SARS-CoV-2: Susceptibility of cats and dogs to infection

SARS-CoV-2: Susceptibility of cats and dogs to infection

Ricardo Alexandre Abreu Barroso

Aplicações em Biotecnologia e Biologia Sintética

Departamento de Biologia e Departamento de Química e

Bioquímica

2021

Orientador

Isabel Fidalgo Carvalho, CEO da Equigerminal S.A/ Professora

Auxiliar na Escola Universitária Vasco da Gama

Coorientador

Agostinho Antunes Pereira, Investigador no CIIMAR / Professor

Auxiliar na Faculdade de Ciências da Universidade do Porto

Todas as correções determinadas

pelo júri, e só essas, foram efetuadas.

O Presidente do Júri,

Porto, ______/______/_________

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FCUP SARS-CoV-2: Susceptibility of cats and dogs to infection

Agradecimentos “What you do makes a difference, and you have to decide what kind of difference you want to make” – Jane Goodall

À minha orientadora, Dra. Isabel. Agradeço pelo acolhimento na Equigerminal, e por todo o apoio e confiança prestados para a concretização deste trabalho. Obrigado por toda a sabedoria científica transmitida, e por me ter feito crescer não só profissionalmente, mas também pessoalmente.

Ao Alexandre. Agradeço o apoio e prontidão de ajuda para o esclarecimento de todas as dúvidas no laboratório, mas principalmente pelas palavras amigas e incentivos.

Ao meu coorientador, Professor Agostinho Antunes. Agradeço por acreditar sempre nas minhas capacidades e, acima de tudo, por ser um excelente professor.

A todos os profissionais das clínicas veterinárias: Ana e os Bichos, Bigodes D’Avenida, Canil municipal de Vila Nova de Famalicão, Canil municipal de Vila de Conde, Centro Clínico Animal de Guimarães, FamaVet, Fernando Soares, Médio Ave, São Francisco, Trio Patinhas, Vet América, Vet Figueiró, Vet Help e Zoo Panda. Agradeço por me receberem de braços abertos e pelo interesse em participar no estudo. Nada seria possível sem a vossa ajuda para a recolha das amostras. Um agradecimento especial à Daniela, por toda a boa disposição e simpatia.

Aos meus amigos, agradeço pela cumplicidade e amizade. Obrigado, Rita, por todo o apoio e memórias inesquecíveis desde pequeninos. Obrigado, Francisca e Mariana, pelas conversas profundas, brincadeiras e aventuras vividas desde caloiros. Obrigado, Raquel, Manuel, Filipa e Rui, pela amizade e por ultrapassarem comigo o desafio que foi este mestrado. Obrigado aos Padrinhos, por tornarem não só a minha vida académica no Porto memorável, mas também pela vossa amizade, que será certamente para a vida toda.

À minha família, permanece o maior agradecimento. Mãe e pai, sem vocês não conseguiria ultrapassar as barreiras mais difíceis da vida e tornar-me na pessoa e profissional que sou hoje. Obrigado por serem o meu porto de abrigo eterno, por acreditarem sempre em mim, e pelos mimos diários, mas também pelos vossos sorrisos face às minhas conquistas, sejam elas grandes ou pequenas. Aos meus avós, obrigado por me darem sempre força para seguir os meus sonhos, vocês são a minha segunda casa. À minha madrinha e ao meu tio, obrigado pela amizade e por me acompanharem sempre desde criança. Ao Vicente e à Benedita, obrigado por serem os meus meninos de ouro. À Mimi, a minha companheira de 4 patas, obrigado por tornar os meus dias melhores.

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Resumo

Betacoronavirus (β-CoV) são vírus (+)ssRNA (do inglês, ‘positive-sense single-stranded

RNA’) que infetam várias espécies de mamíferos. Em 2019, um novo vírus zoonótico

emergiu, o coronavírus da síndrome respiratória aguda grave-2 (SARS-CoV-2), sendo

patogénico para humanos e semelhante a ‘SARS-related-CoVs’ descobertos em

morcegos e pangolins. Porém, a origem animal do vírus permanece desconhecida.

Embora a via de transmissão mais frequente do SARS-CoV-2 seja entre humanos,

foram relatados alguns casos de spillover de humanos para animais domésticos e

selvagens. Neste estudo, uma investigação seroepidemiológica foi realizada em cães e

gatos de Portugal entre dezembro de 2020 e maio de 2021, com o objetivo de

compreender a suscetibilidade dos animais de estimação à infeção por SARS-CoV-2.

Anticorpos contra o ‘receptor binding domain’ (RBD) do SARS-CoV-2 foram detetados

em 15/69 (21,74%) gatos e 7/148 (4,73%) cães, perfazendo 22/217 (10,14%) amostras

positivas nos animais de estimação. A maioria dos cães positivos para o SARS-CoV-2,

6/7 (85,71%), eram assintomáticos, ao contrário dos gatos, em que 9/15 (60,00%) eram

sintomáticos. Os resultados sugerem uma taxa de mortalidade associada à infeção por

SARS-CoV-2 em gatos de 5/15 (33,33%). Dos cães positivos para o SARS-CoV-2, 4/7

(57,14%) estiveram em contato com tutores positivos para o SARS-CoV-2. Dos gatos

positivos para o SARS-CoV-2, 7/15 (46,67%) estiveram em contato com tutores

positivos para o SARS-CoV-2, e 2/15 (13,33%) encontravam-se frequentemente ao ar

livre ou em contato próximo com outros gatos pertencentes a vizinhos positivos para a

COVID-19. Os resultados sugerem que uma abordagem uma só saúde é crucial para

monitorizar as vias de transmissão do SARS-CoV-2 dos humanos para os animais de

estimação, visto que a maioria do spillover de tutores positivos dentro dos lares foi

responsável por 13/22 (59.09%) dos casos positivos nos animais de estimação.

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Resume

Beta-coronavirus (β-CoV) are positive single-stranded RNA viruses known to infect

mammals. In 2019, a novel zoonotic β-CoV emerged, the severe acute respiratory

syndrome (SARS)-CoV-2, being highly pathogenic in humans and with a strong similarity

to bat and pangolin SARS related CoVs. However, the animal origin of the virus is still

unknown. Although the most frequent SARS-CoV-2 transmission route is between

humans, spillover from humans to domestic and wild animals has been reported. In this

study, a seroepidemiologic survey in dogs and cats from Portugal between December

2020 and May 2021 was conducted to understand the susceptibility of pets to SARS-

CoV-2 infection. Antibodies against the receptor binding domain (RBD) of SARS-CoV-2

were detected in 15/69 (21.74%) cats and 7/148 (4.73%) dogs, totalizing 22/217

(10.14%) positive pet samples. Almost all the SARS-CoV-2 positive dogs, 6/7 (85.71%),

were asymptomatic, contrasting to cats, where 9/15 (60.00%) were symptomatic. Data

suggests a mortality rate associated to SARS-CoV-2 infection in cats of 5/15 (33.33%).

Of the SARS-CoV-2 positive dogs, 4/7 (57.14%) had close contact with SARS-CoV-2

positive owners. Of the SARS-CoV-2 positive cats, 7/15 (46.67%) had contact with

SARS-CoV-2 positive owners and 2/15 (13.33%) were frequently outdoors or in close

contact with other cats belonging to neighbors that tested positive for COVID-19. These

results suggest that a one-health approach is crucial to monitor the transmission routes

of SARS-CoV-2 from humans to their pets, since most of the spillover from positive

owners within households were responsible for 13/22 (59.09%) of the positive cases

among pets.

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Keywords Antibodies, antigens, COVID-19, epidemiology, immunity, infection, one-health,

pandemics, pets, RBD, SARS-CoV-2, serum, spillover, susceptibility, zoonosis

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Index Figure List ....................................................................................................................... 7

Table List ........................................................................................................................ 9

Abbreviation List ........................................................................................................... 10

Chapter I. A One Health approach to the pandemic ..................................................... 13

1. Introduction ............................................................................................................ 13

2. COVID-19: the disease .......................................................................................... 13

2.1. Symptomatology of COVID-19 ...................................................................... 14

2.2. Immunity ....................................................................................................... 15

2.3. Diagnosis ...................................................................................................... 18

2.4. Treatment ...................................................................................................... 21

2.5. Vaccines ....................................................................................................... 22

3. SARS-CoV-2 : A zoonotic threat ........................................................................... 23

3.1. Anthropogenic factors causing zoonoses ..................................................... 25

3.2. Strategies to control zoonotic events ............................................................ 26

3.3. Origin of SARS-CoV-2 .................................................................................. 27

4. SARS-CoV-2: a virus of the coronaviridae family .................................................. 30

4.1. CoVs in pets .................................................................................................. 31

4.2. CoVs in humans ............................................................................................ 33

5. SARS-CoV-2: the pandemic virus ......................................................................... 35

5.1. Morphology ................................................................................................... 35

5.2. Genome ........................................................................................................ 37

5.3. Infection cycle ............................................................................................... 38

5.4. Viral evolution and variants ........................................................................... 42

5.4.1. VOCs ..................................................................................................... 45

5.4.2. VOIs ...................................................................................................... 48

5.5. Animal associated variants ........................................................................... 50

6. One Health approach ............................................................................................ 52

6.1. One Health studies ....................................................................................... 53

6.2. Natural and experimental infections in animals ............................................ 59

7. Conclusion ............................................................................................................. 65

Chapter II – A seroepidemiological survey of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Portuguese pets. ....................................................... 66

1. Introduction ............................................................................................................ 66

2. Materials and Methods .......................................................................................... 66

2.1. Study design ................................................................................................. 66

2.2. Animal sampling and processing .................................................................. 67

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2.3. Animal data ................................................................................................... 68

2.4. Survey areas ................................................................................................. 69

2.5. ELISA ............................................................................................................ 70

2.6. Statistics ........................................................................................................ 71

3. Results ................................................................................................................... 72

3.1. Cats signalment ............................................................................................ 72

3.2. Dogs signalment ........................................................................................... 75

3.3. SARS-CoV-2 seroprevalence in pets ............................................................ 78

3.4. SARS-CoV-2 seroprevalence in cats ............................................................ 78

3.5. SARS-CoV-2 morbidity and mortality in cats ................................................ 78

3.6. Risk assessment and clinical outcome of SARS-CoV-2 seropositive cats ... 80

3.7. SARS-CoV-2 seroprevalence in dogs ........................................................... 82

3.8. Risk assessment and clinical outcome of SARS-CoV-2 seropositive dogs .. 84

3.9. Risk factors and susceptibility to SARS-CoV-2 infection .............................. 85

4. Discussion ............................................................................................................. 86

4.1. Wide-scale testing of pets ............................................................................. 86

4.2. SARS-CoV-2 seroprevalence in portuguese pets ......................................... 86

4.3. Risk factors contributing to pets’ infection ..................................................... 87

4.4. SARS-CoV-2 morbidity and mortality in pets ................................................ 87

4.5. Antibody longevity ......................................................................................... 88

5. Conclusion ............................................................................................................. 89

Bibliography .................................................................................................................. 90

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Figure List Fig.1 – Immune response trajectories in Covid-19.

Fig.2 – CD4+ T cell function observed in COVID-19.

Fig.3 – The immune response to SARS-CoV-2 infection after 1 year. Fig.4 – Time intervals estimation of the likelihood of detection of specific SARS-CoV-2

viral RNA and antibodies by different molecular and serological approaches biased on

data from published reports.

Fig.5 – Establishment of zoonotic pathogens within human populations.

Fig.6 – Heat map indicating the probability of the emergence of an infectious disease

event relative to reporting effort.

Fig.7 – Sequence alignment of SARS-CoV-2 and SARSr-CoVs spike (S) protein.

Fig.8 – Representation of the four CoV genera and associated species.

Fig.9 – Genetic features of FCoV and CCoV genotypes.

Fig.10 – Feline Infectious Peritonitis (FIP) in cats.

Fig.11 – Morphology of SARS-CoV-2.

Fig. 12 – SARS-CoV-2 Spike (S) protein.

Fig. 13 – Steps involved in SARS-CoV-2 replication.

Fig. 14 – First lineages and predominant clade of SARS-CoV-2.

Fig. 15 – Combination of SARS-CoV-2 spike mutations occurring in humans and animals

that may be involved in interspecies transmission.

Fig. 16 – Venn diagram illustrating the tree core domains of the One-Health initiative.

Fig. 17 – Venn diagram illustrating the three domains of the present one health study

and their connectivity.

Fig. 18 – Daily new confirmed COVID-19 cases and deaths per million people during the

third wave in Portugal.

Fig. 19 – Total blood samples collected from cats and dogs.

Fig. 20 – Geographic distribution of dog and cat samples.

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Fig. 21 – Anti SARS-CoV-2 RBD ELISA for cats and dogs.

Fig. 22 – Groups formed for cats according to the data collected from the epidemiologic

enquiries.

Fig. 23 – Groups formed for dogs according to the data collected from the epidemiologic

enquiries.

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Table List Table 1. Description of SARS-CoV-2 non-structural proteins (nsp).

Table 2. SARS-CoV-2 VOCs and VOIs.

Table 3. Widescale seroepidemiologic surveys to understand the SARS-CoV-2

transmission routes and prevalence of infection in pets.

Table 4. Epidemiologic information about the positive cases among cats.

Table 5. Epidemiologic information about the positive cases among dogs.

Table 6. Risk factors contributing to seropositivity among cats and dogs.

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Abbreviation List 2019-nCoV : 2019 novel coronavirus

ACE2: Angiotensin-converting enzyme 2

APN: Aminopeptidase N

ARDS: Acute respiratory distress syndrome

BALF: Bronchoalveolar lavage fluid

BMPC: Bone marrow plasma cell

BSA: Bovine serum albumin

BSL: Biosafety level

CCoV: Canine coronavirus

CIRD: Canine infectious respiratory disease

COHERE: Checklist for one heath epidemiological reporting of evidence

COVID-19: Coronavirus disease-19

CRCoV: Canine respiratory coronavirus

Ct: Cycle Threshold

DDP4: Dipeptidyl peptidase 4

E: Envelope

ELISA: Enzyme-linked immunosorbent assay

FCoV: Feline coronavirus

FIP: Feline infectious peritonitis

GD : Guangdong

GX : Guangxi

HCoV: Human Coronavirus

HE: Hemagglutinin esterase

HRP: Horseradish peroxidase

ICTV: International Committee on Taxonomy of Viruses

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IFN: Interferon

LRT: Lower Respiratory Tract

M: Membrane

mAbs: Monoclonal antibodies

MASCp6: mouse-adapted strain at passage 6

MERS-CoV: Middle east respiratory syndrome coronavirus

N: Nucleocapsid

Nsp: Nonstructural proteins

NTD: N terminal domain

OD: Optical Density

orf: Open reading frame

PBST: Phosphate-buffered saline with Tween® detergent

PD: Peptidase domain

pp1ab: Polyprotein 1ab

R0: Basic reproduction number

RBD: Receptor binding domain

RdRp: RNA dependent RNA polymerase

RTC: Replicase/transcriptase complex

RT-PCR: Reverse transcriptase polymerase chain reaction

S: Spike

SARS-CoV: Severe acute respiratory syndrome coronavirus

SARS-CoV-2: severe acute respiratory syndrome coronavirus-2

SARSr-CoV: Severe acute respiratory syndrome related coronavirus

sgRNA: Subgenomic RNA

SLCoV: SARS-CoV-like

ssRNA: Single stranded RNA

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TGV: Transmissible gastroenteritis virus

TMB: 3,3´,5,5´ Tetramethylbenzidine

TMPRSS2: Transmembrane serine protease

TRS: Transcription regulatory sequence

URT: Upper Respiratory Tract

VOC: Variant of concern

VOI: Variant of interest

WGS: Whole genome sequencing

WHO: World health organization

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Chapter I. A One Health approach to the

pandemic

1. Introduction In December 2019, an outbreak of atypical pneumonia of unknown etiology was

detected in the city of Wuhan, located at the Chinese province of Hubei [1]. In record

time, a new coronavirus was isolated in these patients. The new virus was initially

designated as 2019-nCoV and later identified as Severe Acute Respiratory Syndrome

Coronavirus 2 – SARS-CoV-2 - by the International Committee on Taxonomy of Viruses

(ICTV) [2]. On the same day, 11th February of 2020, the World Health Organization

(WHO) named this coronavirus disease as COVID-19 [2].

On 30th January of 2020, WHO declared COVID-19 a public health emergency of

international interest. In the following months, SARS-CoV-2 has spread quickly to

different countries in all continents, leading to an increased number of COVID-19 cases

and deaths in China, Europe, and US. This lead WHO to declare a pandemic on 11th

March of 2020 [3], and the United Nations (UN) to declare, on 1st April of 2020, that the

COVID-19 constitutes the greatest test that humanity has faced since World War II [4].

Since the first occurrence of the disease, it is assumed as a viral zoonosis, which

aroused intense interest in the origin of SARS-CoV-2 and how this new virus emerged

in human populations. For several years, scientists have been warning about the

existence of increasing spillover events, and the possibility of a pandemic occurring in

the 21st century. Despite the scientists' warning, the world was not prepared for the

pandemic.

Herein we present a bibliographical review of the COVID-19 pandemic and SARS-

CoV-2 from a One Health perspective.

2. COVID-19: the disease In human populations, infectious diseases can be considered endemic, i.e., occur

continuously at an expected frequency over a certain period and geographical location,

or epidemic, i.e., occur greater than the expected in a certain geographical region. When

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the epidemic is generalized and involves different countries and a large scale population,

it is defined as a pandemic [5].

In 11th March of 2020, WHO declared the COVID-19 outbreak a public health

emergency of pandemic proportions, owning to the rapid transmission of SARS-CoV-2

within human populations worldwide [6]. COVID-19 has devastated several countries,

resulting in more than 2.9 million deaths and overwhelming healthcare systems. It is the

most significant global health crisis since the era of the 1918 influenza pandemic [6]. In

the face of COVID-19, public health decisions should be focused on the quantification of

disease severity, mortality and immunity to infection, improvement of treatments in

severe cases, and identification and response to novel antigenic variants [7].

2.1. Symptomatology of COVID-19 The median incubation period of SARS-CoV-2 is about 5.1 days, with 97.5% infected

humans developing COVID-19 symptoms within 12 days [8]. Additionally, COVID-19 is

associated with a severe disease course in about 23% and mortality in about 6% of

infected persons [9]. Older males with comorbidities such as immunosuppression,

diabetes and malignancy are more likely to develop severe cases of COVID-19 and need

intensive care [9, 10].

Common symptoms in hospitalized patients include fever, dry cough, fatigue,

shortness of breath, confusion, headache, and sore throat, with diarrhea, abdominal

pain, nausea, and vomiting being less frequent [9-11]. Disturbance of olfactory and

gustatory senses by upper respiratory tract (URT) infection is also common [12]. In more

severe cases, patients develop acute respiratory distress syndrome (ARDS), arrhythmia,

septic shock, and multiple organ failure [10, 11].

Histopathological findings include pulmonary edema with hyaline membrane

formation, indicative of ARDS, together with interstitial mononuclear inflammatory

infiltrates [13].

These findings correlate with experimental studies of SARS-CoV-2 cell tropism,

which could infect and replicate in pulmonary (Calu3), intestinal (Caco2) and neuronal

(U251) cells. Curiously, the virus also infected hepatic (Huh7) and renal (293T) cells

successfully [14].

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2.2. Immunity The immune system is divided into the innate and adaptive immune system, each

consisting of different cell types with different functions. The innate immune system is

essential to restrict viral replication in infected cells, create an antiviral state in the local

tissue and prime an adaptive immune response for an efficient viral clear; while the

adaptive immune system consists of B cells that produce antibodies, CD4+ T cells that

possess a range of helper and effector functionalities, and CD8+ T cells that kill infected

cells, being vital for the control and clearance of almost all viral functions [15].

All viruses causing disease in humans must possess one immune evasion

mechanism. In the case of SARS-CoV-2, the virus is effective at evading the triggering

of early innate immune responses, such as type 1 interferons (INFs) [16]. Most SARS-

CoV-2 infections in humans are asymptomatic or result in clinically mild COVID-19, as

the innate immune response is fast and adequate to provide an effective T cell and

antibody response. However, if the innate immune response is delayed, the virus

replicates more efficiently, as the priming of an adaptive immune response is longer

(Fig.1), resulting in a severest disease outcome. [15]. To compensate the absence of a

T cell response, an excessive innate immune response occurs, characterized innate cell

immunopathology and a plasma cytokine signature of elevated CXCL10, interleukin (IL)-

6 and IL-8 [15, 16].

In the case of COVID-19, CD4+ T cells are of extreme importance due to their ability

to differentiate into a range of helper and effector types (Fig. 2), and the presence of

virus-specific CD8+ T cells are associated with better COVID-19 outcomes due to their

potent cytotoxic effector functions, such as IFNg, granzyme B, perforin and CD107a.

Generally, antibodies mostly stop viruses outside of cells, while T cells stop virus inside

of cells [15]. B cells have a dual role: they produce antibodies that can recognize viral

proteins, and they can present parts of these proteins to specific T cells, or develop into

plasma cells that secrete antibodies in larger quantities [17].

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Fig. 1 – Immune response trajectories in Covid-19. Conceptual schematics of the kinetics of immune

responses in A. a generic viral infection; and in conditions of B. average COVID-19 (non-hospitalized) or

C. severe or fatal COVID-19. The innate immunity line refers to the peak kinetics of innate cytokines and

chemokines in blood. T cells refers to virus specific CD4+ and CD8+ T cells. Antibodies refers to virus-

specific neutralizing antibodies (Adapted from Sette A. & Crotty S., 2021).

Fig. 2 – CD4+ T cell function observed in COVID-19. In response to SARS-CoV-2, CD4+ T cells differentiate into various

cell types, exhibiting a range of helper and effector functions. Tfh cells provide help to B cells for affiniy maturation and

antibody production; Th1 cells have direct antiviral functions through cytokine secretion and recruitment of innate cells;

CD4+ T cells that help CD8+ T cells to proliferate and differentiate; CD4+-CTL have direct cytotoxic activity against virally

infected cells in a class II antigen presentation restricted manner; and CD4+ T cells that produce IL-22, with roles in wound

healing (Adapted from Sette A. & Crotty S., 2021).

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Bone marrow plasma cells (BMPCs) are a persistent and essential source of

protective antibodies, being the best available predictor of long-lasting immunity [17]. In

the case of SARS-CoV-2, BMPCs secreting antibodies against the S protein were

present in 15 of 19 individuals 7 months after infection, remaining stable after 11 months

in all but one of the individuals analyzed. Moreover, a biphasic pattern was observed for

the concentration of antibodies in the blood serum of 77 convalescent individuals, with

anti-SARS-CoV-2 S antibodies declining rapidly in the first 4 months after infection and

then more gradually over the following 7 months, remaining detectable at least 11

months after infection (Fig. 3). Hence, mild infection with SARS-CoV-2 induces robust

antigen-specific, long-lived humoral immune memory in humans [18]. Moreover,

antibody reactivity to the RBD, neutralizing activity, and the number of RBD-specific

memory B cells remain relatively stable between 6 and 12 months after infection (Fig. 3), with vaccination boosting all the components of the humoral response, resulting in

similar or greater serum neutralizing activities against variants of concern compared with

the original Wuhan Hu-1 strain [19].

The main goal of vaccination is to achieve heard immunity, i.e., a high proportion of

individuals in a population with immunity to a specific infectious agent [5]. However,

heard immunity is only relevant if a transmission-blocking vaccine is available. The

current COVID-19 vaccines are extremely effective at preventing symptomatic disease,

but it is still unclear whether they protect people from becoming infected, or from

spreading the virus to others [20].

Fig. 3 – The immune response to SARS-CoV-2 infection after 1 year. During the initial acute phase of the immune

response, antibody levels peak rapidly, being generated by short-lived plasma cells (red line). However, long-lived memory

plasma cells are generated in the bone marrow, providing long-term antibody production that offer stable protection at a

level of 10-20% of that during the acute phase (blue line). Additionally, between 6 and 12 months after infection, the

concentration of neutralizing antibodies remains unchanged. (Adapted from Radbruch, A., & Chang, H. D., 2021).

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2.3. Diagnosis Tests to support or establish a specific diagnosis of a viral infection are of five general

types : (1) those that demonstrate the presence of infectious virus; (2) those that detect

viral antigens; (3) those that detect viral nucleic acids; (4) those that demonstrate the

presence of an agent-specific antibody response; and (5) those that directly visualize the

virus [21].

Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

Allows the real-time detection of viral RNA sequences in clinical specimens in a fast,

sensitive, and specific approach, providing an objective estimate of viral load [21].

However, viral loads are not a measure of disease severity, but a slightly indication of

transmissibility, as the lower the cycle threshold (Ct) value, the higher the viral load and

probability of transmission [22]. In the case of SARS-CoV-2, a Ct value of less than 40 is

clinically reported as PCR positive [23]. The RBD is an optimum target for RT-PCR, but

others, such as the RdRp and E proteins, are also recommended [24].

Nasopharyngeal or throat swabs are more common for viral RNA testing, retrieving

higher viral loads. However, specimens from the lower respiratory tract (LRT), including

sputum and bronchoalveolar lavage fluid (BALF), are likewise viable [12, 25]. Viral RNA

is also present in lower loads in stools, as ACE2 is as well expressed in the

gastrointestinal tract [26]. In a study of 205 patients with confirmed COVID-19 infection,

RT-PCR positivity was higher in BALF specimens (93%), followed by sputum (72%),

nasal swabs (63%), fibrobronchoscope brush biopsy (46%), pharyngeal swabs (32%),

feces (29%) and blood (1%) [23]. On the opposite, SARS-CoV-2 viral RNA was not

detectable in urine [12, 23].

The medium duration of viral shedding goes from 12 to 20 days in humans, with the

viral load peak detectable by RT-PCR between 4 to 6 days after symptom unset (Fig.4) [26]. However, prolonged viral shedding can occur in immunosuppressed patients,

increasing the time scale to obtain a positive RT-PCR result for SARS-CoV-2 [26].

Enzyme-linked immunosorbent assay (ELISA)

Allows the measurement of antiviral specific IgM, IgG and IgA antibodies in serum or

plasma in a rapid, sensitive and specific approach, indicating recent or past infection and

being the pillar of retrospective diagnosis for many clinical and epidemiological purposes

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[21]. However, a positive test does not necessarily correlates with immunity to disease,

as not all antibodies are neutralizing, i.e., block infectivity [27].

Both S and N proteins are the main SARS-CoV-2 immunogens, being commonly

used in ELISA. However, S-based ELISAs are more sensitive, proved by the observation

of an earlier response of IgM antibodies [28-30], and a higher antibody specificity against

the RBD. This RBD-specific antibodies are also expected to be neutralizing due to the

RBD function of attachment to the host ACE2 receptor [31]. For S-based ELISAs, the S1

subunit is more specific and viable for SARS-CoV-2 serologic diagnosis, while the S2

subunit causes more cross-reactivity due to its high conservation state [29].

IgM and IgG against SARS-CoV-2 are usually positive at 11 to 15 dpi, with IgG

persisting beyond 7 weeks after infection and IgM decaying after 5 weeks and practically

disappearing after 7 weeks (Fig.4) [28, 29]. A high percentage of PCR-confirmed SARS-

CoV-2–infected individuals seroconvert by 13-21 days after disease onset [29].

Fig. 4 – Time intervals estimation of the likelihood of detection of specific SARS-CoV-2 viral RNA and antibodies by different molecular and serological approaches biased on data from published reports. a Detection only occurs if

patients are followed up proactively from the time of exposure. b More likely to register a negative result by a nasopharyngeal

swab PCR (Adapted from Sethuraman et al., 2020).

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Neutralization assays

Allows the detection of specific neutralizing antibodies by mixing dilutions of

antibodies with a specific virus, followed by incubation in cultured cells. The highest

dilution of antibody that inhibits the development of cytopathic effect is the end point of

neutralization [32].

For SARS-CoV-2, it is useful to test the neutralization effectiveness of antibodies

from sera of infected or vaccinated individuals against novel variants. However, this

technique is labor intensive and requires biosafety level (BSL)-3 laboratories to deal with

live SARS-CoV-2 virus. Though, if replication-defective pseudotyped virions are used,

the procedures can be performed in BSL-1 and BSL-2 laboratories [33].

Sequencing

High throughput sequencing provides a culture-independent method capable of

quickly identification and molecular characterization of established and novel pathogenic

viral agents. Fortunately, sequencing costs are decreasing, along with the development

of more portable instruments and easier methods for sample preparation and sequence

analysis [34]. A reference genome of SARS-CoV-2 is present in NCBI with the accession

number NC_045512, together with an aggregate of other nucleotide sequences with

crescent expanding [35].

For the detection of a specific SARS-CoV-2 variant, the most common methods are

whole genome sequencing (WGS) or complete/partial S gene sequencing. Although

WGS is better to monitor virus evolution by phylodynamic analysis and is a guide for

vaccine composition development, it is a time-consuming technique, and training in

equipment and bioinformatic analysis is crucial, being unsatisfactory for timely detection

of variants for public health response [33]. However, partial sequencing of the S-gene is

more achievable. To distinguish SARS-CoV-2 variants, at least the entire NTD and RBD

should be covered to detect key mutations. It is suggested to sequence aminoacids 1-

800 (2400 bp) of the entire S-gene to also monitor the S1/S2 cleavage site and other

regions of potential interest [33].

Imaging

COVID-19 pneumonia on lung computed tomographic (CT) scans tends to manifest

as bilateral, subpleural, ground-glass opacities with ill-defined margins, air

bronchogram, smooth or irregular interlobular septal thickening and thickening of the

adjacent pleura, with a slight predominance on the right lower lobe [36]. A rapid evolution

of disease abnormalities can be observed on CT scans from the subclinical period

21


through the first and second weeks after symptom onset, after which they gradually

decrease after the third week [36]. However, CT imaging is nonspecific, usually

overlapping with other infections, limiting the COVID-19 diagnostic value [27].

2.4. Treatment Several strategies were implemented for the treatment of COVID-19, such as anti-

SARS-CoV-2 neutralizing antibodies, antivirals, and targeted immunomodulatory

therapies. In a first phase of infection, antiviral and antibodies treatments are more

helpful to inhibit the greater viral replication before the symptom’s onset. However, as

disease progresses, and in cases of hospitalized patients, immunomodulatory agents

help to attenuate the hyperinflammatory state characterized by cytokine release and

coagulation system activation [6, 27].

Convalescent Plasma

In a systematic review of random clinical trials and matched control studies between

1st January 2020 and 16th January 2021, the mortality rate was lower among transfused

patients, as well in patients receiving earlier transfusion of high-titer plasma [37].

However, in two clinical randomized control trials, patients receiving convalescent

plasma had no significant differences in the distribution of clinical out-comes or overall

mortality compared with placebo within 28-30 days; but all patients had severe or life-

threatening COVID-19, remaining unknown if the treatment would be more effective in

mild-moderate patients [38, 39]. Curiously, higher rates of negative PCR results were

associated with the convalescent plasma group [39].

Monoclonal Antibodies (mAbs)

SARS-CoV-2 mAbs target the S protein and are capable to block viral entry [40].

Nevertheless, mAbs cocktails including multiple antibodies with non-overlapping

epitopes provide a better protection, as even highly potent neutralizing antibodies used

alone do not protect against the rapid emergence of viral escape mutations [41]. Several

neutralizing mAbs were approved by emergency use authorization (EUA) for the

therapeutic treatment of COVID-19. These mAbs include the REGN10933 (Casirivimab)

and REGN10987 (Imdevimab) mAbs cocktail and the LY-CoV555 (Bamlanivimab and

LY-CoV016 (Etesevimab) cocktail [42, 43]. C121, C135 and C144 mAbs are also

potential candidates for clinical development [40].

22


REGN-COV2 cocktail (Casirivimab and Imdevimab) – A randomized

clinical trial involving the administration of placebo, 2.4g of REGN-COV2 and

8g of REGN-COV2 demonstrated that the cocktail reduced viral loads in both

doses together with few and mainly low-grade toxic effects [38]. REGN-COV2

had previously been successful in reducing viral load and preventing virus-

induced pathological sequelae when administered prophylactically or

therapeutically in rhesus macaques, as well in limiting weight loss and

decreasing lung titers and pneumonia in hamsters [44].

Bamlanivimab and Etesevimab (LY-CoV555 and LY-CoV016) – Treatment

with bamlanivimab and etesevimab cocktail in a clinical trial was associated

with a significant reduction in SARS-CoV-2 viral load at day 11 among non-

hospitalized patients with mild to moderate COVID-19 illness compared with

placebo [45].

Antiviral Therapies

Antiviral medications were used in clinical trials against SARS-CoV-2 to test their

efficiency, such as Remdesivir, Hydroxychloroquine/Chloroquine, Lopinavir/Ritonavir

and Ivermectin. However, these therapies had little or no effect on resolution of

symptoms or overall mortality in hospitalized patients compared with placebo [6, 27].

2.5. Vaccines Vaccination is the crucial step to prevent disease and SARS-CoV-2 onward

transmission, triggering the production of potent neutralizing antibodies against the virus

[6]. Currently, three vaccines are authorized and recommended in the United States to

prevent COVID-19. These are safe and effective in reducing the risk of severe illness

[46].

BNT162B2 (Pfizer-BioNTech)

Lipid-nanoparticle-nucleoside-modified RNA-based vaccine encoding the prefusion

stabilized, membrane-anchored full-length S protein [47]. In an ongoing multinational,

placebo-controlled, observer-blinded, pivotal efficacy trial, patients with 16 years old or

older who received two doses of BNT162N2 (30 μg) 21 days apart demonstrated 95%

efficacy in preventing COVID-19, with a safety profile similar to that of other viral vaccines

over a median of 2 months, besides short-term, mild-to-moderate pain at the injection

site, fever, fatigue and headache [47]. The viral load of BNT162N2 vaccinated

23


individuals, measured by Ct values, was substantially reduced compared with non-

vaccinated individuals 12 to 37 days after the first dose [48]. In vitro analyses of serum

samples from 15 patients of the prior clinical trial were tested against the new SARS-

CoV-2 variants. The neutralization of the B.1.1.7 and P.1 variants was roughly equivalent

and efficient, with the B.1.351 variant being robust but lower [49].

mRNA-1273 (Moderna)

Lipid-nanoparticle-encapsulated mRNA-based vaccine encoding the prefusion

stabilized full-length S protein [50]. In a phase 3 randomized, observer-blinded, placebo-

controlled trial, patients who received two doses of mRNA-1273 (100 μg) 28 days apart

demonstrated 94.1% efficacy in preventing COVID-19, with no noticed safety concerns

beside transient local and systematic reactions after the second dose, such as pain at

the injection site, erythema, induration, and tenderness [50]. Serum samples retrieved

from patients vaccinated with mRNA-1273 were tested in vitro against the new SARS-

CoV-2 variants using recombinant vesicular stomatitis virus (rVSV)-based pseudovirus.

The B.1.1.7 variant had no significant effect on neutralization, but B.1.1.7+E484K,

B.1.351, P.1 and B.1.427/B.1.429 showed a decrease in titers of neutralizing antibodies,

with B.1.351 having a larger effect [51].

Ad26.COV2.S (Johnson & Johnson)

Recombinant, replication-deficient human adenovirus type 26 vector vaccine

encoding the prefusion stabilized S protein [52]. In a randomized, double-blind, placebo-

controlled, phase 3 trial, patients who received 1 dose of Ad26.COV2.S (5×1010 viral

particles), including patients from Brazil with a high percentage of P.2 strains and from

South Africa with a high percentage of B.1.351 strains, were protected against moderate

to severe-critical COVID-19. In severe-critical COVID-19, vaccine efficacy was 76.7%

and 85.4% at least 14 and 28 days after administration, respectively. The reactogenicity

of AS26.COV2.S vaccine group were higher compared with placebo, but was generally

mild to moderate and transient, with symptoms ranging from infection-site pain,

headache, fatigue, myalgia and nausea [52].

3. SARS-CoV-2: A zoonotic threat The World Health Organization (WHO) defines a zoonosis as any disease or infection

that is naturally transmissible from vertebrate animals to humans and vice-versa [53]. An

infection is the entrance and development of an infectious agent in a human or animal

24


body, and disease is the illness caused by that specific infectious agent or its toxic

products [5].

Zoonotic pathogens can be viral, bacterial or parasitic [53], and are responsible for

about 75% of the experienced emerging and reemerging infectious diseases in humans

worldwide [54, 55]. However, 94% of the zoonoses documented between 1990 and 2010

were caused by RNA viruses, as wild animals harbor a rich pool of viruses with high host

plasticity [56, 57]. In the last two decades, bat-borne viruses were linked to more serious

zoonotic outbreaks, such as SARS-CoV, Hendra virus, Nipah virus, ebolavirus and

MERS-CoV [58].

The ecological interactions between a pathogen and its hosts, i.e., reservoir (donor),

and recipient (receiver) hosts, are fundamental for zoonotic spillover [59, 60], but

pathogens need to surpass several ecological barriers over many scales of space and

time to efficiently establish infection in humans (Fig. 5a). The odd of zoonotic spillover is

reliant on the pathogen pressure (quantity, dynamics, release, and survival), exposure

(recipient host behavior) and capacity to endure within-host immunological barriers [59,

60]. Furthermore, intermediate hosts can be infected and mediate cross-species

transmission between reservoir and recipient hosts in situations where the contacts

between them are limited or inexistent, securing a critical role in disease emergence [61].

When a pathogen is introduced in human populations, a set of aleatory branched

chains of transmission are generated, but the majority stutters to extinction, as the

pathogen is incapable to further evolve within humans (R0<1). However, the pathogen

can genetically adapt to humans in a few cases, and when it leads to the generation of

a large number of secondary infections (R0>1), the pathogen can spread efficiently within

human populations (Fig. 5b) [62]. Nonetheless, zoonotic spillover is a rare event, and a

minority of zoonoses can trigger large-scale outbreaks without repetitive introductions

from the reservoir host. Although, public health intervention is still necessary to reduce

the spillover risk to humans [59].

25


3.1. Anthropogenic factors causing zoonoses Zoonotic pathogens represent a major public health problem, as they can spread to

human’s through direct contact with domestical, agricultural or wild animals or through

contaminated food, water or environment [53]. Some human activities of higher risk

contribute to the increase of zoonotic events.

Land-use modification

Human population is expanding exponentially over the past few decades, and the

demand for more available land is increasing. As urbanization is growing, some human

activities, such as logging, mining, road construction and agricultural production, resulted

in deforestation and destruction of natural habitats, forcing wild animals harboring

Fig.5 – Establishment of zoonotic pathogens within human populations. a. Ecological barriers that need to be

surpassed by five zoonotic pathogens to establish infection in humans. The gaps’ width is proportional to the capability of

the pathogen to suppress barriers. The question marks represent points lacking crucial data requiring future research. b. Steps involved in the emergence of an infectious disease, demonstrating the introduction of the pathogen from the

reservoir and the subsequent chains of transmission within the human population. An evolved strain gains the ability to

spread and be maintained in the new host (R0>1), representing an epidemic potential. R0 indicates the average number

of secondary infections generated by a single primary infection introduced into a large population of previously unexposed

host, predicting the magnitude of an infectious disease outbreak. Daggers indicate no further transmission (Adapted from

Plowright et al. 2017 and Antia et al. 2013).

b

a

26


potential zoonotic pathogens to migrate to areas of higher human and livestock density,

increasing the risk of zoonotic spillover [53, 63, 64]. Besides, the massive urbanization

contributed to the formation of poorer healthcare systems, inadequate infrastructures,

and accumulated waste, increasing the risk of disease [63, 65].

Climate change

Over the past 50 years, human activities released enough amounts of carbon dioxide

and other greenhouse gases, severely affecting the global climate [66]. Consequently,

insufficient rainfall occurs and cause droughts, leading to poor sanitation and higher

exposure to contaminated water sources, and warmer climates prolong the seasonal

period of activity of ticks and mosquitos. Hence, climate change will increase the risk of

waterborne and vector-borne zoonotic events [54, 63].

Bushmeat

Markets selling the meat of wild animals englobe practices of handling, live

slaughtering, and consumption of wildlife [63, 64]. For example, about 56 species of bats

are hunted and consumed in many regions of Asia [64]. Due to the large number of new

or undocumented pathogens known to exist in some wild animal populations, these

practices increase the emergence of food-borne zoonotic diseases [53, 64].

Antibiotic usage

Agricultural workers using antibiotics in high quantities for farm animals are at an

higher risk of zoonotic spillover by drug-resistant strains of zoonotic pathogens, as these

are more capable to spread rapidly in animal and human populations [53].

3.2. Strategies to control zoonotic events Certain regions worldwide are more susceptible to zoonotic events, including

forested tropical regions with warmer and wetter climates, areas with higher wildlife

biodiversity and locations undergoing high land-use modification. These areas are

mainly located in lower latitudes, such as tropical Africa, Latin America, and Asia (Fig. 6), and should be prioritized for the detection of potential wildlife zoonotic pathogens, as

well to control unsafe and non-sustainable anthropogenic practices [55, 61, 67].

Additionally, global hotspots of potential zoonoses with bat origin are mainly located

in South and Central America and in some regions of Asia, suggesting that higher wildlife

surveillance of bats in these territories is vital to avoid future pandemics [65].

27


Generally, safe and appropriate guidelines for animal care in the agricultural sector,

standards for clean drinking water and waste removal, and education campaigns to

promote handwashing after close contact with animals and other behavioral adjustments

are some of the prevention methods for zoonotic diseases, but can differ for each

pathogen [53]. A successful global mitigation program focused on emerging zoonoses

needs to consider the epidemiologic dynamics and genetic/biochemical properties of the

pathogen and the risk factors contributing to higher infection rates, while contributing for

the development of an effective universal vaccine protecting against novel strains [7].

Currently, in the case of zoonotic viruses, some viral discovery programs, such as

the Global Virome Project and PREDICT, are focused on the catalog of unknown viruses

to simplify viral ecology insight. This will improve infectious disease surveillance and

diagnostics, as well to boost the development of new effective therapeutics [56, 68].

3.3. Origin of SARS-CoV-2 In late December of 2019, a cluster of patients with pneumonia of unknow cause

were epidemiologically related to a seafood wholesale market in Wuhan, Hubei Province,

China. The outbreak was caused by a novel CoV, severe acute respiratory syndrome

(SARS)-CoV-2, which presumably crossed species barriers to infect humans [1, 24, 69].

Fig.6 - Heat map indicating the probability of the emergence of an infectious disease event relative to reporting effort. Tropical Africa, Latin America, and Asia are more susceptible to zoonotic events (Adapted from Allen et al., 2017).

28


Although SARS-CoV-2 is thought as a zoonotic virus and SARS related-CoVs (SARSr-

CoVs) were already found in bats and pangolins, the animal reservoir remains unknown

[70].

Genome identity and the singularity of the Receptor-Binding-Domain (RBD)

SARS-CoV-2 showed a 96.2% genome identity with a SARSr-CoV in bats, RaTG13,

detected in Rhinolophus affinis [24]. Some evidence for past recombination was detected

in the spike (S) gene of RaTG13, but there is no proof that it contributed to the emergence

of SARS-CoV-2 [69]. Furthermore, the neutral sites divergence between SARS-CoV-2

and RaTG13 is 17% (dS=0.17), being much larger than expected [71].

Later, two sub-lineages of SARSr-CoVs were found in samples retrieved from

Malayan Pangolins (Manis javanica) in Guangxi (GX) and Guangdong (GD) provinces of

China, with the GD CoVs clade showing 85.5% to 92.4% genome identity to SARS-CoV-

2 and being extremely similar to the receptor-binding-domain (RBD) region of SARS-

CoV-2, with similar key residues (Fig. 7) [72-74]. However, this higher RBD identity may

be explained by coincidental convergent evolution rather than a recent recombination

event [71].

Earlier in the outbreak, researchers also reported that SARS-CoV-2 has the highest

codon usage bias similarity with snakes, although this method is questionable to

determine initial host origins [75].

Briefly, SARS-CoV-2 holds higher sequence identity in synonymous sites of nine

conserved ORFs with bat RaTG13, followed by GD Pangolin SARSr-CoV, GX Pangolin

SARSr-CoVs, bats ZC45 and ZXC21 and human SARS-CoV [1, 71]. Even If bats are

the probable natural reservoirs of SARSr-CoV, it is possible that SARS-CoV-2 has

entered human populations through an intermediate host, such as pangolins, who are

known to be the most trafficked mammal in the world for bushmeat and harbor a high

species diversity of CoVs [76].

The furin cleavage site

Intriguingly, in SARS-CoV-2, a novel polybasic furin cleavage site (RRAR) was

introduced at the S1/S2 boundary (Fig. 7), being absent in SARS-CoV and other

proximal SARSr-CoVs [77]. This allows efficient cleavage by furin and other proteases

and has a role in determining host range and viral infectivity [70]. Moreover, the insertion

of a leading proline at this site (PRRA) creates a turn, resulting in the addition of O-linked

glycans to S673, T678 and S686, which flank the cleavage site [70]. It was demonstrated

29


that ΔPRRA SARS-CoV-2 mutants diminished infection in Calu3 respiratory cells and

pathogenicity in a hamster disease model [78].

The lab leak origin hypothesis

Although many scientists support the probable animal origin of SARS-CoV-2, the

pandemic could have been ignited by an accidental lab leak followed by infection of a

person in the lab. Researchers might have created SARS-CoV-2 by engineering

coronavirus genomes or collected SARS-CoV-2 from an animal and maintained in the

lab to study [79].

Although there is no clear evidence to back these scenarios, this hypothesis cannot

be ruled out, as it is suspicious by three specific reasons: almost a year and a half into

the pandemic, SARS-CoV-2’s closest relatives still haven’t been found in animals; a top

lab studying coronaviruses is located in the Wuhan Institute of Virology (WIV), closer

where the first COVID-19 cases were detected; and SARS-CoV-2 contains unusual

features and genetic sequences signalling that it was engineered by humans, such as

the furin cleavage site, not found in closest relatives [79].

Fig.7 - Sequence alignment of SARS-CoV-2 and SARSr-CoVs spike (S) protein. The six key residues of the receptor

binding domain (RBD) are highlighted in blue (L455, F486, Q493, S494, N501 and Y505), being equal in SARS-CoV-2

and Pangolin CoV. The O-linked glycan residues are highlighted in dark blue (S673, T678 and S686), and the furin

polybasic cleavage site (RRAR), only detected in SARS-CoV-2, is highlighted in green (R682, R683, A684, R685), with

the insert sequence being PRRA (Adapted from Andersen et al., 2020).

30


4. SARS-CoV-2: a virus of the coronaviridae

family The Coronaviridae family (order Nidovirales and suborder Cornidovirineae) is divided

in two subfamilies – Letovirinae and Orthocoronavirinae [80]. Letovirinae includes the

monotypic genus Alphaletovirus, with a single species, Microhyla letovirus 1, while

Orthocoronavirinae comprises four genera: Alphacoronavirus (α-CoV) and

Betacoronavirus (β-CoV), mostly infecting mammals, and Gammacoronavirus (γ-CoV)

and Deltacoronavirus (δ-CoV), mostly infecting birds (Fig.8) [80, 81].

β-CoV is further divided in five subgenera: Embecovirus, Hibecovirus, Merbecovirus,

Nobecovirus and Sarbecovirus, and α-CoV in twelve, including Duvinacovirus,

Setracovirus and Tegacovirus [6, 80]. CoVs are positive sense single-stranded RNA

(+ssRNA) enveloped viruses with a crown-like appearance (Latin, coronae) containing

the largest genomes among all RNA virus families (25 to 32kb) [6, 82], and are known to

cause respiratory or enteric diseases in humans, mammalians, and avian species [83].

These viruses are prone to frequent recombination during mixed infection of closely

related strains and maintain a large and complex genome with sufficient mutation rates

for adaptation and interspecies transmission due to the proofreading reading mechanism

of CoV replication [84].

Fig.8 – Representation of the four CoV genera and associated species (Adapted from Sharun et al. 2020).

31


4.1. CoVs in pets Three CoVs are known to infect cats and dogs: the Feline Coronavirus (FCoV) and

Canine Coronavirus (CCoV), belonging to the α-CoV genus, and the Canine Respiratory

Coronavirus (CRCoV), belonging to the β-CoV genus [85, 86]. FCoV, CCoV and porcine

transmissible gastroenteritis virus (TGEV) share 96% of sequence identity within

polyprotein (pp)1ab, being grouped in the same species: α-CoV-1 [85, 87, 88].

FCoV and CCoV are grouped into five different genotypes (Fig. 9). CCoV-I contains

an orf3 subjacent to S, and the addition of a new gene leaded to the emergence of CCoV-

II [85, 88]. These two CCoVs share only 54% identity in the S [88]. Subsequently, FCoV-

II emerged as a recombinant form between CCoV-II and FCoV-I [85]. Later, CCoV-II was

subdivided in two subtypes: CCoV-IIa and CCoV-IIb, related to the ancestral CCoV-II

and the recombinant form between CCoV-IIa and TGEV, with a similar S N-Terminal

Domain (NTD), respectively [85, 88].

Recently, a novel feline-canine recombinant α-CoV, CCoV-Hu-Pn-2018, was isolated

from a patient with pneumonia in Malaysia. It belongs to the CCoV-II subtype due to the

absence of orf3, overall similarity with other CCoV-II strains and efficient replication in

A72 canine cells. A 12-aa deletion in the middle portion of N not found in other CCoVs

and α-CoV-1 species is consistent with a recent zoonotic event. However, more studies

are necessary to confirm its pathogenic potential in humans [89].

Fig.9 - Genetic features of FCoV and CCoV genotypes. Blue colour corresponds to feline

sequences, orange to canine sequences, purple to porcine sequences and green to the novel orf3

sequence. Arrows indicate the putative recombination sites (Adapted from Le Poder et al., 2011).

32


FCoV

FCoVs are enterotropic viruses causing asymptomatic infection or diarrhea in cats.

The virus is mainly shed from feces and is transmitted via fecal-oral route. In more severe

conditions, enteric FCoVs mutate within an infected cat and gain the ability to infect

monocytes and macrophages, with cats showing early signs of inactivity, anorexia,

dehydration, and weight loss and causing severe systemic disease, affecting the kidney,

liver, pancreas, eyes, and central nervous system (CNS). This immune complex disease

is known as Feline Infectious Peritonitis (FIP), with kittens being more affected.

The cat’s own immune reaction is the major reason that leads to fatal consequences,

as the severest form of FIP is associated with macrophages, neutrophils, and

lymphocytes aggregations in smaller veins, called pyogranulomas, leading to the

formation of edema and accumulation of large volumes of protein-rich fluids [82, 90]. FIP

can be identified by three forms: an effusive, exudative wet form (Fig.10a), characterized

by ascites, thoracic effusions, or pericardial effusions; a non-effusive, non-exudative, dry,

granulomatous, parenchymatous form (Fig.10b), characterized by granulomatous

changes in different organs, such as eyes and CNS; and a mixed form [90].

Immunity to FCoV is not long-lasting, and cats may be reinfected. The majority of

infected kittens clear FCoV, but about 15% become chronic shredders, being at higher

risk of developing FIP [82].

CCoV

CCoVs are enterotropic viruses causing mild gastroenteritis in dogs, mainly mild to

severe diarrhea in pups, and fatal infections are largely associated with simultaneous

infection by other canine pathogens. The virus is shed at high titers in feces and is

transmitted via the fecal-oral route [88].

Fig.10 – Feline Infectious Peritonitis (FIP) in cats. a. Cat with ascites (effusive

form) and b. cat with uveitis (non-effusive form) (Adapted from Hartmann et al. 2005).

33


However, a higher virulent strain, designated pantropic CCoV, causes fatal

systematic disease in pups, including fever, vomiting, anorexia, hemorrhagic

gastroenteritis, lethargy, ataxia, seizures, and lymphopenia [91].

CRCoV

Belongs to the β-CoV-1 genus, together with bovine coronavirus (BCoV) and HCoV-

OC43, being antigenically distant to enteric CCoVs [85, 88]. This virus causes the canine

infectious respiratory disease (CIRD), where dogs suffer from a mild cough, with some

cases developing to bronchopneumonia [92].

4.2. CoVs in humans Seven CoVs are known to infect humans with different levels of pathogenicity. The

α-CoVs, HCoV-NL63 and HCoV-229E, and the β-CoVs, HCoV-0C43 and HCoV-HKU1,

are endemic in humans and low pathogenic, associated with common colds. However,

three epidemic and highly pathogenic zoonotic β-CoVs, severe acute respiratory

syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV and the novel

β-CoV, SARS-CoV-2, were responsible for outbreaks involving high case fatality rates

[1, 81].

SARS-CoV-2 belongs to the same viral species of SARS-CoV, SARSr-CoV, within

the genus β-CoV and subgenus Sarbecovirus, explained by the 94.4% identity in the

seven conserved orf1a/1b replicase domains [2, 24, 69]. Additionally, the two viruses

share ~80% nucleotide identity at the genome level [24, 86], while MERS-CoV revealed

only 51% genome identity with SARS-CoV-2 [86].

SARS-CoV

In November 2002, an unusual pneumonia emerged in Foshan, Guangdong

Province, caused by a novel CoV, SARS-CoV [93], being responsible for ~8000 cases

and 774 deaths worldwide (~10% fatality rate) [94]. The virus belongs to the subgenus

Sarbecovirus [80] and binds to the ACE2 cell receptor [95].

SARS-CoV-like (SLCoV) viruses were identified in various species of horseshoe bats

(Rhinolophus spp.) with 88% to 92% of genomic identity with SARS-CoV. Given that

horseshoe bats harbor a wide spectrum of genetically diverse SLCoV viruses and a high

seroprevalence against bat-SARS-CoV N, there is strong evidence that these animals

are the natural reservoir of SARS-CoV and SLCoV viruses [96, 97]. Additionally, a single

recombination breakpoint was detected in bat SARSr-CoV RP3 between S and ORF1b,

34


suggesting that SARS-CoV may be a recombinant form between a bat SLCoV and a

strain closely related to human SARS-CoV [98].

SLCoV viruses were also discovered in Himalayan palm civets (Paradoxurus

hermaphroditus) and in one racoon dog (Nyctereutes procyonoides) from Guangdong

province markets, with two virus isolates from palm civets sharing 99.8% of sequence

identity with SARS-CoV, suggesting that these animals could act as amplifying hosts

[99]. There is evidence that viruses in palm civets had evolved to infect humans [100].

Consequently, a massive number of palm civets were culled in Guangdong to remove

sources of SARS reemergence, as, prior to culling, SLCoV viruses were found in many

civets and raccoon dogs [100]. SARS-CoV SZ3 (from palm civets) is capable to bind

more efficiently to palm civet ACE2 compared with hACE2, where SARS-CoV Tor2 (from

humans) utilizes both receptors efficiently, which is consistent with a recent zoonotic

transfer [101]. Martina et al. demonstrated that domestic cats and ferrets are also

susceptible to SARS-CoV experimental infection and the virus is efficiently transmitted

to contact animals [102].

Among infected human patients, cough, myalgia, fever, malaise and watery diarrhea

are experienced in the course of disease, developing to severe respiratory failure,

shortness of breath, multiple organ failure and sepsis in later phases [94].

Histopathological findings included diffuse alveolar damage, inflammatory infiltrates,

edema, pneumocytes desquamation and hyaline-membrane formation [94].

MERS-CoV

In June 2012, a 60-year-old man died as a result of renal and respiratory failure in

Jeddah, Saudi Arabia, due to a novel CoV [103], MERS-CoV, responsible for ~2279

cases and with ongoing outbreaks, mainly reported in Saudi Arabia, together with 806

deaths worldwide (35.5% fatality rate), with the age group 50–59 being at a higher risk

[104]. MERS-CoV belongs to the subgenus Merbecovirus [80] and binds to the DPP4

cell receptor, even when it is exploited to bat cells [105].

There is strong evidence that Old-World insectivorous bats from the Vespertilionidae

family are the original source of MERS-CoV, where the virus probably evolved and

emerged in humans due to a recombination event in the S1 subunit [103, 106, 107].

Additionally, evidence of MERS-CoV spillover from ill dromedary camels (Camelus

dromedarius) to humans [108], as well reports of a high seroprevalence of antibodies

specific to MERS-CoV in dromedaries from the 1980s in Africa, suggest that this animals

have served as a long-term zoonotic reservoir for at least 30 years [109, 110].

35


Among infected human patients, pneumonia, acute respiratory distress syndrome

(ARDS), sepsis and abdominal pain/diarrhea are the most common clinical

manifestations. Mortality is highly associated with septic shock and organ failure, and

hypertension and diabetes are common associated comorbidities among patients [111].

5. SARS-CoV-2: the pandemic virus

5.1. Morphology CoVs virions are pleiomorphic but roughly spherical enveloped particles with ~60-

200 nm of diameter and are constituted by 4 major structural proteins: spike (S),

nucleocapsid (N), membrane (M) and envelope (E) proteins (Fig. 11a). Some CoVs also

have a hemagglutinin esterase (HE) protein, a receptor-destroying enzyme that

facilitates viral progeny release from infected cells [82, 83].

In SARS-CoV-2, the spherical and pleomorphic virions measure 40 to 140 nm of

diameter, with distinct spikes of 9 to 12 nm (Fig. 11b). Furthermore, extracellular free

virions and inclusion bodies filled with virions in membrane bound vesicles in cytoplasm

were detected in the human airway epithelial (Fig. 11c), which is consistent with the

Coronaviridae family [1].

Fig. 11 – Morphology of SARS-Cov-2. a. Virion structure of SARS-CoV-2 with illustrated

interaction between S and ACE2 receptor; Transmission electron microscopy visualization of b. negative stained SARS-CoV-2 virions and c. SARS-CoV-2 virions in the human airway epithelial cell

ultrathin sections (Adapted from Cascella et al. 2021 & Zhu et al., 2020).

a b

c

36


The SARS-CoV-2 S protein is glycosylated and is constituted by two subunits : S1

(N-terminal), containing the RBD, responsible for the attachment to the host cell receptor,

and highly divergent due to host immune responses; and S2 (C-terminal), responsible

for the fusion of the virions to the host cells after S cleavage by host furin-like proteases,

and extremely conserved across the subfamily Coronavirinae (Fig. 12a) [22, 82, 83,

112].

SARS-CoV-2 S trimer exist in distinct multiple conformational states, exhibiting

spontaneously closed and open conformation and establishing asymmetric trimeric

structures (Fig. 12b). When S trimer is in a closed conformation, the RBD is hidden, and

in an open conformation, the RBD is exposed. This is consistent with the S plasticity of

SARS- and MERS-CoV [77, 84, 112]. These conformational changes, together with the

S glycan shielding masking the receptor binding loops, results in decreased recognition

by the hosts immune systems [77, 83].

The N protein packages the viral RNA into a helical ribonucleocapsid. It is also

required for RNA synthesis, as it colocalizes with replicase-transcriptase components

[82], and interacts with other structural proteins throughout virion assembly, leading to

genome encapsidation [113]. The M protein is the most abundant, is tightly associated

with the virus envelope by three hydrophobic domains and it has a short ectodomain

modified by glycosylation that plays a major role in promoting membrane curvature [82].

The E protein is a viroporin responsible for the formation of ion channels in the

membranes, influencing the electrochemical balance in the subcellular compartments

[82]. The interaction of these 3 structural proteins in the ER is crucial for the maturation

and assemble of new virion particles [113].

37


5.2. Genome CoVs genome is constituted by genes that encode the structural, non-structural

(Table 1), and accessory proteins. Additionally, each CoV contain a common 5’ “leader”

transcription regulatory sequence (TRS-L) fused to the “body” RNA sequence and body

TRS (TRS-B) downstream of each ORF for sgRNA negative-strand synthesis via “leader-

to-body-fusion” [82, 114]. The extremities of CoVs genome consist of a 5' CAP structure

and a 3' polyadenylate tract [82].

In SARS-CoV.2, the order of the genes (5′ to 3′) is: orf1a, orf1b, s, orf3a, orf3b, e, m,

orf6, orf7a, orf7b, orf8, n, orf9a, orf9b and orf10, together with a leader TRS and nine

putative TRS-B [69, 115].

Fig. 12 - SARS-CoV-2 Spike (S) protein. a. SARS-CoV-2 S structure colored by domain (SS, signal sequence; NTD,

N-terminal domain; RBD, receptor binding domain; SD1, subdomain 1; SD2, subdomain 2; S1/S2, cleavage site; S2’,

S2’ protease cleavage site; FP, fusion peptide; HR1, heptad repeat 1; CH, central helix; CD, connector domain; HR2,

heptad repeat 2; TM, transmembrane domain; CT, cytoplasmic tail). Arrows indicate protease cleavage sites; b. Side

and top views of the prefusion Cryo-EM structure of SARS-CoV-2 S with a single RBD in the up conformation. The

colors correspond to the schematic in the illustration B (Adapted from Wrapp et al., 2020).

a

b

38


Table 1. Description of SARS-CoV-2 non-structural proteins (nsp).

5.3. Infection cycle The series of steps that SARS-CoV-2 follows to replicate into its host are composed

of attachment, fusion; amplification; assembly and release.

Attachment

The first obstacle of any virus is entry into its host cell.

SARS-CoV-2 binds to the ACE2 receptor by the heavy glycosylated trimeric S protein

[24, 69, 77, 112, 120]. ACE2 is a cell-surface, zinc-binding carboxypeptidase important

Nsp Function Reference

1 Blockage of retinoic acid–inducible gene I–dependent innate immune responses, such

as interferons, trough binding to various ribosomal complexes in the mRNA entry

channels.

[113, 116,

117]

2 Unknown. In SARS-CoV, binds to the prohibitin 1 and 2 (PHB1 and PHB2), with a

possible role in disrupting the host cell environment. [118]

3 Multi-domain membrane-bound protein, including a papain-like protease (PLpro)

domain responsible for releasing nsp1, nsp2, and itself from pp1ab; and a

Macrodomain-X, responsible for removing ADP-ribose from proteins.

[113, 119]

4 Contains a transmembrane domain and is involved in membrane remodeling /

anchoring of the replication-transcription complex to cell membranes. [82]

5 Main protease (Mpro)/ 3C-like protease. It cleaves pp1ab at 11 distinct sites, releasing

nsps 4-12. [69, 113]

6 Contains a transmembrane scaffold and is involved in membrane remodeling /

anchoring of the replication-transcription complex to cell membranes. [82]

7 Forms a large complex with nsp8, performing the RNA primase activity during viral

RNA synthesis. [113]

8 Forms a large complex with nsp7, performing the RNA primase activity during viral

RNA synthesis. [113]

9 Single-stranded RNA-binding protein [82]

10 Scaffold protein forming a mRNA cap methylation complex with nsp14 and nsp16,

enhancing their activities. [113]

12 RNA dependent RNA polymerase (RdRp). Forms a replicase complex with nsp7 and

nsp8 for replication and transcription of the viral RNA genome.

[24, 69, 113,

114]

13 Superfamily 1B helicase with 5’-RNA capping, NTPase and duplex RNA/DNA

unwinding activities. [69, 82, 113]

14 N-terminal exoribonuclease (ExoN) domain with proofreading function, increasing the

fidelity of RdRp during RNA replication and preventing frequent lethal mutations; and a

C-terminal guanine-N7 methyl transferase involved in viral RNA cap methylation.

[58, 69, 82,

84, 113]

15 Endoribonuclease (EndoU). Cleaves both single and double stranded viral RNA

downstream of uridylate residues, promoting immune evasion.

[69, 82, 84,

113]

16 Ribose-2’-O-methyltransferase. Interacts with nsp14 for cap methylation and synthesis. [69, 82, 84]

39


for the regulation of cardiac function and blood pressure, being highly expressed in

epithelial cells of the lung and small intestines, as well as other organs [82]. Other

acknowledged CoV receptors, such as dipeptidyl peptidase 4 (DPP4) and

aminopeptidase N (APN) are not used by SARS-CoV-2 [24, 121].

Curiously, SARS-CoV-2 RBD revealed higher affinity binding to ACE2 compared with

SARS-CoV RBD, which may explain why SARS-CoV-2 can easily enter host cells and

spread efficiently [120-122]. Moreover, SARS-CoV RBD-specific monoclonal antibodies

(mAbs) failed to show appreciable binding to SARS-CoV-2, suggesting limited antibody

cross-reactivity between the two RBDs [112].

Fusion

During the process of viral entry into host cells, the prefusion state of the S protein

exposes the RBD by undergoing a significant structural rearrangement to a less stable

open state, which is necessary to interact with ACE2 [112]. Then, the RBD is recognized

by the extracellular peptidase domain (PC) of the ACE2, mainly through polar residues

[123].

There are two critical steps that precede viral fusion : one cleavage at the S1/S2

boundary and other within S2 (S2’) by host furin-like proteases. In humans, a

transmembrane serine protease (TMPRSS2) is expressed in pneumocytes and binds to

ACE2, putting uncleaved S near a host cell protease upon initial binding [82, 124, 125].

This process results in S1 shedding and S2 refolding for membrane fusion [112].

Amplification

Upon cell entry, RNA synthesis involves two steps: genome replication and

subgenomic RNA (sgRNA) transcription (Fig. 13a). The RNA-dependent RNA

Polymerase (RdRP) is the major responsible for the generation of negative-sense RNA

intermediates that serve as templates for the synthesis of more positive-sense gRNA, as

well as the generation of a nested set of negative-sense 3’co-terminal subgenomic

mRNAs [69, 82, 84, 114].

Genome replication : The orf1a and orf1b from the gRNA are translated into

the polyproteins pp1a and pp1b, respectively. Expression of the orf1b-

encoded part of pp1ab requires a ribosomal frameshift that directs a

controlled shift into the -1-reading frame upstream of the orf1a stop codon.

Both are proteolytically processed to nsps 1-16, some of which will form the

viral replicase/transcriptase complex (RTC). The RTC anchoring to cell

membranes is mediated by the nsp3 and nsp5 viral proteases and the nsp6,

40


which is needed to remodel the host cell membranes towards forming

structures dedicated to the synthesis of additional negative sense RNAs [69,

82, 84, 114, 115].

sgRNA transcription : The RTC generates negative strand sgRNA in a

process of discontinuous transcription by leader-to-body fusion at the TRSs

short motifs, as these sgRNAs canonically contain a sequence present at the

5’ end of the viral genome, known as TRS in the leader (TRS-L), and TRS in

the body (TRS-B) preceding each functional ORF downstream the pp1ab.

RTC pauses when it crosses TRS-B for the negative strand transcription of

the negative-strand sgRNAs intermediates. Then, it switches the template to

the TRS-L, producing positive-strand sgRNAs that are used for the

expression of the structural (E,S,M and N) and accessory proteins dispersed

among the structural genes [69, 82, 114, 115]. Researchers found out that

mutating the accessory proteins have a profound effect on the viral

pathogenesis and compromises the ability of the CoVs to replicate in their

hosts [82].

Assembly and release

Virion assembly occurs in the host cell membranes. The gRNA bound to N combines

with M, E and S in the ER/Golgi intermediate compartments. Then, mature virions are

assembled within golgi vesicles and released by exocytosis from cells (Fig. 13b) [6, 82,

114]. The viroporin E, with ion channel activity, alters the cell secretory pathways to

promote viral release, as it increase the pH of the transport vesicles [82].

41


Fig. 13 – Steps involved in SARS-CoV-2 replication. a. Genome and subgenomic RNA (sgRNA) transcription of

SARS-CoV-2. Orange boxes represent structural proteins (S, E, M and N), yellow boxes represent accessory proteins

and small red boxes represent the TRSs; b. SARS-CoV-2 amplification processes (adapted from Davidson et al.,

2020).

a

b

42


5.4. Viral evolution and variants A virus is defined by a genome sequence capable of autonomous replication inside

cells and dissemination between organisms or cells under suitable conditions, being

harmful or non-harmful to the host. This is the case of RNA viruses, including SARS-

CoV-2 [2].

RNA viruses are prone to high error rates of RNA replication, existing as

quasispecies, i.e., an ensemble of related genomes. A viral quasispecies is formed by a

master sequence and a mutant spectrum containing the molecular record of the

population evolutionary history, englobing a repository of genetic and phenotypic

variants with the ability to become dominant. Therefore, evolutionary selection targets

the RNA virus quasispecies as a whole rather than individual variants, being crucial for

an efficient response to selective environmental constrains previously experienced by

the same viral population. Hence, mutants with higher host tropisms within the

quasispecies memory may be manifested in unusual hosts during inter-species viral

transmission by a bottleneck event, leading to unexpected spillover events [126].

As with all viruses, SARS-CoV-2 continuously adapts to changing environments in

real time via random genome mutations that are subjected to natural selection. Although

these mutations are mostly detrimental or neutral, some may provide a selective

advantage, such as escape from the host immune system and resistance to antiviral

drugs, resulting in increased fitness for transmissibility [127].

To track and clearly identify distinctive SARS-CoV-2 genome sequences, three

nomenclature systems are currently in use: GISAID, Nextstrain and Pango. However, on

31st May 2021, the virus evolution working group of the WHO provided a simplified

nomenclature method for naming SARS-CoV-2 variants of concern (VOCs) and variants

of interest (VOIs) based on the existent nomenclature systems by using Greek alphabet

letters [127, 128]. VOCs are known to cause higher transmissibility, increase in virulence

or change in clinical disease presentation and reduction of diagnostics, vaccines, and

therapeutics effectiveness; and VOIs contain specific genetic markers predicted to affect

viral transmission, disease severity, diagnosis, treatment and response to vaccines

[128].

According to Pango, each descendant lineage (ex: A.1 or B.2) must reveal

phylogenetic evidence of emergence from an ancestral lineage into another

geographically separated population, with considerable onward transmission, by the

following criteria: 1) one or more shared nucleotide differences from the ancestral

43


lineage; 2) at least five genomes with >95% of the genome sequenced within the lineage;

3) genomes within the lineage with at least one shared nucleotide change; 4) a >70%

bootstrap value for the lineage-defining node. Additionally, these lineages act as

ancestors for other emergent lineages detected in a different geographical area (ex:

A.1.1), considering the prior criteria. This logic proceeds until a maximum of three

sublevels (ex : A.1.1.1), after which the new lineage is described with a new letter (ex :

A.1.1.1.1 would be C.1 and A.1.1.1.2 would be C.2) [129].

Initially, two major earlier lineages were identified in humans : lineage A (including

Wuhan/WHO4/2020), being the root of the pandemic and sharing two nucleotides with

other SARSr-CoVs; and lineage B (including Wuhan-Hu-1), more abundant (70%), with

2 single nucleotide polymorphisms (SNPs) in those regions (Fig.14a) [71, 129].

Later, between December 2019 and March 2020, a predominant clade emerged in

Europe with a D614G mutation (Fig.14b) [130, 131]. D614G status was associated with

higher infectious titers and with patients low Ct values, but no correlation was observed

with disease severity [131]. Additionally, higher infectious titers were observed in vitro

[130, 131], probably linked to a higher ACE2 binding fusion-competent state in D614G,

caused by the interprotomer latch disruption between D614 in S1 and T859 in S2, which

increases the distance between the protomers and enhances the ratio of open to closed

S protein trimers. Although a substantial portion of D614G trimers revealed a two-open

conformation (39%) and an all-open state (20%), the neutralization potency of mAbs

targeting the RBD of the D614G mutant was not diminished [130].

Currently, among the vast number of existing variants, four VOCs and four VOIs are

designated, according to the WHO Virus Evolution Working Group (Table 2) [128]. These

are further presented, along with the characterization of the most notable spike

mutations:

44


Fig. 14 – First lineages and predominant clade of SARS-CoV-2. a. Phylogenetic relationship between the first SARS-

CoV-2 lineages: S (currently known as A) and L (currently known as B) lineages of SARS-CoV-2, with other SARSr-CoVs.

Both lineages are separated by two tightly linked SNPs at positions: 8782 (orf1ab: T8517C, synonymous) and 28144 (orf8:

C251T, S84L); (b) Global predominance of SARS-CoV-2 viruses circulating in the human population with the G614 form

of the S protein compared with the D614 form originally identified in the first human cases in Wuhan, China (Adapted from

Tang et al. 2020 and Korber et al. 2020).

a

b

45


Table 2. SARS-CoV-2 VOCs and VOIs (Adapted from WHO: Tracking SARS-CoV-2 variants, accessed 18th August

2021).

5.4.1. VOCs

Alpha (VOC-α)

First documented in England, United Kingdom. The signature spike mutations

include 69del, 70del, 144del, (E484K), (S494P), N501Y, A570D, D614G, P681H, T716I,

S982A, D1118H and (K1191N) [132]. Preliminary results demonstrated that VOC-α is

43% to 82% more transmissible than the wildtype form [133], being associated with an

increased risk of death of 64% [134]. It has overall little reduced neutralization by mAbs,

convalescent plasma and sera from Moderna (mRNA-1273) and Pfizer-BioNTech

(BNT162b2) vaccinated individuals [135, 136], but is resistant to neutralization by mAbs

directed against the NTD supersite [135].

WHO label

PANGO lineage

GISAID clade

Nextstrain Clade

Variant Type

Earliest documented

samples

Date of designation

Alpha B.1.1.7 GRY 20I (V1) VOC United

Kingdom, Sep-2020

18-Dec-2020

Beta B.1.351

B.1.351.2 B.1.351.3

GH/501Y.V2 20H (V2) VOC South Africa, May-2020 18-Dec-2020

Gamma

P.1 P.1.1 P.1.2 P.1.4 P.1.6 P.1.7

GR/501Y.V3 20J (V3) VOC Brazil, Nov-2020 11-Jan-2021

Delta

B.1.617.2 AY.1 AY.2 AY.3

AY3.1

G/478K.V1 21A VOC India, Oct-2020

VOI: 4-Apr-2021 VOC: 11-May-2021

Eta B.1.525 G/484K.V3 21D VOI Multiple

countries, Dec-2020

17-Mar-2021

Iota B.1.526 GH/253G.V1 21F VOI United States of America, Nov-2020

24-Mar-2021

Kappa B.1.617.1 G/452R.V3 21B VOI India, Oct-2020 4-Apr-2021

Lambda C.37 GR/452Q.V1 21G VOI Peru, Dec-2020 14-Jun-2021

Epsilon B.1.427 B.1.429 GH/452R.V1 21C

Alert (previous

VOI)

United States of America, Mar-2020

VOI: 5-Mar-2021 Alert: 6-Jul-2021

46


69-70del is located in the NTD and is associated with some antibody escape

[136, 137], positive selection during treatment of an immunocompromised patient

with convalescent plasma [137] and with S gene target failures (SGTF) in

TaqPath tests [138, 139]. It is also a recurrent deletion region, rapidly rising to

notable abundance, but did not impact the binding of an RBD- and NTD-directed-

mAb. However, together with 144-145del, the binding is impacted [140]. This

deletion also emerged in a chronically infected immunosuppressed patient after

treatment with the rituximab mAb [141].

144del is located in the NTD and is associated with some antibody escape [136],

conferring resistant to most NTD-directed mAbs in B.1.1.7 [135, 142].

Additionally, it emerged as a part of a 4-aa deletion at positions 141-144 in an

immunocompromised patient [143].

N501Y affects one of the six key residues of the RBD and is associated with

increased binding affinity to human and mouse ACE2 [138, 144-151]. This

enhanced binding could be explained by the increases occupancy of the open

state conformation in N501Y viruses relative to other mutations [152]. Curiously,

the N501Y polymorphism contains indication of positive selection , with codon

501 exhibiting a significant amount of non-synonymous substitutions globally

[147]. This was observed in a case of an immunocompromised patient, where

N501Y was found at day 128 p.i. after persistent infection [153]. However, N501Y

does not seem to compromise the antibodies neutralization capacity, as serum

from the mRNA-based vaccine BNT162b2 revealed equal neutralizing titers in

N501 and Y501 viruses [154] and C121, C135 and C144 mAbs were able to

neutralize N501Y [155]. In animals, the mutation N501Y spontaneously emerged

in experimentally infected mice [146].

P681H is instantly upstream of the furin cleavage site in S1/S2 interface, an

important hotspot for epitope signal [78, 133, 136, 138], and may reduce antibody

recognition [136]. Similarly to D614G, P681H is showing a significant exponential

increase in worldwide frequency [156].

Beta (VOC-β)

First documented in Nelson Mandela Bay, South Africa. The signature spike

mutations include D80A, D215G, 241del, 242del, 243del, K417N, E484K, N501Y,

D614G and A701V [132]. It is believed that VOC-β is more transmissible due to the rapid

displacement of other lineages [147]. It exhibits immune evasion from therapeutically

relevant neutralizing mAbs targeting regions in the RBD and NTD [142, 157], as well

from neutralizing antibodies in convalescent plasma [157, 158]. Additionally, a higher

47


reduction in titer to mAbs, convalescent plasma and sera from vaccinated individuals

from Moderna (mRNA-1273) and Pfizer-BioNTech (BNT162b2) was noticed compared

with the alfa variant, which is mainly associated with the E484K mutation [135].

L18F is associated with decreased binding to the S2L28 NTD-directed-mAb.

However, a similar mutation in this site, L18P, was linked with binding to multiple

mAbs [142]. Δ242–Δ244 alone together with R246I is associated with NTD resistance to NTD-

directed-mAbs [135, 157]. Remarkably, recurrent deletions in the 243-244

positions disrupt binding of the mAb 4A8, which defines an immunodominant

epitope within the NTD [140].

K417N and similar mutations in this site are related with immune evasion from

neutralizing mAbs [42, 135, 157] and slightly weaker binding affinity to ACE2

[144, 145, 148].

E484K is located in one of the major antigenic sites within the RBD [159] and is

associated with reduced mAbs [41, 42, 155, 157, 159, 160] and convalescent

serum neutralization [159, 161]. Codon 484 reveals indication of positive

selection, with an overall considerable excess of non-synonymous substitutions

detected globally [147]. In one study co-incubating WT SARS-CoV-2 with an

highly neutralizing convalescent plasma, a variant completely resistant to

neutralization emerged at day 80, with the E484K mutation, together with an

insertion in the NTD N5 loop [162]. There is evidence that it may modestly

enhance binding affinity to ACE2, which may be further increased by the

presence of N501Y and stabilized by the presence of K417N [148, 149, 163]. A

case of reinfection associated with a virus harboring the E484K mutation was

reported in a woman previously infected with a non-E484K variant of SARS-CoV-

2 [164]. E484 binds to the D30 of ACE2 by a salt-bridge, being highly

conservative in ACE2 orthologs [151].

Gamma (VOC-γ)

First documented in Manaus, Brazil, and detected in Japan in Brazilian travelers. The

signature spike mutations include L18F, T20N, P26S, D138Y, R190S, K417T, E484K,

N501Y, D614G, H655Y, T1027I [132]. Modeling work estimates that VOC-γ may be 1.7

to 2.6 times more transmissible [165, 166]. VOC-γ is refractory to multiple neutralizing

mAbs, but also more resistant to neutralization by convalescent plasma (3.4 fold) and

Moderna (mRNA-1273) and Pfizer-BioNTech (BNT162b2) vaccine sera (3.8-4.8 fold)

[167].

48


Delta (VOC-δ)

First documented in Maharashtra state, India. The signature spike mutations include

T19R, (V70F), T95I, G142D, E156del, F157del, R158G, (A222V), (W258L), (K417N),

L452R, T478K, D614G, P681R and D950N [132]. VOC-δ showed to have higher

household transmissibility [168]. Additionally, VOC-δ was resistant to neutralization by

some mAbs targeting the NTD and RBD regions and by convalescent sera (4-fold less

potent against VOC-δ compared with VOC-α) [169]. Post-vaccination sera generated an

efficient neutralizing response against the VOC-δ, but about three- to fivefold less potent

than they are against the VOC-α [169]. Curiously, the effectiveness of the Pfizer and

Moderna vaccines was notably lower among symptomatic patients with the VOC-δ than

among those with the VOC-α [170].

G142D is located in the NTD and is an escape mutant to the mAb COV2-2489

targeting the NTD region [142]. A222V does not seem to have a functional effect on S ability to mediate viral

entry, as pseudotyped lentivirus with the A222V mutation did not have a

measurably different viral titer than those without the mutation [171]. L452R is located in the RBD and is associated with decreased sensitivity to the

X593, P2B-2F6 [172] and the leading Bamlanivimab (LY-COV555) [42]

neutralizing mAbs, as well as 10 out of 34 RBD-specific mAbs [173] and some

convalescent sera [159]. L452R may also increase binding affinity with ACE2

receptor due to the significant free energy changes, leading to the emergence of

more infectious variants [150]. This enhanced infectivity was confirmed as

measured by soluble mACE2 [159].

5.4.2. VOIs

Eta (VOI- η)

First documented in United Kingdom and Nigeria. The signature spike mutations

include A67V, 69del, 70del, 144del, E484K, D614G, Q677H and F888L [132]. The

neutralization of sera from Pfizer-BioNTech (BNT162b2) vaccinated individuals against

VOI- η was only modestly reduced relative to the neutralization of the wild-type virus

[174].

Kappa (VOC-κ)

First documented in Maharashtra state, India. The signature spike mutations include

(T95I), G142D, E154K, L452R, E484Q, D614G, P681R and Q1071H [132]. An

49


experimental infection study with Syrian hamsters demonstrated a higher pathogenicity

of VOC-κ compared with B.1, with more severity lung lesions, but no significant

differences were found in viral load and bodyweight loss [175].

Iota (VOI-ι)

First documented in New York, USA. The signature spike mutations include L5F,

(D80G), T95I, (Y144-), (F157S), D253G, (L452R), (S477N), E484K, D614G, A701V,

(T859N), (D950H), (Q957R) [132]. VOI-ι pseudoviruses with E484K were resistant to

several mAbs, including the two therapeutic mAbs in clinical use : Bamlanivimab (LY-

CoV555) and Casirivimab (REGN10933) [176]. Additionally, it was less susceptible to

neutralization by convalescent plasma and vaccinee sera by 4.1-fold or 3.3-3.6 fold,

respectively [176]. This is consistent with another study, showing reduced neutralization

by vaccinated plasma (Moderna (mRNA-1273) or Pfizer-BioNTech(BNT162b2), 1.3-

month and 6.2-month convalescent plasma by 4.5, 6.0 and 4.8-fold, respectively,

compared with the D614G variant [177]. S447N had a discernible antigenic impact in

booth studies [176, 177].

S447N is associated with enhancing binding to ACE2 [145, 149, 150], resistance

to multiple mAbs [159] and with a possible enhanced infectivity mediated by

soluble mACE2 [159].

Lambda (VOI- λ)

First documented in Lima, Peru. The signature spike mutations include G75V, T76I,

Δ246-252, L452Q, F490S, D614G, T859N [178]. Pseudotyped lentivirus work

demonstrated a reduction in neutralization in post-vaccinated plasma (CoronaVac) by

3.05-fold for the VOI- λ compared to wild-type, as well as an increased infectivity

compared with VOC-α and VOC-γ [179].

F490S is associated with reduced susceptibility to antibody neutralization [155,

159].

Epsilon (VOI-ε)

First detected in July 2020 since its emergence on early May 2020, in California,

EUA, and increased in frequency since November 2020. In contains 3 novel mutations

in the S (S13I, W152C and L452R) [180, 181], with L4523 being of higher biologic

significance [172]. Data shows that VOI-ε is associated with neutralizing titers reduction

in plasma from Moderna (mRNA-1273) and Pfizer-BioNTech (BNT162b2) vaccinated

individuals (2-6 fold) [173, 181] and convalescent plasma (4-6.7 fold) [181] compared

50


with WT, and with completely dropped neutralization by all NTD- and some RBD-specific

mAbs [173]. Additionally, analyses of qPCR Ct values associated with VOI-ε infections

were significantly lower than non-VOI-ε, demonstrating a higher infectivity of this variant

by a 2-fold increase in viral shedding [181]. It was considered a VOI in 5th March 2021,

but is now a variant of alert since 6th July 2021 [128].

5.5. Animal associated variants More than a few SARS-CoV-2 S mutations emerged in animals owing to a variety of

interspecies transmission of the virus, either during natural cases of infections, or during

experimental infection studies (Fig.15) [182]. Therefore, it is important to identify the

adaptation mechanisms by which SARS-CoV-2 adjusts to different animals hosts in order

to avoid potential epizootic threats [183], especially combinations of multiple high-affinity

mutations in the S that could lead to viral adaptation to new susceptible animals species

not previously infected with SARS-CoV-2 [151].

Mink specific variants

Since April 2020, outbreaks of SARS-CoV-2 in Dutch mink farms were reported, with

viruses circulating in minks harboring several novel non-synonymous mutations in the S,

such as N501T, G261D, Y453F and F486L [184, 185]. Later, several variants were also

detected in minks in Denmark in autumn 2020, especially the Cluster 5/ΔFVI-spike,

which contained a specific set of non-synonymous mutations in the S, such as Δ69-70,

Y453F, I692V and M1229I, with Y453F being of higher biologic significance [186].

Preliminary results demonstrated that Cluster 5 was associated with low Ct values,

similar replication to WT Danish viruses, and weakened neutralizing activity against

convalescent plasmas with low and intermediary neutralizing titers. Yet, plasmas with

high neutralizing titers were not affected [186]. Other study confirmed that Cluster 5 did

not affect neutralizing antibody response in convalescent plasma, as well in a vaccine

mouse model, but it had an enhanced 4-fold binding affinity to human ACE2 compared

with WT, suggesting that the rise in frequency of this variant in minks might be correlated

with fitness advantage by receptor adaptation rather than evading immune responses

[187].

N501T, Y453F and F486L mutations appeared multiple times in phylogenetic distant

mink lineages from Danish and Dutch mink farms, and rarely in human genomes,

suggesting a mink-adaptation [188].

51


Y453F may be a mink-specific adaptation that increase binding affinity to mink

ACE2 [151, 186, 189]. It is associated with Casirivimab (REGN10933) mAb

resistance [41, 190], and emerged in a chronically infected immunosuppressed

patient after treatment with the Rituximab mAb [141]. Y453 binds to the ACE2

aa34 histidine (34H) in humans and tyrosine (34Y) in minks [186].

N501T emerged in experimentally infected ferrets [191] and in naturally infected

minks [184, 185, 192], suggesting that it may be a mustelid-specific adaptation

that increase binding affinity to these animals ACE2 [188, 191, 192]. It is also

known to bind with high affinity to human ACE2 [145].

Fig. 15- Combination of SARS-CoV-2 spike mutations occurring in humans and animals that may be involved in interspecies transmission. Variations in the RBD and NTD, near the furin cleavage site

and grouped with D614G are of higher concern, being highlighted in the figure. NTD: Amino-terminal

domain. RBD: Receptor binding domain (Adapted from Garry et al. 2021).

52


6. One Health approach Health is defined by WHO as a state of complete physical, mental, and social well-

being and not merely the absence of disease or infirmity [193]. Therefore, the major goal

of the One Health initiative is to reach an optimum worldwide health safety by

understanding and monitoring health risks at the human-animal-environment interface

(Fig.16), expanding interdisciplinary collaborations between researchers from different

scientific and clinical fields [54, 194, 195]. This approach is crucial to reduce the risk of

emerging and reemerging zoonoses, as more epidemiological information about hazard

levels and exposure routes is obtained in the three ecological domains [196].

However, One Health studies come with a lot of challenges, such as the need to

break barriers between different specialists and to include relevant information in the

three domains [195, 196]. This is important because a multi-disciplinary professional

team, as well as community members with on the ground experience with the issue in

question need to be considered. Importantly, community members living near potential

exposure sites can enhance the research team’s ability to collect and understand data

context. For the planning phase, it is necessary to determine the proper study design,

Fig. 16 - Venn diagram illustrating the tree core domains of the One-Health initiative. A. Epidemiological studies

considering factors between animal and human health, known as “one medicine”; B. Epidemiological studies considering

factors between environmental and human health; C. Epidemiological studies considering factors between animal and

environmental health (Adapted from Davis et al. 2017).

53


informing the data that need to be collected and the analysis methods [196]. If no data

is provided, for example, in the environmental domain, a complete picture of disease

transmission can be lost, leading to weaker control interventions due to unexpected

pathogen dynamics [195]. The inclusion of epidemiologic surveys in One Health studies

provide a better understanding of disease distribution within populations and the

associated risk factors driving this allocations, as diseases often emerge out of human,

animal and environmental interactions [195].

A One Health research requires a well-accepted and globally structured framework

considering at least one element from human, animal, and environmental health, as well

their interactions and the precise questions that need to be answered [196]. For example,

the Checklist for One Health Epidemiological Reporting of Evidence (COHERE) provides

a set of guidelines that must be incorporated in One Health studies through

epidemiological approaches [195]. According to COHERE, it is crucial to identify the

human factors that contribute to the pathogen spillover to animals or vice-versa. Then,

exposure to outdoors and geographical sampling should be defined in the environmental

factors, such as locations where the pathogen prevalence is high. Finally, the appropriate

animals should be aimed in the particular study, together with risk factor hypothesis

associated with the infection [195].

6.1. One Health studies Food Consumption

There is evidence that SARS-CoV-2 can survive from up to 14-21 days in meat, fish,

and animal skin, at both refrigerated (4ºC) and freezing (-20 to -80ºC) temperatures, and

persist in plastic surfaces, without losing infectivity [197]. As a result, meat or animal

products, as well food packaging, can be a possible source of SARS-CoV-2

contamination and transmission [198].

In China, incidents of food contamination by SARS-CoV-2 were reported: viral RNA

was detected in frozen shrimp packages from Ecuador and in frozen chicken imported

from Brazil [197].

Mink Farms

Farmed minks (Neovison vison) are kept in small cages in deteriorating conditions,

contributing to an efficient spread of pathogens between them, either by infectious

droplets, or by feed, bedding, or dust fecal matter. Big outbreaks occurred when these

54


animals were infected by humans, being highly susceptible to SARS-CoV-2 infection

[199, 200]. As the animals are greatly concentrated together, SARS-CoV-2 has more

chances to adapt and gain a more virulent form, as the virus is not penalized if it worsens

the health of the first host animal. It can easily transmit to the next mink [200].

Higher concerns were observed in the Netherlands between March and July of 2020,

the first country to report infection outbreaks of SARS-CoV-2 in mink farms through

genetic tracing. Minks developed severe clinical signs, with high viral loads and interstitial

pneumonia, followed by death. Researchers discovered strains with an animal sequence

signature in farm employees, evidencing animal to human transmission of SARS-CoV-

2, as well as a higher SARS-CoV-2 evolutionary rate within minks [184, 185]. According

to the World Organization for Animal Health (WOAH), mink farm infections were also

reported in Denmark, France, Greece, Italy, Spain, Sweden and USA [198]. The

Denmark government approved the culling of about 17 million minks in order to mitigate

the spread of more dangerous strains, as more than 200 farms were affected and a high

percentage of people were infected with SARS-CoV-2 mink associated strains [186, 199,

201]. Later, the Netherlands also approved the culling of minks in June 2020 to avoid

future outbreaks and a law for banning mink farms as of 2024 [200].

Soon after, in Utah USA, escaped minks were found to be positive for SARS-CoV-2

RNA, making these animals potential spillover agents to susceptible animal species

[202]. Feral cats wandering mink farms were found to be infected by SARS-CoV-2 [184,

200].

Wild Animals

Significant human infections combined with susceptible wildlife hosts at the wildlife-

human interface can lead to potential reverse zoonosis of SARS-CoV-2 into wildlife

populations [203].

Between January and March 2021, antibodies against SARS-CoV-2 were detected

in white-tailed deer in 152 samples (40%) using a surrogate virus neutralization test

[203]. SARS-CoV-2 RNA was also reported in some wild animals in zoos, due to closer

contacts with humans. In March 2020, three lions (Panthera leo) and four tigers

(Panthera tigris) developed respiratory signs, with infected animals shedding virus in

respiratory secretions and feces. The tigers and lions were infected with different

genotypes of SARS-CoV-2, indicating two independent transmission events [204].

Additionally, in January 2021, captive gorillas also exhibited mild clinical signs, such as

coughing and congestion. SARS-CoV-2 RNA was found in their fecal samples [205].

55


Domestic Animals

Many pets live in close contact with humans within households, being expected that

viral spillover of SARS-CoV-2 from humans to pets occur in significant frequencies.

Sporadic cases of SARS-CoV-2 infection in pets were detected worldwide in households

with positive owners, and some developed clinical signs.

For example, two dogs and six cats in Hong Kong were positive for SARS-CoV-2

RNA, with genome sequencing reveling high similarities between pets and owners [206,

207]; a positive SARS-CoV-2 RT-PCR cat in Belgium showed severe clinical sings,

including pronounced lethargy, diarrhea, and poor appetite in the initial days, to harsh

cough, paroxysmal reverse sneezing, breathing complications, and emaciation [208]; a

case of an asymptomatic cat in Spain infected by SARS-CoV-2 [209]; and two cats in

New York developing clinical signs and neutralizing antibodies against SARS-CoV-2

[210]. Contrary to cats and dogs, two rabbits and one guinea pig from COVID-19+

households tested negative for SARS-CoV-2 infection [209].

Widescale one-health seroepidemiological surveys targeting pets are of extreme

importance to monitor cases of SARS-CoV-2 spillover. Except from one study, the results

suggest that pets are susceptible to SARS-CoV-2 infection [211-226] (Table 3).

56


Table 3. Widescale seroepidemiologic surveys to understand the SARS-CoV-2 transmission routes and prevalence of infection in pets. * Luciferase Immunoprecipitation System; ** Indirect

Immunofluorescence Test; ***Microsphere immunoassay; **** Microneutralization test; ***** Automated Western Blot assay

Country (Date) Species (n) Diagnostic Observations Conclusions Reference

China (Jan-Mar 2020)

Cats (102) ELISA

VNT

15/102 (14.7%) cats of animal shelters and hospitals positive for antibodies against RBD

11/102 (10.8%) with neutralizing titers from 1:20 to 1:1080 (highest in cats from COVID-19 positive households)

No serological cross-reactivity detected between SARS-CoV-2 and FCoV

High neutralization titers in cats from COVID-19 positive

households

Unlikely that SARS-CoV-2 cross-reacts with FCoV using

the RBD protein

[220]

China

(Jan-Sep 2021) Dogs (946)

ELISA

PRNT

16/946 (1.75%) dogs positive for antibodies against RBD, all belonging to the 36 sera collected during the pandemic

10/946 (1.09%) with neutralizing titers from 1:20 to 1:180 (highest in dogs from COVID-19 positive households)

No serological cross-reactivity detected between SARS-CoV-2 and CCoV

High neutralization titers in dogs from COVID-19 positive

households

Unlikely that SARS-CoV-2 cross-reacts with CCoV using

the RBD protein

[226]

Spain (Jan-Oct 2020)

Cats (114) ELISA 4/114 (3.5%) stray cats positive for antibodies against RBD

3/4 (75%) of the positive samples seropositive for other pathogens: 1 for T. gondii and FIV, 1 for T.gondii and 1 for FIV

Immunosuppressed cats might be especially susceptible

to SARS-CoV-2 infection [212]

Spain (Jan-Nov 2020)

Ferrets (127) ELISA 2/127 (1.57%) household ferrets positive for antibodies against RBD

Consistent persistence of anti-RBD antibodies in 1 of the positive ferrets beyond 129 days after the first sampling.

Ferrets are susceptible to SARS-CoV-2 spillover from

humans [217]

France

(Feb 2020- Feb 2021)

Dogs (809) ELISA

AWB*****

From 449 pre-pandemic sera, 4 (0.9%) were positive for antibodies against N

From 453 pandemic sera, 25 (4.8%) were positive for antibodies against N

At least 8 ELISA-seroconversions were observed among the 218 dogs during the pandemic

AWB detected 17 (68%) of the 25 ELISA-positive pandemic sera, as well as all doubtful pandemic sera (n = 3)

Possible cross reactivity with other canine CoVs in pre-

pandemic sera using the N protein

Dogs are susceptible to SARS-CoV-2 infections

[216]

France (March 2020)

Cats (9)

Dogs (12) LIPS*

No antibodies detected against the S1 domain and the C-terminal N part of SARS-CoV-2 in 9 cats and 12 dogs living in

close contact with a community of students 1 month after the index case was reported (2 tested positive for SARS-CoV-2

and 11/18 had symptomatology related to COVID-19)

Low rates of SARS-CoV-2 spillover from humans to

pets, even in situations of proximity [221]

Italy (Mar-May 2020)

Cats (191)

Dogs (451) PRNT

15/451 (3.3%) dogs and 11/191 (5.8%) cats positive for neutralizing antibodies

Positive correlation between the recorded load of human COVID-19 infection density and the seropositivity among animals

Dogs more likely to test positive for SARS-CoV-2 antibodies if they came from a COVID-19+ household

Males and adult dogs more susceptible to infection

Companion animals living in areas of high human

infection are exposed to SARS-CoV-2

The link between SARS-CoV-2 household infection and

a pet’s seropositivity was only apparent for dogs

[222]

57


Netherlands (Apr-May 2020)

Cats (544)

Dogs (501)

ELISA

VNT

10/44 (22.7%) stray cats exposed to positive mink farms, 1 dog from a COVID-19+ household, and 2/500 (0.4%) cats and

1/500 (0.2%) dogs with unknown SARS-CoV-2 exposure positive for antibodies against the S1 and RBD

4/500 (0.8%) cat samples were defined as suspected (positive for S1 and RBD by ELISAs but failed to neutralize SARS-

CoV-2 S–bearing VSV pseudovirus infection)

8/9 SARS-CoV-2-free cats infected with FCoV with cross-reactivity with SARS-CoV-2 N protein.

The general prevalence rate was low, indicating that

cats and dogs are probably incidental hosts due to

occasional SARS-CoV-2 spillover from humans

N protein lacks discriminatory power

[211]

USA (Apr-Jun 2020)

Cats (239)

Dogs (510)

ELISA

VNT

19/239 (7.9%) cats and 5/510 (1.0%) dogs positive for antibodies against the N protein

From the N-seropositive cats, 15/19 (78.9%) exhibited neutralizing activity and 7/19 (36.8%) were also RBD-seropositive

From the N-seropositive dogs, none exhibited neutralizing activity or were RBD-seropositive

Cats are more susceptible to natural SARS-CoV-2

infection than dogs

RRD-based IgG ELISAs less sensitive

[218]

Germany (Apr-Sep 2020)

Cats (920)

ELISA

ilFT**

VNT

6/920 (0,69%) domestic cats tested positive for antibodies against the RBD, with 2/6 (33.3%) having neutralizing antibodies

No observed cross-reactivity with FCoV-specific antibodies

Antibodies against SARS-CoV-2 found in cats even

during a period of low incidence of human infection

Unlikely that SARS-CoV-2 cross-reacts with FCoV using

the RBD protein

[223]

Thailand (Apr-Dec 2020)

Cats (1113)

Dogs (2102)

ELISA

sVNT

4/1112 (0.36%) cats and 35/2102 (1.66%) dogs positive for SARS-CoV-2 antibodies against the N protein

All ELISA-positive and suspected samples were negative for neutralizing antibodies

Pets from COVID-19 affected areas with unknown

SARS-CoV-2 exposure develop specific IgG antibodies

N-targeting antibody results do not correlate with

neutralizing antibodies results

[214]

France (May-Jun 2020)

Cats (34)

Dogs (13)

Retrovirus

Based

Pseudopart

icle Assay

MIA***

Antibody prevalence against S1, S2 and N proteins ranged from 21.3% (10/47) (including 23.5% (8/34) of cats and 15.4%

(2/13) of dogs) to 53% in COVID-19+ households (considering that anti-N antibody assays underestimate results in human

seroprevalence studies by 30-45% compared to anti-S antibody assays)

The risk of infection is 8 times higher if pets came from COVID-19+ households

Sex, age, vaccination statue and interaction with other pets within the household were not associated with higher

prevalence of SARS-CoV-2 infection

Infection risk of pets belonging to COVID-19 positive

households is higher

Pet infections mainly occur by spillover from positive

owners rather than from other animals

[225]

Brazil (Jun-Aug 2020)

Cats (49)

Dogs (47) PRNT

2/96 animals, including 49 cats (40 owned and 9 stray) and 47 dogs (42 owned and 5 stray), were positive for SARS-CoV-2

neutralizing antibodies, with PRNT90 titers of 80 and 40 from serum samples of a stray cat and a stray dog, respectively

Stray cats and dogs are exposed to SARS-CoV-2,

possibly by contaminated environments [219]

USA

(Jun-Sep 2020) Cats (17)

Dogs (59)

(qRT-PCR)

VNT

Of 17 cats, live SARS-CoV-2 was recovered from respiratory swab from one (5.9%), 16.7% tested positive by PCR from one

or more swab type, and 41.2% had neutralizing antibodies.

Of 59 dogs, 1.7% tested positive by PCR from one or more swab sample type and 11.9% had neutralizing antibodies.

Across 15 antibody-positive animals, titers increased (33.3%), decreased (33.3%) or were stable (33.3%) over time.

Over 25% of homes with infected humans, pet dogs or

cats had confirmed SARS-CoV-2 infections

The titers of neutralizing antibodies in pets seem to

fluctuate along time

[224]

58


Croatia (Jul-Dec 2020)

Dogs (1147) ELISA

MNT****

In COVID-19 infected households (n=78), 43.9% of dogs positive for antibodies against the RBD, with neutralization

antibodies detected in 25.64%, and 4.69% of dogs in the general population (n=1069) were also positive

The odds of testing ELISA positive were 4 times higher for dogs in infected households than dogs in the general population

Sex (male), breed (Belgian shepherd) and age (1 to 5 years) were identified as significant risk factors

ELISA positive dogs had a 1.97 times greater risk for developing CNS clinical signs

Infection risk of dogs belonging to COVID-19 positive

households is higher

Male, Belgian shepherd and adult dogs are more

susceptible to SARS-CoV-2 infection and at a higher risk

of developing CNS clinical signs

[215]

Netherlands (Aug 2020 – Feb 2021)

Cats (240) ELISA

VNT

5/240 (2.1%) shelter cats were positive for antibodies against the S1 and RBD

2/5 (40.00%) of the positive ELISA samples were also positive for VNT

Shelter cats are low susceptible to SARS-CoV-2

infection [213]

59


6.2. Natural and experimental infections in animals

Severe illness due to SARS-CoV-2 infection has not been well established in

companion animals. A survey in the USA (March to January 2021) found out that a total

of 10/94 (11%) confirmed SARS-CoV-2 positive pets died during or after diagnosis [227].

From these 10 pets, one dog had clinical signs (increased respiratory rate and effort and

dyspnea), significant comorbidities, presence of SARS-CoV-2 in critical tissues and

associated histopathologic changes, suggesting that acute SARS-CoV-2 infection

contributed to this animal’s death. However, the primary cause was chronic interstitial

lung disease. Additionally, one cat also had clinical signs (lethargy, loss of appetite,

respiratory distress), sub-clinical co-morbidities (mild hypertrophic cardiomyopathy),

evidence of SARS-CoV-2 in critical tissues and histopathological changes attributed to

SARS-CoV-2 in critical tissues, suggesting that infection with SARS-CoV-2 was the

primary reason for the animal’s euthanasia. In the remaining eight animals, SARS-CoV-

2 infection was found to be incidental [227].

Moreover, it was reported an increased incidence of myocarditis in pets with no

previous history of heart disease correlated with the emergence of the alfa variant in the

UK in mid-December 2020. Many owners of these pets were PCR positive for SARS-

CoV-2 and had developed COVID-19 3 to 6 weeks before their pets became ill. A total

of 3/11 tested animals (two cats and one dog) were infected by the alfa variant by ddPCR,

with 6/11 being SARS-CoV-2 positive either by ddPCR or by serology. Pets showed

acute onset of lethargy, inappetence, tachypnea/dyspnea and syncopal events, with

cardiac abnormalities characterized by congestive heart failure and ventricular

arrhythmia [228].

Due to SARS-CoV-2 viral-host range and the need to understand which species are

susceptible to infection, as well the development of animal’s models for SARS-CoV-2

infection, several experimental inoculations were performed in domestic and wild animal

species:

Cats

Cats inoculated with SARS-CoV-2 were asymptomatic, with infectious virus detected

in the URT. Viral RNA was found in the nasal turbinate, trachea, lungs, and small

intestine [229-231], with lower levels in tonsils, lymph nodes, olfactory bulbs, spleen,

heart, liver, kidney and bone marrow [230]. Histopathological features included multifocal

lymphocytic and neutrophilic tracheobronchoadenitis of seromucous glands in the lamina

60


propria and submucosa of the trachea and bronchi [230]. Younger cats developed

enormous lesions in nasal and trachea epithelium and lungs [229]. SARS-CoV-2 was

efficiently transmitted through respiratory droplets between inoculated and contact cats

[229-232], with both presenting viral shedding prolonged to multiple days [231] and

measurable high neutralizing antibody titers by 7-13 dpi [229-232].

Dogs

Dogs inoculated with SARS-CoV-2 were asymptomatic, with viral RNA detected in

rectal swabs at 2 to 6 days post infection (dpi). However, viral RNA was not found in any

organ or tissue [229]. Dogs seroconverted with detectable neutralizing antibodies at 14

dpi, peaking at 21 dpi with 1:40 – 1:80 titers [231], but contact dogs were seronegative

for SARS-CoV-2 at 14 dpi [229].

Ferrets

Ferrets inoculated with SARS-CoV-2 had mild symptoms, such as occasional cough,

reduced activity, fever (38.1-40.3 ºC) and reduced appetite [229, 233]. Histopathological

features included inflammatory cell infiltration (mainly neutrophils) in the alveolar septa

and lumen, severe lymphoplasmacytic perivasculitis and vasculitis and mild

peribronchitis in the lungs [229, 233-235]. Viral RNA was detected in higher quantities in

the nasal turbinate, soft palates, tonsils, and trachea, but also in lungs, intestine, kidney,

brain, muscle, skin, lymph nodes and colon [229, 233-235]. Additionally, higher viral

shedding was reported in throat swabs and nasal washes, with mild-lower levels in

saliva, rectal swabs, serum, urine, and fecal samples after 2 dpi [191, 229, 233-235].

However, infectious virus was retrieved only from the URT and nasal washes [229, 233,

234], with viral clearing achieved after 10-14 dpi [233, 234].

SARS-CoV-2 is efficiently transmitted from inoculated to naïve ferrets either by direct

contact or airborne transmission [191, 233, 235]. Viral shedding is similar in both

inoculated, direct, and indirect contact ferrets in terms of duration and SARS-CoV-2 RNA

levels [191]. The gain of non-synonymous mutations in orf1a and S was noted after the

ferret viral passage [191, 235]. High neutralizing antibody titers were detected in booth

ferrets [229, 233, 235].

Mice

Laboratory strains of mice inoculated with SARS-CoV-2 were asymptomatic and

presented very low levels of viral RNA in lungs [236, 237]. Thus, as small animal models

that reproduce the clinical course and pathology observed in diseased patients are highly

61


needed, several strategies were elaborated to use mice as animal models to test for

vaccine or antiviral drug efficacy [236].

Exogenous delivery of human ACE2 with a replication-deficient adenovirus (Ad5-hACE2) in BALB/c mice – Mice exhibited hunching, ruffled fur and

breading difficulties 2 dpi and weigh loss of up to 20% of their body weight in 4-6

dpi [238], with high viral titers detected in lungs. Lower RNA levels were identified

in heart, spleen, and brain [237, 238]. Histopathological lesions included increase

in immune cell infiltration (mainly neutrophils) in perivascular and alveolar

locations, alveolar edema, and necrosis cell debris. Increased vascular

congestion and hemorrhage were observed 5 dpi [238]. Accelerated viral

clearance and inhibition of weight loss and lung tissue histological changes were

noticed after convalescent plasma and remdesivir treatment [237, 238].

Transgenic mice expressing hACE2 receptors – Weight loss was evident in

aged mice [236, 239], together with elevated cytokine production and pulmonary

pathology [236]. Viral RNA was detected in lungs, trachea, brain, heart, and eye

from both young and aged transgenic mice, and in feces of aged mice, with

infectious virus only retrieved from lungs, the major site of infection. Interstitial

pneumonia characterized by immune cell infiltration (macrophages and

lymphocytes) into the alveolar interstitium, distinctive vascular system injury and

alveolar septal thickening were observed [236, 239, 240].

MASCp6 strain – A new mouse strain was obtained by serial passaging of a

SARS-CoV-2 clinical isolate in the respiratory tract of aged BALB/c mice. In the

sixth passage, the mouse-adapted strain (MASCp6) was obtained. This strain

was prone to a higher virulence of SARS-CoV-2 due to a series of adaptive

mutations, such as N501Y, demonstrated by the increased lung infectivity.

Additionally, inoculated aged mice showed mild to moderate interstitial

pneumonia at 3 dpi characterized by thickened alveolar septa, focal hemorrhage

and exudation, alveolar damage and collapsed epithelia cells, together with high

inflammatory responses [146].

Hamsters

Golden Syrian hamsters (Mesocricetus auratus) and Chinese hamsters (Cricetulus

griseus) are highly susceptible to SARS-CoV-2 infection and can serve as a SARS-CoV-

2 infection models [234, 241-245].

Golden Syrian hamsters (M. auratus) - Experimentally inoculated golden

Syrian hamsters developed progressive weight loss, rapid breathing, ruffled furs,

62


lethargy and hunched back posture [234, 241-244], recovering 7 dpi [244].

Histopathological findings included mononuclear inflammatory cell infiltration,

pulmonary edema, alveolar hemorrhage, and lung consolidation [234, 241-244].

Viral RNA was detected in the lungs, trachea, liver, spleen, kidney, large

intestine, olfactory bulb and medulla oblongata [234], with higher viral titers found

in the nasal turbinate’s, trachea, and lungs [243], as well is nasal washed until 14

dpi [241]. Furthermore, hamster ACE2 shows high binding affinity with SARS-

CoV-2 RBD [242, 244] and infected hamsters efficiently transmitted SARS-CoV-

2 to close contact naïve hamsters via aerosols [241, 244]. Primary SARS-CoV-2

infection in golden Syrian hamsters provides protective immunity against

subsequent reinfection [243], with mean neutralizing titers of ≥1:427 developed

14 dpi [244].

Chinese hamsters (C. griseus) – Similar disease outcomes and

histopathological features with M. auratus, but with milder and prolonged

pneumonia and reduced body weight, including prominent acute alveolar

damage until 14 dpi with persistence of viral RNA. C. griseus considerable

smaller size, well-characterized genome, transcriptome and translatome and

availability of molecular tools makes it a more suitable animal model for SARS-

CoV-2 infection studies [245]

Primates

Cynomolgus macaques (Macaca fascicularis) are known to be the closest model to

humans in terms of pathophysiology, and rhesus macaques (Macaca mulatta) have also

been utilized in COVID-19 studies [246].

Cynomolgus macaques (M. fascicularis) - Experimentally infected animals

were asymptomatic or experienced mild symptoms, such as nasal discharge,

fever and reduced body weight, together with pulmonary consolidation, alveolar

and bronchiolar walls thickened by edema fluid, neutrophils and mononuclear

cells, hyaline membrane formation, diffuse hemorrhage, necrosis in the lung and

type II pneumocyte hyperplasia [247, 248]. Viral replication was primarily

restricted to the respiratory tract (bronchi, nasal cavity, lung lobes and trachea),

with the highest levels of viral RNA in lungs. Nasal swabs of aged animals had

higher levels of viral RNA compared with young animals, being detected between

8 to 21 dpi. All animals seroconverted by 14 dpi [246-248], reaching neutralizing

antibody titers of 1:256 at 21 dpi [248].

63


Rhesus macaques (Macaca mulatta) - Experimentally inoculated animals were

asymptomatic or experienced mild and transient clinical signs, such as fever,

weight loss, occasional coughing, hunched posture, pale appearance, reduced

appetite, asthenia, increased respiration and dehydration [248-252], with

histopathological studies showing mild-to-moderate interstitial pneumonia,

inflammatory infiltration (monocytes, lymphocytes, and a few eosinophils),

alveolar septum thickening, epithelia and macrophage degeneration, edema,

pulmonary hemorrhage, alveolar wall necrosis, hyaline membrane formation and

minimal type-II pneumocyte hyperplasia [248-252]. Increased inflammatory

cytokine expression was detected [248], and older macaques experienced a

more severe interstitial pneumonia compared with young animals, characterized

by an extremely thickened alveolar septum and significant infiltration of

inflammatory cells in the alveolar interstitium [249]. Viral RNA was found in the

nose, pharynx, crissum, lungs, trachea, and bronchia, as well in lymphoid tissues,

stomach, spleen, bladder, rectum, and uterus [248, 249, 251]. Viral shedding was

higher from the nose and throat [248, 251], and viral RNA was also detected in

rectal swabs and fecal samples, indicating potential infection in the digestive tract

[248, 250, 251]. Neutralizing antibody titers were high, starting at 10 dpi [251] and

reaching 1:1350 at 14 dpi and 1:4050 at 21 dpi [250], with reinfected macaques

developing immunity against SARS-CoV-2 [252].

Common marmosets (Callithrix jacchus) - Less susceptible to SARS-CoV-2

infection compared with M. mulatta and M. fascicularis, with 3/6 animals being

asymptomatic and 3/6 experiencing a slightly increase in body temperature. Viral

RNA peaks were detected 2 dpi in nasal swabs (lower levels compared with M.

mulatta and M. fascicularis), and the animals evidenced neutralizing activity

against SARS-CoV-2 [248].

Racoon dogs (Nyctereutes procyonoides)

A total of 6/9 racoon dogs were successfully inoculated with SARS-CoV-2, with 2/3

contact animals being further infected. Viral shedding was detected in nasal and

oropharyngeal swabs at 2 dpi. However, no evident clinical signs were noticed, except

for a mild rhinitis associated with the presence of viral antigen in the nasal mucosa. High

throughput sequencing of SARS-CoV-2 re-isolated from nasal swabs showed absence

of viral adaptation. All animals seroconverted [253].

64


Fruit bats (Rousettus aegyptiacus)

A total of 7/9 fruit bats were successfully inoculated with SARS-CoV-2, with 1/3

contact animals being further infected. The animals were asymptomatic, but experienced

oral and fecal shedding. Bats expressed viral RNA in nasal epithelium, trachea, and

lungs, with lower copies in ear, skin, duodenum, and adrenal gland tissues. However,

infectious virus was only retrieved from the nasal epithelium and trachea. No gross

pathological lesions were detected, being associated only with minimal to mild rhinitis,

with epithelial necrosis, infiltrating neutrophils and lymphocytes, edema, and intraluminal

cellular debris. All animals developed SARS-CoV-2 reacting antibodies with variable

neutralizing titers between 1:32 and 1:64 from 8 to 21 dpi [235].

Treeshrew (Tupaia belangeris)

Treeshrews inoculated with SARS-CoV-2 were asymptomatic, except for a slight

increase in body temperature. Viral shedding was low from all age groups, with young

animals experiencing it at earlier stages of infection and older animals with prolonged

shedding. Lungs were the main site of viral replication, followed by digestive tissues and

heart, with the highest viral load detected in pancreas from one adult treeshrew at 14

dpi. Mild histopathological abnormalities were detected in lung tissue, such as hyperemia

of interstitium, airway obstruction, widened pulmonary septum, consolidation of lung

margin, infiltration of inflammatory cells and local hemorrhagic necrosis [254].

White-tailed deer (Odocoileus virginianus)

Intranasal inoculation of deer fawns with SARS-CoV-2 resulted in subclinical viral

infection, with high viral RNA loads detected in nasal turbinate’s, palatine tonsils, and

medial retropharyngeal lymph nodes, and infectious virus shedding in nasal secretions

and feces. Infected animals efficiently transmsitted the virus to contact deer. Microscopic

changes observed 21 dpi included congestion, lymphohistiocytic interstitial pneumonia,

hyaline membranes, and intra-alveolar fibrin. Further, SARS-CoV-2 neutralizing

antibodies were detected by 7 dpi and were consistently observed through 21 dpi [255].

Cattle, poultry, and swine A total of 2/6 cattle inoculated with SARS-CoV-2 had detectable viral loads in nasal

swabs only until 3 dpi, with only one exhibiting detectable antibody titers. However, no

further transmission of SARS-CoV-2 to contact cattle was detected [256]. In addition,

pigs, and several poultry species, such as chickens, ducks, turkeys, geese, and quails

had no detectable SARS-CoV-2 replication, clinical signs, or seroconversion after

65


inoculation [229, 235, 257, 258]. No further transmission of SARS-CoV-2 to contact pigs

and chickens was noted [235, 258].

7. Conclusion This review provides evidence that animals are susceptible to SARS-CoV-2 infection,

either in natural or experimental conditions. Higher concerns were observed in mink

farms, where specific SARS-CoV-2 mink variants originated, such as the Cluster 5/ΔFVI-

spike. Additionally, more than a few SARS-CoV-2 S mutations emerged in animals owing

to several interspecies transmission events of the virus. As SARS-CoV-2 is constantly

mutating to adapt to new environments, viral evolution can be associated to spillover

events to other animal species.

Therefore, more one health studies and monitoring plans are needed to control and

evaluate the impact of SARS-CoV-2 in animal populations. As cats and dogs are in closer

contact with humans, a seroepidemiological survey of SARS-CoV-2 in Portuguese pets

was performed.

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Chapter II – A seroepidemiological survey of

Severe Acute Respiratory Syndrome

Coronavirus 2 (SARS-CoV-2) in Portuguese

pets.

1. Introduction SARS-CoV-2 is a novel betacoronavirus disseminated all over the world, including in

Portugal, being highly pathogenic in humans and causing major economic impacts

worldwide. Although the most frequent transmission route of the virus is between

humans, pets also demonstrated to be susceptible to SARS-CoV-2 infection in natural

conditions [211-216, 218-226]. Consequently, several questions arise about the

vulnerability of pets to SARS-CoV-2 infection, the possible health impacts, and if pets

could become a reservoir of infection with the risk of reverse zoonosis.

Hence, a seroepidemiological survey was conducted in Portugal from December

2020 to May 2021 in cats and dogs, either in SARS-CoV-2-positive households or in

geographic areas severely affected by COVID-19, to increase the knowledge about the

susceptibility of Portuguese pets to infection.

2. Materials and Methods

2.1. Study design A seroepidemiological study was performed to address the susceptibility of cats

(Felis catus) and dogs (Canis lupus familiaris), to SARS-CoV-2 infection.

In this study the three one-health boundaries (Fig.17) were addressed to evaluate

SARS-CoV-2 transmission to pets : 1) from humans to animals by spillover of SARS-

CoV-2 from ‘pet-parents’/humans to pets; 2) from humans to environment by propagation

of SARS-CoV-2 viral particles from infected human’s trough air; and 3) from environment

to pets’ by exposure to outdoors, i.e., contaminated environmental surfaces or air.

Full signalment and clinical history from each animal were collected through

epidemiological enquiries, that included the following information:

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a) detailed animal information, such as sex, age, weight, breed, geographic region,

and clinical signs (asymptomatic, fever, respiratory, digestive, neurological,

others).

b) pet exposure to SARS-CoV-2: pets in close contact with infected humans with a

positive PCR result in the previous two weeks - COVID-19 positive household;

pets in COVID-19 negative household; pets in COVID-19 suspected household;

and pets’ exposure to outdoors in regions with high prevalence of COVID-19

cases.

2.2. Animal sampling and processing A total of 15 veterinarian clinicals, hospitals and animal shelters across different

regions of Portugal (Northern, Center and South regions) have participated in this study.

Informed consents were filled and signed by owner and/or veterinarians to collect animal

data and blood samples.

Whenever possible, when seropositive or borderline results were observed, an

additional sample was collected to address the duration of antibody responses in pets.

Environmental Health

Contamined environments

COVID-19 cases per 100 000 inhabitants in Portugal

Animal Health

Susceptibility to SARS-CoV-2 infection

Signalment, seropositivity and risk factors

Human Health

Principal trasmission route of SARS-CoV-2

Positivity, duration and sevirity of SARS-CoV-2

infection

SARS CoV-2

Spillover within

households

Fig. 17 - Venn diagram illustrating the three domains of the present one health study and their connectivity.

68


All blood specimens were collected in dry biochemical tubes or 2mL microtubes

without anticoagulant. For serum separation, the tubes were centrifugated at 500 RCF

for 10 min and the supernatant was transferred to a clean 2 mL microtube. Sera were

stored at -80ºC until processing

Animal samples were collected between December 2020 and May 2021 and included

the third COVID-19 wave that occurred in January and February 2021. In Portugal the

third wave had one of the highest numbers of COVID-19 cases/millions of people, with

a peak surpassing the 1200 daily cases per million people and 28,61 daily confirmed

deaths per million people on February 1st (Fig.18) [259]. Restricted control measures,

with quarantine and emergency state were in place during this period.

2.3. Animal data A total of 225 blood specimens were collected from 217 pets in 15 animal institutions,

including 13 veterinary hospitals/clinics and two animal shelters, comprising a total of

148 dog samples and 69 cat samples collected between 4th December 2020 and 8th May

2021 (except for four dog samples, collected in June 2020). Additional samples were

collected from five dogs and three cats, totalizing 72 cat samples and 153 dog samples

(Fig.19).

All cats (69/69) included in the survey lived in households.

The dog population surveyed comprised 122 of the 148 total dogs from households

(82.43%), and 26 of the 148 dogs (17.57%) were shelter dogs. 20 dogs from Vila Nova

de Famalicão animal shelter lived in close contact in parks, while the six dogs from Vila

do Conde animal shelter lived in individual cages. Five dog samples and one cat sample

were homolysed, and did not pass the quality control, being excluded from the study.

Fig. 18 – Daily new confirmed COVID-19 cases and deaths per million people during the third wave in Portugal (Source :

Our World in Data).

69


2.4. Survey areas The survey was performed in five different districts of Portugal: Braga, Porto, Aveiro,

Coimbra and Lisbon (Fig.20a).

The sampling was composed by 110 dogs and 56 cats from Braga district; 16 dogs

and nine cats from Porto district; two dogs from Aveiro district; seven dogs from Coimbra

district and 13 dogs and four cats from Lisbon district.

The sample distribution by cities was the following: Braga - cats: n=1 ; dogs: n=1,

Celorico de Basto - cats: n=0; dogs: n=1, Fafe – cats: n=0 ; dogs: n=1, Felgueiras - cats:

n=1 ; dogs: n= 0, Gondomar - cats: n=0 ; dogs: n=1, Guimarães - cats: n=27 ; dogs:

n=44, Penafiel - cats: n=0 ; dogs: n=1, Porto - cats: n=3 ; dogs: n=6, Santo Tirso - cats:

n=4 ; dogs: n=3, Trofa - cats: n=1 ; dogs: n=0, Vila do Conde - cats: n=0 ; dogs: n=5,

Vila Nova de Famalicão - cats: n=28 ; dogs: n=67, Vizela - cats: n=2 ; dogs: n=6, Aveiro-

cats: n=0 ; dogs: n=2, Coimbra - cats: n=0 ; dogs: n=7, and Lisboa cats: n=4 ; dogs:

n=13.

Most pet samples were collected at the Braga district (n=166) (Fig. 20b), especially

in the regions of Vila Nova de Famalicão (n=83) and Guimarães (n=71), followed by

Porto district (n=26) (Fig. 20c)

Fig. 19 - Total blood samples collected from cats and dogs.

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2.5. ELISA Detection of IgG antibodies against SARS-CoV-2 RBD was performed by two ELISA,

one adapted for each species, and both developed in Equigerminal SA, Coimbra

(Fig.21).

The ELISA plates were coated with a recombinant RBD protein, diluted in phosphate-

buffered saline with Tween® detergent (PBST) and blocked using bovine serum albumin

(BSA). All the sera, including positive and negative controls, were screened at a dilution

of 1:100 using a goat anti-cat or anti-dog IgG horseradish peroxidase (HRP) as a

secondary antibody. The reactions ended with the addition of the HRP-substrate

3,3´,5,5´ Tetramethylbenzidine (TMB), followed by 0.2 H2SO4 stop solution, resulting in

switching from a blue color to yellow measurable signs. All the steps were separated by

Faro

Beja

Évora

Portalegre

Castelo Branco

Guarda

Viseu

Vila Real Bragança

Porto

Coimbra

Braga

56 110

9 16

2

7

4 13

Espo

send

e

Barcelos

Guimarães

Vizela

Terras de Bouro

Braga

Fafe

Cabeceiras de Basto

Vieira do Minho

Total = 166 samples

Total = 25 samples

1 1

26 57 27 44

2 6

1

1

Vila do Conde Trofa

Maia

Porto

Vila Nova de

Gaia

Lousada Amarante

Baião

4 3

3 6

5

1

1

1 1

Fig. 20 – Geographic distribution of dog and cat samples; collected across a. Portugal; and in the regions of b. Braga (Braga, Vila

Nova de Famalicão, Guimarães, Vizela, Fafe and Celorico de Basto; and c. Porto (Porto, Vila do Conde, Trofa, Santo Tirso,

Gondomar, Felgueiras and Penafiel).

Number of samples from cats

Number of samples from dogs

Total = 217 samples

a b

c

71


washes in a Wellwash® Microplate Washer. The optical densities (ODs) were measured

at 450 nm. The cutoff values were determined at 3 and 5 times the OD medium value of

reactivity of seronegative samples from a pre-COVID-19 cohort. The test was considered

valid if the quotient between the positive and negative control was above 5.

2.6. Statistics Data obtained from the epidemiologic surveys and results from the serology analyses

were registered in a Microsoft Excel spreadsheet, followed by the calculation of

seroprevalences. Graph Pad Prism 8.0.1 was used for graphic construction and

statistical analyses. Comparison between groups was performed using Fisher’s exact

and Chi-squared tests. Significance was accepted when p<0.05.

Cat IgG

Anti-Cat IgG HRP

RBD protein

Solid Phase

Dog IgG

Anti-Dog IgG HRP

RBD Protein

Solid Phase

Fig. 21- Anti-SARS-CoV-2 RBD ELISA for cats and dogs. The ELISAs were adapted to cats and dogs, by the usage

of anti-cat/dog IgG labeled with Horseradish peroxidase targeting the Fc portion of the antibodies in the serum samples

(adapted from the Mediagnostic ELISA kit illustration (https://mediagnost.de/en/anti-sars-cov-2-elisa/)).

72


3. Results

3.1. Cats signalment A total of sixty-nine cats were analysed, including 33 males (47.83%) and 36 females

(52.17%). The cat groups are summarized on Fig.22 and were distributed accordingly to

sex, age, body weight, breed, clinical signs, COVID-19 household state and outdoors

access.

Distribution by age

The age groups were divided into kittens (<1 year), young adults (1-3 years), mature

adults (4-7 years) and seniors (≥8 years). A total of 7/69 (10.14%) were kittens, 23/69

(33.33%) were young adults, 11/69 (15.94%) were mature adults and 24/69 (34.78%)

were seniors, with 4/69 (5.80%) of cats with unknown age. The mediam age of the sixty-

five (65) cats with know age was 5.88 years (range 0.25-17 years).

Distribution by body weight

A total of 35/69 (50.72%) cats weight less than 4 kg, and 23/69 (33.33%) weight 4 or

more kg, with 11/69 (15.94%) of cats with unknown weight. The mediam weight of the

fifty-eight (58) cats with know weight was 3.83 kg (range 2.4-7.1 kg).

Distribution by breed

Regarding the breed, a total of 11/69 (15.94%) cats were of undetermined breed,

with 48/69 (69.56%) being of pure breed. Of these 44/49 (63.77%) were European

Shortairs, 1/69 (1.45%) were Persian, 1/69 (1.45%) were Ragdoll, 1/69 (1.45%) were

Siamese and 1/69 (1.45%) were Sphynx. The breed was not recorded in 9/69 (13.04%)

of the cats.

Distribution by clinical signs

Of the sixty-nine (69) surveyed cats, twenty-six (26) (37,68%) were symptomatic, and

forty-three (43) (62.31%) were assymptomatic. Of the twenty-six (26) syntomatic cats,

nine (9) (34.61%) showed respiratory signs, nine (9) (34.61%) presented digestive signs,

three (3) (11.54%) had neurologic signs, three (3) (11.54%) showed fever, one (1)

(3.85%) was seropositve to Feline Immunodeficiency Virus (FIV) and 10 (38.46%)

reported other medical conditions.

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Distribution by COVID-19 positive and negative households

Fifteen (15) of the sixty-nine (69) (21.74%) surveyed cats come from COVID-19

positive households, and had contact with owners with a positive PCR result in the

previous two weeks before sampling. One of the sixty-nine (69) (1.45%) were suspected

to have contact- COVID-19 suspected household. Forthy (40) (57.97%) had no contact

with COVID-19 infected humans - COVID-19 negative household. In twelve (12) of the

sixty-nine (69) cats (17.39%) we has no information regarding contact with COVID-19

infected persons.

Distribution by outdoors access

A total of 28/69 cats (40.58%) had contact with outdoors, with 30/69 (43.48%) having

no contacts with outdoors. No information was available for 11/69 (43.48%) cats

regarding contact with outdoors.

74


Fig. 22- Groups formed for cats according to the data collected from the epidemiologic enquiries.

75


3.2. Dogs signalment A total of one-hundred-and-forty-eight (148) dogs were analysed, including

seventeen (70) males (47.30%) and seventeen-eight (78) females (52.70%).

The dogs groups are summarized on Fig.23 and were distributed accordingly to sex,

age, body weight, breed, clinical signs, COVID-19 household state and outdoors access.

Distribution by age

The age groups were divided into puppies (<1 year), young adults (1-3 years), mature

adults (4-7 years) and seniors (≥8 years). A total of 2/148 (1.35%) were puppies, 45/148

(30.40%) were young adults, 48/148 (32.43%) were mature adults and 48/148 (32.43%)

were seniors, with 5/148 (3.38%) of dogs with unknown age. The mediam age of the

one-hundred-and-forty-three (143) dogs with know age was 6.16 years (range 0.42-18

years).

Distribution by body weight

The body weight groups were divided in intervals of 10kg. A total of 53/148 (35.81%)

of dogs weight less than 10 kg, 39/148 (26.35%) weight between 10 to 20 kg, 17/148

(11.49%) weigh between 20 to 30 kg, and 15/148 (10.13%) weigh more than 30 kg, with

24/143 (16.22%) of dogs with unknown weight. The mediam weight of the one-hundred-

and-forty-four (124) dogs with know weight is 15.60 kg (range 2.00-64.90 kg).

Distribution by breed

Regarding the breed, a total of 63/148 (42.57%) dogs were of undetermined breed,

with 64/148 (43.24%) being of pure breed: 2/64 (63.77%) are Beagles, 1/64 (1.56%) are

Bichon Maltese, 3/64 (4.69%) are Boxers, 3/64 (4.69%) are Caniche, 2/64 (63.77%) are

Chihuahua, 3/64 (4.69%) are Cocker Spaniels, 1/64 (1.56%) are Dalmatian, 1/64

(1.56%) are Deutcshe Dogue, 2/64 (63.77%) are English Bulldogs, 2/64 (63.77%) are

French Bulldogs, 5/64 (7.81%) are German Sherperds, 4/64 (6.25%) are Golden

Retrievers, 1/64 (1.56%) are Huskys, 1/64 (1.56%) are Jack Russels, 10/64 (15.62%)

are Labrador Retrievers, 5/64 (7.81%) are Pinschers, 1/64 (1.56%) are Pitbulls, 4/64

(6.25%) are Podengos, 1/64 (1.56%) are Rafeiro Alentejano, 1/64 (1.56%) are

Rotweillers, 1/64 (1.56%) are Schnauzers, 1/64 (1.56%) are West Highland White

Terriers and 9/64 (14.06%) are Yorkshires Terriers. The breed was not recorded in

21/148 (14.19%) of dogs.

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Distribution by clinical signs

A total of 26/148 (17.57%) of dogs were symptomatic, including 3/26 (11.54%) with

respiratory signs, 14/26 (53.85%) with digestive signs, 3/26 (11.54%) with neurologic

signs, 1/26 (3.85%) with fever, and 9/26 (34.61%) with other medical conditions. On the

contrary, a total of 122/143 (85.31%) dogs were assymptomatic.

Distribution by COVID-19 positive or negative households.

A total of 23/148 (15.54%) dogs had contact with owners with a positive PCR result

in the previous two weeks before sampling - COVID-19 positive household, with 61/148

(41.89%) having no contact - COVID-19 negative household. Of the with one-hundred-

and-forty-eight (148) surveyed dogs, only thee (3) (2.03%) were suspected to have

contact with COVID-19 infected persons. A total of 60/148 (40.54%) has no information

regarding contact with positive households, including the twenty-six (26) shelters dogs.

Distribution by outdoors access

A total of 111/143 (75.00%) dogs had contact with outdoors, with 15/143 (10.49%)

having no contacts with outdoors. No information was available for 22/143 (14.86%) dogs

regarding contact with outdoors.

77


Fig. 23- Groups formed for dogs according to the data collected from the epidemiologic enquiries.

78


3.3. SARS-CoV-2 seroprevalence in pets Antibodies against the RBD of SARS-CoV-2 were detected in the serum of 22/217

pet samples, indicating a 10,14% seroprevalence in the pet population surveyed.

A total of 191/217 (88.02%) pet samples tested seronegative for SARS-CoV-2 RBD,

and 4/217 (18.43%) had doubtful results.

3.4. SARS-CoV-2 seroprevalence in cats Antibodies against the RBD of SARS-CoV-2 were detected in the serum of 15/69

(21.74%) cat samples. A total of 55/69 (79.71%) cat samples tested seronegative for

SARS-CoV-2 RBD, and 1/69 (1.45%) had doubtful results.

From these positive cat cases, 6/15 (40.00%) are males and 9/15 (60.00%) are

females, with a total of 0/0 (0.00%) aged <1, 6/15 (40.00%) aged 1-3, 5/15 (33.33%)

aged 4-7 and 4/15 (26.67%) aged +8 years.

A total of 9/15 (60.00%) seropositive cats lived in COVID-19 positive households or

had contact with COVID-19 cases in the vicinity. Seven seropositive cats (46.67%)

cohabited with COVID-19 positive owners and two seropositive cats (13.33%) were

frequently outdoors in close contact with other cats from neighbours that tested positive

for COVID-19. Data suggests that animal exposure to COVID-19 households can explain

most of the SARS-CoV-2 transmission to cats.

From these cats, 7/15 (46.67%) had contact with outdoors. Data suggests that

access to outdoors could play a risk in SARS-CoV-2 infection of cats.

3.5. SARS-CoV-2 morbidity and mortality in cats A total of 9/15 (60.00%) cats exhibited clinical signs, with the majority experiencing

more than one of the following clinical signs: 4/9 (44.44%) showed respiratory signs, 2/9

(22.22%) had neurologic signs, 2/9 (22.22%) showed apathy and appetite reduction, 3/9

(33.33%) had digestive signs and 1/9 (11.11%) had fever.

Moreover, 10/15 (66.67%) seropositive cats recovered from the clinical signs, while

5/15 (33.33%) were euthanized or succumbed to death.

Information about the seropositive cats is presented on Table 4.

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Table 4. Data from SARS-CoV-2 seropositive cats. *Contact with neighbors with a positive PCR result and cats belonging to COVID-19+ households. ** Other viral infection. *** Same household

Sample ID

Date of collection Sex Age Breed Weight

(kg)

Contact with positive

PCR case

Contact with

outdoors Clinical Signs Date of reported

clinical signs Recovery Location Risk (cases per 100000 inhabitants)

3 15th December 2020 M 3 European

Shorthair 5.0 Yes* Yes Apathy Reduced appetite

End of January 2021 No (Death) Guimarães Very High

(751)

41 15th December 2020 F 10 European

Shorthair 3.0 Yes No

Apathy Reduced appetite

Pyometra FIV**

1st December 2020 No (Death) Vizela High

(343)

63 15th March 2021 M 4 European Shorthair 4.6 Yes Yes - - Yes Vila Nova de

Famalicão Moderate

(94)

65 18th March 2021 F 4 European Shorthair 3.0 Yes No Respiratory 18th March 2021 Yes Vila Nova de

Famalicão Moderate

(58)

66 17th March 2021 F 10 Ragdoll 5.3 Yes* Yes Neurologic Respiratory 17th January 2021 Yes Vila Nova de

Famalicão Moderate

(58)

69 26th February 2021 F 2 European

Shorthair 4.5 No Yes Digestive

Seborrhea Ulcers

18th February 2021 Yes Guimarães Moderate

(49)

71 26th February 2021 F 2 European

Shorthair 3.8 No Yes Digestive 25th February 2021

No (Euthanized) Guimarães Moderate

(49)

130 11th March 2021 F 5 European Shorthair 5.0 No Yes

Fever Respiratory Digestive

5th March 2021 Yes Vila Nova de Famalicão

Moderate (94)

192*** 15th March 2021 F 16 Undetermined 4.0 No No - - No (Death) Guimarães Moderate (42)

200 19th March 20201 F 2 European

Shorthair 3.4 Yes No - - Yes Vila Nova de Famalicão

Moderate (58)

213 18th March 2021 F 1 European Shorthair 3.3 Yes No - - Yes Braga Moderate

(25)

219*** 27th March 2021 M 4 Undetermined 2.9 No No Neurologic 27th March 2021 No (Death) Guimarães Moderate (20)

221 29th March 2021 M 2 Undetermined 3.5 No Yes - - Yes Guimarães Moderate (20)

232 8th April 2021 M 7 Persian 3.7 Yes No - - Yes Guimarães Moderate (55)

516 19th April 2021 M 15 - - Yes - Respiratory - Yes Lisbon Moderate (96)

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3.6. Risk assessment and clinical outcome of

SARS-CoV-2 seropositive cats Seropositive cases were followed to ascertain the risk assessment and evaluate the

impact of SARS-CoV-2 in cat health.

Herein we summarize the data obtained from SARS-CoV-2 seropositive cats.

Case 3 – A three-year-old European short-haired male cat from Vila Nova de

Famalicão. The cat lived indoors and outdoors and in contact with other neighbors and

nearby cats. It was usually involved in fights with other cats in the vicinity. The cat did

not belong to a COVID-19 positive household, but interestingly, the neighbors of the cat's

owners who were also cat owners were RT-PCR positive for SARS-CoV-2. The cat

showed clinical signs in late January (shortly after sample collection), with decreased

appetite and apathy, followed by death. We suspect that this cat was infected by human-

to-cat or cat-to-cat transmission. However, the owner related that the cat did not like

cuddles from humans, being the second hypothesis (cat-to-cat) more reliable.

Case 41 - A ten-year-old European shorthaired cat from Vizela had close contact

with the owner who was COVID-19 positive, with a positive RT-PCR result for SARS-

CoV-2 for two months (October 15 to December 15) . The cat was also positive for feline

immunodeficiency virus (FIV), exhibiting visible apathy and decreased appetite in early

December. The clinical status later evolved to pyometra. The cat subsequently died on

December 23, 2020. We suspect that FIV coinfection played an important role in the

susceptibility to SARS-CoV-2 infection and clinical outcome.

Case 63 – A four-year old European Shorthair male cat from Vila Nova de Famalicão

had contact with owners with a positive RT-PCR result for SARS-CoV-2 and had contact

with outdoors. However, the cat was asymptomatic and stayed healthy.

Case 65 – A four-year old European Shorthair female cat from Vila Nova de

Famalicão had contact with owners with a positive RT-PCR result for SARS-CoV-2. After

18th March 2021, the cat began sneezing, but ended up recovered. The cat was not in

contact with outdoors, suggesting that infection occurred by human-to-cat spillover of

SARS-CoV-2 within the household.

Case 66 – A ten-year old Ragdoll female cat from Vila Nova de Famalicão had no

contact with positive owners within households but was in contact with neighbors who

had a positive RT-PCR result for SARS-CoV-2. The cat was constantly outdoors. The

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cat developed neurological clinical signs and sneezing since 17th January 2020, two

months before the blood sample collection, but ended up recovering.

Case 69 - A two-year old European Shorthair female cat from Guimarães had no

contact with owners with a positive RT-PCR result for SARS-CoV-2. However, the cat

had contact with other visitors with unknown SARS-CoV-2 status, as well with outdoors.

The cat had seborrhea, ulcers, and digestive signs since 18th February 2021, one week

before the blood sample collection, but ended up recovering.

Case 71 – A two-year old European Shorthair female cat from Guimarães had no

contact with owners with a positive RT-PCR result for SARS-CoV-2 but had contact with

outdoors. The cat came from a household with several cats, with all going outside as

well. The cat exhibited digestive signs since 25th February 2021, one day before

sampling, being euthanized in the same month. The cat was probably infected indoors

by an asymptomatic cat, suggestive of cat-to-cat transmission, or outdoors.

Case 130 – A five-year old European Shorthair female cat from Vila Nova de

Famalicão had no contact with owners with a positive RT-PCR result for SARS-CoV-2,

neither with outdoors, but was occasionally in a veterinary clinic. The cat developed

fever, respiratory and digestive signs since 5th March 2020, but ended up recovering. On

11th June, two months after the first sampling, a second sampling was collected from the

cat, but the results were negative, suggesting that antibody levels decayed after the first

infection.

Case 192 – A sixteen-year-old female cat of indeterminate breed from Guimarães

had no contact with owners with a positive RT-PCR result for SARS-CoV-2, neither with

outdoors, being asymptomatic. However, the cat died in March 2021. It is unknown how

the cat has been infected by SARS-CoV-2, but the cat had contact with another cat in

the same household - case 219- a deadly seropositive case of SARS-CoV-2 . We cannot

discard that this cat (case 192) has died due to advanced age or due to fulminant SARS-

CoV-2 infection.

Case 200 – A two-year old European Shorthair female cat from Vila Nova de

Famalicão had contact with owners with a positive RT-PCR result for SARS-CoV-2 and

with outdoors. However, the cat was asymptomatic.

Case 213 – A one-year old European Shorthair female cat from Braga had contact

with owners with a positive RT-PCR and serologic result for SARS-CoV-2 in September

2020. However, the cat was asymptomatic and had no contact with outdoors. The cat

was probably infected indoors by the owners.

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Case 219 – A four-year old male cat of indeterminate breed from Guimarães had no

contact with owners with a positive RT-PCR result, neither with outdoors. The cat

developed neurological signs since 27th March 2021, followed by death in the same

month. The cat had contact with another cat in the same household (case 192), who was

also positive for SARS-CoV-2 infection and died but was asymptomatic.

Case 221 - A two-year old male cat of indeterminate breed from Guimarães had no

contact with owners with a positive RT-PCR result but had contact with outdoors.

Case 232 – A seven-year-old Persian male cat from Guimarães had contact with

owners with a positive RT-PCR result in October 2020 who developed COVID-19, but

not with outdoors. However, the cat was asymptomatic. The cat was probably infected

indoors by the owners.

Case 516 – A fifteen-year-old male cat from Lisbon had contact with owners with a

positive RT-PCR result. The cat developed respiratory signs but ended up recovering

after clinical treatment.

3.7. SARS-CoV-2 seroprevalence in dogs Antibodies against the RBD of SARS-CoV-2 S protein were detected in the serum of

7/148 (4.73%) dog samples. A total of 137/148 (92.57%) dog samples tested

seronegative for SARS-CoV-2, and 4/148 (2.70%) had doubtful results.

From these positive dog cases, 3/7 (40.00%) are males and 4/7 (60.00%) are

females, with a total of 0/0 (0.00%) aged <1, 1/7 (14.29%) aged 1-3, 2/7 (28.57%) aged

4-7 and 3/7 (42.86%) aged +8 years (1/7 (14.29%) of unknown age.

A total of 4/7 (57.14%) had contact with COVID-19 positive households or secondary

contacts, with only 1/7 (14.29%) exhibiting digestive clinical signs.

From these dogs, the majority, 6/7 (85.71%), had contact with outdoors (1/7 (14.29%)

of unknown exposure).

Information about the seropositive dogs is presented on Table 5.

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Table 5. Data from SARS-CoV-2 seropositive dogs.

Sample ID

Date of collection Sex Age Breed Weigh

t (kg)

Contact with

positive PCR case

Contact with

outdoors

Clinical Signs

Date of reported clinical signs Recovery Location

Risk (nº cases per 100000 inhabitants)

1 5th June 2020 M 8 - - No Yes - - - Lisbon Very High (501)

190 14th March 2021 F 10 Labrador Retriever 34 No Yes Digestive 14th March 2021 - Guimarães Moderate (109)

218 26th March 2021 F 12 Undetermined 9.5 Yes Yes - - Yes Guimarães Moderate (20)

220 26th March 2021 M 7 Undetermined 7.0 Yes Yes - - Yes Guimarães Moderate (20)

89S 4th February 2021 F - - - Yes Yes - - - Coimbra Extremely High

(1579)

90S 14th February 2021 F 4 - - Yes Yes - - - Coimbra Very High (841)

501 9th February 2021 M 1 - - - - - - - Lisbon Extremely High

(2123)

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3.8. Risk assessment and clinical outcome of

SARS-CoV-2 seropositive dogs After ELISA analyses, each positive case was studied again in order to ascertain the

possible cause of infection in each dog:

Case 1 – A eight-year-old male dog from Lisbon had no contact with owners with a

positive RT-PCR result but had contact with outdoors. The dog was asymptomatic. The

first sample retrieved on 5th June 2020 was positive for ELISA. However, a second

sampling collected on 25th March 2021, nine months after the first sampling, had negative

results, suggesting that antibody levels decayed after the first infection.

Case 190 – A ten-year-old Labrador Retriever female dog from Guimarães had no

contact with owners with a positive RT-PCR result but had contact with outdoors. The

dog was symptomatic, exhibiting digestive signs since 14th March 2021. It is unknown if

the dog recovered from the clinical signs. The dog was probably infected outdoors.

Case 218 – A twelve-year old female dog of undetermined breed from Guimarães

had contact with owners with a positive RT-PCR result, as well with outdoors. The dog

was asymptomatic. The dog was probably infected indoors by human-to-dog

transmission or outdoors.

Case 220 – A seven-year-old male dog of undetermined breed from Guimarães had

contact with a child in the household with a positive RT-PCR result, as well with outdoors.

The dog was asymptomatic. The dog was probably infected indoors by SARS-CoV-2

spillover from the owner or by contact with contaminated environments.

Case 89S – A female dog from Coimbra was asymptomatic and had contact with

owners with a positive RT-PCR result . The first sample, retrieved on 4th February 2021,

was positive for ELISA. However, a second sampling collected on 2nd March 2021,

approximately one month after the first sampling, had negative results, suggesting that

antibody levels decayed after the first infection.

Case 90S – A four-year old female dog from Coimbra was asymptomatic and had

contact with owners with a positive RT-PCR result. The first sample, retrieved on 4th

February 2021, was negative for ELISA. However, a second sampling collected on 14th

February 2021, ten days after the first sample, had positive results. Data suggest that

the dog was recently infected by SARS-CoV-2 in the moment of the first sampling, with

antibodies against SARS-CoV-2 beginning to form in the 10-day time interval before the

second sampling.

85


Case 501 – A one-year old male dog from Lisbon was asymptomatic. It is unknown

if the dog had contact with positive owners or with outdoors.

3.9. Risk factors and susceptibility to SARS-CoV-2

infection All pets were susceptible to SARS-CoV-2 infection, with cats being more susceptible

than dogs (p=0.0004), as 15/69 (21.75%) of cats were infected by SARS-CoV-2, with

only 7/148 (4.73%) of dogs being infected. The resumed seropositivity among cats and

dogs are represented in Table 6, with the associated percentages and p values for each

risk factor, including household, sex, age and outdoors.

Table 6. Risk factors contributing to seropositivity among cats and dogs. The data is divided into risk factors,

including household contact, sex, age, and outdoor contact.

According to data from the statistical analysis, cats (p=0.006) and dogs (p=0.020)

were significantly more likely to test positive for SARS-CoV-2 antibodies if they came

from a COVID-19 positive household. No correlation with seropositivity was found

regarding sex, age or contact with outdoors for both species

Risk Factor Cats Dogs

Nº+ (Total) % p Nº+

(Total) % p

Household 0.006 0.020 COVID19+ 9 (15) 60.00% 4 (23) 17.39% COVID19- 6 (40) 15.00% 3 (62) 4.83% Suspected 0 (1) 0.00% 0 (3) 0.00% Unknown 0 (12) 0.00% 1 (60) 1.67%

Sex 0.567 >0.999 Male 6 (33) 18.18% 3 (70) 42.86%

Female 9 (36) 25.00% 4 (78) 51.28% Age 0.816 0.125 <1 0 (7) 0.00% 0 (2) 0.00% 1-3 6 (23) 26.09% 1 (45) 2.22% 4-7 5 (11) 36.36% 2 (48) 4.17% 8+ 4 (24) 16.67% 3 (48) 6.25%

Unknown 0 (4) 0.00% 1 (5) 20.00% Outdoors 0.448 0.586

Yes 7 (30) 23.33% 6 (108) 5.56 % No 7 (28) 25.00% 0 (15) 0.00 %

Unknown 1 (11) 9.09% 1 (25) 4.00 %

86


4. Discussion

4.1. Wide-scale testing of pets Cats and dogs are known to be infected by α-CoVs (FCoV and CCoV) and β-CoVs

(CRCoV). As SARS-CoV-2 is a β-CoV, and cats and dogs demonstrated to be

susceptible to natural and experimental infection, pet contamination by infected owners

or contaminated environments may be more common than expected, due to the

numerous opportunities for spillover. Henceforth, wide-scale testing of susceptible pets,

as well of other animal species, is highly needed in the COVID-19 pandemics.

One health is important in the COVID-19 pandemics, as pets can possibly be infected

either by : 1) spillover from ‘pet-parents’/humans; 2) transmission from other pets, or 3)

exposure to contaminated environments.

4.2. SARS-CoV-2 seroprevalence in portuguese

pets In this wide-scale seroepidemiologic study, a total of 22/217 (10.14%) pets were

seropositive for SARS-CoV-2, including 15/69 (21.74%) cats and 7/148 (4.73%) dogs.

The seropositivity among animals was higher than the seropositivity rates recorded

in humans in Portugal between May and June 2020 - 2.9% [260], demonstrating that

pets are being infected with SARS-CoV-2 at considerable rates. However, the survey in

humans was accomplished in the first months of the pandemic, where the incidence of

infection in humans was considerably lower than the incidence during this study

(December 2020 to May 2021), coincident with the third COVID-19 wave [259].

Cats were significantly more susceptible to SARS-CoV-2 infection compared to dogs

(p=0.0004). These results are according to experimental studies, evidencing cats as

more predisposed to infection [229-232]. Other wide-scale studies evidenced the same

hypothesis. In Wuhan, 15/102 (14.70%) cats and 1.69% (16/946) dogs were seropositive

to SARS-CoV-2 [220, 226]. In Italy, 11/191 (5.8%) cats and 15/451 (3.3%) dogs were

seropositive to SARS-CoV-2 [222]. In Texas, 41.2% of 17 cats and 11.9% of 59 dogs

had detectable neutralizing antibodies against SARS-CoV-2 [224].

87


4.3. Risk factors contributing to pets’ infection In this study, no correlation with seropositivity was found regarding sex, age or

contact with outdoors for both species.

None of the seven kittens and two puppies (aged <1 year) were seropositive for

SARS-CoV-2, suggesting that adult animals are being more prone to infection. Patterson

et al. also demonstrated that none of the nine kittens and 20 puppies (aged <1 year)

were infected by SARS-CoV-2 [222], revealing that younger pets may be less likely to

be infected.

Both cats and dogs were more likely to be infected by SARS-CoV-2 if they came from

positive COVID-19 households (p=0.006 and p=0.020, respectively), as 9/15 (60.00%)

of the infected cats and 4/7 (57.14%) of the infected dogs had contact with infected

humans, corresponding to more than a half, 13/22 (59.09%), of the positive pet samples.

Fritz et al. reported a much greater seroprevalence in animals from COVID-19+

households (21% to 53%, depending on the positivity criteria chosen), and infection was

not associated with the number of pets in the households, suggesting mainly spillover of

SARS-CoV-2 from the owners [225]. Additionally, the highest neutralization titers were

observed in cats and dogs from COVID-19 positive households in Wuhan surveys [220,

226], revealing that pets are at a higher risk in conditions of close proximity with infected

owners.

All dogs belonging to animal shelters, 0/25 (0.00%), were seronegative to SARS-

CoV-2 infection. 20 of these dogs lived in close contact in parks, and six were kept in

separated cages. Hence, it is unlikely that dogs from shelters are prone to SARS-CoV-2

spillover, being more common within COVID-19 positive households

From the seropositive cats, 1/15 (6.67%) was infected with FIV, and had associated

clinical signs. Villanueva-Saz et al. reported that 3/4 (75.00%) cats seropositive for

SARS-CoV-2 were coinfected with Toxoplasma gondii, FIV or both [212]. Hence, it is

hypothesized that FIV coinfection played an important role in the susceptibility to SARS-

CoV-2 infection and clinical outcome.

4.4. SARS-CoV-2 morbidity and mortality in pets It is extremely difficult to identify if clinical signs in animals are linked to SARS-CoV-

2 infection. A report in the United Kingdom in March 2021 demonstrated that pets

infected with the SARS-CoV-2 VOC-α were suffering from myocarditis [228]. However,

88


a longitudinal study of dogs and cats living with at least one SARS-CoV-2 infected human

in Texas reported that the majority (82.4%) of SARS-CoV-2 infected cats and dogs were

asymptomatic [224], revealing variable results.

In this study, a higher percentage of seropositive cats were symptomatic, including

9/15 (60.00%) of cats, with the majority experienced more than one clinical sign. From

these 15 cats, five (33.33%) were euthanized or succumbed to death. On the contrary,

from the seven positive dogs, only one (14.29%) dog was symptomatic. Hence, cats may

be more disposed to pathologies associated with SARS-CoV-2 infection, but more

studies are needed to confirm this hypothesis, as clinical signs may be associated with

other diseases or infections.

4.5. Antibody longevity Results from the second sampling in pets suggest that antibody longevity against

SARS-CoV-2 in cats and dogs may not be very lasting, as one cat was seronegative

after 3 months of the first sampling, and dogs were seronegative after 1 month.

Curiously, one dog was negative for SARS-CoV-2 antibodies in the first sampling,

but after 10 days, in the second sampling, antibodies against SARS-CoV-2 were

detectable. This suggest that the animal was recently infected by SARS-CoV-2 in the

moment of the first sampling, with antibodies against SARS-CoV-2 beginning to form in

the 10-day time interval before the second sampling.

In experimental studies, cats had detectable neutralizing antibodies against SARS-

CoV-2 at 7-12 dpi [229, 231], and dogs seroconverted at 14 dpi, with a peak at 21 dpi

[231]. This is similar to humans, where PCR confirmed individuals seroconverted at 13-

21 dpi [29]. Therefore, for animal serologic testing, it is recommended to test pets 14

days after contact with owners positive for SARS-CoV-2 RT-PCR.

Hamer et al. reported that the titers of neutralizing antibodies in pets seem to

fluctuate along time. Across 15 antibody-positive animals, titers increased (33.3%),

decreased (33.3%) or were stable (33.3%) over time.

Curiously, consistent persistence of SARS-CoV-2 antibodies was reported in 1 pet

ferret beyond 129 days after the first sampling [217]. More studies are needed to

understand the persistence of SARS-CoV-2 antibodies in cats and dogs.

89


5. Conclusion It is particularly difficult to determine animals’ involvement in zoonotic diseases

exposure, with a lot of data of potential viral spillover lacking in many zoonotic viruses.

The best way to elucidate animal exposure is trough wide scale seroepidemiological

studies to understand important activities at high-risk interfaces that contribute to

animals’ infection in areas severely affected by the pandemic.

This study had some challenges, such as breaking down barriers between different

fields, and the fact that some veterinarian clinicals did not want to alarm their clients

about the hypothesis of their pets being infected by SARS-CoV-2, preventing

abandonment of animals during the COVID-19 pandemics. Limitations included the

biased sampling, as it was not randomized, with veterinarians preferring to sample

animals who were easier to handle and those undergoing surgical operations. However,

sampling was made by several clinics from different regions of Portugal, demonstrating

a wider geographic infection in pets.

In this study, 22/217 (10.14%) pets were seropositive for SARS-CoV-2, including

15/69 (21.74%) cats and 7/148 (4.73%) dogs. Cats are at a higher risk of infection

compared with dogs, and exposure to positive COVID-19 households is a significant risk

factor. To the best of knowledge, this is the first wide-scale seroepidemiological study

report of animals being infected by SARS-CoV-2 under natural conditions in Portugal.

90


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SARS-CoV-2: Susceptibility of cats and dogs to infection

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