Histopathologic and molecular characterization of Erethizon dorsatum papillomavirus 1 and Erethizon dorsatum papillomavirus 2 infection in North American porcupines (Erethizon dorsatum).

Erethizon dorsatum papillomavirus 1 (EdPV1) and Erethizon dorsatum papillomavirus 2 (EdPV2) are associated with cutaneous papillomas in North American porcupines (Erethizon dorsatum). This study defined gross, histopathologic, and molecular characteristics of viral papillomas in 10 North American porcupines submitted to the New York State Animal Health Diagnostic Center. Investigation for the presence of EdPV1 and EdPV2 DNA via polymerase chain reaction (PCR) was performed in 9 of the 10 (90.0%) porcupines, and all porcupines were investigated for the detection and localization of EdPV1 and EdPV2 E6 and E7 nucleic acid via chromogenic in situ hybridization (CISH). Next-generation sequencing (NGS) was performed in 2 porcupines. Papillomas were diagnosed on the muzzle (n = 4), caudal dorsum (n = 1), upper lip (n = 1), chin (n = 1), gingiva (n = 2), and nasal planum (n = 1). Histologically, the lesions consisted of hyperplastic epidermis or epithelium with orthokeratotic keratin, prominent keratohyalin granules, and intranuclear inclusion bodies. PCR identified EdPV1 in 6 of 9 samples and EdPV2 in the remaining 3 samples. NGS resulted in 100% genome coverage of EdPV1 and 76.20% genome coverage of EdPV2 compared with GenBank reference sequences, with 99.8% sequence identity to the complete EdPV2 L1 gene of a novel subtype recently identified in France. Hybridization patterns in 9 of the 10 (90.0%) porcupines were characterized by strong nuclear signals in the superficial epidermis, with strong nuclear and punctate cytoplasmic signals in the stratum spinosum and basale. In one animal, CISH suggested dual EdPV1 and EdPV2 infection.

White-tailed deer (Odocoileus virginianus) may serve as a wildlife reservoir for nearly extinct SARS-CoV-2 variants of concern.

The spillover of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to white-tailed deer (WTD) and its ability to transmit from deer to deer raised concerns about the role of WTD in the epidemiology and ecology of the virus. Here, we present a comprehensive cross-sectional study assessing the prevalence, genetic diversity, and evolution of SARS-CoV-2 in WTD in the State of New York (NY). A total of 5,462 retropharyngeal lymph node samples collected from free-ranging hunter-harvested WTD during the hunting seasons of 2020 (Season 1, September to December 2020, n = 2,700) and 2021 (Season 2, September to December 2021, n = 2,762) were tested by SARS-CoV-2 real-time RT-PCR (rRT-PCR). SARS-CoV-2 RNA was detected in 17 samples (0.6%) from Season 1 and in 583 samples (21.1%) from Season 2. Hotspots of infection were identified in multiple confined geographic areas of NY. Sequence analysis of SARS-CoV-2 genomes from 164 samples demonstrated the presence of multiple SARS-CoV-2 lineages and the cocirculation of three major variants of concern (VOCs) (Alpha, Gamma, and Delta) in WTD. Our analysis suggests the occurrence of multiple spillover events (human to deer) of the Alpha and Delta lineages with subsequent deer-to-deer transmission and adaptation of the viruses. Detection of Alpha and Gamma variants in WTD long after their broad circulation in humans in NY suggests that WTD may serve as a wildlife reservoir for VOCs no longer circulating in humans. Thus, implementation of continuous surveillance programs to monitor SARS-CoV-2 dynamics in WTD is warranted, and measures to minimize virus transmission between humans and animals are urgently needed.

Infection and Transmission of SARS-CoV-2 B.1.617.2 Lineage (Delta Variant) among Fully Vaccinated Individuals.

The emergence of the SARS-CoV-2 B.1.617.2 lineage (Delta variant) in 2021 was associated with increased case numbers and test positivity rates, including a large number of infections in fully vaccinated individuals. Here, we describe the findings of an investigation conducted in Tompkins County, New York, to evaluate factors underlying a significant uptick in the number of coronavirus disease 2019 (COVID-19) cases observed in the months of July and August 2021. We performed genomic surveillance and genotyping as well as virological assessments to determine infectivity of the virus in a select number of clinical diagnostic samples. Genomic sequence analyses revealed complete replacement of the B.1.1.7 lineage (Alpha variant) with the B.1.617.2 lineage (Delta variant) between July 1 and August 4 2021. We observed a strong association between viral RNA loads detected by real-time reverse transcriptase PCR and infectious virus detected in respiratory secretions by virus titration. A marked increase in positive cases among fully vaccinated individuals was observed. The sequence divergence between two index Delta variant cases in April and May, and the cases after July 1st, revealed independent Delta variant introductions in Tompkins County. Contact tracing information enabled the detection of clusters of connected cases within closely related phylogenetic clusters. We also found evidence of transmission between vaccinated individuals and between vaccinated and unvaccinated individuals. This was confirmed by detection and isolation of infectious virus from a group of individuals within epidemiologically connected transmission clusters, confirming shedding of high viral loads and transmission of the virus by fully vaccinated individuals. IMPORTANCE The SARS-CoV-2 lineage B.1.617.2 (Delta variant) emerged in Asia and rapidly spread to other countries, becoming the dominant circulating lineage. Worldwide infections with B.1.617.2 peaked at a time in which vaccination rates were increasing. In this study, we present data characterizing the emergence of SARS-CoV-2 lineage B.1.617.2 (Delta variant) in Tompkins County, New York, which has one of the highest vaccination rates in the state. We present evidence demonstrating infection, replication, and transmission of SARS-CoV-2 lineage B.1.617.2 (Delta variant) between fully vaccinated individuals. Importantly, infectious virus loads were determined in a subset of samples and demonstrated shedding of high viral titers in respiratory secretions of vaccinated individuals.

The Omicron Variant BA.1.1 Presents a Lower Pathogenicity than B.1 D614G and Delta Variants in a Feline Model of SARS-CoV-2 Infection.

Omicron (B.1.1.529) is the most recent SARS-CoV-2 variant of concern, which emerged in late 2021 and rapidly achieved global predominance by early 2022. In this study, we compared the infection dynamics, tissue tropism, and pathogenesis and pathogenicity of SARS-CoV-2 D614G (B.1), Delta (B.1.617.2), and Omicron BA.1.1 (B.1.1.529) variants in a highly susceptible feline model of infection. Although D614G- and Delta-inoculated cats became lethargic and showed increased body temperatures between days 1 and 3 postinfection (pi), Omicron-inoculated cats remained subclinical and, similar to control animals, gained weight throughout the 14-day experimental period. Intranasal inoculation of cats with D614G- and the Delta variants resulted in high infectious virus shedding in nasal secretions (up to 6.3 log10 TCID50.Ml-1), whereas strikingly lower level of viruses shedding (<3.1 log10 TCID50.Ml-1) was observed in Omicron-inoculated animals. In addition, tissue distribution of the Omicron variant was markedly reduced in comparison to the D614G and Delta variants, as evidenced by lower in situ viral RNA detection, in situ viral immunofluorescence staining, and viral loads in tissues on days 3, 5, and 14 pi. Nasal turbinate, trachea, and lung were the main-but not the only-sites of replication for all three viral variants. However, only scarce virus staining and lower viral titers suggest lower levels of viral replication in tissues from Omicron-infected animals. Notably, while D614G- and Delta-inoculated cats presented pneumonia, histologic examination of the lungs from Omicron-infected cats revealed mild to modest inflammation. Together, these results demonstrate that the Omicron variant BA.1.1 is less pathogenic than D614G and Delta variants in a highly susceptible feline model. IMPORTANCE The SARS-CoV-2 Omicron (B.1.1.529) variant of concern emerged in South Africa late in 2021 and rapidly spread across the world causing a significant increase in the number of infections. Importantly, this variant was also associated with an increased risk of reinfections. However, the number of hospitalizations and deaths due to COVID-19 did not follow the same trends. These early observations suggested effective protection conferred by immunizations and/or overall lower virulence of the highly mutated variant virus. In this study we present novel evidence demonstrating that the Omicron BA.1.1 variant of concern presents a lower pathogenicity when compared to D614G- or Delta variants in cats. Clinical, virological, and pathological evaluations revealed lower disease severity, viral replication, and lung pathology in Omicron-infected cats when compared with D614G and Delta variant inoculated animals, confirming that Omicron BA.1.1 is less pathogenic in a highly susceptible feline model of infection.

The Omicron variant BA.1.1 presents a lower pathogenicity than B.1 D614G and Delta variants in a feline model of SARS-CoV-2 infection.

Omicron (B.1.1.529) is the most recent SARS-CoV-2 variant of concern (VOC), which emerged in late 2021 and rapidly achieved global predominance in early 2022. In this study, we compared the infection dynamics, tissue tropism and pathogenesis and pathogenicity of SARS-CoV-2 D614G (B.1), Delta (B.1.617.2) and Omicron BA.1.1 sublineage (B.1.1.529) variants in a highly susceptible feline model of infection. While D614G- and Delta-inoculated cats became lethargic, and showed increased body temperatures between days 1 and 3 post-infection (pi), Omicron-inoculated cats remained subclinical and, similar to control animals, gained weight throughout the 14-day experimental period. Intranasal inoculation of cats with D614G- and the Delta variants resulted in high infectious virus shedding in nasal secretions (up to 6.3 log10 TCID 50 .ml -1 ), whereas strikingly lower level of viruses shedding (<3.1 log10 TCID 50 .ml -1 ) was observed in Omicron-inoculated animals. In addition, tissue distribution of the Omicron variant was markedly reduced in comparison to the D614G and Delta variants, as evidenced by in situ viral RNA detection, in situ immunofluorescence, and quantification of viral loads in tissues on days 3, 5, and 14 pi. Nasal turbinate, trachea, and lung were the main - but not the only - sites of replication for all three viral variants. However, only scarce virus staining and lower viral titers suggest lower levels of viral replication in tissues from Omicron-infected animals. Notably, while D614G- and Delta-inoculated cats had severe pneumonia, histologic examination of the lungs from Omicron-infected cats revealed mild to modest inflammation. Together, these results demonstrate that the Omicron variant BA.1.1 is less pathogenic than D614G and Delta variants in a highly susceptible feline model. Author Summary: The SARS-CoV-2 Omicron (B.1.1.529) variant of concern (VOC) emerged in South Africa late in 2021 and rapidly spread across the world causing a significant increase in the number of infections. Importantly, this variant was also associated with an increased risk of reinfections. However, the number of hospitalizations and deaths due to COVID-19 did not follow the same trends. These early observations, suggested effective protection conferred by immunizations and/or overall lower virulence of the highly mutated variant virus. In this study we present novel evidence demonstrating that the Omicron BA.1.1 variant of concern (VOC) presents a lower pathogenicity when compared to D614G- or Delta variants in cats. Clinical, virological and pathological evaluations revealed lower disease severity, viral replication and lung pathology in Omicron-infected cats when compared to D614G and Delta variant inoculated animals, confirming that Omicron BA.1.1 is less pathogenic in a highly susceptible feline model of infection.

From Deer-to-Deer: SARS-CoV-2 is efficiently transmitted and presents broad tissue tropism and replication sites in white-tailed deer.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) in humans, has a broad host range, and is able to infect domestic and wild animal species. Notably, white-tailed deer (WTD, Odocoileus virginianus), the most widely distributed cervid species in the Americas, were shown to be highly susceptible to SARS-CoV-2 in challenge studies and reported natural infection/exposure rates approaching 30-40% in free-ranging WTD in the U.S. Thus, understanding the infection and transmission dynamics of SARS-CoV-2 in WTD is critical to prevent future zoonotic transmission to humans, at the human-WTD interface during hunting or venison farming, and for implementation of effective disease control measures. Here, we demonstrated that following intranasal inoculation with SARS-CoV-2 B.1 lineage, WTD fawns (~8-month-old) shed infectious virus up to day 5 post-inoculation (pi), with high viral loads shed in nasal and oral secretions. This resulted in efficient deer-to-deer transmission on day 3 pi. Consistent a with lack of infectious SARS-CoV-2 shedding after day 5 pi, no transmission was observed to contact animals added on days 6 and 9 pi. We have also investigated the tropism and sites of SARS-CoV-2 replication in adult WTD (3-4 years of age). Infectious virus was detected up to day 6 pi in nasal secretions, and from various respiratory-, lymphoid-, and central nervous system tissues, indicating broad tissue tropism and multiple sites of virus replication. The study provides important insights on the infection and transmission dynamics of SARS-CoV-2 in WTD, a wild animal species that is highly susceptible to infection and with the potential to become a reservoir for the virus in the field.

SARS-CoV-2 B.1.1.7 Variant Infection in Malayan Tigers, Virginia, USA.

We report infection of 3 Malayan tigers with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.1.7 (Alpha) variant at a zoologic park in Virginia, USA. All tigers exhibited respiratory signs consistent with SARS-CoV-2 infection. These findings show that tigers are susceptible to infection with the SARS-CoV-2 B.1.1.7 variant.

Severe SARS-CoV-2 Infection in a Cat with Hypertrophic Cardiomyopathy.

Coronavirus disease 19 (COVID-19), has claimed millions of human lives worldwide since the emergence of the zoonotic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China in December 2019. Notably, most severe and fatal SARS-CoV-2 infections in humans have been associated with underlying clinical conditions, including diabetes, hypertension and heart diseases. Here, we describe a case of severe SARS-CoV-2 infection in a domestic cat (Felis catus) that presented with hypertrophic cardiomyopathy (HCM), a chronic heart condition that has been described as a comorbidity of COVID-19 in humans and that is prevalent in domestic cats. The lung and heart of the affected cat presented clear evidence of SARS-CoV-2 replication, with histological lesions similar to those observed in humans with COVID-19 with high infectious viral loads being recovered from these organs. The study highlights the potential impact of comorbidities on the outcome of SARS-CoV-2 infection in animals and provides important information that may contribute to the development of a feline model with the potential to recapitulate the clinical outcomes of severe COVID-19 in humans.

Susceptibility of white-tailed deer (Odocoileus virginianus) to SARS-CoV-2.

The origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing the global coronavirus disease 19 (COVID-19) pandemic, remains a mystery. Current evidence suggests a likely spillover into humans from an animal reservoir. Understanding the host range and identifying animal species that are susceptible to SARS-CoV-2 infection may help to elucidate the origin of the virus and the mechanisms underlying cross-species transmission to humans. Here we demonstrated that white-tailed deer (Odocoileus virginianus), an animal species in which the angiotensin converting enzyme 2 (ACE2) - the SARS-CoV-2 receptor - shares a high degree of similarity to humans, are highly susceptible to infection. Intranasal inoculation of deer fawns with SARS-CoV-2 resulted in established subclinical viral infection and shedding of infectious virus in nasal secretions. Notably, infected animals transmitted the virus to non-inoculated contact deer. Viral RNA was detected in multiple tissues 21 days post-inoculation (pi). All inoculated and indirect contact animals seroconverted and developed neutralizing antibodies as early as day 7 pi. The work provides important insights into the animal host range of SARS-CoV-2 and identifies white-tailed deer as a susceptible wild animal species to the virus.IMPORTANCEGiven the presumed zoonotic origin of SARS-CoV-2, the human-animal-environment interface of COVID-19 pandemic is an area of great scientific and public- and animal-health interest. Identification of animal species that are susceptible to infection by SARS-CoV-2 may help to elucidate the potential origin of the virus, identify potential reservoirs or intermediate hosts, and define the mechanisms underlying cross-species transmission to humans. Additionally, it may also provide information and help to prevent potential reverse zoonosis that could lead to the establishment of a new wildlife hosts. Our data show that upon intranasal inoculation, white-tailed deer became subclinically infected and shed infectious SARS-CoV-2 in nasal secretions and feces. Importantly, indirect contact animals were infected and shed infectious virus, indicating efficient SARS-CoV-2 transmission from inoculated animals. These findings support the inclusion of wild cervid species in investigations conducted to assess potential reservoirs or sources of SARS-CoV-2 of infection.

From People to Panthera: Natural SARS-CoV-2 Infection in Tigers and Lions at the Bronx Zoo.

Despite numerous barriers to transmission, zoonoses are the major cause of emerging infectious diseases in humans. Among these, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and ebolaviruses have killed thousands; the human immunodeficiency virus (HIV) has killed millions. Zoonoses and human-to-animal cross-species transmission are driven by human actions and have important management, conservation, and public health implications. The current SARS-CoV-2 pandemic, which presumably originated from an animal reservoir, has killed more than half a million people around the world and cases continue to rise. In March 2020, New York City was a global epicenter for SARS-CoV-2 infections. During this time, four tigers and three lions at the Bronx Zoo, NY, developed mild, abnormal respiratory signs. We detected SARS-CoV-2 RNA in respiratory secretions and/or feces from all seven animals, live virus in three, and colocalized viral RNA with cellular damage in one. We produced nine whole SARS-CoV-2 genomes from the animals and keepers and identified different SARS-CoV-2 genotypes in the tigers and lions. Epidemiologic and genomic data indicated human-to-tiger transmission. These were the first confirmed cases of natural SARS-CoV-2 animal infections in the United States and the first in nondomestic species in the world. We highlight disease transmission at a nontraditional interface and provide information that contributes to understanding SARS-CoV-2 transmission across species.IMPORTANCE The human-animal-environment interface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important aspect of the coronavirus disease 2019 (COVID-19) pandemic that requires robust One Health-based investigations. Despite this, few reports describe natural infections in animals or directly link them to human infections using genomic data. In the present study, we describe the first cases of natural SARS-CoV-2 infection in tigers and lions in the United States and provide epidemiological and genetic evidence for human-to-animal transmission of the virus. Our data show that tigers and lions were infected with different genotypes of SARS-CoV-2, indicating two independent transmission events to the animals. Importantly, infected animals shed infectious virus in respiratory secretions and feces. A better understanding of the susceptibility of animal species to SARS-CoV-2 may help to elucidate transmission mechanisms and identify potential reservoirs and sources of infection that are important in both animal and human health.

Detection of avian metapneumovirus subtype A from wild birds in the State of São Paulo, Brazil / Detecção de metapneumovirus aviário subtipo A em aves silvestres no estado de São Paulo, Brasil

The present study investigated the circulation of avian metapneumovirus (aMPV) in wild birds in Brazil. To do so, 131 samples from 366 oropharyngeal or cloacal swabs collected from 18 species of birds were tested individually or in pools by RT-PCR. Samples detected by RT-PCR were selected for DNA sequencing. Thirteen (9.9%) samples were detected by the RT-PCR targeting the N gene and four out of 13 samples were sequenced. Sequencing results showed a high identity with the aMPV subtype A. Our results confirm the circulation of the aMPV subtype A in wild birds in Brazil even five years after its last detection.(AU)

O presente estudo investigou a circulação de metapneumovírus aviário em aves silvestres no Brasil. Para tanto, 131 amostras de 366 suabes orofaringeanos ou cloacais coletados de 18 espécies de aves foram testadas individualmente ou na forma de pools por RT-PCR. As amostras detectadas por RT-PCR foram selecionadas para sequenciamento. Treze (9,9%) das amostras foram detectadas por RT-PCR tendo o gene N como alvo; destas, quatro foram sequenciadas com sucesso. Resultados do sequenciamento mostraram alta identidade com o aMPV de subtipo A. Nossos resultados confirmam a circulação de aMPV subtipo A em aves silvestres no Brasil mesmo cinco anos após sua última detecção.(AU)

Avian coronavirus isolated from a pigeon sample induced clinical disease, tracheal ciliostasis, and a high humoral response in day-old chicks.

The detection of avian coronaviruses (AvCoV) in wild birds and the emergence of new AvCoV have increased in the past few years. In the present study, the pathogenicity of three AvCoV isolates was investigated in day-old chicks. One AvCoV isolated from a pigeon, which clustered with the Massachusetts vaccine serotype, and two AvCoV isolated from chickens, which grouped with a Brazilian genotype lineage, were used. Clinical signs, gross lesions, histopathological changes, ciliary activity, viral RNA detection, and serology were evaluated during 42 days post infection. All AvCoV isolates induced clinical signs, gross lesions in the trachea, moderate histopathological changes in the respiratory tract, and mild changes in other tissues. AvCoV isolated from the pigeon sample caused complete tracheal ciliostasis over a longer time span. Specific viral RNA was detected in all tissues, but the highest RNA loads were detected in the digestive tract (cloacal swabs and ileum). The highest antibody levels were also detected in the group infected with an isolate from the pigeon. These results confirm the pathogenicity of Brazilian variants, which can cause disease and induce gross lesions and histopathological changes in chickens. Our results suggest that non-Galliformes birds can also play a role in the ecology of AvCoV.

Complete Genome Sequence of an Avian Metapneumovirus Subtype A Strain Isolated from Chicken (Gallus gallus) in Brazil.

We report here the complete genome sequence of an avian metapneumovirus (aMPV) isolated from a tracheal tissue sample of a commercial layer flock. The complete genome sequence of aMPV-A/chicken/Brazil-SP/669/2003 was obtained using MiSeq (Illumina, Inc.) sequencing. Phylogenetic analysis of the complete genome classified the isolate as avian metapneumovirus subtype A.

Coronaviruses Detected in Brazilian Wild Birds Reveal Close Evolutionary Relationships with Beta- and Deltacoronaviruses Isolated From Mammals.

This study showed that the most of the coronaviruses (CoVs) detected in Brazilian wild birds clustered with the mouse hepatitis virus A59 strain, belonging to the BetaCoV group. Furthermore, CoV detected in two different bird species, Amazona vinacea and Brotogeris tirica, clustered with a CoV isolated from Sparrow (SpaCoV HKU17) belonging to a monophyletic group related with the CoVs isolated from swines (PorCoV HKU15), both belonging to the DeltaCoV genus, previously unreported in South America. Considering the risk of inter-species host switching and further adaptation to new hosts, detection in bird species of CoVs closely related to mammal CoVs should warn for the potential emergence of new threatening viruses.

Experimental infection of inbred BALB/c and A/J mice with Massachusetts and Brazilian strains of infectious bronchitis virus (IBV).

The ability of avian coronaviruses to replicate in mice was investigated to investigate interspecies transmission. Two inbred mouse strains (BALB/c and A/J) with different genetic backgrounds were inoculated with the avian coronavirus strains Mass and BR-I and monitored for at least 10 days. Analysis of viral RNA, histopathological examinations, immunohistochemistry and serology were performed. After virus inoculation, neither clinical signs nor evident gross lesions were observed. Viral RNA, histopathological changes, and viral nucleoprotein were observed in the lung, trachea and sinus of all inoculated mice. Our study demonstrates the importance of elucidating the epidemiology of coronaviruses, including in rodents that are pests in poultry production.

Phylogenetic and phylogeographic mapping of the avian coronavirus spike protein-encoding gene in wild and synanthropic birds.

The evolution and population dynamics of avian coronaviruses (AvCoVs) remain underexplored. In the present study, in-depth phylogenetic and Bayesian phylogeographic studies were conducted to investigate the evolutionary dynamics of AvCoVs detected in wild and synanthropic birds. A total of 500 samples, including tracheal and cloacal swabs collected from 312 wild birds belonging to 42 species, were analysed using molecular assays. A total of 65 samples (13%) from 22 bird species were positive for AvCoV. Molecular evolution analyses revealed that the sequences from samples collected in Brazil did not cluster with any of the AvCoV S1 gene sequences deposited in the GenBank database. Bayesian framework analysis estimated an AvCoV strain from Sweden (1999) as the most recent common ancestor of the AvCoVs detected in this study. Furthermore, the analysis inferred an increase in the AvCoV dynamic demographic population in different wild and synanthropic bird species, suggesting that birds may be potential new hosts responsible for spreading this virus.