Avian Polyomavirus Among Psittacine Birds in Iran: Molecular Detection Rate and Associated Risk Factors.

Avian polyomavirus (APV) infection causes various health problems in psittacine species, including death. The present study was conducted to investigate the prevalence of APV among psittacine birds in Iran. We also aimed to evaluate the impact of age, sex, species, season, and origin of the birds on the prevalence of APV. This study investigated the presence of APV among 1050 individual birds from 7 psittacine species over a 1-year period in Iran, namely, green-cheeked parakeets (Pyrrhura molinae), rosy-faced lovebirds (Agapornis roseicollis), monk parakeets (Myiopsitta monachus), sun conures (Aratinga solstitialis), Senegal parrots (Poicephalus senegalus), cockatiels (Nymphicus hollandicus), and grey parrots (Psittacus erithacus). The overall prevalence of APV in all studied species was 25% (263/1050, 95% confidence interval [CI]: 22.5-27.8). Results of the study showed that age and the season of the year were 2 important determinant factors in the prevalence of APV in psittacine birds. Young psittacine birds <6 months old were 2.94 (95% CI: 1.19-7.27) times more likely to be infected with APV than birds >1 year old, and there was a significant interaction between season and species in the multivariate analysis. In the winter season, rosy-faced lovebirds and green-cheeked parakeets were 15.6 (95% CI: 4.20-57.95) and 4.76 (95% CI: 1.4-16.21) times more likely to be infected with APV than in other seasons, respectively. This is the first report on the detection rate of APV in psittacine birds in Iran.

DEVELOPMENT AND VALIDATION OF A NOVEL DUPLEX PROBE-HYBRIDIZATION QUANTITATIVE PCR FOR LYMPHOMA-ASSOCIATED MIROUNGINE GAMMAHERPESVIRUS 3 IN NORTHERN ELEPHANT SEALS (MIROUNGA ANGUSTIROSTRIS).

Recently, a novel gammaherpesvirus, miroungine gammaherpesvirus 3 (MirGHV3), was described in two juvenile elephant seals (Mirounga angustirostris) with diffuse large B-cell lymphoma. We developed and validated a quantitative (q)PCR for rapid detection of MirGHV3 and investigated its potential association with lymphoma. We developed a duplex probe-hybridization qPCR with MirGHV3 DNA polymerase (pol) as the target gene. Each primer-probe combination was cross-validated against the others. Interference was not seen when they were run in the same well as a duplex assay. Twenty-three samples from seven northern elephant seals were tested using the duplex assay. Viral DNA was detected by the assay in 9 of 9 (100%) tissues affected by lymphoma and in 6 of 14 (43%) samples from tissues unaffected by lymphoma. There was a strong correlation between viral copies detected with each of the assays (P=0.0002). Viral load was significantly higher in tissues affected by lymphoma than in those unaffected (P<0.0001). Excluding the virus-negative samples, viral load was still significantly higher in tissues affected by lymphoma than in those unaffected (P=0.0004). This is consistent with a potential role of MirGHV3 in oncogenesis in northern elephant seals, although more studies are needed to determine this definitively. The qPCR developed has utility for further investigations of MirGHV3.

Shope fibroma of the eyelid margin in a domestic rabbit.

OBJECTIVE: To describe the clinical and histopathologic features as well as response to treatment of a solitary Shope fibroma affecting the eyelid margin of a domestic rabbit. ANIMAL STUDIED: A seven-year-old female intact domestic rabbit with a progressively enlarging firm, pedunculated, and encrusted inferior eyelid mass of the left eye of 1-month duration. PROCEDURES: Under general anesthesia, the crust was removed revealing an ulcerated mass that was excised via a house-shaped resection and submitted for histopathology. Purulent discharge associated with the mass was swabbed for aerobic and anaerobic bacterial culture and sensitivity testing. Histopathology revealed intraepithelial, cytoplasmic leporipoxviral inclusion bodies consistent with Shope fibroma virus. There was no growth on aerobic or anaerobic bacterial culture. The lesion was completely excised, and no recurrence was noted during a 3-month follow-up period. CONCLUSIONS: The solitary nature and clinical appearance of this eyelid margin Shope fibroma are unique. Shope fibroma should be considered a differential diagnosis for eyelid masses in rabbits even in the absence of other cutaneous masses. Thorough systemic evaluation to attempt to distinguish Shope fibroma from malignant myxomatosis should be performed.

Tumor Viruses and Their Associated Cancers: Remain on the Track with the Latest Advances.

Infection with certain types of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) viruses, known as tumor viruses or oncogenic viruses, can lead to cancer [...].

First report of fibropapillomatosis (FP) and Chelonid alphaherpesvirus 5 (ChHV5) in a green sea turtle (Chelonia mydas) from the historically fibropapillomatosis-free Fernando de Noronha Archipelago, Northeastern Brazil / Primeiro caso de fibropapilomatose (FP) e de Chelonid alphaherpesvirus 5 (ChHV5) em tartaruga-verde (Chelonia mydas) no Arquipélago de Fernando de Noronha, Nordeste do Brasil

Fibropapillomatosis (FP) is an infectious disease caused by Chelonid alphaherpesvirus 5 (ChHV5). Nevertheless, its clinical manifestations are considered multifactorial. Due to its relevance, FP is currently monitored in sea turtle populations in the United States, Australia, Caribbean, and Brazil. Between 2000 and 2020, the TAMAR Project/ TAMAR Project Foundation analyzed the prevalence of FP in nine states and oceanic islands along the Brazilian coast, including Fernando de Noronha Archipelago (FNA), a historically FP-free area. A total of 4,435 green sea turtles (Chelonia mydas) were monitored from 2010 to 2016. Additionally, in 2012 and 2014, 43 FP-free skin samples were analyzed for ChHV5 using a qualitative PCR for the UL30 polymerase (pol) sequence. In 2015, a bilateral ocular nodule characterized as an FP tumor was reported in one of the monitored individuals undergoing rehabilitation. Tissue samples were collected following surgical removal of the tumor. Characterization of a 454 bp UL30 polymerase gene revealed a ChHV5 sequence previously reported in other areas of the Atlantic Brazilian coast. In the years following this finding from January 2017 to March 2020, a total of 360 C. mydas were monitored in the same area and no FP tumors were detected. This is the first report of FP and the first detection of ChHV5 in FNA, a finding of great concern considering this site's historical absence of FP occurrence. This study highlights the importance of monitoring this disease in historically FP-free areas of the Brazilian Atlantic coast.(AU)

A fibropapilomatose (FP) é uma doença infecciosa causada pelo Chelonid alphaherpesvirus 5 (ChHV5). No entanto, as manifestações clínicas da doença são consideradas multifatoriais. Esta doença é monitorada atualmente em populações de tartarugas marinhas nos EUA, Austrália, Caribe e Brasil. Desde 2000, o Projeto TAMAR/Fundação Projeto TAMAR analisa a presença de FP em nove estados da costa brasileira e ilhas oceânicas, incluindo o arquipélago de Fernando de Noronha, uma área historicamente livre de FP. Um total de 4.435 indivíduos de Chelonia mydas foram monitorados de 2010 a 2016 e 43 amostras de pele foram analisadas para detectar ChHV5 em 2012 e 2014 com o objetivo de avaliar a presença do vírus em tecidos sem FP, usando uma PCR qualitativa para detecção de sequências do gene da UL30 polimerase. Em 2015, uma tartaruga verde (C. mydas) foi relatada com um nódulo ocular bilateral caracterizado como FP. Amostras de tecido foram coletadas durante sua reabilitação e procedimento cirúrgico para remover o tumor. A caracterização parcial de uma sequência de 454 bp do gene UL30 polimerase detectou ChHV5 anteriormente relatado em outras áreas da costa atlântica brasileira. Após estes achados, de janeiro de 2017 a março de 2020, um total de 360 indivíduos de C. mydas foram monitorados e nenhum caso de FP foi registrado. Este é o primeiro relato de FP e a primeira caracterização de ChHV5 no arquipélago de Fernando de Noronha, uma questão preocupante e que ressalta a importância do monitoramento desta doença em áreas historicamente livres de FP na costa atlântica brasileira.(AU)

The expression profile of miR-222b-5p/MAPK10 in spleens of SPF chickens infected with REV-SNV at 28-42 dpi.

Reticuloendotheliosis virus (REV) is an avian oncogenic retrovirus that causes atrophy of immune organs, such as the spleen, thymus, and bursa of Fabricius, leading to severe immunosuppression. However, there is limited information describing the genes or microRNAs (miRNAs) that play a role in replicating REV-spleen necrosis virus (SNV). Our previous miRNA and RNA sequencing data showed that the expression of gga-miR-222b-5p was significantly upregulated in REV-SNV-infected chicken spleens of 7, 14, and 21 dpi compared to non-infected chicken spleens, but mitogen-activated protein kinase 10 (MAPK10), which is related to innate immunity, had the opposite expression pattern. To understand chicken cellular miRNA function in the virus-host interactions during REV infection, we used quantitative reverse transcription PCR (qRT-PCR) to determine whether the expression of gga-miR-222b-5p and MAPK10 in the spleen of specific-pathogen-free chickens at 28, 35, and 42 dpi was consistent with the first 3 time points, and dual-luciferase reporter assay was used to determine the targeting relationship between gga-miR-222b-5p and MAPK10. Results show that MAPK10 was downregulated at all 3 time points; however, significant difference (p⟨0.01) was noted only at 35 dpi. Moreover, the expression of gga-miR-222b-5p was upregulated; however, significant difference (p⟨0.01) was observed only at 28 and 35 dpi. A dual-luciferase reporter assay showed that MAPK10 is a direct target of gga-miR-222b-5p. This study suggests that gga-miR-222b-5p may target MAPK10 to promote the REV-SNV-induced tumorigenesis via the RLRs signaling pathway.

Rapid detection of avian leukosis virus subgroup J by cross-priming amplification.

Avian leukosis virus subgroup J (ALV-J) causes oncogenic disease in chickens in China, resulting in great harm to poultry production, and remains widespread in China. Herein, we employed a cross-priming amplification (CPA) approach and a nucleic acid detection device to establish a visual rapid detection method for ALV-J. The sensitivity of CPA, polymerase chain reaction (PCR) and real-time PCR (RT-PCR) was compared, and the three methods were used to detect ALV-J in the cell cultures which inoculated with clinical plasma. The result showed when the amplification reaction was carried out at 60 °C for just 60 min, the sensitivity of CPA was 10 times higher than conventional PCR, with high specificity, which was comparable with RT-PCR, based on detection of 123 cell cultures which inoculated with clinical plasma, the coincidence rate with real-time PCR was 97.3% (71/73). CPA detection of ALV-J does not require an expensive PCR instrument; a simple water bath or incubator is sufficient for complete DNA amplification, and the closed nucleic acid detection device avoids aerosol pollution, making judgment of results more intuitive and objective. The CPA assay would be a promising simple, rapid and sensitive method for identification of ALV-J.

Development of a SYBR Green-based real-time quantitative polymerase chain reaction assay to detect enzootic nasal tumor virus in goats.

Enzootic nasal adenocarcinoma is a contagious respiratory disease in goats that is caused by the enzootic nasal tumor virus 2 (ENTV-2). In order to increase the number of available detection methods for ENTV-2, we developed a SYBR Green real-time polymerase chain reaction (SGrPCR) assay that targets the gag gene of ENTV-2. The low limit of detection of the assay was 3.68 × 101 copies/µL, a hundredfold more sensitive than conventional PCR. The melt curve showed a single sharp melt peak at 83°C, which indicated that there was no non-specific amplification or primer dimer formation. The intra-assay and inter-assay coefficients of variation were 1.58% and 1.82%, respectively. There was no cross-reactivity with closely related goat viruses (i.e., orf virus, peste des petits ruminants virus, goatpox virus, foot-and-mouth disease virus) and endogenous retroviruses. In conclusion, the SGrPCR assay is specific for the gag gene of ENTV-2 and provides a rapid and sensitive approach for detecting ENTV-2 in clinical samples.

L'adénocarcinome nasal enzootique est une maladie respiratoire contagieuse chez les chèvres qui est causé par le virus de la tumeur nasale enzootique 2 (ENTV-2). Afin d'augmenter le nombre de méthodes de détection disponibles pour ENTV-2, nous avons développé un test de réaction en chaîne par polymérase en temps réel SYBR Green (SGrPCR) qui cible le gène gag de ENTV-2. La limite basse de détection du test était de 3,68 × 101 copies/µL, cent fois plus sensible que la PCR conventionnelle. La courbe de fusion montrait un seul pic de fusion net à 83 °C, ce qui indiquait qu'il n'y avait pas d'amplification non spécifique ou de formation de dimère d'amorce. Les coefficients de variation intra-essai et inter-essai étaient respectivement de 1,58 % et 1,82 %. Il n'y avait pas de réactivité croisée avec les virus caprins étroitement apparentés (c'est-à-dire le virus orf, le virus de la peste des petits ruminants, le virus de la variole caprine, le virus de la fièvre aphteuse) et les rétrovirus endogènes. En conclusion, le test SGrPCR est spécifique du gène gag de l'ENTV-2 et fournit une approche rapide et sensible pour la détection d'ENTV-2 dans des échantillons cliniques.(Traduit par Docteur Serge Messier).

Compounds Identified from Marine Mangrove Plant (Avicennia alba) as Potential Antiviral Drug Candidates Against WDSV, an In-Silico Approach.

Walleye dermal sarcoma virus (WDSV) is a type of retrovirus, which affects most of the adult walleye fishes during the spawning time. The virus causes multiple epithelial tumors on the fish's skin and fins that are liable for more than 50% of the mortality rate of fish around the world. Till now, no effective antiviral drug or vaccine candidates have been developed that can block the progression of the disease caused by the pathogen. It was found that the 582-amino-acid (aa) residues long internal structural gag polyprotein of the virus plays an important role in virus budding and virion maturation outside of the cell. Inhibition of the protein can block the budding and virion maturation process and can be developed as an antiviral drug candidate against the virus. Therefore, the study aimed to identify potential natural antiviral drug candidates from the tropical mangrove marine plant Avicennia alba, which will be able to block the budding and virion maturation process by inhibiting the activity of the gag protein of the virus. Initially, a homology modeling approach was applied to identify the 3D structure, followed by refinement and validation of the protein. The refined protein structures were then utilized for molecular docking simulation. Eleven phytochemical compounds have been isolated from the marine plant and docked against the virus gag polyprotein. Three compounds, namely Friedlein (CID244297), Phytosterols (CID12303662), and 1-Triacontanol (CID68972) have been selected based on their docking score -8.5 kcal/mol, -8.0 kcal/mol and -7.9 kcal/mol, respectively, and were evaluated through ADME (Absorption, Distribution, Metabolism and Excretion), and toxicity properties. Finally, molecular dynamics (MD) simulation was applied to confirm the binding stability of the protein-ligands complex structure. The ADME and toxicity analysis reveal the efficacy and non-toxic properties of the compounds, where MD simulation confirmed the binding stability of the selected three compounds with the targeted protein. This computational study revealed the virtuous value of the selected three compounds against the targeted gag polyprotein and will be effective and promising antiviral candidates against the pathogen in a significant and worthwhile manner. Although in vitro and in vivo study is required for further evaluation of the compounds against the targeted protein.

Measuring the Humoral Immune Response in Cats Exposed to Feline Leukaemia Virus.

Retroviruses belong to an important and diverse family of RNA viruses capable of causing neoplastic disease in their hosts. Feline leukaemia virus (FeLV) is a gammaretrovirus that infects domestic and wild cats, causing immunodeficiency, cytopenia and neoplasia in progressively infected cats. The outcome of FeLV infection is influenced by the host immune response; progressively infected cats demonstrate weaker immune responses compared to regressively infected cats. In this study, humoral immune responses were examined in 180 samples collected from 123 domestic cats that had been naturally exposed to FeLV, using a novel ELISA to measure antibodies recognizing the FeLV surface unit (SU) glycoprotein in plasma samples. A correlation was demonstrated between the strength of the humoral immune response to the SU protein and the outcome of exposure. Cats with regressive infection demonstrated higher antibody responses to the SU protein compared to cats belonging to other outcome groups, and samples from cats with regressive infection contained virus neutralising antibodies. These results demonstrate that an ELISA that assesses the humoral response to FeLV SU complements the use of viral diagnostic tests to define the outcome of exposure to FeLV. Together these tests could allow the rapid identification of regressively infected cats that are unlikely to develop FeLV-related disease.

Transcriptomic Profiling of Fibropapillomatosis in Green Sea Turtles (Chelonia mydas) From South Texas.

Sea turtle fibropapillomatosis (FP) is a tumor promoting disease that is one of several threats globally to endangered sea turtle populations. The prevalence of FP is highest in green sea turtle (Chelonia mydas) populations, and historically has shown considerable temporal growth. FP tumors can significantly affect the ability of turtles to forage for food and avoid predation and can grow to debilitating sizes. In the current study, based in South Texas, we have applied transcriptome sequencing to FP tumors and healthy control tissue to study the gene expression profiles of FP. By identifying differentially expressed turtle genes in FP, and matching these genes to their closest human ortholog we draw on the wealth of human based knowledge, specifically human cancer, to identify new insights into the biology of sea turtle FP. We show that several genes aberrantly expressed in FP tumors have known tumor promoting biology in humans, including CTHRC1 and NLRC5, and provide support that disruption of the Wnt signaling pathway is a feature of FP. Further, we profiled the expression of current targets of immune checkpoint inhibitors from human oncology in FP tumors and identified potential candidates for future studies.

A polyomavirus detected in American black bear (Ursus americanus).

Polyomaviruses are ancient DNA viruses that infect several species of animals. While recognition of the family Polyomaviridae has grown rapidly, there are few studies that consider their potential association with disease. Carnivora are a diverse and widespread order affected by polyomaviruses (PyVs) that have co-evolved with their hosts for millions of years. PyVs have been identified in sea lions, raccoons, badgers, Weddell seals, and dogs. We have discovered a polyomavirus, tentatively named "Ursus americanus polyomavirus 1" (UaPyV1) in black bears (Ursus americanus). UaPyV1 was detectable in various tissues of six out of seven bears submitted for necropsy. Based on viral phylogenetic clustering and detection of the virus in multiple individuals, we suggest that black bears are the natural hosts for UaPyV1. In this albeit small group, there is no clear relationship between UaPyV1 infection and any specific disease.

Genomic evolution of avian polyomaviruses with a focus on budgerigar fledgling disease virus.

Gammapolyomaviruses may cause serious inflammatory diseases in a broad range of avian hosts. In this study we investigated genomic evolution of and selection constraint acting on avian polyomaviruses (APyVs). Our analyses suggested that goose haemorrhagic polyomavirus (GHPV) evolves more slowly (3.03 × 10-5 s/s/y mean evolutionary rate) than budgerigar fledgling disease virus (BFDV), finch polyomavirus (FPyV) and canary polyomavirus (CaPyV) (1.39 × 10-4 s/s/y, 2.63 × 10-4 s/s/y and 1.41 × 10-4 s/s/y mean evolutionary rate, respectively). In general, purifying selection seems to act on the protein coding regions of APyV genomes, although positive Darwinian selection was also predicted in a few positions (e.g., in the large tumor antigen coding region of BFDV and GHPV and in the capsid protein sequences of BFDV). The importance of these aa changes remains elusive. Overall, a better understanding of adaptive changes in the genome of APyVs requires additional data from various incidental hosts and reservoir species.

Feline leukemia virus in owned cats in Southeast Asia and Taiwan.

Feline leukaemia virus (FeLV) is a retrovirus associated with fatal disease in cats with infection in its progressive form. Although there are numerous reports on the occurrence of FeLV in the feline population worldwide, there is a paucity of data in Asia. In this study, we assessed the circulation of FeLV by ELISA and nested PCR in cats from different countries in Southeast Asia (i.e., Thailand, Malaysia, Singapore, Philippines, Indonesia and Vietnam) and Taiwan during 2017-2018. Forty-seven cats were positive to FeLV by antigen or provirus detection, but 32 samples were considered truly positive on the basis of positive molecular testing. Frequency of occurrence of FeLV proviral DNA ranged from 0% (0/43 positive samples) in Indonesia to 18.5% (22/119 positive samples) in Thailand. A statistically significant association (p < 0.05) was found between country of cats origin, age, lifestyle, abnormal oral mucosa, and FeLV molecular positive results. In-depth studies are needed in other countries in Southeast Asia to elucidate the mosaic of knowledge about FeLV epidemiology.

Identification of African Elephant Polyomavirus in wild elephants and the creation of a vector expressing its viral tumor antigens to transform elephant primary cells.

Wild elephant populations are declining rapidly due to rampant killing for ivory and body parts, range fragmentation, and human-elephant conflict. Wild and captive elephants are further impacted by viruses, including highly pathogenic elephant endotheliotropic herpesviruses. Moreover, while the rich genetic diversity of the ancient elephant lineage is disappearing, elephants, with their low incidence of cancer, have emerged as a surprising resource in human cancer research for understanding the intrinsic cellular response to DNA damage. However, studies on cellular resistance to transformation and herpesvirus reproduction have been severely limited, in part due to the lack of established elephant cell lines to enable in vitro experiments. This report describes creation of a recombinant plasmid, pAelPyV-1-Tag, derived from a wild isolate of African Elephant Polyomavirus (AelPyV-1), that can be used to create immortalized lines of elephant cells. This isolate was extracted from a trunk nodule biopsy isolated from a wild African elephant, Loxodonta africana, in Botswana. The AelPyV-1 genome contains open-reading frames encoding the canonical large (LTag) and small (STag) tumor antigens. We cloned the entire early region spanning the LTag and overlapping STag genes from this isolate into a high-copy vector to construct a recombinant plasmid, pAelPyV-1-Tag, which effectively transformed primary elephant endothelial cells. We expect that the potential of this reagent to transform elephant primary cells will, at a minimum, facilitate study of elephant-specific herpesviruses.

Molecular characterization of a marine turtle tumor epizootic, profiling external, internal and postsurgical regrowth tumors.

Sea turtle populations are under threat from an epizootic tumor disease (animal epidemic) known as fibropapillomatosis. Fibropapillomatosis continues to spread geographically, with prevalence of the disease also growing at many longer-affected sites globally. However, we do not yet understand the precise environmental, mutational and viral events driving fibropapillomatosis tumor formation and progression.Here we perform transcriptomic and immunohistochemical profiling of five fibropapillomatosis tumor types: external new, established and postsurgical regrowth tumors, and internal lung and kidney tumors. We reveal that internal tumors are molecularly distinct from the more common external tumors. However, they have a small number of conserved potentially therapeutically targetable molecular vulnerabilities in common, such as the MAPK, Wnt, TGFß and TNF oncogenic signaling pathways. These conserved oncogenic drivers recapitulate remarkably well the core pan-cancer drivers responsible for human cancers. Fibropapillomatosis has been considered benign, but metastatic-related transcriptional signatures are strongly activated in kidney and established external tumors. Tumors in turtles with poor outcomes (died/euthanized) have genes associated with apoptosis and immune function suppressed, with these genes providing putative predictive biomarkers.Together, these results offer an improved understanding of fibropapillomatosis tumorigenesis and provide insights into the origins, inter-tumor relationships, and therapeutic treatment for this wildlife epizootic.

Molecular detection of myxoma virus in the environment of vaccinated rabbitries.

Myxoma virus (MYXV) is the aetiological agent of myxomatosis, a systemic, mostly lethal disease that affects European rabbits. Vaccination against it, although widespread, has not been completely effective and disease outbreaks still take place on farms which carry out vaccination programmes. Since some of these cases have been attributed to airborne transmission or the spread of the virus via inanimate vectors, the aims of this study were to determine MYXV contamination levels and distribution in the environment of vaccinated farms and to ascertain whether the detected virus corresponded to field strains. For that, environmental samples from several areas, tools and employees from four (three infected and one uninfected) rabbitries were taken and analysed by qPCR. MYXV was detected in the environment of all the infected farms, whereas all the samples from the non-infected farm were negative. Furthermore, all the positive samples contained viral DNA compatible with field strains of the virus. These results lead us to believe that the administration of currently available commercial vaccines does not prevent infected animals from shedding the field virus. Moreover, viral DNA was also found in items that are not in direct contact with the animals, which could play a role in the transmission of the infection throughout the farm and to other farms. Therefore, this study proves that current vaccination schemes on their own are not sufficient to prevent this disease and should be accompanied by adequate biosecurity measures.

Monitoring of emerging myxoma virus epidemics in Iberian hares (Lepus granatensis) in Spain, 2018-2020.

Myxomatosis is an infectious disease caused by the myxoma virus (MYXV), which has very high mortality rates in European wild rabbits (Oryctolagus cuniculus). While sporadic cases of myxomatosis have also been reported in some hare species, these lagomorphs are considered to have a low susceptibility to MYXV infection. In the present study, we describe the spatiotemporal evolution and main epidemiological findings of novel hare MYXV (ha-MYXV or MYXV-Tol) epidemics in Iberian hares (Lepus granatensis) in Spain. In the period 2018-2020, a total of 487 hares from 372 affected areas were confirmed to be MYXV-infected by PCR. ha-MYXV outbreaks were detected in most of the Spanish regions where the Iberian hare is present. The spatial distribution was not homogeneous, with most outbreaks concentrated in the southern and central parts of Spain. Consecutive outbreaks reported in the last two years suggest endemic circulation in Spain of this emerging virus. A retrospective study carried out just after the first epidemic period (2018-2019) revealed that the virus could have been circulating since June 2018. The number of outbreaks started to rise in July, peaked during the first half of August and October and then decreased sharply until January 2019. The apparent mean mortality rate was 55.4% (median: 70%). The results indicated high susceptibility of the Iberian hare to ha-MYXV infection, but apparent resistance in the sympatric hare species present in Spain and less infectivity in European rabbits. The novel ha-MYXV has had significant consequences on the health status of Iberian hare populations in Spain, which is of animal health and conservation concern. The present study contributes to a better understanding of ha-MYXV emergence and will provide valuable information for the development of control strategies. Further research is warranted to assess the impact of this emerging virus on wild lagomorph populations and to elucidate its ecological implications for Iberian Mediterranean ecosystems.

An outbreak in three-yellow chickens with clinical tumors of high mortality caused by the coinfection of reticuloendotheliosis virus and Marek's disease virus: a speculated reticuloendotheliosis virus contamination plays an important role in the case.

Both reticuloendotheliosis and Marek's disease are neoplastic diseases of chickens caused by reticuloendotheliosis virus (REV) and Marek's disease virus (MDV), respectively. The infection of REV or MDV may lead to clinical tumors and also result in immunosuppression and easily allow secondary infection by other pathogens. Here, we investigated a breeder flock of three-yellow chickens in southern China that had been vaccinated with CVI988/Rispens at hatching and had experienced depression, weakness, reduction in weight gain, and an increased death rate after 120 d of age. The morbidity and mortality were 20% and 10%, respectively, at 140 d of age when this infection was diagnosed. The necropsy of the birds revealed significant tumor-like lesions in the heart, liver, spleen, and ceca. Peripheral blood lymphocytes and tumor-like tissues were sampled for PCR detection and for histopathological observation, for virus isolation and the subsequent immunofluorescent assay on the cell cultures and for gene sequencing of the isolated viruses. A REV isolate GX18NNR1 and a MDV isolate GX18NNM5 were both recovered from the sampled bird. Further phylogenetic analysis based on the env gene of REV and the meq gene of MDV demonstrated that GX18NNR1 was closely related to the reference REV strain MD-2, which was isolated from a contaminated commercial turkey herpesvirus vaccine. In addition, the GX18NNM5 was found to belong to the Chinese very virulent MDV strains' cluster. The coinfection of REV and MDV may contribute to tumor outbreaks with high morbidity and mortality in three-yellow chicken flocks.

Comparative In Vitro and In Vivo Studies on Feline, Canine, and Human Merkel Cell Carcinoma.

Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine tumor, and most human MCC cases are infected by Merkel cell polyomavirus (MCPyV). However, the underlying pathogeneses of MCC in animals remain unclear. In the present study, newly established cell lines from feline and canine MCC, a MCPyV-positive human MCC cell line, and MCC tissues from 25 cats and 1 dog were examined and compared pathologically. Feline and canine MCCs were composed of tumor cells arranged in trabeculae and solid packets. Twenty out of 25 feline MCC cases (80%) had other proliferative cutaneous lesions, such as carcinoma in situ and squamous cell carcinoma. Among the 25 feline MCC cases, tumor cells were immunopositive for cytokeratins (CKs), including CK5/6 (4/25 cases, 16%), CK7 (5, 20%), CK18 (25, 100%), CK19 (20, 80%), and CK20 (20, 80%). The tumor cells of feline MCC were also immunopositive for synaptophysin (24/25, 96%) and CD56 (22/25, 88%). The tumor cells of canine MCC were immunopositive for CK18, CK19, CK20, and synaptophysin. Cultured feline and canine MCC cells grew in adherent monolayers and exhibited diffuse cytoplasmic immunoreactivity for CKs, whereas human MCC cells grew in suspension and exhibited dot-like cytoplasmic immunoreactivity for CKs. Differences in the distribution of CKs between human and animal MCC may be attributed to cell adhesion propensities. MCPyV genes and antigen were not detected in feline or canine MCC, suggesting a different etiology from human MCC.