R i i I f iRespiratory virus Infections&&

Laboratory testingLaboratory testing

The Common Cold

The common cold is a viral infection of the upper respiratory tract Usually last approximately 7 daysrespiratory tract Usually last approximately 7 days

Common Cold Syndrome includes rhinitis, tonsilitis,Common Cold Syndrome includes rhinitis, tonsilitis, pharyngitis, laryngitis (including croup), pharyngo-laryngitis etc. Sometimes Influenza (the flu) and sinusitis are characterized as a common coldsinusitis are characterized as a common cold syndrome

Season of the year, age, and prior exposure are y g p pimportant factors in the type of virus causing the infection and the type of symptoms that may occur

RISK FACTOR FOR MORE SEVERRISK FACTOR FOR MORE SEVER COMMON COLD

LOW NEUTRALIZING AbLOW NEUTRALIZING AbCHRONIC LUNG DISEASEEXTREMES AGEASTHMA

ALLERGYIg EIg ECYTOKINE PRODUCTION

I F N -gammaI L-5

Common Cold Viruses% COLD% COLDSEROTYPESEROTYPEVIRUSESVIRUSES % COLD% COLDSEROTYPESEROTYPEVIRUSESVIRUSES

60+100RHINOVIRUS

152CORONAVIRUS

< 103INFLUENZA

< 104PARA INFLUENZA

<102R S V <102R S V

< 1047ADENOVIRUS

< 10+40ENTROVIRUS

Variables used to distinguish viral fromVariables used to distinguish viral from bacterial pneumonia

CoronavirusCoronavirus

Coronavirus classification

• Order Nidovirales• Order Nidovirales• Family Coronaviridae• Genus Coronavirus• Genus Coronavirus• Non-segmented (+) ssRNA virus•There are 4 major groups within There are 4 major groups within coronavirus:

group 1- HCoV-229E (6 species)g p pgroup 2- HCoV-OC43 (7 species)group 3- Avian CoV (1 specie)SARS- CoV (1 specie)- responsible

for SARS epidemiology in 2003

Environmental stabilityEnvironmental stabilityEnvironmental stabilityEnvironmental stability

Viable virus on surfaces after 24 hrs

Easily inactivated by…H tHeatDryingLi id l tLipid solvents Oxidizing agentsDetergentsDetergentsUV radiation

Coronavirus proteins with immunoevasive properties

protein Virus source function

NSP1 SARS HKU4 a)Suppresses host protein expression throughNSP1 SARS,HKU4,HKU9

a)Suppresses host protein expression through direct inhibition of translation or by promoting

degradation of host mRNA including IFN-mRNAb)Inhibits IFN induction and signaling

NSP3(PLP) SARS,NL63 Blocks IRF3 activation and NFkB signaling

NSP3(ADRP) SARS 229E )I t f ith IFN i d d i l ti itNSP3(ADRP) SARS,229E a)Interfere with IFN induced viral activityb)Enhances host proinflammatory cytokine

expression

ORF 3b i SARS I hibi IFN h i d i liORF 3b protein SARS Inhibits IFN synthesis and signaling

ORF 6 SARS Inhibits STAT1 nuclear translocation

N SARS Inhibits IFN inductionInterfere with 2’,5’ OAS RNase L activity

M SARS Inhibits IRF3 activation

SARS pathogenesisSARS pathogenesis3 phases :

1. Viral replication2. Immune hyperactivity3. Pulmonary destruction

Pathology in the lung :Diffuse alveolar damage Epithelial cell proliferation p pIncrease of MQ

Multinucleate giant-cell have been associated with

putative syncytium like formation that is characteristic of

many coronaviruses infections

Symptoms y p• Clinical course:

short incubation period (6 days)time period from exposure to onset of symptoms ranging

from 2 to 16 daysintensive care usually required about 10 days after onsetintensive care usually required about 10 days after onset

of symptoms•There are generally 3 phases:

k 1 ld lik t f l i hill dweek 1: cold like symptoms,fever, myalgia, chill and a sore throat

week 2, recurrence of fever. Onset of diarrhea, and d t tioxygen desaturation

only 20% of patients reach this phase, requires ventilatory support

The fatality rate is about 10% upon infection

Risk factorsRisk factorsAgeHigh peak LDHg pHigh absolute neutrophil count

SARS-CoV Laboratory Testing: Specimens for PCRSpecimens for PCR

Lower respiratory tract specimensSputum, broncheoalveolar lavage, tracheal aspirate

Upper respiratory tract specimensNasopharyngeal aspirate/washp y g pNasopharyngeal/oropharyngeal swab

Place in dry sterile containeryDacron or rayon with plastic shaft

Blood5-10ml in EDTA tube (Purple top)5 10ml in EDTA tube (Purple top)

Stool10 - 50cc in stool cup or urine container, no preservatives

14

Store all specimens at 4oC prior to shipping

SARS-CoV Laboratory TestingSARS CoV Laboratory Testing

Real-time PCR

Respiratory specimens

SerologySerologyELISA for total antibody (IgG, IgM, and IgA)Considered the “Gold Standard”

Viral CultureTo rule-out other respiratory virusesNOT for SARS-CoV

SARS-CoV Laboratory Testing:

Real-time RT-PCRSensitivity: 1-10 copies in the reaction mixtureSensitivity: 1 10 copies in the reaction mixtureMultiple samples and specimen types can increase

sensitivity

Highly specific for SARS-CoV, but <50% sensitive during 1st week of infectionduring 1st week of infection

Low viral titers early in infection

Potential for false negatives(Poor samples, degradation during transport, low titers)

False positives are always a concern

SARS-CoV Laboratory Testing: S i f S lSpecimens for Serology

Acute and convalescent seraAcute and convalescent sera

approx. 30% positive within 6-10 days post-onset

Convalescent serum----Collect >28 days post-onset

Most patients positive at 2 weeksMost patients positive at 2 weeks

6-14 days before antibody detectable

Serology cannot be considered negative until >28 days

post onset

Biosafety in the LaboratoryBiosafety in the Laboratory

Untreated specimenspBSL-2 facilities using BSL-3 practices

Biosafety cabinet (BSC)

Centrifuge using sealed rotors or sample cups

Perform risk assessmentMay want to refer to another laboratory

Viral culture of suspect SARS specimens should be performed in BSL-3 facilityperformed in BSL 3 facility

ADENOVIRUSESADENOVIRUSES

Adenovirus Genome

Late genesE1 E3

Late genes

E2 E4

36 kb linear ds DNA

Early genes for host and viral transcription y g pcontrol, viral DNA replication

Late genes for virion structureLate genes for virion structure

Adenovirus Genome

Late genesE1 E3

Late genes

E2 E4

36 kb linear ds DNA

Earl genes for host and iral transcription control iralEarly genes for host and viral transcription control, viral DNA replication

Late genes for virion structureg

CLINICAL SYNDROMES

A. Respiratory diseases: p y

B. Eye infections:

C. Gastrointestinal disease

D. Other diseases:

E. Adenoviral infections of the immune compromised host

Respiratory diseases:Respiratory diseases:

The most important etiological association of adenoviruses is with the respiratory diseases.

They are responsible for 5% of acute respiratory diseases in:young children and much less in adults.

Respiratory diseases:Respiratory diseases:Four different syndromes of respiratory infection haveFour different syndromes of respiratory infection have been linked to Adenoviruses.

Acute febrile pharyngitis: most commonly seen in infants and young children,symptoms include cough, stuffy nose, fever and sore throat.

Pharyngo conjunctival fever:Pharyngo conjunctival fever:symptoms are similar to those of acute febrile pharyngitis but

conjunctivitis is also present. It tends to occur in outbreaks such as at children's summer camps

(swimming pool conjunctivitis)(swimming pool conjunctivitis).

Respiratory diseases:p y

Acute respiratory disease:is characterized by pharyngitis, fever, cough and

malaisemalaise. It occurs in an epidemic form among young

recruits under conditions of fatigue and overcrowdingovercrowding

Pneumonia: a complication of acute prespiratory disease in both children and adults.

Cli i l t tiClinical presentations

Clinical Syndromesy1. Pharyngitis 1, 2, 3, 5, 72 Pharyngoconjunctival fever 3 72. Pharyngoconjunctival fever 3, 7 3. Acute respiratory disease of recruits 4, 7, 14, 21 4. Pneumonia 1, 2, 3, 7 5. Follicular conjunctivitis 3, 4, 116. Epidemic keratoconjunctivitis 8, 19, 37 7. Pertussis-like syndrome 5 y8. Acute haemorrhaghic cystitis 11, 21 9. Acute infantile gastroenteritis 40, 41 10 Intussusception 1 2 510.Intussusception 1, 2, 511.Severe disease in AIDS and other immunocompromized patients 5, 34, 3512. Meningitis 3, 7

Adenoviral infections of theAdenoviral infections of the immune compromised host

The most common clinical manifestations are:

pneumoniapneumonia

hepatitis p

gastroenteritis

Laboratory Diagnosisy g

Direct detection:Direct detection:

I l tiIsolation

Serology

Laboratory Diagnosis

Direct detection:

Virus particle by EM can be detected by direct examination

Detection of adenoviral antigens by ELISA.Enteric Adenoviruses

D t ti f d i l NA b PCR b d f di iDetection of adenoviral NA by PCR: can be used for diagnosis of Adenovirus infections in tissue samples or body fluids.

Laboratory DiagnosisLaboratory Diagnosis

I l tiIsolation:

Adenovirus may be isolated from most body fluids and ti b th t b i f dsecretions; eye swabs, throat swabs, urine, faeces, and

CSF. Human embryonic kidney cells Hep-2 cells Primary monkey kidney cells 293 cellsPrimary monkey kidney cells 293 cells

CPE includes rounding, clustering of cells with CPE includes rounding, clustering of cells with refractilerefractileintranuclearintranuclear inclusion bodies inclusion bodies

Isolation is much more difficult from the stool or rectal swabs

Laboratory Diagnosisy g

SerologySerologyHaemagglutination inhibition Neutralization testsNeutralization tests

can be used to detect specific antibodies following Adenovirus infection.

CytomegalovirusCytomegalovirus

ClassificationSubfamily Growth &

CytopathologyLatent

infectionsGenus Official

name (herpesv

irus)

Common name

Alphaherpesvirinae Short, cytolytic Neurons Simplexvirus 12

HSV-1HSV-2

Vaicellvirus 3 VZV

B t h i i L Gl d C t l i 5 CMVBetaherpesvirinae Long, cytomegalic

Glands, kidneys

Cytomegalovirus 5 CMV

Long, lymphoproliferati

Lymphoid tissue

Roseolovirus 67

HHV-6HHV-7

ve

Gammaherpesvirinae Long, lymphoproliferative

Lymphoid tissue

Lymphocryptovirus 4 EBV

Rh di i 8 K i’

Herpesviridae 37

Rhadinovirus 8 Kaposi’ sarcoma virus

Pathogenesis & Pathology inNormal hosts

CMV transmission: person to person by close contactCMV transmission: person-to-person by close contact

Incubation period 4-8 weeks

Infectious mononucleosis-like

Most infections are subclinicalMost infections are subclinical

Lifelong latent infections

Virus shed from pharynx & urine for months to years

Prolonged kidney and salivary gland infections

Cell-mediated immunity is depressed

Pathogenesis & Pathology inimmunosupressed hosts

Primary CMV infections in immunosupressedPrimary CMV infections in immunosupressed

hosts are more severe

Viral excretion is increased & prolonged

Infection is disseminated

Pneumonia is most commonPneumonia is most common

Ductal epithelial cells are usually infected

Laboratory Diagnosis (1)Laboratory Diagnosis (1)Direct detection

In this assay, leukocytes are separated from other bloodelements, spotted onto a microscope slide, and stainedusing a monoclonal antibody to pp65 the CMV lowerusing a monoclonal antibody to pp65, the CMV lowermatrix phosphoprotein

The pp65 CMV antigenaemia test is now routinely usedpp g yfor the rapid diagnosis of CMV infection inimmunocompromised patients.

C f C A i d i b hPCR for CMV-DNA is used in some centers but theremay be problems with interpretation.

CMV pp65 antigenaemia testCMV pp65 antigenaemia test

(Virology Laboratory, New-Yale Haven Hospital)

Laboratory Diagnosis (2)y g ( )Virus Isolation

conventional cell culture is regarded as gold standard butconventional cell culture is regarded as gold standard butrequires up to 4 weeks for result.

More useful are rapid culture methods such as the DEAFF(Detection of early antigen fluorescent foci) testwhich can provide a result in 24-48 hours.

Cultures of blood are more helpful diagnostically, becausepositive blood cultures are virtually never found in

t ti i i d i di id lasymptomatic, non immunocompromised individuals.

Cytopathic Effect of CMVCytopathic Effect of CMV

(Courtesy of Linda Stannard, University of Cape Town, S.A.)

DEAFF test for CMV

(Virology Laboratory, New-Yale Haven Hospital)

Laboratory Diagnosis (3)y g ( )Serology:

the presence of CMV IgG antibody indicates past infection. The detection of IgM is indicative of primary infection

C tomegalo ir s serolog is of less al e for the diagnosis ofCytomegalovirus serology is of less value for the diagnosis ofinfections in immunocompromised patients than direct tests forthe virus.

is useful, however, for the diagnosis of CMV infection in thenormal host and for defining CMV immune status, for example inblood donors or for transplant donors and recipients.

EIA is more sensitive than the complement fixation test,which was widely used in the past.

CMV avidity assays

Specimens for Laboratory Diagnosis

Site for virus culture Serology Urine Saliva Blood Tissue affected IgG IgM Neonates + + - - - + Adults + - + - + +Adults + - + - + + Pregnant women - - - - + + Immunocompromised + + + + + -

Cytomegalovirusy g

Cytomegalovirus in the Normal Host:y g

a mononucleosis-like illness. the heterophile antibody t t i titest is negative.

It should be noted that acute EBV infection can also yield a false positive finding in the CMV IgM test.a false positive finding in the CMV IgM test.

In patients with CMV mononucleosis, CMV is regularly detected in the urine by culture, and often in the blood by

lt th 65 ti i PCRculture, the pp65 antigenemia, or PCR

Cytomegalovirusy gCytomegalovirus in the Immunocompromised Host:

important cause of opportunistic infection in patients who havereceived either solid organ or hematopoietic stem celltransplants and AIDStransplants and AIDS.

quantitative test performed on blood. PCR is the mostsensitive test.

The hybrid capture assay and the pp65 antigenemia assaybecome positive several days to 1 week after PCR andoccasionally fail to detect CMV antigenemia before the onset ofsymptoms.

Culture is the least sensitive, and often the last test to becomepositive.positive.

BocavirusBocavirus

VIRUS STRUCTURE

Fam

ily Parvoviridae

Sub

fam

ily Parvovirinae

Gen

us Boca virus

Li DNA f b 5 5kb i i• Linear, ssDNA genome of about 5.5kb in size• Non-enveloped capsid of icosahedral symmetry and

diameter of ~25 nm• Proteins Non-structural NS1

NP1(nuclear phosphoprotein)Structural VP1Structural VP1

Introduction

4 species of human bocavirus :4 species of human bocavirus :Respiratory pathogen

Stool pathogen

HBoV1 HBoV2

Important

HBoV3

Importantcause of LRTI HBoV4

RISK FACTORSproduce a long-lasting , high-avidity IgG antibody response

l k f t ti B ll i it i ht i th i k f HB Vlack of protecting B-cell immunity might increase the risk of HBoV

infection

Risk factors for severe HBoV1-associated illness have been

considered to be similar to those for common respiratory viral

infections:

cardiac disease (congenital heart lesions or heart failure)pulmonary disease (asthma or chronic obstructive pulmonary disease)pulmonary disease (asthma or chronic obstructive pulmonary disease)Prematurity with chronic lung disease ImmunosuppressionYoung age

maternal smokingmaternal smokingwinter birth timeAsthma predisposition

Signs and symptoms S g s a d sy p o srhinitis, pharyngitis , cough, wheezing, pneumonia, acute otitis media, fever, nausea, p , , , ,vomiting, and diarrhea

Diagnostic approach Quantitative PCRSerologySerology

RhinovirusRhinovirus

I t d tiIntroduction y Picornaviridae s Entrovirus s A

Fam

il

Gen

u

Spe

cie B

C

S

URTI Otitis media Si itiSinusitis Chronic pulmonary disease Asthma development Severe bronchiolitis in infants and childrenFatal pneumonia in elderly and immunocompromised adults

Rhinovirus

Belong to the picornavirus familySmall 20-30nmIcosahedral symmetryNon enveloped-acid-labileSi l t d d iti RNASingle stranded positive sense RNAat least 100 serotypes are known

ssRNA , 7.2 Kb Single gene : 11 proteins , cleaved by proteasesVP1,2,3,4 : viral capsid VP1,2,3 : antigenic diversity

60 copies of each made an p g y

VP4 : anchors the RNA core to the capsidNS : assembly and replication

icosahedral structure

Functions of Viral ProteinsDerived from one polyprotein precursor

Processed by post-translational cleaving

Structural proteinsStructural proteinsresponsible for host tropismsprotection of genome

ti i itantigenicityNon-structural proteins

proteasesRNA polymeraseinhibitors of normal host cell functions

Canyon in VP1 : site of attachment to cell surface receptors :Major group :ICAM-1 , Heparan sulfatej g p , pMinor Group :LDLR

Di i i f th i i l 99 t i i t 3 iDivision of the original 99 strains into 3 species:HRV-A (74 serotypes)HRV-B (25 serotypes)HRV B (25 serotypes)HRV-c (50 serotypes)

Each serotype (>115) is distinctnot cross-reactivelittl t tilittle cross-protection

RhinovirusRhinovirusRhinovirus infections are chiefly limited to the upper

respiratory tract but may include otitis media and sinusitissinusitis.

Rhinovirus plays a role in exacerbations of asthma, cystic fibrosis, chronic bronchitis, and serious lower

i ill i i f ld l drespiratory tract illness in infants, elderly persons, and patients who are immunocompromised

Although infections occur year-round, the greatest incidence is in the fall and spring.

Of persons exposed to the virus, 70-80% have symptomatic disease.symptomatic disease.

PathophysiologyPathophysiology

Rhinoviruses are transmitted to susceptible individuals by :direct contact

aerosol particles

infecting both ciliated areas of the nose and nonciliated areas of the nasopharynx through receptors, most frequently ICAM-1 (found in high quantities in the posterior nasopharynx)in high quantities in the posterior nasopharynx).

Few cells are actually infected by the virus, and the infection involves only a small portion of the epitheliumonly a small portion of the epithelium.

Pathophysiologylocalized infections of the nose

symptoms occur 2 to 4 days after exposure and last about one week There is no feverone week. There is no fever.

The virus has a limited temperature range in which it can p ggrow (33-35°C) and cannot tolerate an acidic environment.Thus finding the virus outside of the nasopharynx is unlikelyThus, finding the virus outside of the nasopharynx is unlikely because of the acidic environment of the stomach and the temperature elevation in both the lower respiratory and gastrointestinal tracts. g

LABORATORY DIAGNOSISDirect detection of rhinovirus antigen: an ELISA has been

developed for the detection of rhinovirus antigen in nasal washings.

Culture: Nasal washing is the best clinical specimen for recovering the virus.

Rhinoviruses grow ONLY IN in vitro on:Rhinoviruses grow ONLY IN in vitro on:Cells of primate origin,Human diploid fibroblast cells

The virus CPE, which consists of the rounding of cells similar to that induced by enteroviruses should appear within 8 d f i l tidays of inoculation.

The identity can be confirmed by acid lability tests. (pH3) Serology:Serologic testing to document rhinovirus infection is

i lnot practical.virus neutralization tests remain the best method.

Normal Cells Infected Cells

Laboratory testingLaboratory testing

Respiratory Viral Infectionsp yA. Culture:The influenza and parainfluenza viruses replicate best in primary monkey kidney cells, although continuous cell lines (e.g., Madin-Darby canine kidney) can be used for influenza and LLC-MK2 for parainfluenza

without producing CPE and hemadsorption with guinea pig erythrocytes is used for detection (24 hours)pig erythrocytes is used for detection (24 hours)

RSV replicates optimally in HEp-2 cells very labileRSV replicates optimally in HEp 2 cells. very labile.delayed transport result in failure to isolate RSV.

Respiratory Viral InfectionsAdenoviruses replicate optimally in human embryonic kidney cells.

p y

Rhinoviruses replicate only in human diploid fibroblast cells. Incubation at 33°C to 34°C.human metapneumovirus grown in monkey kidneyhuman metapneumovirus grown in monkey kidney cells (e.g., LLC-MK2), but growth is slow and clearly less sensitive than detection by molecular methods.SARS replicates in Vero and other monkey kidney cells, including fetal rhesus monkey kidney cells.R tl th f lt i i t iRecently, the process of culturing respiratory viruses has been simplified by the development of mixed cell culture systems

Respiratory Viral InfectionsB. Antigen Detection:be used to achieve rapid diagnosis of RSV, influenza, parainfluenza and adenovirus respiratory tract infection

p y

parainfluenza, and adenovirus respiratory tract infection.Viral antigens are visualized by FA staining of respiratory epithelial cells.pnasopharyngeal aspirate, wash, or swab, Tracheal aspirate and bronchoalveolar lavage specimens.Aspirates and washes provide a higher cellular yield and have higher viral titers than swabs.Rhinoviruses and coronaviruses are not detected byRhinoviruses and coronaviruses are not detected by antigen detection methods because of antigenic diversity and lack of availability of appropriate antibodies.

Respiratory Viral InfectionsC. Nucleic Acid Amplification:The advantages of these assays are increased

p y

sensitivity compared with conventional methods and the ability through multiplex methods to detect multiple viruses in the same sampleviruses in the same sample.nucleic acid amplification can detect viruses such as HMPV, coronaviruses, and human bocavirus that are difficult or impossible to detect by other methods.A advantage is that nucleic acid amplification facilitates the detection of respiratory viruses from non respiratorythe detection of respiratory viruses from non respiratory sites.

Other Possible Diarrhoeal Viruses

CoronavirusesRNA viruses with a crown-like appearance

Not convincing associated with gastroenteritis at present

Small Round Virusesll i lik i l i h h f 22 28 i dismall virus-like particles with a smooth surface, 22-28nm in diameter

may possibly be parvoviruses, enteroviruses, or cubic bacteriophages

occasionally seen in the faeces of endemic or epidemic cases ofoccasionally seen in the faeces of endemic or epidemic cases of gastroenteritis