrow, and joints (Carbone and Rubin, 2007). Virulent strains of

MuV commonly target CNS and result in seizures or remain asymptomatic (Azimi et al., 1969).

Pancreatitis (Feldstein et al., 1974), arthritis (Gordon and Lauter, 1984), abortion (Philip et al.,

1959), renal dysfunction (Utz et al., 1964), deafness (Kirk, 1987), obstructive hydrocephalus

(Bistrian et al., 1972), and transient electrocardiogram abnormalities (Arita et al., 1981) may also

occur.

?DV is one of the most important avian pathogens known. The clinical signs of NDV

varies depending on the tissue tropism, strain virulence and susceptibility of the avian order

(Cross, 1991). NDV strains display a spectrum of virulence ranging from mild, inapparent

infections to severe disease with high mortality (Brugh and Beard, 1984; Cross, 1991).

Viscerotropic velogenic NDV causes 100% morbidity and mortality and considered to be the

most devastating disease in poultry industry. Infected birds rapidly become anorectic and listless

with cyanotic and edematous combs and wattles, followed by severe respiratory distress and

conjunctivitis (Cross, 1991). Neurotropic velogenic NDV is another acute, generally lethal

infection of chickens of all ages affecting respiratory and neurologic tissues. It can casue 100%

morbidity but mortality is generally far less with extremes of 50 percent in adult birds and 90

percent in young chickens (Cross, 1991). Mesogenic NDV is less virulent and result in

respiratory signs, CNS signs and reduction in egg production (Cross, 1991). Lentogenic NDV is

9

milder than mesogenic NDV infection and CNS signs are rarely present (Brugh and Beard,

1984).

The records of paramyxoviruses isolated from pigs with encephalomyelitis are extensive

from many parts of the world including Japan (Sasahara et al., 1954), Canada (Greig et al.,

1971), and Israel (Lipkind et al., 1986). LPMV, responsible for blue eye disease, is the most

thoroughly studied neurotropic paramyxovirus isolated from pigs. LPMV was first isolated in

central Mexico in the early 1980s (Moreno-Lopez et al., 1986), and it has become endemic in

Mexico (Linne et al., 1992). Clinical signs include reproductive failure, interstitial pneumonia ,

encephalitis and corneal opacity in infected pigs (Stephano, 1990). Nursing piglets are most

susceptible. Morbidity is between 20% and 50% and mortality is between 87% and 90% (Linne

et al., 1992).

Hendra virus was discovered in 1994 in Australia and caused the death of 13 horses and

a trainer (Murray et al., 1995). In horses, infection caused pulmonary oedema and congestion,

while, in humans, hemorrhage and edema of the lungs, and encephalitis were seen. A total of

nine outbreaks of Hendra virus have occurred since 1994, all involving infection of horses. Four

of these outbreaks have spread to humans as a result of direct contact with infected horses.

?ipah virus was identified in 1999 when it caused an outbreak of neurological and respiratory

disease on pig farms in peninsular Malaysia, resulting in 105 human deaths and the culling of

one million pigs (Chua et al., 1999). Symptoms of infection from the Malaysian outbreak were

primarily encephalitic in humans and respiratory in pigs. Later outbreaks have caused respiratory

illness in humans, increasing the likelihood of human-to-human transmission and indicating the

existence of more dangerous strains of the virus (Chadha et al., 2006; Hsu et al., 2004).

10

Several paramyxoviruses have been isolated from rodents since 1960s. Mossman virus

(MoV) was first isolated from pooled organs of trapped native rats (Miller et al., 2003)and J-

virus (J-V) was isolated from trapped moribund Mus musculus (Jun et al., 1977) both in 1970s in

Queensland, Australia. Beilong virus (BeV) was first isolated from the human mesangial cell

line. However, the presence of BeV in a rat mesangial cell line used in the same laboratory prior

to the isolation from the human mesangial cells suggests it to be of rodent origin (Li et al.,

2006b). Tupaia paramyxovirus (TPMV) was isolated from the kidneys of an apparently healthy

tree shrew captured in Thailand (Tidona et al., 1999). Phylogenetic analysis places these four

viruses between or within the genera Morbillivirus, Respirovirus and Henipavirus, but the

taxonomic status is still unclear and they have not been officially classified into any of the

existing genera (Li et al., 2006b; Tidona et al., 1999).

Atlantic salmon paramyxovirus (ASPV) was first isolated in 1995 from Atlantic

salmon (Salmo salar L.) experiencing respiratory disease, proliferative gill inflammation (PGI)

and the outbreaks increased in past years (Nylund et al., 2008). PGI shows signs of inflammation

and proliferation and cell death of gill tissue in Atlantic salmon. Affected fish had reduced

growth rate and even mortality in severe cases (Nylund et al., 2008). ASPV was one of the

agents isolated from some PGI cases although the primary causative agent has not yet been

identified (Kvellestad et al., 2003; Kvellestad et al., 2005).

T. truncates parainfluenza virus type I (TtPIV-1) was isolated from 19-year old male

Atlantic bottlenose dolphin suffering from respiratory illness including raspy, foul-odored breath

and cream-colored exudates from the blowhole (Nollens et al., 2008). Foamy mononuclear cells

were observed in blood smear and neutrophils and monocytes were detected in blowhole swabs

11

(Nollens et al., 2008). Lung showed partial consolidation by percutaneous ultrasound.

Phylogenetic analysis classified TtPIV-1 in the cluster of Paramyxovirinae, genus Respirovirus,

and significant relationship with bPIV3 (Nollens et al., 2008).

1.5 Molecular Biology of Paramyxovirus

1.5.1 Virion Structure and Genome Organization

The family Paramyxoviridae has a typical negative-stranded RNA virus structural pattern

(Figure 1). The enveloped virus particle contains a left-handed, helically symmetrical

ribonucleoprotein core, called nucleocapsid (NC), which consists of non-segmented single-

negative-stranded viral RNA genome surrounded by nucleocapsid proteins and associated with

phosphoproteins and large polymerase protein (Lamb and Parks, 2007). The NC of

Paramyxovirinae, 18 nm in diameter, 1µm in length, and a pitch of 5.5 nm in diameter, is

enclosed by an outer lipoprotein envelope (Lamb and Parks, 2007). The helical NC of

paramyxoviruses presents “herring bone” morphology when viewed under an electron

microscope. Pneumovirinae subfamily can be differentiated from Paramyxovirinae subfamily

morphologically because they have narrower nucleocapsids (Lamb and Parks, 2007). The virions

of Paramyxoviridae are pleiomorphic in shape being spherical or filamentous (Figure 1). In

general, spherical virions are 150-350 nm in diameter (Lamb and Parks, 2007).

The genome length of family Paramyxoviridae ranges from 15,000 to 19,000 nucleotides

(nt), encoding six to ten tandemly linked genes in the order 3'-N-P/C/V-M-F-HN-L-5'.

Interestingly, the genome length of most members of Paramyxoviridae is divisible by six, known

12

as “rule of six” (Calain and Roux, 1993; Kolakofsky et al., 1998; Kolakofsky et al., 2005), i.e.

their genomes must be of polyhexameric length (6n+0) to replicate efficiently. Each nucleoprotein

subunit associates with six nucleotides and that makes the RNA chain length as an even multiple

of six when completely encapsidated. There is a ~50 nt 3’ extracistronic leader region and a 50 to

171 nt 5’ extracistronic trailer region (or [–] leader) at the two ends of the genome. The leader and

trailer are considered to be cis-acting signals and are essential for transcription and replication.

Between the gene boundaries, there are intergenic regions (IGS), which are strictly conserved

trinucleotides in Respirovirus, Henipavirus and Morbillivirus but quite variable in length for the

Rubulavirus and Pneumovirinae, ranging from 1 to 56 nt. The newly discovered

paramyxoviruses, such as TioV, have an even longer IGS, which may be up to 70 nt in length.

The glycoprotein complexes are stalk-like spikes on the surface of virions. Fusion protein

(F) and an attachment protein, called hemagglutinin-neuraminidase (HN) or hemagglutinin (H) or

glycoprotein (G) depending on the virus genera (Lamb and Parks, 2007), are the major

components of the spikes. The F and HN/H/G proteins may be in the form of trimers (F) and

tetramers (attachment protein), respectively. The envelope glycoproteins are responsible to

mediate virus attachment and penetration during infection. The internal helical nucleocapsid core

contains the RNA genome and nucleocapsid (N), phospho- (P) and large (L) polymerase proteins.

Another structural protein, matrix (M) protein, is located between the envelope and the core, and

is important in virion architecture and in infection process. Accessory proteins are mostly

generated from overlapping open reading frames within P gene transcriptional units by a process

called RNA editing or by alternative translation initiation. These accessory proteins are important

in viral morphogenesis, RNA synthesis, and pathogenesis (Lamb and Parks, 2007).

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1.5.1.1 %ucleocapsid Protein

The nucleocapsid protein (N) is one of the most abundant proteins in paramyxoviruses

and is present as the first transcribed gene, adjacent to the leader sequence at the 3’ end of viral

genomic RNA for all paramyxoviruses, except the pneumoviruses, in which two more

nonstructural genes, NS1 and NS2 will be transcribed first. The N protein is composed of 489 to

553 amino acids (aa) with the predicted molecular weight of 53-58 kDa. The N-terminal region

of the N protein is involved with encapsidation of viral RNA, coats full-length of viral [-] sense

genomic and [+] sense antigenomic RNAs to form the helical nucleocapsid. This helical

structure serves several functions including protection from nuclease digestion, alignment of

distal RNA segments to create a functional 3’ end promoter and providing interaction with P and

L proteins during transcription and replication, and association with M protein during virus

assembly (Lamb and Parks, 2007).

1.5.1.2 The P gene and its encoded proteins

Paramyxovirinae P gene encoding multiple viral proteins can be considered as a

remarkable example of exploiting the coding capacity of viruses. Sendai virus (SeV) P gene can

direct the expression of at least seven polypeptides, including P, V, W, C’, C, Y1 and Y2. Other

paramyxoviruses may express fewer proteins from the P gene, however this capability is always

seen. Generally, two mechanisms involve production of multiple mRNA species from the P

gene. One is known as alternative initiation, by which the family of C proteins is produced from

an alternative initiation translation codon. The second mechanism involves pseudotemplated

14

insertion of multiple G residues at a specific position, known as mRNA editing site, to produce

mRNAs of P, V proteins and virus-specific proteins variously called W/I/D proteins, depending

on the virus genus. P, V, W/I/D are a set of proteins that are amino co-terminal and the mRNAs

are only different by inserted G nucleotides that shift the translation reading frame after the

insertion site (Lamb and Parks, 2007).

Viral accessory proteins and their interactions with the host

Type I interferon (IFN) is one of the most important antiviral response which has two

general phases: a primary transcriptional phase of induction of IFN synthesis, and a secondary

transcriptional phase through type I IFN signaling pathway (Biron and Sen, 2007).

Paramyxovirus accessory proteins can counteract the host cell IFN pathways at both levels

(Biron and Sen, 2007). The paramyxovirus V protein cysteine-rich domain can limit dsRNA-

induced activation of the IFN-beta promoter by direct interaction with melanoma differentiation-

associated gene 5 (MDA5) (Komatsu et al., 2004). The expression of C proteins in SeV and W

proteins in NiV can also reduce IFN-beta promoter activation in response to dsRNA (Komatsu et

al., 2004). IFN signaling is initiated by binding of secreted IFN and its cognate receptor on the

cell surface leading to phosphorylation of latent transcription factors, STAT1 and STAT2, which

are signal transducers and activators of transcription (Karron and Collins, 2007; Lamb and Parks,

2007). The V protein of some paramyxoviruses such as PIV5, MuV, SV41, hPIV2, NDV can

target one of the STATs for degradation to block the IFN signaling (Biron and Sen, 2007) while

some like henipaviruses and measles virus can form a V and STAT protein binding complex and

prevent its translocation to the nucleus (Palosaari et al., 2003; Rodriguez et al., 2003; Shaw et al.,

15

2004). The C protein of respiro- and morbilliviruses are involved in blocking IFN signaling by a

different mechanism (Garcin et al., 2001; Garcin et al., 2002; Gotoh et al., 2003; Takeuchi et al.,

2001). SeV and hPIV3 can alter STAT phosphorylation patterns (Gotoh et al., 2003), interact

with STAT1 (Garcin et al., 2001; Takeuchi et al., 2001), and induce ubiquitination and

degradation of STAT1 in some mouse cells (Garcin et al., 2002).

Phosphoprotein

The paramyxovirus P protein is generally 400 to 600 aa long. It is heavily phosphorylated

at serine and threonine residues predominantly within the N-terminal region (Lamb and Parks,

2007). The C-terminal module and N-terminal domain of P protein play important roles as

polymerase cofactor and in nascent chain assembly, respectively. The C-terminal polymerase

cofactor module is conserved in the predicted secondary structure for all Paramyxovirinae

members. It contains domains for P-P multimerization, for L protein interactions and for N-RNA

template binding (Horikami et al., 1992). The N-terminal region is believed to act as a chaperone

to facilitate interactions with unassembled N in order to prevent N aggregation and to prevent

uncontrolled encapsidation of non-viral RNA (Curran et al., 1995).

V protein

The V protein is a 25 to 30 kDa polypeptide that is amino co-terminal with P protein but

with a variable C-terminal domain. The C-terminal V-specific domain is highly conserved

among paramyxoviruses with invariantly spaced histidine and cysteine residues forming a

16

domain (cys-rich domain) that binds two zinc molecules per V protein (Fukuhara et al., 2002; Li

et al., 2006a; Liston and Briedis, 1994; Paterson et al., 1995). The V protein plays important

roles in virus replication (Baron and Barrett, 2000; Curran et al., 1991), functions as a negative

regulator to inhibit RNA synthesis (Baron and Barrett, 2000; Delenda et al., 1997; Durbin et al.,

1999), and inhibits host cell antiviral response by interacting with cellular proteins (Andrejeva et

al., 2002; Lin et al., 1998).

W/D/I protein

The W and D proteins are expressed by inserting 2 G residues at the P gene mRNA editing

site in respiro-, morbilli-, and henipaviruses. The W protein in SeV is found to interact with

unassembled N0, suggesting an inhibitory role in viral RNA synthesis (Horikami et al., 1996). In

PIV3, insertion of 2 G residues at the editing site produces a protein called D protein (Galinski et

al., 1992). In rubulaviruses, insertion of one or four G residues during RNA editing produces I

protein (Paterson and Lamb, 1990; Thomas et al., 1988). The role of W/D/I in viral growth cycle

has not been understood (Lamb and Parks, 2007).

C protein

The C proteins are generated using alternative translation initiation codons in Respiro-

,Henipa-, Morbillivirus (Lamb and Parks, 2007). In SeV, C’, C, Y1 and Y2 comprise a nested set

with a shared C-terminal. The C proteins are small basic polypeptides that may involve in the

17

viral growth cycle, control of viral RNA synthesis (Lamb and Parks, 2007), counteracting host

cell antiviral pathways (Komatsu et al., 2004) and facilitating the release of virus from infected

cells (Garcin et al., 1997; Kato et al., 2001).

1.5.1.3 Matrix Protein

The M protein is the most abundant protein in the virion, comprising of 341 to 375

residues with a molecular weight of 38.5-41.5 kDa. The net charge at neutral pH is +14 to 17

making it a basic protein. Although there is no transmembrane domain that has sufficient length

to span a lipid bilayer, M protein is somewhat hydrophobic and peripherally associated with

membranes (Lamb and Parks, 2007). The M protein is considered to be the central organizer of

viral morphogenesis interacting with the cytoplasmic tails of integral membrane protein, F and

HN, the lipid bilayer and the nucleocapsids (Blumberg et al., 1984; Cathomen et al., 1998;

Sanderson et al., 1993; Schmitt et al., 1999; Stricker et al., 1994).

1.5.1.4 L Protein

The L protein, located at the most promoter distal part in the genome, is the least abundant

protein in virus particles. A paramyxovirus particle contains only ~50 copies of L (Lamb et al.,

1976). Overexpression of L protein may inhibit virus growth (Banerjee and Barik, 1992). It is an

essential subunit of paramyxovirus RNA-dependent RNA polymerase (RdRP). The L gene

consists of ~2200 aa with a molecular weight of 220-250 kDa. The L protein possesses all the

18

enzymatic activities needed for polymerization, 5’ end capping, methylation and 3’ end

polyadenylation of mRNA (Grdzelishvili et al., 2005; Ogino et al., 2005). The L protein

associates with P protein to form the active viral polymerase and the polymerase complex can

recognize the helical N-RNA template (Hamaguchi et al., 1983; Poch et al., 1990).

1.5.1.5 Viral Glycoproteins

Fusion Protein

The F glycoprotein is a type I integral membrane protein mediating viral penetration by

fusion between the virion envelope and the host cell plasma membrane at neutral pH. The F

protein is composed of 540 to 580 aa residues. It is synthesized as an inactive precursor, F0,

which must be proteolytically cleaved to produce the active fusion protein, which consists of

disulfide-linked F1 and F2 polypeptides to be biologically functional (Scheid and Choppin, 1974).

This cleavage event is required for progeny virions to become infectious (Garten et al., 1980;

Nagai et al., 1976). The cleavage of F0 is a candidate to be a key determinant for infectivity and

pathogenicity for certain viruses. The Paramyxoviridae has a cleavage signal sequence located

at the N-terminal of F with multibasic or single basic residue at the cleavage site. The cleavage

of F containing multibasic residues at the cleavage site occurs intracellularly by furin, a

subtilisin-like endoprotease (Klenk and Garten, 1994), while F with a single basic residue at the

cleavage site must be expressed at the cell surface and incorporated into released virions and

then can be cleavage activated by the addition of exogenous protease (Scheid and Choppin,

1974). For NDV, the nature of the cleavage site correlates with the virulence of the virus strains.

19

The ones with multibasic residues at the cleavage site show virulence and readily disseminate

through the host whereas strains with single basic residue at the cleavage site are avirulent and

tend to be restricted to the respiratory and alimentary tracts where the necessary secreted

protease is present (Nagai and Klenk, 1977). The C-terminal of F has a hydrophobic

transmembrane (TM) domain that anchors the protein into the membrane leaving a short

cytoplasmic tail (~20-40 residues). A 4-3 (heptad) pattern of hydrophobic repeats, designated

heptad repeats HRA and HRB can be found between the fusion peptide and the TM domain, with

approximately 250 aa apart from each other (Lamb and Parks, 2007). Biophysical data and

crystallographic studies have shown that HRA and HRB can form a helical hairpin or six-helix

bundle (6HB) structure (core trimer) in F protein homotrimers and this 6HB formation is tightly

linked to the merger of lipid bilayers and is believed to couple the free energy released on protein

refolding to membrane fusion (Melikyan et al., 2000; Russell et al., 2003) .

Attachment Protein

Attachment protein is another glycoprotein in Paramyxoviridae involved in cell

attachment process, which is also an integral membrane protein (Lamb and Parks, 2007). The

attachment protein of Respirovirus, Avulavirus and Rubulavirus binds to cellular sialic acid-

containing receptors and are capable of agglutinating erythrocytes. Besides, these attachment

proteins also have neuraminidase activity that cleaves sialic acid from the progeny virus particles

to prevent viral self-aggregation (Scheid and Choppin, 1974) and enable release of virions from

the cell membrane. Thus, the protein has been designated hemagglutinin-neuraminidase (HN).

HN is a type II membrane protein composed 565 to 582 residues that span the membrane once. It

20

contains an N-terminal cytoplasmic tail, a single N-terminal transmembrane domain (TM

domain), a membrane-proximal stalk domain that supports a C-terminal globular head domain.

The globular head domain contains both receptor binding and enzymatic activity (Hiebert et al.,

1985a). HN presents on the surface of virions and infects cells as a tetramer consisting of pairs of

homodimers (Thompson et al., 1988).

In Morbillivirus, attachment protein, H, can cause agglutination of primate erythrocytes

but lacks neuraminidase activity. This restricted host range, representative in measles virus for

primate cells, makes the sialic acid unlikely to be the primary receptor. It is now believed that

CD150, also known as signaling lymphocyte activation molecule (SLAM), is the principal

receptor for unadapted isolates of lymphotropic measles virus (Oldstone et al., 2002; Yanagi et

al., 2002). The attachment protein, G, in Henipavirus has neither hemagglutinating nor

neuraminidase activity. Ephrin B2 is considered as the cellular receptor for both HeV and NiV.

The widespread occurrence of ephrin B2 in vertebrates, particularly in arterial endothelial cells

and in neurons, provides an explanation for the wide host range of henipaviruses and their ability

to cause systemic infections (Eaton et al., 2006).

The Pneumovirus, RSV, does not cause detectable HA. The cellular receptor for RSV is

not completely understood yet, but it is believed that it involves interactions with heparan sulfate,

a glucosaminoglycan that is part of the extracellular matrix. The structure of G protein in

Pneumovirus is quite different from the attachment protein of Paramyxovirinae. Both membrane

bound and proteolytically cleaved soluble forms can be found in infected cells. Extensive

carbohydrate modification is one distinguishing feature of the G protein of RSV, which makes

the protein migration on SDS-PAGE with a dramatic increase in molecular weight than that of

21

the predicted polypeptide. These studies also show that G protein is not essential for virus

assembly or growth in tissue culture or animals, although, it does confer a growth advantage

(Lamb and Parks, 2007). Interestingly, the observation that G protein deletion mutant of RSV

and HN deleted virus-like particle (VLP) of SeV can infect cells via the asialoglycoprotein

receptor suggests that some paramyxovirus F proteins may also show binding activity (Leyrer et

al., 1998).

For most paramyxoviruses, the receptor binding protein (HN, N, or G) is required to

mediate the fusion reaction (Lamb et al., 2006; Morrison, 2003). The precise role of HN/N/G in

stimulating the F conformational change remains to be understood. However, HN may execute

its function by stabilizing the F prefusion stalk in a receptor-dependent manner (Lamb and Parks,

2007). Several models were studied to rationalize the involvement of HN in fusion promotion

(Lamb, 1993; Lamb and Parks, 2007). A model in NDV proposed a second sialic acid binding

site in addition to the active site (Crennell et al., 2000). The location of this new site at the HN

dimer interface, together with observation that changes in the HN structure that appear to be

generated by catalysis alters the association of the HN dimer or tetramer and may also propagate

a change in the stalk region that triggers the fusion protein, suggests a model for how sialic acid

binding by HN might trigger conformational changes in F (Zaitsev et al., 2004).

Other Envelope Proteins

The rubulaviruses PIV5 and mumps virus contain a small gene located between F and

HN genes, designated “small hydrophobic” (SH) gene (Hiebert et al., 1985b; Hiebert et al.,

22

1988). Mutants lacking SH of PIV5 is attenuated in vivo, and can induce apoptosis in L929 and

MDCK cells through a tumor necrosis factor alpha-mediated extrinsic apoptotic pathway (He et

al., 2001; Lin et al., 2003). Mumps SH may have a similar role as in PIV5 (Wilson et al., 2006).

Avian paramyxovirus type 6 (APMV6), a member in Avulavirus also contains the SH gene in its

genome. However, NDV and other members in the genus Avulavirus do not encode SH. NDV

was used to be classified in Rubulavirus. The presence of the SH gene in APMV6 to some extent

justifies the close relationship between this two genera and also suggests that APMV-6 might

play an intermediate role between the evolution of Avulavirus and members of the genus

Rubulavirus (Chang et al., 2001). Members of the Pneumovirinae also encode a SH protein,

however, it does not necessarily mean a commonality in function with Rubulavirus SH and the

role of SH in the RSV life cycle is not completely understood (Lamb and Parks, 2007). M2 gene,

encoding two partially overlapping ORFs, M2-1 and M2-2, can be found in members of the

Pneumovirinae and two more nonstructural proteins, NS1 and NS2, can be exclusively found in

RSVs. M2-1 and M2-2 play an important role in transcription and RNA replication

(Bermingham and Collins, 1999; Fearns and Collins, 1999) and NS1, NS2 show importance in

suppression of type I interferon induction and RNA synthesis (Atreya et al., 1998; Bossert et al.,

2003; Spann et al., 2004; Teng and Collins, 1999; Whitehead et al., 1999).

J-V possesses two unique proteins that are not observed in other paramyxoviruses.

Putative transmembrane protein (TM) is 258 aa long protein located between SH and G protein

genes in J-V genome (Jack et al., 2005). J-V “G” gene has an extraordinarily long putative 3’

UTR in which a second open reading frame (ORF), termed ORF-X, has been identified

commencing immediately after the stop codon for the putative G protein (Jack et al., 2005).

ORF-X encodes a putative protein of 704 aa within which the first methionine is the 30th aa

23

residue (Jack et al., 2005). No significant amino acid sequence homology was identified between

TM or X and other known proteins in sequence data banks by BLASTp search and this

sequencing based prediction need to be experimentally verified (Jack et al., 2005).

1.5.2 Strategy of replication

Paramyxoviruses encode and package their own RNA dependent RNA polymerase

(RdRP). The synthesis process is activated only after the virus being uncoated in the infected cell

and the replication occurs in the cytoplasm. In cell cultures, a single cycle of growth takes ~14-

30 hours for most paramyxoviruses, but with virulent NDV strains, this can be as short as 10

hours. Viral intracellular replication begins with viral RdRP packaged in the virion transcribing

the N encapsidated genome RNA into 5’ capped and 3’ polyadenylated mRNA. This virion RNA

being encapsidated with the N and associated with P and L proteins forms the transcriptase

complex which is the minimum transcriptional unit of paramyxovirus (Lamb and Parks, 2007).

The transcription signals are located at gene-start (GS) and gene-end (GE), which border

individual genes. Starting from the 3’ end of the genome, viral RdRP begins the transcription

process in a sequential manner to produce capped and polyadenylated viral mRNAs by

terminating and reinitiating at each gene junction, also known as a start-stop-restart mechanism.

A controlled attenuation of transcription occurs in each intergenic region, where a fraction of

elongating polymerases are released from the genomic templates, producing mRNA levels that

progressively decrease from N to L (Lamb and Parks, 2007), resulting in a transcriptional

gradient, commonly referred to as “polarity of transcription”. After primary transcription and

translation, as unassembled N protein accumulates, genomic RNA synthesis will be activated.

24

The RdRP will then ignore all the gene junctions to produce an exact complementary antigenome

chain in a fully assembled nucleocapsid.

1.6 Parainfluenza viruses

1.6.1 History of parainfluenzaviruses

Human parainfluenza virus contains 4 serotypes, hPIV1 to hPIV4. They were first

recovered from infants and children with lower or upper respiratory tract diseases, referred as

“croup associated” (CA) viruses at that time (Chanock, 1956; Chanock et al., 1958; Johnson et

al., 1960). The hPIV1, 2, and 3 viruses are second to human respiratory syncytial virus (hRSV)

as a common cause of lower respiratory tract disease in young children, which are major causes

of croup (hPIV1 to 3) and pneumonia bronchiolitis (hPIV3). However, hPIV4 causes less severe

disease at less frequency. Bovine parainfluenza virus type 3 is one of the major pathogens in

bovine shipping fever complex and it is a close relative of hPIV3.

Sendai virus (SeV) was first isolated from mice inoculated with an autopsy specimen

from an infant with respiratory disease and it appeared to be a murine relative of hPIV1 rather a

natural human pathogen. Simian virus 5 (SV5) and Simian virus 41 (SV41) were all originally

isolated from primary monkey kidney tissue culture (Hull et al., 1956; Nishio et al., 1990). Both

could be detected in a proportion of human population indicating capability for human infection.

SV5 can also cause croup in dogs and is a canine relative of hPIV2. However, SV41 shows more

close relationship to hPIV2 than SV5 in antigenic and structural properties (Tsurudome et al.,

1990).

25

Newcastle Disease was recognized as an emerging poultry disease in Indonesia and

England in 1926 (Doyle, 1927) and is now known as serotype 1 of nine distinct serotypes

(Alexander, 1982; Alexander et al., 1983) of avian PIV (now redesignated avian

paramyxoviruses (APMV)). Besides, mumps virus was also historically considered as a member

of PIV group due to the morphologic and antigenic relativities. These viruses were classified in

a single genus historically and then identified into distinguished genera after the addition of

related viruses and molecular confirmation. Nowadays, it is widely acknowledged that within

PIV group there are two divisions of mammalian PIV genera: Respirovirus and Rubulavirus, and

one avian viruses genus Avulavirus (Ruth A. Karron, 2007).

1.6.2 Bovine parainfluenzavirus type 3

1.6.2.1 bPIV3 as a pathogen

bPIV3 is a respiratory pathogen associated with bronchopneumonia in cattle and it is one

of the important pathogens of the bovine respiratory disease syndrome called “shipping fever”,

which causes severe economic losses to the cattle industry in North America and other parts of

the world. Clinical signs of shipping fever include rapid fever, respiratory symptoms, nasal

discharge, cough, lacrimation, conjunctivitis and inappetence (Andrewes et al., 1959; Reisinger

et al., 1959). bPIV3 can be isolated from clinically normal cattle, (Reisinger et al., 1959). Gross

lesions in cattle experimentally infected with bPIV3 include consolidation of the anteroventral

portions of the lungs (Bryson et al., 1979; Marshall and Frank, 1975), congestion of respiratory

mucosa, and enlargement of the bronchial and retropharyngeal lymph nodes (Marshall and

26

Frank, 1975). Mononuclear and polymorphonuclear cells and multinucleated giant cell can be

observed in affected lung lobes by histopathologic study (Tsai and Thomson, 1975). It is

generally accepted that co-infection of bPIV3 along with other viruses and

Mannheimia/Pasturella species in addition to a variety of other factors as environmental

temperature, transportation, hygiene, stocking density, co-mingling and host immune status

could cause shipping fever and can contribute to increased susceptibility to secondary bacterial

infection and severity of clinical disease (Battrell, 1995; Woods, 1968).

1.6.2.2 Cross-species infection of bPIV3

PIV3 infections have been serologically demonstrated in a wide variety of mammals

including cattle, human, sheep (Lyon et al., 1997), goats (Yener et al., 2005), bison (Zarnke and

Erickson, 1990), guinea pigs (Ohsawa et al., 1998), black and white rhinoceros (Fischer-

Tenhagen et al., 2000), moose (Thorsen and Henderson, 1971), bighorn sheep (Parks et al.,

1972) and camels (Eisa et al., 1979). A novel parainfluenza virus isolated from bottlenose

dolphin (Nollens et al., 2008) was phylogenetically closely related to PIV3.

Besides, cross-species infection has also been reported in numerous instances, including

hPIV3-GPv isolated from lung homogenates of sentinel guinea pigs placed together with

seropositive asymptomatic individuals (Ohsawa et al., 1998), and bPIV3 in a child experiencing

pneumonia (Ben-Ishai et al., 1980). bPIV3 in lambs can cause rapid, shallow respiration,

dyspnea and sporadic coughing (Stevenson and Hore, 1970). No respiratory signs were observed

in calves experimentally infected with ovine PIV3 (Stevenson and Hore, 1970). Macroscopic

27

lesions of pneumonia were present in bPIV3 inoculated lambs and ovine PIV3 inoculated calves

(Stevenson and Hore, 1970).

1.6.2.3 Virion Structure and Genome organization

PIV3 has spherical to pleiomorphic virions of approximately 150-200 nm in diameter and

morphologically indistinguishable from paramyxoviruses (Karron and Collins, 2007). The

genetic organization of bPIV3 is very similar to hPIV3, composed of 6 genes in the order of 3’-

N-P-M-F-HN-L-5’, and the overall length and nt and amino acid sequences are also broadly

conserved among PIV3 viruses. There is a high degree of identity between bPIV3 and hPIV3,

ranging from 58.6% for P protein to 89.7% for M protein (Bailly et al., 2000). They also share

extensive sequence identity in their cis-acting noncoding regulatory elements, 3’ leader (86.3%),

5’ trailer (93.2%) and trinucleotide intergenic sequence (100%). All bPIV3 and hPIV3 have a

semi-conserved gene-start sequence and semi-conserved U-rich gene-end sequence.

Interestingly, majority of variable amino acid residues found in bPIV3 and hPIV3 showed

polymorphisms, either showing host specific or variable within the species at the given position

(Bailly et al., 2000; Coelingh and Winter, 1990; Swierkosz et al., 1995). These residues might be

products of distinct selective pressures during PIV3 evolution in their respective host. The host-

specific sequences are likely responsible for the host range differences and growth efficiency in

different hosts. Using reverse genetics to import these sequences from bPIV3 into hPIV3 was

shown to be very useful in the development of vaccines (Bailly et al., 2000).

28

The complete nucleotide sequence for four isolates of bPIV3 has previously been

determined (Bailly et al., 2000; Horwood et al., 2008; Sakai et al., 1987; Suzu et al., 1987). With

the increasing availability of the sequence information, two distinct bPIV3 genotypes were

proposed, designated as bPIV3-a and bPIV3-b (Horwood et al., 2008). However, the genotype b

is an isolated variant lineage that has only been identified in Australia and could hypothetically

be a lineage from a strain that recently crossed from another host species into cattle (Horwood et

al., 2008). The newly identified genotypes have implications for the development of bPIV3

molecular detection methods and may also impact on bPIV3 vaccine formulations.

1.6.2.4 Antigenic relatedness with other parainfluenzaviruses

bPIV3 and hPIV3 shared approximately 25% antigenic relatedness. The Kansas/1562/84

(Ka) and Shipping Fever (SF) strains of bPIV3 replicated 100 to 1000 times less efficiently than

hPIV3 in the upper and lower respiratory tract of rhesus monkeys and chimpanzees compared

with hPIV3 (van Wyke Coelingh et al., 1988). Thus, bPIV3 was evaluated as a live vaccine

against hPIV3. The bPIV3-Ka strain was highly attenuated in humans and was used in clinical

trials as a candidate vaccine against hPIV3 (Karron et al., 1996). Although intranasally

administered bPIV3 vaccine gave seroconversion rate to bPIV3 (57–65%), it did not give a

robust response to the heterotypic human strain. However, bPIV3 vaccine being used as an

attenuated backbone for insertion of human PIV3 HN and F proteins and F protein of RSV is

promising and the effectiveness of this vaccine against both PIV3 and RSV challenge has been

demonstrated in African green monkeys (Sato and Wright, 2008).

29

Monoclonal antibodies (Mabs) were used to identify the antigenic properties of hPIV3

and bPIV3 (Coelingh et al., 1986; van Wyke Coelingh et al., 1985). HI-Mabs, which are Mabs

that have HI and neutralizing activities, can define 11 operationally unique HN epitopes which

are organized into two topologically nonoverlapping antigenic sites (A and B) and a third

bridging site (C) (van Wyke Coelingh et al., 1985). Using HN-Mabs, which has no known

biological activity, three more sites (D to F) were identified (Coelingh et al., 1986). Two

epitopes in site A and all epitopes in sites D and F are highly conserved among human clinical

isolates examined as well as bovine strains (Coelingh et al., 1986). Nucleotide substitutions in

the HN genes of antigenic variants selected with NI-Mabs were identified representing epitopes

in site A, which are shared by human and bovine PIV3. The deduced amino acid substitutions in

the variants were located in separate hydrophilic stretches of HN residues which are conserved in

the primary structures of the HN proteins of both human and bovine PIV3 strains (Coelingh et

al., 1986).

1.6.2.5 Diagnostic methods for bPIV3

A diagnosis of bPIV3 requires laboratory confirmation. Virus may be isolated when

animals that are in the incubation or acute phases of infection are sampled. In live animals, virus

can be isolated from nasopharyngeal swabs or broncho-alveolar washings, and in dead animals,

from the lungs and trachea and their associated lymph nodes. The virus can be grown in a variety

of cell cultures, such as bovine and ovine fetal lung, kidney and testis, and as well as in cell lines,

such as MDBK and RK-13. Cytopathic effects are characterized by syncytia, intracytoplasmic

and intranuclear inclusion bodies and cell destruction. Antigen detection methods such as

30

immunofluorescence, immunoperoxidase staining or enzyme immunoassay can be used (Haines

et al., 1992). RT-PCR-based diagnostic tests enable accurate and timely detection of bPIV3 from

nasopharyngeal swabs. Seroconversion can be detected 7-10 days following the onset of clinical

signs by HI or virus neutralization or plaque reduction tests.

1.7 Swine Paramyxoviruses

1.7.1 History

The first record of the outbreak of one of two swine paramyxoviruses, ISU92, was

described by Janke et al. (1992). A group of 400 pigs in a continuous-flow finishing barn showed

signs of respiratory and CNS disease. Sick pigs showed mild cough with temperature from 103

to 104.5 °F. Injectable dexamethasone and tylosin were administered and total of seventeen pigs

died within four days after the first appearance of clinical signs. Continued treatment with a

combination of penicillin, dexamethasone, spectinomycin and atropine were used and no more

deaths occurred. Wet, heavy, congested lungs with small areas of consolidation on the ventral

tips of the lung lobes were observed at necropsy.

The disease continued to spread throughout the operation and infected the second

finishing all-in/all-out unit. The entire 400 pigs in the finishing barn were infected within a few

days. Severe dyspnea, coughing and CNS disturbance were observed. Several sick pigs showed

persistent squealing, head pressing, whole body tremors, and hind-limb ataxia (Janke et al.,

2001). In the breeding, farrowing, nursery and grower buildings, located ¼ mile north of the

finishing barns, nursery age pigs showed dyspnea and less cough than finishing pigs; clinical

31

signs in nursing piglets in farrowing barn were more severe; the grower buildings were only mild

affected; few sows and gilts became dyspneic, two or three abortions and two premature

farrowing occurred, however, the cause was not investigated. Injectable antimicrobials were

administered to all affected pigs immediately after first onset of clinical signs and there were no

more death losses due to the outbreak (Janke et al., 2001).

1.7.2 Laboratory Examination

Two live pigs from the second finishing barn were sent to the Veterinary Diagnostic

Laboratory of Iowa State University. CNS disorder was the major signs observed similar to those

seen during the first outbreak. Pigs exhibited depression, whole body tremors, continuous

squealing, and rear-end ataxia. No respiratory symptoms were observed. 20% of lung volume

showed bilateral consolidation on ventral portions of cranial and middle lung lobes. No gross

lesions were noticeable in brain or other internal organs. In histopathologic examination,

moderate to severe bronchointerstitial pneumonia with thickening of alveolar septa containing

mixed inflammatory cells and swollen pneumocytes and endothelial cells were observed. Mild

diffuse gliosis and widespread, mild lymphocytic perivasculitis were seen in brain from both

pigs.

No pseudorabies (PRV), Swine influenza virus (SIV), porcine reproductive and

respiratory syndrome virus (PRRSV), porcine respiratory coronavirus (PRCV) were detected by

either virus isolation or fluorescent antibody test. No significant bacteria were recovered from

the collected tissue samples. Paramyxo-like virus particles were isolated from fetal porcine

32

kidney (FPK) cell culture, which was inoculated with a pooled tissue homogenates. Cytopathic

effect (CPE) was observed in infected cell cultures from second passage onwards (Janke et al.,

2001).

1.7. 3 Pathogenicity studies

Specific pathogen free (SPF) pigs were inoculated with the lung homogenate

(Battrell,1995). No clinical illness, gross or microscopic lesions were observed. No antibody

against ISU92 was detected in the serum of before inoculation or at necropsy. Experimental

inoculation of three-day-old gnotobiotic piglets with cell-culture propagated ISU-92 showed

temporary and slight increase of rectal temperature in some infected pigs. Irregular tan-gray

discoloration was observed in the lung lobes of infected pigs. Lungs with thickened alveolar

septa and proliferation of pneumocytes and brain with glial nodules were observed on

microscopic examination (Janke et al., 2001).

1.7.4 Prevalence studies

To identify the extent of prevalence of swine paramyxoviruses in USA, a virus

neutralization test was developed (Battrell, 1995). Antibodies against ISU92 strain were detected

in the serum of SPF pigs experimentally infected with ISU92 by 7 days post inoculation (DPI),

and remained at detectable levels for the duration of the trial, 24 weeks PI. A seroprevalence

study was performed with 876 serum samples collected from 36 swine farms in Iowa between

33

1988 and 1989. Only 6 samples, representing 5 swine herds, were shown to have anti-ISU92

antibody titers and the seroprevalence of sPMV was believed to be very low (Battrell, 1995).

1.7.5 Texas81 history

Texas81 virus was isolated from the brain of pigs that exhibited respiratory and

neurological disease in Texas State, in 1981 (cited in (Janke et al., 2001). Further information on

the history of this virus is not available.

1.8 Paramyxoviruses as vaccine vector

The first reverse genetic system of nonsegmented negative-strand RNA viruses (NNSV)

was established in 1994, recovering infectious rabies virus from cloned cDNA (Schnell et al.,

1994) (Figure 2). This milestone event made it possible to explore the great potential of NNSVs

to be developed as vaccine vectors (Bukreyev et al., 2006). NNSV as vaccine vectors have many

advantages to become promising viral vector candidates. First, a large number of well-

understood NNSVs belong to this group, such as NDV, and Vesicular stomatitis virus (VSV).

Some of them have the host range restrictively exclusive in human or much attenuated in

primates making them widely acceptable for immunization in human population (Bukreyev et

al., 2006). Many NNSV can induce cell-mediated protective immune responses, systemic IgG

antibody as well as effective local IgA immune response via intranasal infection. Therefore, the

vaccines could be intranasally administrated which will be safer and easier during an epidemic

34

and especially superb in respiratory tract infection, such as SARS and influenza (Bukreyev et al.,

2006). Due to the nature of NNSV replication strategy and genome organization, the possibility

of vaccine genome integrating into host genome and recombination between vaccine and

circulating viruses is extremely low (Bukreyev et al., 2006).

Three strategies were generally used (Figure 3): (i) Wild-type NNSV expressing foreign

genes. Insert a foreign gene into a wild-type NNSV, which is either a naturally attenuated due to

the host range restriction or a genetically engineered mutant. (ii) Antigenic chimeric viruses. A

construct of the chimeric virus use foreign gene/genes in a pathogenic virus to replace the major

protective surface antigen (s) of the vector. (iii) Antigenic chimeric virus expressing additional

foreign gene/genes. It is a combination of the first two strategies that are commonly employed to

produce bivalent vaccines (Bukreyev et al., 2006).

Antigenic chimeric virus B/HPIV3 is an example of application of the second strategy. It

uses bPIV3-Ka, attenuated in human due to the host range restriction (Bailly et al., 2000;

Skiadopoulos et al., 2003a; van Wyke Coelingh et al., 1988), as vector backbone with the

replacement of F and HN glycoprotein from hPIV3, and has been evaluated clinically by the

National Institutes of Allergy and Infectious Diseases as vaccine against hPIV3 (Haller et al.,

2000; Pennathur et al., 2003). Besides, bPIV3 as backbone for replaced F and HN of hPIV3 and

inserted F of RSV was also constructed. The effectiveness of this vaccine against both PIV3 and

RSV challenge has been demonstrated in African green monkeys and is being evaluated in

clinical trials (Sato and Wright, 2008).

35

1.9 Objectives

As new viruses emerge that cause severe disease in pigs and have the potential to cross

species barrier and infect other hosts including humans, it is important that these new viruses

need to be studied further. As a NNSV, the sPMV could be developed as a vaccine vector for

many swine pathogens and even for other species. Understanding the significance of these two

strains of sPMV for swine health and their molecular characterization are the essential steps to

reach these goals. Therefore, the objective of this study is to undertake the biological and

molecular characterizations of these two novel paramyxoviruses, and to study their pathogenicity

in conventionally reared pigs, thus to establish a foundation for development of a reverse

genetics system. Our longterm goal is to develop them into vaccine vectors using the reverse

genetics system.

Objectives:

1. To undertake biological and molecular characterization of two strains of novel swine

paramyxoviruses.

2. To examine the pathogenicity of the two viruses in conventionally reared pigs.

36

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61

1.11 Tables

Table 1. Examples of Members of the Paramyxoviridae family

Family Paramyxoviridae

Subfamily Paramyxovirinae

Genus Rubulavirus

Mump virus (MuV)

Parainfluenza virus 5 (PIV5/SV5)

Human parainfluenza virus type 2, type 4a and 4b (hPIV2/4a/4b)

Porcine rubulavirus (La-Piedad-Michoacán virus, LPMV)

Genus Avulavirus

Newcastle disease virus (avian paramyxovirus 1, NDV)

Genus Respirovirus

Sendai virus (mouse parainfluenza virus type 1, SeV)

Human parainfluenza virus type 1 and type 3 (hPIV1/3)

Bovine parainfluenza virus type 3 (bPIV3)

Atlantic Salmon Paramyxovirus (ASPV) (?)

T. truncates parainfluenza virus type1 (TtPIV-1) (?)

Genus Henipaviruses

Hendra virus (HeV)

Nipah virus (NiV)

Genus Morbillivirus

Measles virus (MeV)

Canine distemper virus (CDV)

Peste-des-Petits ruminants virus (PPRV)

Rinderpest virus (RPV)

Subfamily Pneumovirinae

Genus Pneumovirus

Human respiratory syncytial virus A2, B1, S2 (hRSV)

Bovine respiratory syncytial virus (bRSV)

Pneumonia virus of mice (PVM)

Genus Metapneumovirus

Human metapneumovirus (hMPV)

Avian metapneumovirus (aMPV)

Unclassifed paramyxoviruses

Fer-de-Lance virus (FDLV)

Tupaia paramyxovirus (TPMV)

Menangle virus (MenV)

Tioman virus (TioV)

Beilong virus (BeV)

J virus (J-V)

Mossman virus (MoV)

1.12 Figures

(A)

(B)

Figure 1. Virion structure of a Paramyxovirus.

(A) Schematic diagram of a paramyxovirus (not drawn to scale) (from Field’s virology, 5

(B) Electron micrograph of paramyxovirus virion.

62

Figure 1. Virion structure of a Paramyxovirus.

Schematic diagram of a paramyxovirus (not drawn to scale) (from Field’s virology, 5

Electron micrograph of paramyxovirus virion.

Schematic diagram of a paramyxovirus (not drawn to scale) (from Field’s virology, 5th edition)

63

Figure 2. Reverse genetics system of ??SV (from (Bukreyev et al., 2006)).

A plasmid encoding a full length antigenomic RNA of a parainfluenza virus is shown at the top. The

extragenic 3’ leader and 5’ trailer regions, individual genes, GS and GE are shown, respectively. The

antigenomic cDNA is flanked at the left by a T7 promoter (T7 pr.), and a self-cleaving ribozyme and T7

terminator (T7 tr.) at the right. Three other T7 expression plasmids encode the N, P, and L support

proteins needed to reconstitute a biologically active nucleocapsid. The T7 RNA polymerase is supplied

from a cotransfected eukaryotic expression plasmid. The plasmids are transfected into a cell monolayer in

which the plasmid-expressed viral RNA and protein components assemble into a functional nucleocapsid

and launch a productive infection. Recovered virus is amplified by passage and can be plaque purified.

64

Figure 3. Strategies for designing a ??SV vector (from (Bukreyev et al., 2006)).

(A) Wild-type NNSV expressing foreign genes.

(B) Antigenic chimeric viruses.

(C) Antigenic chimeric virus expressing additional foreign genes.

65

Chapter 2

Molecular Characterization of Glycoprotein Genes and

Phylogenetic Analysis of Two Swine Paramyxoviruses Isolated From

United States

Dan Qiao1, Bruce H. Janke

2, Subbiah Elankumaran

1.

1Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and

State University, Blacksburg, VA.

2Dept. of Veterinary Diagnostic & Production Animal Medicine, College of Veterinary

Medicine, Iowa State University, Ames, IA.

This manuscript has been published in the journal Virus Genes (2009) 39:53-65.

66

Abstract

Two swine Paramyxoviruses (sPMV) (81-19252 (Texas81), and 92-7783 (ISU92) were

isolated from encephalitic pigs in the United States in 1981 and 1992. Antigenic, morphologic

and biological characteristics of these two viruses were essentially similar to members of the

family Paramyxoviridae. Antigenic analysis by indirect fluorescent antibody, immunoblot and

one-way cross-neutralization tests placed these viruses along with bovine parainfluenza 3

(bPIV3) viruses. Purified virions were 50-300 nm in size and morphologically indistinguishable

from other paramyxoviruses. These two viruses hemagglutinated red blood cells and had

neuraminidase activity. The gene junctions of fusion (F) and hemagglutinin (HN) glycoprotein

genes of these viruses contained highly conserved transcription start and stop signal sequences

and trinucleotide intergenic regions similar to other Paramyxoviridae. The F gene of ISU92 was

longer than Texas-81 due to insertion of a 24 nucleotide “U” rich 3’ UTR. Structure based

sequence alignment of glycoproteins of these two sPMVs indicated that they are essentially

similar in structure and function to parainfluenzaviruses, The Texas81 strain was closely related

to bPIV3 Shipping Fever (SF) strain at nucleotide and amino acid level, while the ISU92 strain

was more closely related to bPIV3-910N strain. The envelope glycoproteins of ISU92 had only

~92% and ~96% identity at nucleotide and amino acid levels with bPIV3-SF strain, respectively.

The high sequence identities to bPIV3 indicated cross-species infection in pigs. Phylogenetic

analyses based on both F protein and HN protein suggested the classification of these viruses

into the subfamily Paramyxovirinae, genus Respirovirus and genotype A of bPIV3.

Keywords: Paramyxovirus, Bovine Parainfluenzavirus 3, Glycoproteins, Cross-species

infection, Pigs.

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2.1 Introduction

Paramyxoviruses are established pathogens of the central nervous and respiratory systems

in many host species. In the last few decades, many novel paramyxoviruses have emerged

causing catastrophic illnesses in different aquatic and terrestrial species of animals and some of

them also made the species jump to humans. Members of the family Paramyxoviridae are

enveloped viruses possessing a non-segmented negative-strand genome and are divided into two

subfamilies, Paramyxovirinae and Pneumovirinae. Currently, there are five genera within the

subfamily Paramyxovirinae: Rubulavirus, Avulavirus, Respirovirus, Morbillivirus, and

Henipavirus (Lamb RA, 2005). La Piedad Michoacán paramyxovirus (LPMV) is the only well

studied neurotropic paramyxovirus isolated from pigs prior to the 1990s. LPMV was first

isolated in central Mexico in the early 1980s (Moreno-Lopez et al., 1986), and it has become

endemic in Mexico (Linne et al., 1992). This virus induced interstitial pneumonia and

encephalitis in pigs. There were extensive records of paramyxoviruses derived from the brain or

nasal swabs of sick pigs in Japan, in 1950s (Philbey et al., 1998), Canada, in 1971 (Ellis et al.,

1998), in Israel in 1986 (Janke et al., 2001) as well as the United States, in 1960s and 1980s in

Texas (Janke et al., 2001). There was also concurrent infection of porcine reproductive and

respiratory syndrome virus and a paramyxovirus in Germany in the 1990s (Heinen et al., 1998)

that has been subsequently named “SER” virus (Tong et al., 2002).

Since 1994, four bat-associated paramyxoviruses have emerged, three of which caused

disease in animals and humans (Wang et al., 2001). In late 1994 in Australia, Hendra virus

(HeV) caused an outbreak of severe respiratory disease resulting in the death of 13 horses and

their trainer (Murray et al., 1995), followed by sporadic HeV outbreaks in horses and humans

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(Field et al., 2007a). A closely related virus, Nipah virus (NiV) from Malaysia caused severe

febrile encephalitis and death in pigs and humans (Chua et al., 2000), which spread to

Bangladesh and India (Chadha et al., 2006; Hsu et al., 2004). In 1997, Another paramyxovirus,

named Menangle virus (MenV) has been isolated in Australia from still-born pigs with

deformities (Philbey et al., 1998), and associated human illness (Chant et al., 1998). In 2000,

Tioman virus (TioV) was isolated from urine collected beneath a fruit bat colony on Tioman

Island, Malaysia (Chua et al., 2001). Molecular characterization revealed that MenV and TioV

are closely related novel members of the genus Rubulavirus (Bowden and Boyle, 2005; Chua et

al., 2001; Chua et al., 2002). Mapuera virus (MPRV) was isolated in 1979 from the salivary

glands of an apparently healthy fruit bat (Sturnira lilium), captured in the tropical rainforest of

Brazil (Karabatsos, 1985). J-virus (JV), isolated from wild mice in Australia, and Beilong virus

(BeV), originally isolated from human mesangial cells in China and subsequently detected in rat

mesangial cells, represent a new group of paramyxoviruses (Basler et al., 2005; Jack et al., 2005;

Jun et al., 1977; Li et al., 2006b; Schomacker et al., 2004). Recently, novel Paramyxoviruses

were also isolated from Atlantic bottlenose dolphins and Atlantic salmons and characterized

(Nollens et al., 2008; Nylund et al., 2007).

Two swine paramyxoviruses (sPMV) (81-19252 (Texas81), and 92-7783 (ISU92)) were

isolated from the brain of pigs that experienced respiratory and central nervous system disease in

the 1980s and 1990s from South and North Central United States, respectively. ISU92 virus was

isolated from a swine operation in the 1990s in north central United States. The outbreak started

in a continuous-flow finishing barn of 400 pigs with signs of respiratory disease. Affected pigs

showed high fever and mild cough and seventeen pigs died within 4 days. Ten days later, the

epizootic spread to a second finishing barn and affected all pigs of different ages. Encephalitic

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signs observed from several pigs with persistent squealing, head pressing, whole body tremors,

and hind-limb ataxia (Janke et al., 2001). Texas-81 virus was isolated from the brain of pigs that

exhibited respiratory and neurological disease in Texas State, in 1981. Further information on the

history of this virus is not available. Antigenic analysis by indirect fluorescent antibody assay

(IFA) at the National Veterinary Services Laboratory (NVSL), Ames, Iowa indicated that these

two viruses are closely related to human parainfluenza virus (hPIV) type 1 and 3 and bovine

parainfluenza virus type 3 (bPIV3).

As new viruses emerge that cause severe diseases in pigs and have the potential to cross

species barrier and infect other hosts including humans, it is important that these new viruses

need to be studied further. Although paramyxoviruses have been isolated from swine in United

States and other parts of the world, their importance as swine pathogens or zoonotic agents

remains to be determined. To understand the significance of these two strains of sPMV for swine

health and to determine their taxonomic status, we have initiated molecular characterization

studies. Here, we describe the biological and molecular characterization of the fusion (F) and

hemagglutinin (HN) glycoprotein genes and their phylogenetic identity with other

paramyxoviruses.

2.2 Materials and Methods

2.2.1 Viruses and Cells

Texas-81 and ISU-92 were received from the NVSL, Animal and Plant Health Inspection

Service (APHIS), United States Department of Agriculture (USDA), Ames, Iowa. Porcine

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Kidney (PK15) cells, tested free of porcine circovirus (PCV) were obtained from Dr. X. J. Meng,

Virginia Polytechnic Institute and State University, and used to propagate these swine

paramyxoviruses. Vero cells (ATCC #C1008) were used for fusion and plaque assays.

2.2.2 Virus purification

Iodixanol gradients (OptiPrep, Sigma) were prepared in phosphate buffered saline (PBS)

(14% to 26%). Cell lysates from virus stock (plaque purified) were layered onto the top of the

gradient and centrifuged for 1.5 h at 250,000 × g in a SW41 Ti rotor. Virus fraction was

collected from the gradient and was either examined by transmission electron microscopy or

analyzed for protein content by immunoblot.

2.2.3 Transmission Electron Microscopy

Purified virus was adsorbed to glow discharged carbon films on 400 mesh grids. The grids were

then washed in PBS and fixed with 2% paraformaldehyde (10 min) and extensively washed with

distilled water before negatively stained with 1% phosphotungstic acid. All specimens were

observed in Philips EM 420 transmission electron microscope operating at 100 KV.

71

2.2.4 Plaque Assay

Confluent Vero cells in 6-well plates were infected with virus stocks prepared from the

third limiting dilution passage in PK15 cells. Viruses were diluted in Dulbecco's Modified Eagle

Medium (DMEM) (Invitrogen) , incubated at 37 °C, in a 5% CO2 incubator for 1 h, washed with

PBS, and then overlaid with 0.8% methyl cellulose in DMEM with 2% FBS. The cells were

fixed with methanol-acetone (1:1) at 6 days post-infection (dpi) and stained with 1% crystal

violet. The mean number of plaque forming units (pfu) ml-1

for each virus was determined.

2.2.5 Hemagglutination (HA) Assay

Swine red blood cells (sRBCs) were washed 3 times with PBS and resuspended in PBS

as a 1% suspension. Texas81 and ISU92 virus stocks prepared in Vero cells were titrated in

doubling dilutions against sRBC in V-bottom microtiter plates (Nunc).

2.2.6 Fusion Index Assay

The fusogenicity of Texas81 and ISU92 viruses was examined as described by Kohn

(Kohn and Fuchs, 1969). Viruses were inoculated into confluent Vero cells in 6-well plates at a

MOI of 0.1. Cells were maintained in DMEM with 2% FBS at 37 °C in a 5% CO2 environment.

After 72 h, the medium was removed, and cells were washed once with 0.02% EDTA and then

incubated with 1 ml of 0.02% EDTA for 2 min at room temperature. The cells were then washed

with PBS and fixed with methanol and stained with hematoxylin-eosin (Hema 3, Sigma). Fusion

72

was quantitated by expressing the fusion index as the ratio of the total number of nuclei to the

number of cells in which these nuclei were observed (i.e., the mean number of nuclei per

cell). The formation of syncytia was visualized by staining virus-infected cells at 48 h with Hema

3 without EDTA treatment.

Fusogenicity of Texas81 and ISU92 F genes were also tested in a plasmid-based system.

The open reading frames of the F and HN genes of Texas81 and ISU92 viruses were cloned into

pCAGGS plasmid and transfected into Vero cells (1µg of each plasmid) using Lipofectamine

2000 (Invitrogen). At 72 h post-transfection, fusion index was calculated as described above.

2.2.7 3euraminidase (3A) activity

The NA activity of the Texas81 and ISU92 strains was determined by a fluorescence-

based NA assay according to the procedures of Potier et al. (Ferraris et al., 2005; Potier et al.,

1979). Newcastle Disease Virus (NDV) Beaudette C strain was used as a standard control virus.

Briefly, serial dilutions of virus strains in dilution buffer (32.5mM MES (2-(%-morpholino)

ethanesulfonic acid, sodium salt, Sigma–Aldrich) pH 5.8, 4mM CaCl2) were prepared in a 96-

well black flat bottom plates. Then, the same volume of substrate buffer (2’-(4-

methylumbelliferyl)-α-D-%-acetylneuraminic acid, sodium salt (MUN, Sigma–Aldrich) prepared

in 32.5mM MES pH 5.8, 4mM CaCl2 buffer) was added to each well. The plate was gently

shaken on a mechanical vibrator and then incubated for 1 h at 37 °C. The reaction was

terminated by adding 150ul of 50mM glycine buffer pH 10.4. The results were read in a

73

VIKTOR multilabel reader (TECAN Safire2) with an excitation wavelength of 360nm and an

emission wavelength of 450nm.

2.2.8 Indirect Fluorescent Antibody (IFA) Test

Virus stocks were inoculated into confluent Vero cells in 8-well chambers at a MOI of

1.0, 0.1, and 0.01. Cells were maintained in DMEM with 2% FBS at 37 °C in a 5% CO2

environment for 48 h and observed for cytopathic effects (CPE). The cells were then washed

with PBS and fixed with methanol-acetone followed by 3 washes with PBST (0.1% Tween 20 in

PBS). The cell monolayers were blocked overnight at 4 °C with PBST containing 5% skim milk

powder (PBSM). After 3 brief washes with PBST, the cells were incubated with anti-bovine

PIV3 polyclonal antibody (NVSL) diluted 1:100 in PBSM and incubated overnight at 4 °C. Then

the cells were washed 3 times with PBST and incubated with FITC-anti bovine IgG (KPL) (1:32

dilution in PBSM) at 37 °C in a moist environment for 1 h. After 3 washes with PBST, the slides

were layered with glycerol-PBS (1:1) and observed under a Nikon Eclipse TS-100

epifluorescence microscope.

2.2.9 Immunoblot

OptiPrep purified virions were subjected to immunoblotting using standard procedures.

Briefly, the proteins were separated in a 4-20% sodium-dodecyl sulphate polyacrylamide gel

electrophoresis (SDS-PAGE) and transferred to Immobilon-P (Millipore) membranes. The viral

74

proteins were probed with anti-bovine PIV3 polyclonal bovine antibody (NVSL) (1:100

dilution), followed by HRP labeled anti-bovine IgG (KPL) (1:500 dilution) secondary antibodies.

The blots were visualized with chemiluminiscent ECL Western blotting system (GE Healthcare).

2.2.10 One-way Cross-neutralization Test

Monospecific convalescent sera were obtained from pigs inoculated intranasally with

5x107TCID50 of Texas81 or ISU92 virus stock. One way-cross neutralization test was

performed in 96-well plates against 500 TCID50 of respective viruses and heat-inactivated, two-

fold diluted sera. The cells were fixed on day 5 and stained with Hema 3 to determine the virus-

neutralization titer.

2.2.11 Reverse Transcription (RT)-Polymerase Chain Reaction (PCR) and Sequencing

RNA extraction and reverse transcription (RT) reactions were performed using standard

procedures. Briefly, virus-infected PK15 cells were scraped into the medium and subjected to

three cycles of freezing and thawing. After initial clarification at 3000 x g for 15 min,

polyethylene glycol (PEG) 8000 (Sigma) was added to the cell lysate to a concentration of 10 %

and the lysate was incubated for 4 h at 4 °C. The virus was pelleted at 12, 000 x g for 60 min at 4

°C, and the viral genomic RNA was extracted from the virus pellet using RNeasy Mini Kit

(QIAgen). Oligonucleotide primers were designed based on consensus bPIV3 F and HN gene

nucleotide sequences (primer sequences available upon request). The complementary DNA

75

(cDNA) copies of the genomic RNA of the two virus strains were synthesized using the specific

oligonucleotide primers and Superscript III reverse transcriptase (Invitrogen). Subsequently,

Platinum Taq DNA Polymerase (Invitrogen) was used to amplify the F or HN gene. The PCR

products were purified using Qiaquick PCR purification kit (Qiagen). The PCR products were

cloned using the TA cloning system (Invitrogen). Both purified PCR products and at least 10

TA clones were sequenced in both directions in an automated sequencer (Core Laboratory

Facility at Virginia Bioinformatics Institute, Virginia Tech). Based on the sequencing results,

primer-walking strategy was employed and new sets of oligonucleotide primers were designed

for sequencing the middle part of the genes.

2.2.12 Determination of antigenic relatedness

The antigenic relatedness of sPMV strains were expressed by R value, calculated using

the following formula created by Archetti and Horsfall (1950)(Archetti and Horsfall, 1950).

where r1 is the ratio of the heterologous titer obtained with virus 2 to the homologous

titer obtained with virus 1; r2 is the ratio of the heterologous titer obtained with virus 1 to the

homologous titer obtained with virus 2; R is a geometric mean of ratio r1 and r2, which is used to

express the antigenic relatedness between two viruses when both antigens and antisera were used

in indirect FA test.

76

R values were interpreted by the method of Brooksby (Brooksby, 1967) for similarity

comparison of two viruses. The criteria are as follows: 1) R value of 1 or close to 1 indicates

antigenic identity between two tested viruses; 2) R value >70% means little or no difference

between two viruses tested; 3) R value between 33% and 70% indicates a minor subtype

difference; 4) R value between 11% and 32% represents a major subtype difference; 5) R value

between 0% and 10% represents a serotype difference.

2.2.13 Data Analysis

Nucleotide sequence editing, alignments and prediction of amino acid sequence and

analyses were conducted using the software package DNASTAR (Lasergene). Phylogenetic

relationships of these two viruses within Paramyxoviridae family were constructed by

Phylogenetic Analysis using Parsimony (PAUP 4.01) software with 1000 bootstrap replicates.

The protein functional analyses of deduced amino acid sequences were done using online

resources available at ExPASy Proteomics tools (http://us.expasy.org/tools/).

2.2.14 Accession numbers for sequence analyses

The sequences of ISU-92 and Texas-81 were deposited in GenBank under accession #

EU439428 and EU439429, respectively. The accession numbers for other viral sequences used

for phylogenetic analysis are: Atlantic salmon paramyxovirus (ASPV), EF646380; Avian

metapneumovirus (aMPV), NC_007652; Avian paramyxovirus 6 (aPMV6), NC_003043;

77

Beilong virus (BeV), NC_007803; Bovine parainfluenza virus 3-910N (bPIV3-910N), D84095;

Bovine parainfluenza virus 3 strain Kansas/15626/84 (bPIV3-Ka), AF178654; Bovine

parainfluenza virus Q5592 (bPIV3-Q5592), EU277658; Bovine parainfluenza virus 3 strain

Shipping Fever (bPIV3-SF), AF178655; Bovine parainfluenza virus 3 strain SK-217 (bPIV3-

SK217), U31671; bovine respiratory syncytial virus (bRSV), NC_001989; Canine distemper

virus (CDV), NC_001921; Dolphin morbillivirus (DMV), NC_005283; Fer-de-lance virus

(FDLV), NC_005084; Hendra virus (HeV), AF017149; Human metapneumovirus (hMPV),

NC_004148; Human parainfluenza virus 1 strain Washington/1964 (hPIV1-Wa), NC_003461;

Human parainfluenza virus 2 (hPIV2), NC_003443; Human parainfluenza virus 3 (hPIV3),

AB012132; Human parainfluenza virus 3-GPv (hPIV3-GPv), NC_001796; Human parainfluenza

virus 3-JS (hPIV3-JS), Z11575; Human respiratory syncytial virus (hRSV), NC_001781; J-virus

(J-V), NC_007454; Measles virus (MeV), NC_001498; Menangle virus (MenV), NC_007620;

Mossman virus (MoV), NC_005339; Mumps virus (MuV), NC_002200; Newcastle disease virus

(NDV), NC_002617; Nipah virus (NiV), NC_002728; Peste-des-petits-ruminants virus (PPRV),

NC_006383; Pneumonia virus of mice, AY573818; Porcine Rubulavirus (PoRV/LPMV),

NC_009640; Rinderpest virus (RPV) (strain Kabete O), NC_006296; Sendai virus (SeV),

NC_001552; Simian parainfluenza virus 5 (SV5), NC_006430; Tioman virus (TioV),

NC_004074; Tupaia paramyxovirus (TPMV), NC_002199.

78

2.3 Results and Discussion

2.3.1 Morphological and Biological Properties

Transmission electron microscopy revealed spherical to pleiomorphic virions

approximately 50-300 nm in diameter morphologically indistinguishable from paramyxoviruses

(Robert A. Lamb, 2007). Intact virions were enveloped and densely packed with surface

projections representing viral glycoprotein spikes. Nucleocapsids were visible and exhibited a

typical “herringbone” pattern (Figure 1). Both Texax81 and ISU92 viruses were able to

agglutinate sRBCs. The HA titers of Texas-81 and ISU-92 were 24 and 2

5, respectively. The

agglutinated sRBCs eluted after 1 h suggesting neuraminidase function. Neuraminidase assay

also confirmed this. bPIV3 has been shown to have hemagglutinating and neuraminidase

functions (Breker-Klassen et al., 1996). The fusion index of Texas81 was 63 nuclei per cell and

ISU92 was 60 nuclei per cell (72 hpi, MOI, 0.1), indicating that these viruses are highly

fusogenic (Figure 2). The fusion index with Texas81 and ISU92 F plasmids with homologous

HN plasmids was 22.7 and 13.5 respectively. The viruses were able to form plaques in Vero cells

at 6 dpi and grew to 2.62×107

pfu ml-1

(Texas81) and 5.75×107

pfu ml-1

(ISU92). Both viruses

produced similar sized plaques in Vero cells (Figure 2).

2.3.2 Antigenic analysis

Antigenic analysis at the National Veterinary Services Laboratory (NVSL), Ames, Iowa

indicated that they were closely related to human parainfluenza virus (hPIV) type 1 and 3 and

bPIV3 (Table 1). Cross-reactivity in several epitopes in F and HN proteins with hPIV3 and

79

bPIV3 has been reported earlier (Coelingh et al., 1986). Bovine anti-bPIV3 serum was able to

detect the cells infected by these viruses (Figure 2) by IFA. Besides, using bovine anti-bPIV3

serum, all the viral proteins were detectable in OptiPrep purified virion preparations (Figure 2).

The protein designations described were based on studies with virion proteins of PIV3 (Storey et

al., 1984; Wechsler et al., 1985). Homologous sera could neutralize virus infectivity of similar

titers and heterologous serum (bPIV3-SF) neutralized virus infectivity of Texas81 at 1:32 and

ISU92 at 1:8, suggesting antigenic variation. The R values of Texas81 and ISU92 strains with

bPIV3-SF strain were 100% and 35%, respectively by IFA. These observations suggest that

Texas81 and ISU92 were antigenically closely related to bPIV3. However, antigenic analysis by

IFA also suggested that ISU92 is a minor subtype of bPIV3, according to the criteria of

Brooksby (1967) (Brooksby, 1967).

2.3.3 Transcriptional start and stop sequences

The nucleotide sequence of F and HN genes of both Texas81 and ISU92 viruses were

determined from RT-PCR products amplified from viral genomic RNA. Individual genes are

transcribed by a start-stop mechanism controlled by conserved sequences at the gene borders in

members of the family Paramyxoviridae. The F and HN genes of Texas81 and ISU92 shared the

universal pattern of paramyxovirus genome structure, flanked at 3’ end with a conserved gene-

start (GS) sequence and at 5’ end with a gene-end (GE) sequence with an intergenic sequence

(IGS) in between. The GS and GE sequences of the F and HN genes were essentially similar to

bPIV3. Only the variable fifth and sixth positions of the GS sequence showed host-specificity

(Table 2). All of them terminated with U-rich GE sequences. The trinucleotide IGS (3’-GAA) of

80

these two viruses were also identical to members of the genera Respirovirus, Morbillivirus, and

Henipavirus (Bailly et al., 2000). A long U-rich sequence was identified in the 3’ UTR of F gene

of ISU92. This is a characteristic feature commonly found in bPIV3 and hPIV3. This has been

shown to result in read-through transcripts of M gene in bPIV3 and hPIV3 (Sakai et al., 1987;

Suzu et al., 1987).

2.3.4 F gene

The F gene of Texas81 strain was 1869 nucleotides (nt) in length with a single ORF of

1620 nt beginning at position 211 (Table 3), capable of encoding a 540 amino acid protein. The

F gene of ISU92 strain was 1893 nt in length with a 1620 nt ORF but with a longer 3’

untranslated region (235 nt). The long 3’ UTR in ISU92 strain had a 24 nt “U” rich insertion

compared to the Texas81 strain. The F protein of ISU92 had a predicted molecular weight of

60,039 Da and an estimated isoelectric point (pI) of 6.797. For Texas-81 strain, the uncleaved F0

protein had a predicted molecular weight of 60,189 Da and an estimated pI of 6.539. The size

determination by immunoblotting confirmed this (Figure 2). The Texas81 strain has 100%

identity with bPIV3-SF strain both in nucleotide and deduced amino acid sequences. ISU92

strain had 98.7% identity in nucleotide sequence and 99.1% identity in amino acid sequences

with bPIV3-SK217 strain, while it had 90.9% and 95% identity with bPIV3-SF strain in

nucleotide and amino acid sequences, respectively (Table 4) for F gene.

The F protein mediates fusion of virus and cell membrane in paramyxoviruses. Fusion

activation is dependent on the cleavage of F0 protein into disulfide-linked subunits F2-s-s-F1. The

81

furin, a subtilisin-like endoprotease, is believed to be one of the proteases that cleaves most F

proteins intracellularly (Klenk and Garten, 1994; Ortmann et al., 1994). In Texas81 and ISU92

viruses, the F cleavage motif L/SRTKR was located between amino acid residues 105 to 109,

and cleavage occurs between residues 109(R) and 110(F). This cleavage site conformed to the

pattern of consensus motif for cleavage by furin (Hosaka et al., 1991), R-X-K/R-R, which is

conserved in the majority of Paramyxovirinae (Robert A. Lamb, 2007). The predicted F cleavage

site was immediately followed by a 25-aa hydrophobic fusion peptide, highly conserved in all

paramyxovirus F proteins (Horvath et al., 1992; Robert A. Lamb, 2007). The cleaved F1 protein

was approximately 51kDa in size. The F protein of both strains, like the F protein of other

paramyxoviruses, was predicted to be a type I membrane protein. The transmembrane region was

near the carboxyl terminal (amino acid residues 497-517), which was thought to serve as an

anchor in the viral envelope, leaving a 23 amino acid cytoplasmic tail (Figure 3).

The F proteins of swine paramyxoviruses are essentially similar in structure and function

to other paramyxoviruses, and especially to BPIV3. Peptides corresponding to the heptad repeat

regions A and B (HRA and HRB) from SV5 (Baker et al., 1999), hRSV (Zhao et al., 2000) and

hPIV3 (Yin et al., 2005), were conserved in the F protein of sPMV. F proteins assembled into

stable six helical bundles (6HBs) (the F1 core), and their structure in hPIV3 had been determined.

Structure based sequence alignment of the sPMV F protein sequences with these 6HB fragments

revealed only minor differences. The conserved blocks in F1 and F2 (CBF1, CBF2) were also

preserved in sPMV as in other paramyxoviruses which strengthens the hypothesis that these

conserved blocks had conserved functions, either in the membrane fusion or in the folding and

processing of the F protein (Gardner and Dutch, 2007). Cysteines, which were important for

disulfide bond formation and secondary structure, were also identical to bPIV3 and hPIV3.

82

Among the five potential conserved N-linked glycosylation sites (N 101, N238, N359, N446, and

N508) in the F protein (Suzu et al., 1987), all but one had the N-X-T motif. The one at position

446 had the N-X-S motif. It should be noted that only one potential glycosylation site (at N101)

was located before the F protein cleavage site, and one site was located in the transmembrane

domain (N508). The other three were located prior to the transmembrane domain in the F1

protein and were, therefore, likely to be exposed on the surface, as in other members of the genus

Respirovirus (Robert A. Lamb, 2007).

2.3.5 H3 gene

The HN gene was 1888 nt in length with a single ORF beginning at position 74 that could

encode a 572 amino acid protein (Table 3). Texas81 strain shared 99.8% identity with bPIV3-SF

strain both in nucleotide and predicted amino acid sequences. ISU92 strain had 98.4% nucleotide

sequence identity and 99.1% amino acid sequence identity with bPIV3-SK217 strain. Identity of

ISU92 with bPIV3-SF strain is 91.7% at nucleotide level and 95.8% at amino acid level (Table

4). Comparing with hPIV3, the two swine viruses had 80.1% ~ 82.7% identities in the HN gene

nucleotide sequences and 76.4% ~ 77.6% identity at deduced amino acid sequences.

The deduced amino acid sequence of the HN protein was 572 residues in length. The HN

protein of ISU92 had a predicted molecular weight of 64,652 Da and an estimated pI of 7.343.

The HN protein of Texas81 strain had a predicted molecular weight of 64,624 Da and an

estimated pI of 7.720. The active site residues predicted from NDV or hPIV3 HN crystal

structure were also conserved in the swine viruses. The major transmembrane region of HN

83

protein was predicted to be from amino acid residues 36 to 54 of the protein as in other type II

membrane glycoproteins. The transmembrane domain at the N-terminal end of the HN protein

contained several conserved substitutions among the examined viruses (Figure 4).

The disulfide bonds in the HN protein C190–C214 (A), C256–C269(C), C355–C469 (D),

C463–C473(E), C535–C544(F), C159–C571(W) and C350–C363(X) were conserved as in NDV

HN or hPIV 3 HN crystal structures (Crennell et al., 2000; Lawrence et al., 2004). Predicted N-

glycosylation sites were conserved in HN proteins as in other members of Paramyxovirinae. In

swine viruses, potential N-linked glycan sites observed at all predicted sites (N8, N308, N351,

N448 and N523) were similar to hPIV3. The counterpart to N351 in NDV HN is N341, which is

glycosylated in that molecule. NDV HN contains a N-linked glycosylation site at N481, but in

hPIV3 HN the sequence at this site is N-P-T and in swine viruses, it is N-P-S, the asparagine

moiety of which is thus not expected to be glycosylated. Texas81 strain had an additional (N15)

N-linked glycosylation acceptor site in the HN protein, which appears to be host specific.

Whether all of the potential N-linked glycan sites are glycosylated awaits future studies. Both

Texas81 and ISU92 viruses contained the conserved NRKSCS neuraminidase active site motif

(Jorgensen et al., 1987). Up to date, all analyzed members of Respirovirus and Rubulavirus had

this sequence (Langedijk et al., 1997).

The sequence analysis showed that Texas81 and ISU92 had higher levels of identity with

bPIV3 than hPIV1 or hPIV3. The 100% identity at nucleotide level between the F protein of

Texas81 and the SF strain of bPIV3 indicated that cross species transmission might occur among

different hosts. However, further studies will be needed to confirm this. On the other hand,

ISU92, which was closely related to the SK217 and 910N strains of bPIV3, possessed host-

84

specific amino acid residues in the F and HN proteins differing from both bPIV3 strains. There

were three host specific amino acid residues (Y100, A437, and V501) in F protein and 3 in the

HN protein (K70, S88 and S387) of ISU92 strain, compared to bPIV3 and hPIV3 (Figures 3 and

4). bPIV3 strains SK217 and SF/Ka were shown to differ in virulence (Breker-Klassen et al.,

1996; Shibuta et al., 1981). This virulence difference had been associated with a change in amino

acid 193 in HN protein which had a dramatic effect on syncytium inducing activity,

neuraminidase activity and hemagglutinating activity (Breker-Klassen et al., 1996). ISU92 had I

at this position, unlike SK217 or 910N, but identical with Texas81 and bPIV3-SF/Ka. Further,

ISU92 strain was more fusogenic than the Texas81 strain. The importance of these residues in

host specificity awaits future reverse genetic studies.

2.3.6 Phylogenetic analysis

Phylogenetic analyses of the full length F and HN genes of these viruses with other

members of the Paramyxoviridae were performed by parsimony analysis. Phylogenetic analysis

of Texas81 and ISU92 based on the deduced amino acid sequences of the F and HN proteins

placed them on the same clade along with bPIV3 (Figure 5). Recently, it has been reported that

there are two genotypes of bPIV3: genotype A and genotype B (Horwood et al., 2008). However,

two subgenotypes of bPIV3 were discernible in genotype A; one represented by 910N-like

viruses and the other represented by Ka, and SF viruses. The Texas81 and SF/Ka strains formed

the first genetic group while ISU92 and 910N strains formed the second genetic group.

Phylogenetic reconstruction with HN gene also confirmed these groupings. Antigenic analysis by

IFA has also confirmed that there are subtypic differences between bPIV3 and sPMV strains as

85

well. The above facts support the classification of these two swine viruses in the genus

Respirovirus, Paramyxovirinae subfamily.

The phylogenetic analyses also indicated that these two strains are very likely the host-

adapted variant strains transmitted from cattle to pigs. The host-specific sequence elements

might contribute to the growth of the virus in heterogeneous hosts (Bailly et al., 2000). Cross-

species infection of bPIV3 in a human (Ben-Ishai et al., 1980), bPIV3 in sheep and ovine PIV-3

in cattle (Stevenson and Hore, 1970) has been reported. This happens to be the first report of

cross-species infection of bPIV3 in pigs. Several studies using reverse genetic system to recover

recombinant PIV3 have been successfully constructed and employed including hPIV3 and bPIV3

(Skiadopoulos et al., 1998; Skiadopoulos et al., 1999). Similar approaches can be undertaken to

determine the importance of the individual or combined effects of host-specific sequence

changes in the pathogenesis and host-adaptation of bPIV3. Further, this would also help in

developing an attenuated phenotype as a vaccine candidate for these viruses.

2.4 Acknowledgments

The authors thank Dr. X. J. Meng, Dr. Laure Deflube, Shobana Raghunath, Thomas

Rogers-Cotrone and Vrushali Chavan and for their excellent technical assistance.

86

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2.6 Tables

Table 1. Antigenic relationship of ISU92 and Texas81 with other paramyxoviruses

Antibody Titer of Antisera to Viruses by IFA*

Virus ISU92 hPIV

1 hPIV2 hPIV3

hPIV4

A

hPIV

4 B

hPIV

5 LPMV bRSV Texas81

bPIV3

-SF

ISU92 1:1280 1:640 < 1:20 1:320 < 1:20 1:20 1:20 < 1:20 < 1:20 1:320 1:640

hPIV1 < 1:20 < 1:20

hPIV2 < 1:20 < 1:20 1:320 < 1:20 1:80 ≥ 1:80 ≥ 1:80 < 1:20 < 1:20 < 1:20 < 1:20

hPIV3

hPIV4A < 1:20 < 1:20 ≥ 1:80 < 1:20 1:640 ≥ 1:80 ≥ 1:80 < 1:20 < 1:20 < 1:20 < 1:20

hPIV4B < 1:20 1:40 1:320 < 1:20 1:640 1:640 1:640 < 1:20 < 1:20 < 1:20 < 1:20

hPIV5 < 1:20 < 1:20 ≥ 1:160 < 1:20 ≥ 1:80 ≥ 1:80 1:640 < 1:20 < 1:20 < 1:20 < 1:20

LPMV < 1:20 < 1:20 < 1:20 < 1:20 < 1:20 < 1:20 < 1:20 1:1280 < 1:20 < 1:20 < 1:20

bRSV < 1:20 < 1:20 < 1:20 < 1:20 < 1:20 < 1:20 < 1:20 < 1:20 1:2560 < 1:20 < 1:20

Texas81 1:640 1:320 < 1:20 1:640 < 1:20 < 1:20 < 1:20 < 1:20 < 1:20 1:640 1:1280

bPIV3-SF 1:640 < 1:20 < 1:20 1:1280 < 1:20 < 1:20 < 1:20 < 1:20 < 1:20 1:1280 1:2560

*Indirect fluorescent antibody test was performed at the National Veterinary Services Laboratory,

Ames, IA against a panel of paramyxoviruses.

93

Table 2. Comparison of the gene-start and gene-end sequences of sPMV, bPIV3 and

hPIV3.

Gene Strain Gene-start Gene-end

F

Texas81 UCCUAGUUUCa UUCAUGUUUUUU

ISU92 UCCUAGUUUC UUCAUGUUUUUU

bPIV3-SF/Ka UCCUAGUUUC UUCAUGUUUUUU

bPIV3-910? UCCUAGUUUC UUCAUGUUUUUU

hPIV3 UCCUGUUUUC CUAAUAUUUUUU

H?

Texas81 UCCUUGUUUC UUAUUAUUUUUUb

ISU92 UCCUUGUUUC UUAAUGUUUUUU

BPIV3-Ka/SF UCCUUGUUUC UUAUUAUUUUUU

BPIV3-910? UCCUUGUUUC UUAAUGUUUUUU

hPIV3 UCCUCAUUUC UUUAUAUUUUUU

Consensus: UCCUNNUUUC NUNNUNUUUUUU

a. Host-specific nucleotide residues between hPIV3 and bPIV3 are underlined;

b. Positions that display variability within the host group are in bold-face type.

94

Table 3. Comparison of nucleotide sequences between sPMV, bovine and human PIV3

viruses.

Gene Virus ?ucleotide Amino acid 3'UTR(nt) ORF(nt) 5' UTR(nt)

F

Texas81 1869 540 211 1620 38

ISU92 1893 540 235 1620 38

bPIV3-SF/Ka 1869 540 211 1620 38

bPIV3-910N 1893 540 235 1620 38

hPIV3 1851 539 193 1617 41

H?

Texas81 1888 572 73 1716 99

ISU92 1888 572 73 1716 99

bPIV3 1888 572 73 1716 99

hPIV3 1888 572 73 1716 99

95

Table 4. ?ucleotide and Amino acid identities between sPMV, bPIV3 and hPIV3

Virus Strain F H?

?ucleotide Amino acid ?ucleotide Amino acid

Texas81

bPIV3-910N 91.4% 95.2% 91.8% 96.2%

bPIV3-SK217 92.2% 95.9% 92.1% 96.2%

bPIV3-SF 100.0% 100.0% 99.8% 99.8%

bPIV3-Ka 97.8% 98.3% 98.3% 98.3%

hPIV3 82.2% 82.7% 80.1% 76.4%

ISU92

bPIV3-910N 98.0% 98.9% 98.3% 98.8%

bPIV3-SK217 98.7% 99.1% 98.4% 99.1%

bPIV3-SF 90.9% 95.0% 91.7% 95.8%

bPIV3-Ka 91.0% 95.6% 91.4% 94.9%

hPIV3 82.0% 82.5% 82.7% 77.6%

2.7 Figures

Figure 1. Ultrastructure of swine paramyxoviruses.

with 1% phosphotungstic acid and viewed under a transmission electron microscope. (A

ISU92 virus, (B, D) Texas81 virus.

representing the viral glycoprotein spikes. Highly ple

nucleocapsids were indicated by arrow b. Nucleocapsids exhibit a typical “herringbone” pattern.

A

b

C

96

Figure 1. Ultrastructure of swine paramyxoviruses. Purified virions were negatively stained

ith 1% phosphotungstic acid and viewed under a transmission electron microscope. (A

ISU92 virus, (B, D) Texas81 virus. Intact virion with fine surface projections (arrow a)

representing the viral glycoprotein spikes. Highly pleiomorphic viral particles with extruded

nucleocapsids were indicated by arrow b. Nucleocapsids exhibit a typical “herringbone” pattern.

B

a

96K

a

b

D

57K

96K

96K

Purified virions were negatively stained

ith 1% phosphotungstic acid and viewed under a transmission electron microscope. (A, C)

Intact virion with fine surface projections (arrow a)

with extruded

nucleocapsids were indicated by arrow b. Nucleocapsids exhibit a typical “herringbone” pattern.

E D

A B

G H

J 1 2

97

C

F

I H

3

98

Figure 2. (A-C) Syncytium formation in Vero cells by Texas81 and ISU92. The fusogenicity

of Texas81 and ISU92 viruses was examined in Vero cells. A. ISU92; B. Texas81; C. Mock-

infected Vero cells. (D-F) Plaque Assay. Confluent Vero cells in 6-well plates were infected

with serially diluted virus stocks and then overlaid with 0.8% methyl cellulose in DMEM with

2% FBS. Plaque sizes of Texas81 and ISU92 viruses were similar D. ISU92; E. Texas81; F.

mock infected. (G-J) Antigenic analysis of swine paramyxoviruses. (G-I) Indirect fluorescence

antibody assay was performed by infecting Vero cells with swine paramyxoviruses at a MOI of

1.0, 0.1, and 0.01. Virus-specific antigens to the swine viruses were probed with anti-BPIV3

polyclonal bovine antibody (NVSL) (1:100 dilution) followed by anti-bovine FITC-conjugated

antibody. (G) ISU-92, (H) Texas-81, (I) mock infected Vero cells. (J) Optiprep purified virions

of Texas81 (Lane 2) and ISU92 (Lane 3) were used for immunoblotting. The viral proteins were

probed with anti-bPIV3 polyclonal bovine antibody (NVSL), followed by HRP labeled anti-

bovine IgG (KPL) secondary antibodies.

99

Figure 3. Schematic of the predicted domain structure of the swine paramyxovirus F0

protein. Identification of conserved block in F1 (CBF1), conserved block in F2 (CBF2) and

fusion peptide (FP) (Gardner and Dutch, 2007; Yin et al., 2005) regions were obtained from

structure based sequence alignment of the F protein of sPMV with other paramyxoviruses.

Domains are indicated as DI to DIII. TM indicates transmembrane domain. HRA, HRB, and

HRC indicate heptad repeat regions. Conserved amino acid residues are shaded.

CBF1

Texas81 GGLIANCITTTCTCNGIDNRINQSPDQGIKIIT 413

ISU92 GGLIANCITTTCTCNGVDNRINQSPDQGIKIIT 413

bPIV3-SF GGLIANCITTTCTCNGIDNRINQSPDQGIKIIT 413

bPIV3-910N GGLIANCITTTCTCNGVDNRINQSPDQGIKIIT 413

hPIV3 GGVVANCITTTCTCNGIGNRINQPPNQGVKIIT 413

NDV GSVIANCKMTTCRCVNPPGIISQNYGEAVSLID 421

Mump GTIVANCRSLTCLCKSPSYPIYQPDHHAVTTID 406

SV5 GIVYANCRSMLCKCMQPAAVILQPSSSPVTVID 406

Sendai GGVVANCIASTCTCGTGRRPISQDRSKGVVFLT 420

NiV GVLFANCISVTCQCQTTGRAISQSGEQTLLMID 413

HeV GVLFANCISVTCQCQTTGRAISQSGEQTLLMID 413

400 200 300 500 100

HRA D III D I D II HRB TM FP D I D III HRC

110 134 497 517

Texas81 LILSLIPKIENS-HSCGDQQINQYKKLLDRLIIPLYDGLK 95

ISU92 LILSLIPKIENS-HSCGDQQINQYKKLLDRLIIPLYDGLK 95

bPIV3-SF LILSLIPKIENS-HSCGDQQINQYKKLLDRLIIPLYDGLK 95

bPIV3-910N LILSLIPKIENS-HSCGDQQINQYKKLLDRLIIPLYDGLK 95

hPIV3 LILSLIPKIEDS-NSCGDQQIKQYKRLLDRLIIPLYDGLR 95

NDV IIVKLLPNLPKDKEACAKAPLDAYNRTLTTLLTPLGDSIR 102

Mump IVVKLLPNIQPTDNSCEFKSVTQYNKTLSNLLLPIAENIN 88

SV5 IVVKLMPTIDSPISGCNITSISSYNATVTKLLQPIGENLE 88

Sendai IVLSLVPGIDLE-NGCGTAQVIQYKSLLNRLLIPLRDALD 102

NiV IVIKMIPNVSNM-SQCTGSVMENYKTRLNGILTPIKGALE 95

HeV IVIKMIPNVSNV-SKCTGTVMENYKSRLTGILSPIKGAIE 95

CBF2

F2 F1 S - S C63 C192

Texas81 LRTKRF 110

ISU92 SRTKRF 110

bPIV3-SF LRTKRF 110

bPIV3-910N SRTKRF 110

hPIV3 PRTKRF 110

NDV GRQGRL 117

Mump RRHKRF 103

SV5 RRRRRF 103

Sendai VPQSRF 117

NiV VGDVRL 110

HeV VGDVKL 110

Cleavage site

aa

100

(A)

(B)

Conserved Active Site Residues

hPIV3 ?DV SV5 sPMV

R1 192 174 163 192

R4 424 416 405 424

R5 503 498 495 503

Y6 530 526 523 530

E4 409 401 390 409

E6 549 547 544 549

D1 216 198 187 216

Figure 4. Schematic of the H? protein globular head region domain structure of swine

paramyxovirus. (A) The transmembrane domain, sialic acid site (NRKSCS motif) and the

predicted alpha helix regions (Garnier-Robson) of swine paramyxovirus were given as colored

boxes (Crennell et al., 2000). Disulfide bonds followed those used in a structure-based sequence

alignment and the crystal structure of NDV HN protein (Crennell et al., 2000). (B) The

corresponding position of the active site residues in the deduced amino acid sequence of the

sPMV and other paramyxoviruses.

400 200 300 500 100

Y

Z A A

C C

Y

E

E

D

F F Z

D

Sialic acid binding site (aa 252-257)

Transmembrane domain (aa 36-54)

C159 C190 C214 C256 C269 C350 C355

C363

C463 C469

C473

C535 C544 C571

α2 α3 TM α 1

I193

aa

(A)

Figure 5. Phylogenetic analysis.

4.01) software with 1000 bootstrap replicates. (A) Phylogenetic analysis based on F gene

deduced amino acid sequences compared to other

Phylogenetic analysis of PIV3 genotypes based on F and HN gene sequences, respectively.

101

. Phylogenetic analysis. Phylogenetic analysis was performed using Parsimony (PAUP

4.01) software with 1000 bootstrap replicates. (A) Phylogenetic analysis based on F gene

deduced amino acid sequences compared to other Paramyxoviridae family members. (B, C

Phylogenetic analysis of PIV3 genotypes based on F and HN gene sequences, respectively.

Unclassified

Phylogenetic analysis was performed using Parsimony (PAUP

4.01) software with 1000 bootstrap replicates. (A) Phylogenetic analysis based on F gene

family members. (B, C)

Phylogenetic analysis of PIV3 genotypes based on F and HN gene sequences, respectively.

102

(B)

(C)

hPIV3-GPv

hPIV3-JS bPIV3-Q5592

bPIV3-SF

Texas81

bPIV3-Ka

bPIV3-910?

ISU92

bPIV3-SK217

100 100

100

100

90

100

bPIV3 Genotype B

hPIV3

bPIV3

Genotype A

bPIV3-

910?

ISU92 bPIV3-

SK217 hPIV3-

JS

hPIV3-

GPv

bPIV3-

Q5592

bPIV3-

SF

Texas81

bPIV3-

Ka

100

100

100

77

93 bPIV3 Genotype B

hPIV3

bPIV3 Genotype A

103

Chapter 3

Complete Genome Sequence and Pathogenicity of Two Swine

Parainfluenzaviruses Isolated From Pigs in the United States

Dan Qiao1, Bruce H. Janke

2, Subbiah Elankumaran

1.

1Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and

State University, Blacksburg, VA.

2Dept. of Veterinary Diagnostic & Production Animal Medicine, College of Veterinary

Medicine, Iowa State University, Ames, IA.

This manuscript is under review in Journal of Virology

104

Abstract

Two novel paramyxoviruses 81-19252 (Texas81) and 92-7783 (ISU92) were isolated

from pigs that experienced respiratory and central nervous system disease in the 1980s and 1990s

from South and North Central United States. The complete genome of Texas81 virus was 15456

nucleotides (nt) and ISU92 was 15480 nt in length consisting of six non-overlapping genes

coding for the nucloeo- (N), phospho- (P), matrix (M), fusion (F), hemagglutinin-neuraminidase

(HN) and large polymerase (L) protein in the order 3'-N-P/C/V-M-F-HN-L-5'. The features

related to virus replication and found to be conserved in most members of Paramyxoviridae were

also found in swine viruses. These include: conserved and complementary 3’ leader and 5’ trailer

regions, trinucleotide intergenic sequences, highly conserved gene start and gene stop signal

sequences. The length of each gene of these two viruses were similar except for the F gene, in

which ISU92 had an additional 24 nt “U” rich 3’ untranslated region (UTR). The P gene of these

viruses were predicted to express P protein from the primary transcript and edit a portion of its

mRNA to encode V and D proteins, and the C protein was expected to be expressed from an

alternate translation initiation codon from the P gene as in respiroviruses. Sequence specific

features related to virus replication and host specific amino acid signatures in viral proteins

indicated that these viruses probably originated from bovine parainfluenzavirus 3. Pairwise

comparison of deduced amino acid sequences of swine viral proteins with members of

Paramyxoviridae and phylogenetic analysis based on the individual gene as well as predicted

amino acid sequences suggested that these swine parainfluenzaviruses (sPIV3) are novel

members of the genus Respirovirus of the Paramyxovirinae subfamily and can be grouped into

two subgenotypes of genotype A of bovine parainfluenza virus 3. The mild clinical signs and

undetectable gross and microscopic lesions observed in sPIV3-infected pigs indicate the

105

inapparent nature of these viruses in conventionally reared pigs. Seroprevalence studies in serum

samples collected from pig farms in Minnesota and Iowa in 2007-2008 by indirect ELISA

revealed that sPIV3 are not circulating in these farms. The mild pathogenicity of sPIV3 can

facilitate its development as a vaccine vector.

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3.1 Introduction

Outbreaks of many novel paramyxoviruses causing catastrophic illnesses have been

reported all over the world in the last few decades. A large and diverse host species are involved,

including avian, porcine, canine, bovine, equine, ovine, human, reptiles and aquatic species

(Franke et al., 2001; Horwood et al., 2008; Nollens et al., 2008; Nylund et al., 2007; Nylund et

al., 2008; Robert A. Lamb, 2007). Cases of cross-species transmission and pathogen jumping to

humans were also reported (Chua et al., 1999; Field et al., 2007a), demonstrating the value of

characterizing new animal pathogens, even if their pathogenic potential was currently unknown.

Prior to 1990s, only La Piedad Michoacán paramyxovirus (LPMV) has been well studied as a

neurotropic paramyxovirus isolated from pigs. Many paramyxoviruses as porcine pathogen have

been reported since 1950s all over the world, including Japan (Philbey et al., 1998), Canada

(Ellis et al., 1998), Israel (Janke et al., 2001) as well as the United States in Texas (Janke et al.,

2001). There was also concurrent infection of porcine reproductive and respiratory syndrome

virus (PRRSV) and a paramyxovirus in Germany in the 1990s (Heinen et al., 1998) that has been

subsequently named “SER” virus (Tong et al., 2002). Four bat-associated paramyxoviruses were

reported to cause animals and human diseases in 1994 (Wang et al., 2001). Hendra virus (HeV)

and Nipah virus (NiV), which were identified to cause an outbreak of severe respiratory disease

and death in horses and their trainer, and severe febrile encephalitis and death in pigs and

farmers, respectively, have been classified as Henipavirus as a genus in subfamily

Paramyxovirinae (Chadha et al., 2006; Chua et al., 2000; Field et al., 2007a; Hsu et al., 2004;

Murray et al., 1995).

Some recently isolated viruses, such as Menangle virus (MenV) (Philbey et al., 1998),

107

Tupaia paramyxovirus (TPMV) (Tidona et al, 1999), Tioman virus (TioV) (Chua et al., 2001),

Mossman virus (MoV) (Miller et al, 2003) J-virus (J-V) (Jack et al., 2005; Jun et al., 1977),

Beilong virus (BeV) (Li et al., 2006b), Mapuera virus (MPRV) (Karabatsos, 1985), T. truncates

parainfluenza virus type 1 (TtPIV-1) isolated from bottlenose dolphins (Nollens et al., 2008), and

Atlantic salmon paramyxovirus (ASPV) (Nylund et al., 2008) remain unclassified below

subfamily level. Most of them have been well characterized genetically, and complete genome

sequences are available. All members of the subfamily Paramyxovirinae, have six invariant

genes in the order 3′– N – P – M – F – A – L –5′, indicating the nucleocapsid, phospho-, matrix,

fusion, attachment and large polymerase proteins, respectively (Lamb and Parks, 2007).

Recently, we have reported the antigenic and molecular characterization of glycoprotein

genes of two novel swine parainfluenza type 3 viruses (sPIV3) isolated from the pigs that

experienced respiratory and central nervous system (CNS) disease (Qiao et al., 2009). These two

sPIV3 strains were antigenically and genetically closely related to bovine parainfluenzavirus 3

(bPIV3) and were closely related to the genus Respirovirus (Qiao et al., 2009). However, the

pathogenicity of these sPIV3 in conventionally reared pigs and the complete genome sequences

of these isolates are presently unknown.

In cattle, bPIV3 infection results in asymptomatic infection to severe respiratory disease,

but no neurological disease has been reported due to bPIV3 in cattle (Dinter and Morein, 1990).

Limited sequence polymorphism has been reported among bPIV3 strains (Coelingh and Winter,

1990; Swierkosz et al., 1995). Recently, after analysis of Australian isolates of bPIV3, two

distinct genotypes of bPIV3: genotypes A and B, have been proposed (Horwood et al., 2008). In

this study, we have performed a complete genome sequence analysis of these novel sPIV3 and

108

determined their pathogenicity in conventionally reared pigs. Our analysis indicated that there

are two distinct genetic groupings discernible within genotype A, represented by bPIV3 shipping

fever strain (bPIV3-SF)-like and bPIV3 910N strain (bPIV3-910N)-like viruses with one swine

virus strain in each of these groups. Several species-specific amino acid residues were identified

that may dictate host range. But, both swine viruses induced a very mild respiratory illness

without any neurological signs in young piglets, suggesting that co-infection with other

infectious agents or the presence of other environmental factors may be required to precipitate

clinical disease.

3.2 Materials and Methods

3.2.1 Viruses and Cells

Texas 81-19252 (Texas81), and ISU-92-Minnesota isolate 92-7783 (ISU92) viruses were

obtained from the National Veterinary Services Laboratory (NVSL), Animal and Plant Health

Inspection Service (APHIS), United States Department of Agriculture (USDA), Ames, Iowa.

Porcine Kidney (PK15) cells, tested free of porcine circovirus (PCV) were obtained from Dr. X.

J. Meng, Virginia Polytechnic Institute and State University, and used to propagate these viruses

for the first three passages. Vero cells (ATCC #CCL-81) were used for all other tests as

described earlier (Qiao, Janke et al, 2009).

109

3.2.2 Reverse Transcription (RT)-Polymerase Chain Reaction (PCR) and Sequencing

RNA extraction and reverse transcription (RT) reactions were performed using standard

procedures. Briefly, virus-infected PK15 cells were scraped into the medium and subjected to

three cycles of freezing and thawing. After initial clarification at 3000 x g for 15 min,

polyethylene glycol (PEG) 8000 (Sigma) was added to the cell lysate to a concentration of 10 %

and the lysate was incubated for 4 h at 4 °C. The virus was pelleted at 12, 000 x g for 60 min at 4

°C, and the viral genomic RNA was extracted from the virus pellet using RNeasy Mini Kit

(QIAgen). Oligonucleotide primers were designed based on consensus bPIV3 and hPIV3

nucleotide sequences (Table 1). Based on the sequencing results, new sets of oligonucleotide

primers were designed and primer-walking strategy was employed for sequencing the gap areas.

The leader and trailer sequences were determined according to the RACE method of Li et al (Li

et al., 2005). Briefly, a single set of adaptor and identical set of reagents were used. The two

RACE methods differ only in the order of adaptor ligation and cDNA synthesis. In the 5’ RACE,

adaptor ligation was carried out after cDNA synthesis whereas in the 3’ RACE the order was

reversed (Figure 1).

The complementary DNA (cDNA) copies of the genomic RNA of the two virus strains

were synthesized using the specific oligonucleotide primers and Superscript III reverse

transcriptase (Invitrogen). Subsequently, Platinum Taq DNA Polymerase (Invitrogen) was used

to amplify each fragment with the following cycling parameters: 94◦C for 4 min, followed by 30

cycles of 94◦ C for 30 seconds, 55

◦ C for 30 seconds, 72

◦ C for 1 minute, and a final 7 minutes

extension at 72◦ C. The PCR products were purified using Qiaquick PCR purification kit

(Qiagen). To ensure the consensus sequence, every nucleotide in the genome was sequenced

110

once from purified PCR products and at least from 3 independent TA clones in both directions in

an automated sequencer (Core Laboratory Facility at Virginia Bioinformatics Institute, Virginia

Tech).

3.2.3 Data Analysis

Nucleotide sequence editing, alignment, prediction of amino acid sequences and analyses

were conducted using the software package DNASTAR (Lasergene). Phylogenetic

reconstructions of these two viruses were performed using Parsimony (PAUP 4.01) (Swofford,

2002) software with 1000 bootstrap replicates.

3.2.4 Accession 3umbers for Sequence Analysis

The sequences of ISU92 and Texas81 were deposited in GenBank under accession #

EU439428 and EU439429, respectively. The accession numbers for other viral sequences used

for phylogenetic analysis are: Atlantic salmon paramyxovirus (ASPV), EF646380; Avian

metapneumovirus (aMPV), NC_007652; Avian paramyxovirus 2 (aPMV2), EU338414; Avian

paramyxovirus 6 (aPMV6), NC_003043; Beilong virus (BeV), NC_007803; Bovine

parainfluenza virus 3 strain 910N (bPIV3-910N), D84095; Bovine parainfluenza virus 3 strain

Kansas/15626/84 (bPIV3-Ka), AF178654; Bovine parainfluenza virus strain Q5592 (bPIV3-

Q5592), EU277658; Bovine parainfluenza virus 3 strain Shipping Fever (bPIV3-SF), AF178655;

Bovine respiratory syncytial virus (bRSV), NC_001989; Canine distemper virus (CDV),

111

NC_001921; Dolphin morbillivirus (DMV), NC_005283; Fer-de-lance virus (FDLV),

NC_005084; Hendra virus (HeV), AF017149; Human metapneumovirus (hMPV), NC_004148;

Human parainfluenza virus 1 strain Washington/1964 (hPIV1-Wa), NC_003461; Human

parainfluenza virus 2 (hPIV2), NC_003443; Human parainfluenza virus 3 (hPIV3), AB012132;

Human parainfluenza virus 3-GPv (hPIV3-GPv), NC_001796; Human parainfluenza virus 3-JS

(hPIV3-JS), Z11575; Human respiratory syncytial virus (hRSV), NC_001781; J-virus (J-V),

NC_007454; Measles virus (MeV), NC_001498; Menangle virus (MenV), NC_007620;

Mossman virus (MoV), NC_005339; Mumps virus (MuV), NC_002200; Newcastle disease virus

(NDV), NC_002617; Nipah virus (NiV), NC_002728; Peste-des-petits-ruminants virus (PPRV),

NC_006383; Pneumonia virus of mice, AY573818; Porcine Rubulavirus (PoRV/LPMV),

NC_009640; Rinderpest virus (RPV) (strain Kabete O), NC_006296; Sendai virus (SeV),

NC_001552; Simian parainfluenza virus 5 (SV5), NC_006430; Tioman virus (TioV),

NC_004074; Tupaia paramyxovirus (TPMV), NC_002199.

The consensus nucleotide sequence of hPIV3 Wash/47885/57 (hPIV3-Wa) was

assembled from individual gene sequences from GenBank (Bailly et al., 2000) including,

M11849 (N gene), M14552 (N gene), X04612 (N gene), M14890 (P gene), X04721 (P gene),

D00130 (M gene), M16458 (M gene), M16569 (M gene), Y00119 (M gene), M14892 (F gene),

S82195 (F gene), M21649 (F, HN, L gene and 5’ genomic termini), Z26523 (HN gene), M17641

(HN gene), M20402 (HN and partial L gene), X03967 (3’ genomic termini).

112

3. 2.5 Pathogenicity Studies in Pigs

A total of eighteen 6-weeks old Yorkshire crossbred pigs (less than 25 lbs body weight) were

obtained from the Swine facility at Virginia Tech and randomly divided into 3 groups of 6

piglets. They were determined to be free of antibodies to PRRSV, Swine influenza virus, and

PCV-2. The pigs were allowed to acclamatize for a week before the start of the experiment. The

rectal temperatures were recorded two times a day (morning and evening). Clinical signs were

observed three times a day (every 8 h) after inoculation until day 10. Treatment groups were

inoculated intranasally with 2 ml (5x107TCID50/ml) of virus stock with 1 ml in each nostril. The

control group was inoculated intranasally with 2 ml phosphate buffered saline (PBS) with 1 ml in

each nostril. From each of these groups, three pigs were euthanized on day 6 and 10,

respectively. The clinical signs were scored on a 0 to 3 scale for physical appearance (normal,

dull, lameness, recumbency), activity (alert, anorectic, mild response, no response), respiratory

signs (none, sneezing, heavy nasal discharge, dyspnea) and other signs/body weight loss (none,

<10% weight loss, 10-15% weight loss and >15% weight loss).

Blood was collected before inoculation and at necropsy. Serum was tested against the

respective virus strains by virus neutralization (VN) test as described (Battrell, 1995). Trachea,

lung, heart, brain, liver, spleen, and other internal organs were collected at necropsy, fixed in

10% neutral buffered formalin and used for histopathologic examination. Paraffin embedded

tissue sections were employed for immunohistochemistry using bovine anti-bPIV3-SF

polyclonal antiserum (NVSL, Ames, IA). Nasal and fecal swabs, blood, and 20% suspensions of

lung and brain were subjected to virus isolation in Vero cells. The serum samples were examined

by VN test in 96-well plates using homologous viruses.

113

3.2.6 Development of indirect Enzyme-linked immunosorbent assay (ELISA) for serology

The ISU92 virus stock prepared in Vero (1x108 pfu/ml) cells was used as an antigen to

coat the polystyrene ELISA plates (Nunclon). The optimum dilution of antigen and serum were

determined by checkerboard titration. The indirect ELISA is performed as follows: Briefly, a

pre-determined concentration of antigen in coating buffer (KPL, Kirkegaard & Perry

Laboratories, Inc.) was used to coat each well. The plates were then covered with parafilm and

incubated overnight at 4°C. The plates were washed once with wash buffer (KPL) in an ELISA

plate washer (TECAN, HydorFLEX). After blocking the unbound sites with a commercial

blocking buffer (KPL) for 1 h, 100ul of 1 in 50 dilution of serum was added and the plates were

incubated at 37°C for 1 h. Duplicate wells were set up for each sample and blank wells were set

up with only diluent buffer. The plates were then washed three times with a brief soak and shake

between each wash. The bound antigen-antibody complexes were probed with anti-species IgG

horseradish peroxidase (HRP) secondary antibody diluted 1:500 and the plate was incubated for

a further 1 h at 37°C. After washing the plates 3 times, 100 ul of TMB microwell substrate

(KPL) was used to develop color for 10 min and then 100 ul BlueSTOPTM

Solution (KPL) was

added to each well to stop the reaction. The absorbances were read at 650 nm immediately

(TECAN, Safire2). A cut-off value of two times the mean of negative serum absorbance + 1

standard deviation was chosen based on the results of checkerboard titrations with known

positive and negative sera samples. One hundred randomly selected pig serum samples from

different age groups obtained from 5 swine farms in Minnesota and Iowa in 2007-2008 were

screened for the presence of sPIV3 antibodies. The sera samples from pigs experimentally

inoculated with ISU92 or Texas81 were also subjected to ELISA. The mean values from

duplicate wells were used for analysis.

114

3.3 Results

3.3.1 Genome Features

The complete genome of Texas81 is 15456 nucleotides (nt) in length and 15480 nt for

ISU92. The genome length is divisible by six and consistent with the “rule of six” as described

for most other members of Paramyxoviridae (Calain and Roux, 1993; Kolakofsky et al., 1998;

Kolakofsky et al., 2005). The genome contains six discrete, non-overlapping transcription units.

The coding capacity of the genome is 93.3% in Texas81 and 93.2% in ISU92. Both viruses have

conserved gene start (GS), gene end (GE) and strictly conserved trinucleotide intergenic

sequence (IGS), for all the 6 genes compared to bPIV3 (Table 2). ISU92 shared the identical GS

and GE with bPIV3-910N strain, while Texas81 shared the identical GS and GE with bPIV3-

SF/Ka strain, except the GS of L gene, which is identical with bPIV3-910N strain.

The genomic temini of members of Paramyxoviridae are observed to have

complementarity between the 3′ and 5′ termini (Li et al., 2005). These conserved terminal

sequences, especially the first 12~13 nt, are believed to contain the genome and anti-genome

promoters essential for replication and transcription (Robert A. Lamb, 2007). The swine viruses

have a 55 nt 3’ leader before the transcription start site for the N gene, the length of which is

conserved among almost all of the members of subfamily Paramyxovirinae. The 5’ trailer of

swine viruses is 44 nt long following the L gene transcription stop site and the length is variable

among Paramyxovirinae subfamily but conserved among parainfluenza type 3 (PIV3) viruses.

Overall, the swine viruses possess a 96.4% (53 nt out of 55 nt) identity in the leader region with

bPIV3, while a 92.7% (51 nt out of 55 nt) identity with hPIV3. In trailer region, there is a 97.7%

(43 nt out of 44 nt) identity between swine viruses and bPIV3, and 90.9% (40 nt out of 44 nt)

115

identity with hPIV3. The exact complementarity at the first 14 nt of 3’ genomic leader and

overall 65.9% (ISU92)/63.6% (Texas81) complementarity (29(ISU92)/28 (Texas81) nt out of 44

nt) between the 3’ leader and 5’ trailer termini suggest conserved elements in the 3’ promoter

regions of the genome and antigenome (Figure 2).

3.3.2 Genome organization

The genome organization of swine viruses can best be described as 3’-N-P/V/D-M-F-

HN-L-5’ and can potentially encode 9 proteins. By inserting a broad distribution of Gs at the

mRNA editing site, V and D proteins were predicted to express from the P gene of both ISU92

and Texas81. The features of six genes and corresponding proteins of two viruses were shown in

Table 3. The identity of Texas81 with ISU92 genome is 94.1% at nucleotide level. The identity

between swine viruses and bPIV3 is 98.2% at the highest (between Texas81 and bPIV3-SF)

level, while the identity between swine viruses and hPIV3 is 80.1% at the highest (between

Texas81 and hPIV3-JS) level. Analysis of the start sites of each gene and the P gene editing site

of Texas81 and ISU92, and the hexamer phasing positions of “2,1,1,1,1,2,2”, which is shown to

be genus-specific within the Paramyxovirinae (Harcourt et al., 2001; Kolakofsky et al., 1998) are

identical with hPIV3 and bPIV3 (Table 4).

116

3.3.2.1 The %ucleoprotein (%) Gene

The N gene of both Texas81 and ISU92 is 1646 nt long and encodes a N protein of 515

amino acid (aa) long, with a predicted molecular weight (MW) of 57.3 k daltons (kDa) and pI of

5.0 (Texas81) and 5.2 (ISU92), respectively. The N protein tightly binds to the entire length of

genomic and antigenomic RNA to form the nucleocapsid and it is also associated with the

polymerase complex during transcription and replication. A highly conserved motif located near

the middle of all members of Paramyxovirinae N protein, which is thought to be essential in N-N

self assembly and N-RNA interaction process, is F-X4-Y-X3-Ø-S-Ø-A-M (where X is any

residue and Ø is an aromatic amino acid) (Myers et al., 1997b; Robert A. Lamb, 2007). This

motif is also seen within the central domain of the N protein of swine viruses, presented as

323FAPGNYPALWSYAM

336. In SeV and other paramyxoviruses, the first residue of this motif

F324 (annotated in SeV) is needed for correct self-assembly and another residue Y260

(annotated in SeV) (Myers et al., 1997b) is critical for N-viral RNA binding. These two residues

are conserved in swine viruses as F323 and Y259, respectively. The last 24% sequence of

carboxy terminal of N (aa 394 to 515) has a very low identity with other members of

Paramyxoviridae. This region with consistently low similarity is where most of the

phosphorylation and antigenic sites of the protein (Robert A. Lamb, 2007; Ruth A. Karron, 2007)

are located. The N protein of ISU92 and Texas81 has 97.1% amino acid sequence identity with

each other. When aligned with selected members from Paramyxoviridae, the identities decreased

in order: subfamily Paramyxovirinae, Respirovirus (60.6%-100%); Morbillivirus (18.9%-

23.4%); Henipavirus (19.8%-20.2%); Avulavirus (17.7%-20.6%); Rubulavirus (17.4%-19.2%);

subfamily Pneumovirinae, Metapneumovirus (11.5%-12.2%); Pneumovirus (11.1%-11.8%);

unclassified viruses, ASPV (23.4%-24.8%), BeV (23.3%-23.8%), J-V (20.2%-21.5%) (Table 5).

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3.3.2.2 The Phosphoprotein (P) Gene and P/C/V Editing

The P gene of the two strains of sPIV3 is 1995 nt long with a major ORF of 1788 nt

encoding the large P protein of 596 aa long with a calculated size of 66.3 kDa (ISU92) and 69.2

kDa (Texas81) and pI of 5.4 and 5.6, respectively (Table 3). The sequence identity between two

sPMV P genes is 87.6% and when compared with other Paramyxoviruses P proteins, it is poorly

conserved (Table 5).

The N terminal of P protein of paramyxoviruses are heavily phosphorylated at serine and

threonine residues (Robert A. Lamb, 2007). There are 44 serine and 16 threonine residues in

Texas81 P protein and 50 serine and 17 threonine residues in ISU92 P protein identified as

potential phosphorylation sites using NetPhos 2.0 server (Blom et al., 1999)

(http://www.cbs.dtu.dk/services/%etPhos/).

The P gene contains an mRNA editing site, 5’AAAAAAGGG3’(mRNA sense) in both

Texas81 and ISU92 (794-802 nt), which is identical to those of other respiroviruses (Figure 3).

By sequencing at least 40 clones of cDNA from the mRNA of two strains, we found that

Texas81 had 1-4 G insertions while ISU92 had a broader distribution of G insertions, (1-9 Gs) at

the editing sites. The insertion of G residues during mRNA synthesis can shift the translational

reading frame and thus potentially generate V (+1/4/7 Gs) protein, predicted to be 412 aa long of

47.8 kDa in ISU92 and 46.7 kDa in Texas81, with identity of 83.3% between two strains, and D

(+2/5/8 Gs) protein, 367 aa long of 41.3 kDa in ISU92 and 41.5 kDa in Texas81 with identity of

84.0% between two strains. The predicted V and D proteins will be amino co-terminal with P

(+0/3/6/9 Gs) protein (first 241 aa). In sPIV3, the C protein is predicted to be initiated 10 nt

downstream of the P protein start codon by alternate translation initiation AUG codon and

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predicted to be 201 aa long with MW of 23.6 kDa in ISU92 and 23.7 kDa in Texas81 with

identity of 91.1% between two strains.

At the C-terminal of V protein, sPIV3 contains all seven conserved invariantly placed

cysteine residues, highly conserved motifs H-R-R-E and W-C-N-P, known among

paramyxovirus V proteins (Figure 4). This cysteine-rich C-terminal is shown to be involved in

coordinating two zinc molecules per V protein and may play an important role in viral

pathogenesis and blocking of host interferon defense mechanisms (He et al., 2002; Patterson et

al., 2000; Poole et al., 2002; Robert A. Lamb, 2007).

3.3.2.3 The Matrix protein (M) Gene

The M gene is 1149 nt long with a single ORF of 1053 nt. The encoded protein is 351 aa

long with a predicted MW of 39.3 kDa and a pI of 9.5 (Texas81) and 9.6 (ISU92). The M protein

is the most abundant virion structural protein located in the inner surface of envelope. It interacts

with the cytoplasmic tails of the integral membrane proteins, lipid bilayer and the nucleocapsids,

and plays an important role in virion assembly, budding, release as well as transport of viral

components (Robert A. Lamb, 2007; Ruth A. Karron, 2007). M protein is considered to be the

most conserved parainfluenza viral protein (Spriggs et al., 1987). The nuclear localization signal

(NLS) (245

KMGRMYSVEYCKQKIEK261

) of M protein in swine viruses is highly conserved in

PIV3 (Coleman and Peeples, 1993; Peeples et al., 1992). The ISU92 virus M protein has 96.6%

amino acid sequence identity with Texas81. The amino acid sequence identity with members of

the other genera of Paramyxovirinae decreased in the following order: Respiroviruses (63.1%-

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100%); Morbilliviruses (33.5%-37.2%); Henipaviruses (31.9%-32.7); Avulaviruses (17.9%-

22.9%); Rubulaviruses (18.1%-19.9%); with unclassified viruses, ASPV (39.2%-40.1%), J-V

(35.7%-36%), BeV (35.7%-36%). There was only less than 10% identity when sPIV3 were

compared with the members of Pneumovirinae.

3.3.2.4 The Fusion Protein (F) Gene and Hemagglutination-%euraminidase Protein (H%) Gene

We have recently reported the molecular characterization of the envelope glycoprotein

genes of these two swine viruses (Qiao et al., 2009). The molecular features of these two genes

are shown in Table 3. There were several amino acid changes in the antigenic sites of the F

protein differing from hPIV3 but conserved as in bPIV3. They include: E101N, V/T367I (only in

ISU92), S418Q, T492A, T513V.

3.3.2.5 The Large Polymerase Protein (L) Gene

The L gene of sPIV3 is 6795 nt long, with a major 6699 nt long ORF encoding a 2233 aa

protein with a pI of 6.3 for Texas81 or 6.2 for ISU92, and MW of 25.6 kDa. The L protein of

parainfluenzaviruses is the major RNA polymerase (RNAP) component and it is responsible for

nucleotide polymerization, mRNA capping, methylation and polyadenylation (Robert A. Lamb,

2007). It is proposed that there are six highly conserved domains (DI-DVI) in nonsegmented

negative-strand virus (NNSV) and each domain may be individually responsible for each of the

L protein multiple functions (Poch et al., 1990; Sidhu et al., 1993; Svenda et al., 1997). Pairwise

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alignment of sPIV3 along with the other paramyxoviruses revealed all the six domains, highly

conserved A to D subdomains within DIII and a highly variable hinge region between DII and

DIII (Figure 5). A highly conserved stretch (positions 543 to 562) within DII, proposed to be a

template recognition site, was present in sPIV3 L protein and matched the pattern of basic and

hydrophobic amino acid pair repeats every four residues as described (Poch et al. 1990). The

highly conserved 772

GDNQ775

motif, which is believed to be the active site for nucleotide

polymerization (Chattopadhyay and Shaila, 2004; Malur et al., 2002), was present in subdomain

C in DIII. The L protein of swine viruses also contained a putative ATP binding site with motif

1786K-X21-G-E-G-A-G

1810 (Poch et al., 1990) in DVI. The L protein of Texas81 has 99.2%

amino acid sequence identity with ISU92, 59.7%-98.7% with Respiroviruses, 38.8%-39.7% with

Henipaviruses, 37.2%-42.5% with Morbilliviruses, 28.3%-29.8% with Rubulaviruses, 26.2%-

27.2% with Avulaviruses, 48.3%-48.5% with ASPV, 38.6%-38.8% with J-V, 37.9%-38% with

BeV, 16.7%-17.0% with Metapneumoviruses, and 15.1%-15.5% with Pneumoviruses.

3.3.3 Phylogenetic analysis

Phylogenetic trees were generated based on N, P, M, F, HN, L nucleotide sequence,

deduced amino acid sequences, and complete genome sequence of sPIV3 with other

representative members of all the five genera of family Paramyxoviridae and found to be similar.

The sPIV3 strains were phylogenetically closely related to genus Respirovirus within

Paramyxovirinae subfamily, and Texas81 and ISU92 belonged to two subgenotypes of genotype

A and the Australian bPIV3 strain Q5592 formed the genotype B (Figure 6). The phylogenetic

reconstructions of representative trees are shown (Figure 6).

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3.3.4 Pathogenicity

Conventionally reared pigs infected intranasally with either of the two viruses resulted in

mild respiratory signs only. None of the infected pigs developed neurological signs or elevated

body temperatures throughout the experimental period (Figure 7). Two pigs from each of the

infected groups developed mild respiratory signs at 2 days post inoculation (DPI) (mean clinical

score 1). One pig from Texas81 group had diarrhea on day 2 and another on day 3 (mean clinical

score 1). The gross lesions at necropsy on 6 and 10 DPI from virus-infected pigs were

unremarkable, such as segmented, thickened small intestine and greenish milky intestinal

contents. Histologically, no microscopic lesions were observed. Virus isolation attempts were

unsuccessful on swab and tissue samples collected at day 6 and 10 DPI except from one nasal

swab sample from Texas81 virus infected pig on day 6. None of the lung tissues examined by

immnohistochemistry using polyclonal anti-bPIV3-SF serum revealed the presence of virus-

specific antigen. Pre-inoculation serum samples were negative for sPIV3-specific antibodies by

VN test. The homologous neutralization titers of sera samples at 10 DPI ranged from 1:8 to1:16,

but none of the infected pigs had VN antibodies at 6 DPI.

3.3.5 Serum antibody responses to sPIV3 in pigs detected by indirect ELISA

None of the 100 serum samples obtained from pigs aged 19 days to 70 days from the 5

swine farms designated A, B, C, D, E in Minnesota and Iowa in 2007-2008 were positive by

ELISA suggesting the absence of prevalence of sPIV3 in these farms. The sera samples from

experimentally inoculated pigs were positive at 6 and 10 dpi and the pre-inoculation samples and

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sera from mock-infected pigs were negative. These results confirmed the neutralization test

results of sera from experimentally infected pigs. However, the fact that 6 dpi samples also

showed positive result in ELISA but not in neutralization test suggests that ELISA is capable of

detecting non-neutralizing antibodies. A large number samples need to be screened by both tests

to determine the relative merits of ELISA over virus neutralization test for prevalence studies.

But, the indirect ELISA can be used as a simple screening test for the presence of sPIV3

antibodies.

3.4 Discussion

The recent insurgence of novel paramyxoviruses of zoonotic importance necessitates the

close monitoring of viral pathogens with respiratory and neurological tropism in animals.

Although, several paramyxoviruses have been isolated from swine in USA and other parts of the

world, many except a few have shown the potential to be extremely virulent and infect across

species (Field et al., 2007b). The epidemiologic significance of many of the swine

paramyxovirus isolates has never been examined except for the initial case reports (Janke et al.,

2001; Philbey et al., 1998).

In this study, we have performed detailed analysis of two swine viruses isolated in the

United States between 1980 and early 1990s. Our preliminary study of the morphologic,

antigenic characteristics and sequence analysis of the envelope glycoproteins indicated that these

two viruses belonged in the genus Respirovirus, subfamily Paramyxovirinae. We have further

characterized these isolates for their complete genomic sequence and features, and their

123

pathogenicity in conventionally reared pigs. Our comprehensive studies revealed that these

sPIV3 are host-adapted variants of bPIV3, possibly transferred from cattle to pigs but failed to

establish an active enzootic state.

The mild clinical signs and undetectable gross and microscopic lesions observed in

sPIV3-infected pigs indicate the inapparent nature of these viruses in pigs. It has been reported

that bPIV3 can be isolated from clinically normal cattle (Rosenberg et al., 1971). It is generally

accepted that co-infection of bPIV3 along with other viruses and Mannheimia/Pasturella species

could precipitate the classical shipping fever syndrome (Battrell, 1995; Woods, 1968). A variety

of factors such as environmental temperature, transportation, hygiene, stocking density, co-

mingling, and host immune status can contribute to increased susceptibility to secondary

bacterial infections and severity of clinical disease (Battrell, 1995; Woods, 1968). The outbreak

of disease in 1980s and 1990s in pigs with these sPIV3, therefore, might have resulted from

coinfection with other pathogens. Initial pathogenicity studies in gnotobiotic piglets with ISU92

strain also resulted in mild respiratory disease and microscopic pathology (Janke et al., 2001),

suggesting this possibility. Further, our limited serological prevalence studies in sera samples

collected between 2007 and 2008 also indicated that sPIV3 are not prevalent in the field. An

earlier study examining the seroprevalence of pigs from Iowa indicated that there was a very low

seroprevalence. From 876 serum samples collected from 36 swine farms in 1988 to 1989, only 6

samples, representing 5 swine herds, were detected to have anti-ISU92 antibody titers (Battrell,

1995).

The complete genome of sPIV3 is 15456 nt in Texas81 and 15480 nt in ISU92, which is

typical of genome sizes (15500 nt) in paramyxoviruses. However, the recently determined

124

sequences of aPMV6 (16236 nt) (Chang et al., 2001), aPMV3 (16272 nt) (Kumar et al., 2008),

NiV (18246 nt) (Harcourt et al., 2001; Wang et al., 2001), HeV (18234 nt) (Wang et al., 2001), J-

V (18954 nt) (Jack et al., 2005), BeV (19212 nt) (Li et al., 2006b), MoV(16650 nt) (Miller et al.,

2003), TPMV (17904 nt) (Tidona et al., 1999) and ASPV (16965 nt) (Nylund et al., 2008) show

a greater genome length ranging from ~16300 nt to ~19200 nt. The lengths of the genome of

sPIV3 conform to the “rule of six” which is consistent in members of subfamily

Paramyxovirinae (Calain and Roux, 1993) and this is thought to be associated with nucleocapsid

organization in which each N protein monomer bind to six nucleotides of the viral genome.

Recombinant PIV with a genome length that was not an even multiple of six has been shown to

mutate to conform to the rule (Skiadopoulos et al., 2002; Skiadopoulos et al., 2003b).

The genome organization of these two strains of sPIV3 is essentially consistent with

members of the subfamily Paramyxovirinae. The transcriptional start signal is conserved as in

the consensus sequence of genus Respirovirus. The trinucleotide IGS between the gene

boundaries is an exact resemblance to the ones of Respirovirus, Morbillivirus and Henipavirus,

but differ from the variable-length intergenic regions found in the Rubulavirus (1-47nt),

Avulavirus (1-63nt) and Pneumovirus (1-56nt) (Robert A. Lamb, 2007). The length of the 3’

leader is consistent in most known paramyxoviruses (55nt), except hPIV2 (70nt) (Robert A.

Lamb, 2007). The length of 5’ trailer in Paramyxoviridae is more variable, ranging from the

shortest 21 nt in hPIV2 (Ruth A. Karron, 2007) to 707 nt in aPMV3 (Kumar et al., 2008) as the

longest trailer among members of family Paramyxoviridae. The 5’ trailer of sPIV3 is 44 nt

within the 25-58 nt range seen in Respirovirus, Morbillivirus, Rubulavirus and Henipavirus

genera. The overall complementarity between 3’ and 5’ termini was as high as 65.9%

125

(ISU92)/63.6% (Texas81) in sPIV3, and this high complementarity was also seen in most

members of the Paramyxovirinae.

The total coding percentage of the sPIV3 is 93% which is similar to what is found in

other Paramyxovirinae with an average of 92% coding capacity (Wang et al., 2000). The

genome contains six genes: N, P, M, F, HN, L, as found in most members of Paramyxoviridae.

In some of the members, instead of HN, the H or G proteins serve as attachment proteins and

some Rubulaviruses, Avulaviruses (APMV6) and all Pneumoviruses also encode a third integral

membrane protein, SH, in the genome. The genome of swine viruses can potentially encode 9

proteins including the six invariant structural proteins and three accessory proteins, V, D, C,

which are all derived from P/C/V gene by a mechanism known as mRNA editing (V and D) or

alternative translation initiation (C).

The mRNA editing in P/C/V gene is a common feature in Respiroviruses,

Morbilliviruses, Henipaviruses and Avulaviruses, and is considered as a remarkable example of

exploiting the coding capacity of viruses (Robert A. Lamb, 2007). The V protein plays important

roles in virus replication (Baron and Barrett, 2000; Curran et al., 1991), function as a negative

regulator to inhibit RNA synthesis (Baron and Barrett, 2000; Delenda et al., 1997; Durbin et al.,

1999), and inhibit host cell antiviral response by interacting with cellular proteins (Andrejeva et

al., 2002; Lin et al., 1998). The C-terminal V-specific domain is highly conserved among

paramyxoviruses with invariantly spaced histidine and cysteine residues forming a domain (cys-

rich domain) that binds two zinc molecules per V protein (Fukuhara et al., 2002; Li et al., 2006a;

Liston and Briedis, 1994; Paterson et al., 1995). In sPIV3, the C-terminal of V protein contains

all of the seven conserved cysteine residues and motifs H-R-R-E and W-C-N-P. The presence of

126

these domains suggests a similar function for the V protein of swine viruses as in other

paramyxoviruses. The role of D in viral growth cycle has not been understood (Lamb and Parks,

2007). The C protein ORF is commonly observed in Respiroviruses and Morbilliviruses (Robert

A. Lamb, 2007). C proteins are small basic polypeptides that may involve in the viral growth

cycle, control of viral RNA synthesis (Lamb and Parks, 2007), counteracting host cell antiviral

pathways (Komatsu et al., 2004) and facilitating the release of virus from infected cells (Garcin

et al., 1997; Kato et al., 2001). Both the editing site and the predicted size of accessory proteins

of sPIV3 highly resemble that of hPIV3 and bPIV3 and may have similar functional roles.

The amino acid identities with the corresponding proteins of other Paramyxoviruses

clearly place the sPIV3 as novel viruses in the genus Respirovirus with a high level of identity to

bPIV3. The overall conservation of homologous proteins in paramyxoviruses is generally

consistent with the description in the most recent ICTV report, V-C≥L>M>F>N, and HN>V>P

(Lamb, 2005). Limited sequence polymorphism has been reported among bPIV3 strains (Bailly

et al., 2000; Coelingh and Winter, 1990; Swierkosz et al., 1995). The host specific amino acids

identified in bPIV3 (Bailly et al. 2000) were invariable between bPIV3 and sPIV3 but, further

amino acid changes noticed in envelope glycoproteins (Qiao, Janke et al, 2009) and P/N473H in

P gene and amino acid residues A326T and I924M in the L gene, suggest that the sPIV3 is

adapting to the swine host. The bPIV3-910N strain was a plaque-type variant and reported to

have intermediate fusion capabilities but avirulent in mice (Shibuta et al., 1981). The bPIV3-SF

strain was originally obtained from a calf showing signs of shipping fever (Breker-Klassen et al.,

1996). The antigenic site amino acid residues in F protein (Van Wyke Coelingh and Winter,

1990) were conserved in both strains of sPIV3 as in bPIV3. The fusogenicity of both Texas81

127

and ISU92 were comparable (Qiao, Janke et al, 2009). Detailed epitope mapping studies with F

and HN monoclonal antibodies are needed to elucidate host-specific antigenic site variations.

The genomic organization, amino acid identities of homologous proteins, phylogenetic

analysis based on the genome sequences and antigenic analysis all support the classification of

these two novel strains of sPIV3 into Respirovirus genus in Paramyxovirinae subfamily and

Paramyxoviridae family.

This mild pathogenicity of sPIV3 can facilitate its development as a vaccine vector. The

bovine/hPIV3 chimeric virus (Bailly et al., 2000; van Wyke Coelingh et al., 1988), as a vector

backbone with the replacement of F and HN glycoprotein from hPIV3 has been evaluated as a

vaccine against hPIV3 (Haller et al., 2000; Pennathur et al., 2003). Besides, bPIV3 as backbone

with replaced F and HN of hPIV3 and inserted F of RSV were also constructed. The

effectiveness of this vaccine against both PIV3 and RSV challenge has been demonstrated in

African green monkeys and is being evaluated in clinical trials (Sato and Wright, 2008). The

promising outcome of the vectored vaccines using paramyxoviruses and the biological and

molecular characteristics of swine viruses described here suggest a great potential for sPIV3 to

be developed as a vaccine vector. To fulfill this goal, the prevalence of sPIV3 in human

population needs to be ascertained and potential risk of increased pathogenicity by co-infection

need to be further studied. The screening ELISA method developed by us with sPIV3 antigen

can be used to detect seroprevalence in human and animal populations.

128

3.5 Acknowledgments

The authors thank Dr. X. J. Meng, Dr. Lijuan Yuan, Dr. Kevin Myles and Dr. Chris

Roberts for their constructive crtiticism and lab members Dr. Laure Deflube, Shobana

Raghunath, Vrushali Chavan, Thomas Rogers-Cotrone and Gopakumar Moorkanat for their

excellent technical assistance and help.

129

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158

3.7 Tables

Table 1. Primer list

Primer ID Sequence (from 5’ to 3’)a

Primers

for

RT-PCR

BPN-GS-f 44

AAAAATTAACTTAGGATTAAAGAACTTTACCGAA77

BPN-M-f 785

ACAGAGCTTGGTAACACTCATGGTTGAAAC814

BPN-GE-r 1721

TAATTTCCATTAATCCTAAGTTTTTCTTACTTGTTG1686

BPP-GS-f 1688

ACAAGTAAGAAAAACTTAGGATTAATGGAAATTATC1723

BPP-M-f 2368

CAGTACATTTGATTCAGGATATACCAGTATAGTGAC2403

BPP-GE-r 3718

TTAGTCCTTTCATCCTAAGTTTTTCTTAATCATCTATG3671

BPM-GS-f 3672

ATAGATGATTAAGAAAAACTTAGGATGAAAGGACTAA3717

BPM-GE-r 4870

TGATCCCTTTGATCCTAAGTTTTTGATTTTTCTCCTG4834

BPF-GS-f 4835

AGGAGAAAAATCAAAAACTTAGGATCAAAGGGAT4838

BPF-M-f 5423

ATAGGGATAGCCACTTCAGCACAAATCACC5452

BPF-GE-r 6742

GAACAACTTTGTTCCTAAGTTTTTTGTACTTTTTAT6707

BPHN-GS-f 6714

GTACAAAAAACTTAGGAACAAAGTTGTTC6742

BPHN-M-f 7325

CCAGGACCAGGTTTATTAGCAACATCTACTACAG7358

BPHN-GE-r 8638

TTTCTTGCACACTTTTCTCCTAAGTTTTTTATTATTTC8601

BPL1-GS-f 8602

AAATAATAAAAAACTTAGGAGAAAAGTGTGCAAG8635

BPL1-M-r 10046

AGTTAAGTCTTCATCCAATTGAGGCTCTATAAAC10013

BPL2-M-f 9993

AATTTGACAAGTTTATAGGAGCCTCAATTGGATGAAG10038

BPL2-M-r 11385

TATTTTGGGTTATAGTTGGGTTTATATTCATTC11352

BPL3-M-f 11307

GTATGCTCAATCTTCAAAAATATCCAACAG11336

BPL3-M-r 12706

GTACTCTCCTCTAACCTAAATAGATATTCAAATACACT12669

BPL4-M-f 12594

ATATGTCTATTAAAGAAGCAAATGAAACTAAAGATACA12632

BPL4-M-r 14130

AATTATAATAATTTATTGCAGGACCGAGTGTAGC14097

BPL5-M-f 14054

CCTTTTTAGGAGAAGGAGCAGGAGCTATGTTAG14086

BPL5-GE-r 15430

GTATATATATATTACATGTTTTTCTTACTTTTCATTTTGTTG15389

Primers

for

sequencing

BPN-M-f-seq 1420

ATGAGCCTCGGTCATCCATAGTTCC1444

BPP-M-r-seq 2139

GTTCTACTATCTGGGCTGCTTCCTCTCC2112

BPM-M-f-seq 4704

GATGCAGTTCTCCAGCCTTCATTACC4729

BPHN-M-r-seq 7065

GTTCCAATGTCATCCGAAGTCCTCTG7039

BPHN-M-f-seq 8368

CAGAACACTTCCAGCTGCATATACAACAAC8437

BPL1-M-r-seq 9645

CATTAGGTATTTCCTCTGTTGTACACTCAGC9615

BPL2-M-f-seq 10573

CTACAGATATATACAATGATGGATACGAAACCG10605

BPL3-M-r-seq 11734

CCAGATAGTAATGGGTTTGGTGAGTCC11708

Pimers for

RACE

DT89 CCAAA ACGCCATTTCCACCT TCTCT TC

DT88 GAAGAGAAGGTGGAAATGGCGTTTTGG

ISU-5-1 14780

GATAATGAGGCCATATCATACAGCACT14806

ISU-5-2 14911

CAATTAAAGATGTTATGTTCGAATGGGTC14939

ISU-5-3 15034

ATAAGGGAAAACTAAGATTATTATCACGAAG14994

ISU-3-1 499

CAGTCAGTTGTCTTTTCATAAACCATCTC471

ISU-3-2 166

GATTTTGTTATATTCTCTTGCCTACGC140

TX-5-1 14925

CATGACAATAAAGACATAAATTAGGAGGAAG14956

TX-5-2 15025

GGATATCATTATCTTTATCAACCAGATTACTG15056

TX-5-3 15158

TATTGTCAAAAGTTACAAAGAACACATAGG15187

TX-3-1 405

CATATTTAACATCTGCATTACTACCATTTG376

TX-3-2 166

GATTTTGTTATGTTCTCCTGCCTACG141

a. All primer sequences were given from 5’ to 3’. The numbers in superscript indicate the expected

position of the primer in bPIV3-SF/Ka genome. DT88 and DT89 were the primers used in Li et al., 2005

for RACE.

159

Table 2. Comparison of the gene-start and gene end signals of sPIV3, bPIV3 and hPIV3.

Gene Strain Gene-start Gene-end

N

Texas81 UCCUAAUUUC UUCAUUCUUUUUa

ISU92 UCCUAAUUUC UUCAUUCUUUUU

bPIV3-Ka/SF UCCUAAUUUC UUCAUUCUUUUU

bPIV3-910N UCCUAAUUUC UUCAUUCUUUUU

bPIV3-Q5592 UCCUAAUUUC UUUAUUCUUUUU

hPIV3 UCCUAAUUUC UUUAUUCUUUUU

P

Texas81 UCCUAAUUACb UUCAUUCUUUUU

ISU92 UCCUAAUUGC UUCAUUCUUUUU

bPIV3-Ka/SF UCCUAAUUAC UUCAUUCUUUUU

bPIV3-910N UCCUAAUUGC UUCAUUCUUUUU

bPIV3-Q5592 UCCUAAUUGC CUAAUUCUUUUUc

hPIV3 UCCUAAUUUC UUCAUUCUUUUU

M

Texas81 UCCUACUUUC UUUUU-----------------AGUUUUU

ISU92 UCCUAUUUUC UUUUU-----------------AGUUUUU

bPIV3-Ka/SF UCCUACUUUC UUUUU-----------------AGUUUUU

bPIV3-910N UCCUAUUUUC UUUUU-----------------AGUUUUU

bPIV3-Q5592 UCCUUAUUUC UUUUU-----------------AGUUUUU

hPIV3 UCCUAAUUUC UUUAUUCCCUAUUAGUUUUU

F

Texas81 UCCUAGUUUC UUCAUGUUUUUU

ISU92 UCCUAGUUUC UUCAUGUUUUUU

bPIV3-Ka/SF UCCUAGUUUC UUCAUGUUUUUU

bPIV3-910N UCCUAGUUUC UUCAUGUUUUUU

bPIV3-Q5592 UCCUAGUUUU UUUAUCUUUUUU

hPIV3 UCCUGUUUUC CUAAUAUUUUUU

HN

Texas81 UCCUUGUUUC UUAUUAUUUUUU

ISU92 UCCUUGUUUC UUAAUGUUUUUU

bPIV3-Ka/SF UCCUUGUUUC UUAUUAUUUUUU

bPIV3-910N UCCUUGUUUC UUAAUGUUUUUU

bPIV3-Q5592 UCCUUGUUUC UUAUUAUUUUUU

hPIV3 UCCUCAUUUC UUUAUAUUUUUU

L

Texas81 UCCUCCUUUC UUCAUUCUUUUU

ISU92 UCCUCCUUUC UUCAUUCUUUUU

bPIV3-Ka/SF UCCUCUUUUC UUCAUUCUUUUU

bPIV3-910N UCCUCCUUUC UUCAUUCUUUUU

bPIV3-Q5592 UCCUCUUUUC UUUAUUCUUUUU

hPIV3 UCCUCGUUUC UUCAUUCUUUUU

Consensus: UCCUNNUUNC NUNNUNNUUUUU

a. Host-specific nucleotide residues between hPIV3 and bPIV3 are in red;

b. Positions that display variability within the host group are in blue;

c. Positions that unique in bPIV3-Q5592 are in bold.

160

Table 3. Comparison of gene sequences between swine, bovine and human PIV3 viruses

Gene Virus

mRNA Deduced protein

Length 3’ UTR

(nt)

ORF

(nt)

5' UTR

(nt) Length Calculated MW pI

N

Texas81 1646 55 1545 46 515 57303.70 5.009

ISU92 1646 55 1545 46 515 57340.89 5.217

bPIV3a 1646 55 1545 46 515 57303.70 5.009

hPIV3 1646 55 1545 46 515 57840.25 5.212

P

Texas81 1995 79 1788 128 596 69185.44 5.789

ISU92 1995 79 1788 128 596 66290.29 5.718

bPIV3 1995 79 1788 128 596 66459.46 5.576

hPIV3 2013 79 1809 125 603 67794.65 5.292

M

Texas81 1149 32 1053 64 351 39327.09 9.473

ISU92 1149 32 1053 64 351 39252.06 9.610

bPIV3 1149 32 1053 64 351 39327.09 9.473

hPIV3 1155 32 1059 64 353 39571.39 9.582

F

Texas81 1869 211 1620 38 540 60188.74 6.539

ISU92 1893 235 1620 38 540 60039.42 6.797

bPIV3

1869 211

1620

38

540

60188.74 6.539

1893(910N) 235 38 60007.41 7.025

1887(Q5592) 223 44 60128.69 6.796

hPIV3 1851 193 1617 41 539 59978.35 7.499

HN

Texas81 1888 73 1716 99 572 64624.47 7.720

ISU92 1888 73 1716 99 572 64652.46 7.343

bPIV3 1888 73

1716 99 572 64623.48 7.869

1912(Q5592) 97 64431.41 7.504

hPIV3 1888 73 1716 99 572 64313.66 7.546

L

Texas81 6795 22 6699 74 2233 255734.70 6.281

ISU92 6795 22 6699 74 2233 255814.76 6.161

bPIV3 6795 22 6699 74 2233 255801.85 6.220

hPIV3 6795 22 6699 74 2233 256071.48 6.394

a bPIV3 indicates bPIV3 shipping fever stain if not specified.

161

Table 4. Subunit hexamer phasing positions for gene start sites and P editing sites of a

selection of Paramyxovirinae

Genus Virus(es) N P/V M F HN L P Edit

Respirovirus

sPIV3 2 1 1 1 1 2 2

hPIV3, bPIV3 2 1 1 1 1 2 2

SeV 2 1 1 1 1 2 1

Rubulavirus SV5 2 1 1 2 1 6 3

MuV 2 1 6 1 1 6 3

Morbillivirus CDV,DMV 2 2 4 2 3 2 6

MeV, RPV 2 2 4 3 3 2 6

Avulavirus NDV 2 4 4 4 3 6 1

Henipavirus HeV 2 3 4 4 4 3 5

Unclassified

ASPV 2 2 1 2 3 4 2

TPMV 2 2 1 3 3 2 6

FDLV 2 2 4 3 6 3 2

162

Table 5. Amino acid identities of swine parainfluenzaviruses with analogous proteins in

other paramyxoviruses.

Paramyxovirus

genus Species

% Amino acid identiy between sPIV3 and other paramyxovirus proteins

N P M F Aa L

Tb Ib T I T I T I T I T I

Respirovirus

sPIV3b 97.1 87.6 96.6 95.0 96.0 99.2

bPIV3 100 97.1 100 87.6 100 96.6 100 95.0 99.8 95.8 98.7 98.2

hPIV3 86.2 86.4 64.3 65.4 92.6 92.0 82.6 81.9 77.1 77.6 90.6 90.6

hPIV1 60.6 61.0 24.1 24.5 64.6 63.1 42.7 42.7 45.8 45.6 60.7 60.7

SeV 62.6 62.6 23.7 24.5 64.0 63.7 43.1 42.9 46.7 46.0 59.7 59.7

Rubulavirus

MenV 19.2 19.2 9.2 9.2 19.9 19.9 23.4 23.0 15.2 15.4 28.5 28.5

SV5 18.5 18.8 8.8 8.5 18.4 18.1 24.8 24.0 25.9 25.5 29.4 29.4

hPIV2 18.5 18.6 6.4 5.9 18.7 18.4 25.2 25.0 24.0 24.4 28.4 28.3

LPMV 18.3 18.8 8.5 8.8 18.2 18.8 24.4 24.4 25.6 25.6 28.5 28.4

Avulavirus

NDV 20.1 20.3 5.9 5.9 19.9 20.5 25.0 24.9 23.2 23.0 26.2 26.3

aPMV2 20.6 20.0 10.7 10.2 17.9 18.2 22.0 22.2 21.0 21.2 27.2 27.1

aPMV6 17.9 17.7 7.6 8.8 22.9 22.3 24.2 24.6 20.9 20.5 26.8 26.8

Morbillivirus MeV 20.8 20.8 9.4 9.4 37.2 37.2 25.7 25.7 11.6 11.8 37.6 37.6

RPV 19.8 19.4 9.5 9.7 37.2 37.2 24.8 24.2 11.6 11.3 37.4 37.6

Henipavirus NiV 20.2 19.8 10.0 10.0 32.5 31.9 27.7 28.1 18.9 19.4 39.7 39.7

HeV 20.2 19.8 10.6 10.0 32.7 32.5 26.9 26.7 18.9 19.6 38.8 38.8

Metapneumovirus hMPV 12.2 11.5 13.9 13.9 10.6 9.8 15.3 15.3 10.6 11.0 16.7 16.8

aMPV 12.0 11.5 14.3 15.0 9.8 9.1 14.6 14.8 8.8 9.1 17.0 17.0

Pneumovirus hRSV 11.1 11.5 12.0 12.0 9.4 9.4 13.2 12.9 11.7 12.3 15.1 15.1

bRSV 11.1 11.8 12.9 14.0 9.8 9.8 12.9 13.2 12.0 12.0 15.4 15.1

Unclassified virus

ASPV 24.8 23.4 9.5 10.5 39.2 40.1 31.7 31.7 37.0 37.4 48.3 48.5

BeV 23.8 23.3 8.9 9.1 36.0 35.7 28.0 27.6 24.2 24.4 37.9 38.0

J-V 20.5 21.5 7.5 7.8 36.0 35.7 28.9 29.3 24.1 24.8 38.6 38.8

FDLV 21.4 21.7 11.3 12.3 36.0 35.8 30.1 30.3 36.4 36.6 42.4 42.5

a A, attachment protein. The attachment protein for morbilliviruses is H; for henipaviruses,

metapneumoviruses, pneumoviruses, BeV, J-V is G; for remaining is HN.

b T indicates Texas81, I indicates ISU92, the identities in the row sPIV3 indicates the identities between

the two swine strains.

163

3.8 Figures

Figure 1. Strategy for rapid Amplification of cD?A Ends (RACE) (adapted from Li et al.

2005). For 5′RACE, cDNA synthesis was carried out first using a virus-specific primer-1 (VSP5-

1), followed by RNaseH treatment, then ligation of adaptor (DT88). In 3′ RACE, adaptor ligation

was carried out first directly with the viral RNA, followed by cDNA synthesis using the DT89

primer complementary to adaptor DT88. The remaining steps of the primary and hemi-nested

PCRs are the same for both RACE methods with DT89 and VSPs using standard procedures.

164

(A) 3’ leader

bPIV3-910N 3’-UGGUUUGUUCUCUUCUCUGAACGAACCCUUAUAAUUAAGUCUAUUUUUAAUUGAA

bPIV3-Ka 3’-UGGUUUGUUCUCUUCUCUGAACGAACCCUUAUAAUUAAGUUUAUUUUUAAUUGAA

bPIV3-SF 3’-UGGUUUGUUCUCUUCUCUGAACGAACCCUUAUAAUUAAGUUUAUUUUUAAUUGAA

bPIV3-Q5592 3’-UGGUUUGUUCUCUUCUCUGAACGAACCUUUAUGUUUAAGUUUAGUUUUAAUUGAA

hPIV3-Wash 3’-UGGUUUGUUCUCUUCUUUGAACAAACCUUUAUAUUUAAAUUUAAUUUUAAUUGAA

hPIV3-JS 3’-UGGUUUGUUCUCUUCUUUGAACAGACCCUUAUAUUUAAAUUGAAAUUUAAUUGAA

ISU92 3’-UGGUUUGUUCUCUUCUCUGAACAAACCCUUAUAAUUAAGUUUAUUUUUAAUUGAA

Texas81 3’-UGGUUUGUUCUCUUCUCUGAACAGACCCUUAUAAUUAAGUUUAUUUUUAAUUGAA

(B) 5’ trailer bPIV3-910N GUAUAUUAUAUAUAUAUGGUUUGUCUCAAAAAGAGAACAAACCA-5’

bPIV3-Ka GUACAUUAUAUAUAUAUGGUUUGUCUCAAAAAGAGAACAAACCA-5’

bPIV3-SF GUAUAUUAUAUGUAUAUGGUUUGUCUCAAAAAGAGAACAAACCA-5’

bPIV3-Q5592 GUAUUUUAUAUAUAUAUGGUUUGUCUCAAAAAGAGAACAAACCA-5’

hPIV3-Wash GUACAUUAUAUAUAUAUGGUUUGUCUCAAGAAGAGAACAAACCA-5’

hPIV3-JS GUACAUUAUAUAUAUAUGGUUUGUCUCAAGAAGAGAACAAACCA-5’

ISU92 GUAUAUUAUAUAUAUAUGGUUUGUCUCAAAAAGAGAACAAACCA-5’

Texas81 GUAUAUUAUAUGUAUAUGGUUUGUCUUAAAAAGAGAACAAACCA-5’

(C) Genomic termini ISU92

3’- UGGUUUGUUCUCUUCUCUGAACAAACCCUUAUAAUUAAGUUUAUUUUUAAUUGAA

5’- ACCAAACAAGAGAAAAACUCUGUUUGGUAUAUAUAUAUUAUAUG

Texas81

3’- UGGUUUGUUCUCUUCUCUGAACAGACCCUUAUAAUUAAGUUUAUUUUUAAUUGAA

5’- ACCAAACAAGAGAAAAAUUCUGUUUGGUAUAUGUAUAUUAUAUG

Figure 2. ?ucleotide sequence of the 3’ leader (A), 5’ trailer (B) regions and genomic

termini (C) of PIV3 genomic R?A. Sequences are 3’ to 5’ in negative sense. (A,B) Nucleotide

residues in blue are host-specific (bPIV3 or hPIV3). Host-specific positions are those where

bPIV3 strains share an assignment and hPIV3 strains share a different assignment. Positions that

display variability within a host species are in red. (C) Shading indicates complementary base

pairs.

165

Figure 3. Schematic of P protein structure, P gene expression strategy and R?A editing site

sequence of swine viruses compared with other paramyxoviruses. The scale bar at the top

indicates the amino acid of P protein, and the boxes below indicate possible reading frames of

protein P, V, D and C. known functional domains are indicated for chaperoning uassembled N

proteins during the nascent chain assembly of genome replication (N), self-assembly as a

tetramer (4’mer), L protein-binding site (L), and N-RNA-binding site (N:RNA) based on the

study of sendai virus (Robert A. Lamb, 2007). In the editing site sequences, AnGn elements are

spacing between An and Gn to facilitate visual comparison (Hausmann et al., 1999; Robert A.

Lamb, 2007).

aa 400 200 300 500 100

N 4’mer N:RNA L

Editing Site (794-802 nt)

V protein (412 aa) +1G

D protein (367 aa) +2G

C protein (201 aa) Alternative initiation

Cys-Rich

sPIV3 AAAAAA GGG

Respirovirus AAAAAA GGG

Morbillivirus AAAAA GGG

Rubulavirus AAGA GGGG(n)

Avulavirus AAAAA GGG

Henipavirus AAAAA GGG

ASPV AAAAAA GG

J-V AAAAAA GG

BeV AAAAA GG

FDLV AUAA GGGGGG

TPMV AAAAAA GG

Polymerase cofactor Nascent chain assembly P protein

(596 aa)

166

Figure 4. Amino acid sequence alignment of the conserved cysteine-rich C-terminal region

of selected paramyxovirus V proteins. Positions of the conserved seven conserved cysteine

residues that are involved in Zn binding are underlined.

Texas81 SWCNPICSKIRPIPRQESCVCGECPKQCRYCI

ISU92 SWCNPICSKIRPIPRQESCVCGECPKQCGYCI

bPIV3-SF SWCNPICSKIRPIPRQESCVCGECPKQCRYCI

hPIV3 SWCNPICSKIRPVPRQESCVCGECPKQCGYCI

SeV SWCNPVCSRIRIIPRRELCVCKTCPKVCKLCR

MenV EWCNPICHPISQSTFRGSCRCGNCPGICSLCE

SV5 EWCNPSCSPITAAARRFECTCHQCPVTCSECE

hPIV2 EWCNPRCAPVTASARKFTCTCGSCPSICGECE

LPMV EWCNPACSPISMEPRYYQCTCGTCPAKCPQCA

NDV SWCNPSWSPIKAEPKQYPCFCGSFPPTCRLCA

aPMV2 SWCNPVCSPIRSEPRREKCTCGTCPESCILCR

aPMV6 EWCNPVCAPVTPEPRAFKCICGRCPRVCINCR

MeV EWCNPICHPISQSTFRGSCRCGNCPGICSLCE

RPV RWCNPTCSKVTVGTVRAKCICGECPRVCEQCI

Nipah EWCNPACSRITPLPRRQECQCGECPTECFHCG

HeV EWCNPVCSRITPQPRKQECYCGECPTECSQCC

J-V EWCNPQCAPITVTPTQSRCTCGECPKVCARCI

BeV EWCNPQCAPLTVTPTQKECSCGECPRQCARCI

ASPV SWCNPQCAPIRRTPYREVCRCGGCPEECPDCS

TPMV SWCNPMCARIRPLPIREICVCGRCPLKCSKCL

FDLV EFCNPMCSRITPDFKPKICVCGECPRYCPRCK

167

Figure 5. Schematic overview of important features and conserved domains found in the

predicted L-protein amino acid sequences. Positions of the conserved RNA-dependent RNA

polymerase major domains DI-DVI, and subdomains A–D within DIII (Poch et al., 1990) are

indicated. In addition, the template recognition site in DII, conserved GDNQ sequence in

subdomain C, and a putative ATP-binding site motif (K–X21–G–E–G–A–G) in major domain

VI (Harcourt et al., 2001) are shown.

1600 800 1200 2000 400

Template recognition site

(543-562)

ʬ

225-417 653-876 927-1092 1129-1378 1770-1851

ATP binding site 1786

K-X21-G-E-G-A-G1810

Hinge

DIII DIV DVI DI DII DV

503-607

B C A D

659-671 768-777 838-851 730-754

Polymerization active site 772

G-D-N-Q775

aa

(A)

Figure 6. Phylogenetic analysis.

4.01) analysis with 1000 bootstrap replicate

compared with other members

genotypes based on N and M gene nucleotide sequences, respectively. The numbers over the

branches indicate the percentage of 10

branch. Strain information and GenBank accession numbers are presented in Materials and

Methods.

168

. Phylogenetic analysis. Phylogenetic analysis was performed using Parsimony (PAUP

with 1000 bootstrap replicates, based on M gene (A) nucleotide sequences

of Paramyxoviridae. (B, C) Phylogenetic analysis of PIV3

genotypes based on N and M gene nucleotide sequences, respectively. The numbers over the

branches indicate the percentage of 1000 bootstrap replicates that support each phylogenetic

branch. Strain information and GenBank accession numbers are presented in Materials and

Phylogenetic analysis was performed using Parsimony (PAUP

s, based on M gene (A) nucleotide sequences

C) Phylogenetic analysis of PIV3

genotypes based on N and M gene nucleotide sequences, respectively. The numbers over the

00 bootstrap replicates that support each phylogenetic

branch. Strain information and GenBank accession numbers are presented in Materials and

(B)

(C)

169

170

Figure 7. Rectal temperature in experimentally inoculated pigs. The rectal temperatures of

Texas81 and ISU92 or mock-inoculated pigs were recorded two times per day during the

experiment and the mean + standard deviation of temperatures for each group at each time point

were plotted.

98

99

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)Temperature Record

Control

Texas81

ISU92

171

General Conclusion

In conclusion, the complete genome of Texas81 virus was 15456 nucleotides (nt) and ISU92

was 15480 nt in length consisting of six non-overlapping genes coding for the nucloeo- (N),

phospho- (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN) and large

polymerase (L) protein in the order 3'-N-P/C/V-M-F-HN-L-5'. The features related to virus

replication and found to be conserved in most members of Paramyxoviridae were also found

in swine viruses. These include: conserved and complementary 3’ leader and 5’ trailer

regions, trinucleotide intergenic sequences, highly conserved gene start and gene stop signal

sequences. The length of each gene of these two viruses were similar except for the F gene,

in which ISU92 had an additional 24 nt “U” rich 3’ untranslated region (UTR). The P gene of

these viruses were predicted to express P protein from the primary transcript and edit a

portion of its mRNA to encode V and D proteins and the C protein was expected to be

expressed from alternate translation initiation from the P gene as in Respiroviruses. Sequence

specific features related to virus replication and host specific amino acid signatures in N, F,

HN, L proteins indicated that these viruses probably originated from bovine

parainfluenzavirus 3. Phylogenetic analysis based on the individual genes as well as

predicted amino acid sequences suggested that these swine parainfluenzaviruses are novel

members of the genus Respirovirus of the Paramyxovirinae subfamily, genotype A of bovine

parainfluenza virus type 3 and distributed into two subgenotypes of genotype A. The mild

clinical signs and undetectable gross and microscopic lesions observed in sPIV3-infected

pigs indicate the inapparent nature of these viruses in pigs. The mild pathogenicity and

limited replication of sPIV3 in the lungs of pigs can facilitate its development as a mucosal

vaccine vector.