Discovery of Novel Viruses in Culicoides Biting Midges in Chihuahua, Mexico.

Biting midges (Culicoides) are vectors of many pathogens of medical and veterinary importance, but their viromes are poorly characterized compared to certain other hematophagous arthropods, e.g., mosquitoes and ticks. The goal of this study was to use metagenomics to identify viruses in Culicoides from Mexico. A total of 457 adult midges were collected in Chihuahua, northern Mexico, in 2020 and 2021, and all were identified as female Culicoides reevesi. The midges were sorted into five pools and homogenized. An aliquot of each homogenate was subjected to polyethylene glycol precipitation to enrich for virions, then total RNA was extracted and analyzed by unbiased high-throughput sequencing. We identified six novel viruses that are characteristic of viruses from five families (Nodaviridae, Partitiviridae, Solemoviridae, Tombusviridae, and Totiviridae) and one novel virus that is too divergent from all classified viruses to be assigned to an established family. The newly discovered viruses are phylogenetically distinct from their closest known relatives, and their minimal infection rates in female C. reevesi range from 0.22 to 1.09. No previously known viruses were detected, presumably because viral metagenomics had never before been used to study Culicoides from the Western Hemisphere. To conclude, we discovered multiple novel viruses in C. reevesi from Mexico, expanding our knowledge of arthropod viral diversity and evolution.

Molecular characterization of canine circovirus based on the Capsid gene in Thailand.

BACKGROUND: Canine circovirus (CanineCV) is a single-stranded circular DNA virus that infects domestic and wild canids in many countries. CanineCV is associated with gastroenteritis and diarrhea, respiratory disease, and generalized vasculitis leading to a fatal event. The Capsid protein (Cap) is a structural protein of the virus which has high genetic variability and plays a role in the canine immune response. In this study, we cloned the full-length CanineCV Capsid gene (Cap). In-silico analyses were used to explore the genomic and amino acid variability and natural selection acting on the Cap gene. The immune relevance for T-cell and B-cell epitopes was predicted by the immunoinformatic approach. RESULTS: According to the Cap gene, our results showed that CanineCV was separated into five phylogenetic groups. The obtained CanineCV strain from this study was grouped with the previously discovered Thai strain (MG737385), as supported by a haplotype network. Entropy analyses revealed high nucleotide and amino acid variability of the Capsid region. Selection pressure analysis revealed four codons at positions 24, 50, 103, and 111 in the Cap protein evolved under diversifying selection. Prediction of B-cell epitopes exhibited four consensus sequences based on physiochemical properties, and eleven peptide sequences were predicted as T-cell epitopes. In addition, the positive selection sites were located within T-cell and B-cell epitopes, suggesting the role of the host immune system as a driving force in virus evolution. CONCLUSIONS: Our study provides knowledge of CanineCV genetic diversity, virus evolution, and potential epitopes for host cell immune response.

Recombinant expression and characterization of Canine circovirus capsid protein for diagnosis.

Canine circovirus (CanineCV) is a contagious virus that causes severe gastroenteritis, diarrhea, respiratory disease, and vasculitis, often resulting in fatality among infected dogs. In this study, a recombinant Capsid protein (rCap) of CanineCV was expressed in the Escherichia coli (E. coli) Rosetta (DE3) pLysS host cell, followed by affinity purification, and then analyzed by SDS-PAGE, revealing a molecular weight of approximately 31 kDa. The antigenicity of the CanineCV rCap protein was confirmed through recognition by a rabbit anti-CanineCV rCap protein polyclonal antibody (PoAb). Additionally, the reactivity and specificity of this PoAb were assessed using indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis before applying in an immunohistochemistry (IHC), namely, immunoperoxidase detection. The immunoperoxidase assay using rabbit anti-CanineCV rCap protein PoAb demonstrated that the CanineCV Cap protein was predominantly located in immune cells, especially lymphocytes and macrophages, within the spleen, lung, tracheobronchial lymph nodes, small intestine, and kidney. Similarly, the Cap protein was also found in pneumocytes in the lung and renal tubular epithelial cells in the kidney. These findings reflected the biological activity and cell tropism of the virus. Therefore, the recombinant Cap protein and its PoAb could be used for the development of a valuable diagnostic tool for CanineCV detection.

SARS-CoV-2 Transmission from Human to Pet and Suspected Transmission from Pet to Human, Thailand.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been the cause of human pandemic infection since late 2019. SARS-CoV-2 infection in animals has also been reported both naturally and experimentally, rendering awareness about a potential source of infection for one health concern. Here, we describe an epidemiological investigation of SARS-CoV-2 infection in 639 cats and 224 dogs throughout multiple waves of COVID-19 outbreaks in Thailand. To indicate the potential source of infection, we performed SARS-CoV-2 genomic sequencing of samples obtained from pets and contacted humans, combined with in-depth interviews to support the epidemiological investigation. In the tested animals, SARS-CoV-2 RNA was present in 23 cases (19 cats and 4 dogs). Whole-genome sequencing of selected samples showed various SARS-CoV-2 variants of concern, which included the original European lineage (B.1), Alpha (B.1.1.7), Delta (B.1.617), and Omicron (BA.2). Among SARS-CoV-2-positive pets, 34.78% had evidence of contact with infected humans. Together with genomic analysis and an overlapping timeline, we revealed evidence of viral transmission from infected humans as the primary source, which spread to household cats via an undefined mode of transmission and most likely circulated between cohoused cats and caretakers within the weeks before the investigation. The SARS-CoV-2 surface glycoprotein (spike gene) obtained from caretakers of individual cats contained sequence signatures found in the sequences of infected cats, indicating possible exposure to the virus excreted by cats. Although pet-to-human transmission of SARS-CoV-2 is considered relatively rare, our study provides suspected episodes of human infection from animals that were initially infected through contact with infected humans.

Epidemiology, genetic diversity, and association of canine circovirus infection in dogs with respiratory disease.

Although canine circovirus (CanineCV)-associated with gastroenteritis has been well documented, the virus is also detectable in the respiratory discharge of dogs with respiratory disease. In this study, an epidemiological approach was used to explore the association between the presence of CanineCV and respiratory symptoms in dogs. Respiratory swabs were collected from 76 healthy dogs and 114 dogs with respiratory illness and tested for CanineCV using conventional PCR (cPCR). Furthermore, lung tissues collected from 15 necropsied dogs showing pneumonia were tested using the real-time PCR (qPCR) and in situ hybridization (ISH) technique. A total of 8.95% (17/190) of dogs were CanineCV positive, with a significant association (p = 0.013) in dogs with respiratory signs. Four necropsied dogs were qPCR positive with the CanineCV-DNA labeling localized in tracheobronchial lymphoid cells (3/4), pulmonary parenchyma, capillary endothelia, and mononuclear cells harboring in alveoli (2/4). Full-length genome sequences of seven CanineCV strains were analyzed, indicating that the detected CanineCV genome clustered in the CanineCV-4 genotype. Genetic recombination was also evident in the replicase (Rep) gene. Although the role of CanineCV primarily affecting lung lesions could not be determined from this study, the presence of CanineCV DNA in pulmonary-associated cells indicated the potential association of the virus with canine respiratory disease; thus, linking causality must be examined in further studies.

Domestic cat hepadnavirus associated with hepatopathy in cats: A retrospective study.

BACKGROUND: Whether domestic cat hepadnavirus (DCH) infection is associated with clinical disease remains to be determined. OBJECTIVES: To determine the relationship between DCH detection, hematology, serum bichemistry and liver histology in DCH-positive cats. ANIMALS: One thousand twenty-two cats in Thailand without concurrent diseases and not undergoing treatments adversely affecting the liver. METHODS: Retrospective cross-sectional study. Samples derived from cats with concurrent virus detection were excluded. DCH detection was determined in blood and fresh-frozen liver by quantitative polymerase chain reaction (qPCR) and further investigated in liver sections showing histological parenchymal disorders (HPD) and normal liver (HNL) using in situ hybridization (ISH). Proliferative/apoptotic activities were determined using immunohistochemistry and ISH panels. Biochemical variables and risk factors for DCH infection were investigated. RESULTS: Six hundred sixty-one (557 blood and 119 liver samples) cats were included. DCH was detected in 18.50% (103/557), 13.85% (9/65), and 3.70% (2/54) of the blood, HPD, and HNL groups, respectively. Cats with DCH revealed abnormally high activity of aspartate aminotransferase (AST) (P = .001) and alanine aminotransferase (ALT) (P < .001). Among DCH-positive HPD case 2/9 an 7/9 were acute and chronic hepatitis, of which 4/7 had hepatitis. Log viral copy number (LVCN) was positively correlated with ALT (P < .001), triglyceride (P < .001), and gamma-glutamyl transpeptidase (GGT) (P = .022). The LVCN also had a positive association with degree of hepatitis (P < .05). There was hepatocyte proliferation activity in DHC positive cats. CONCLUSION AND CLINICAL IMPORTANCE: Domestic cat hepadnavirus infection was associated with high serum activity of liver enzymes and chronic lymphoplasmacytic hepatitis (LPH).

Feline morbillivirus-1 in dogs with respiratory diseases.

Feline morbillivirus-1 (FeMV-1) is a viral pathogen associated with kidney disease in domestic cats and wild felids. We initially identified the FeMV-1 from the lung of a necropsied dog with severe pulmonary disease by the reverse transcription polymerase chain reaction (RT-PCR). Thereafter, we investigated FeMV-1 in nasal and oral swab samples from 73 healthy and 113 dogs with respiratory illnesses. We found polymerase chain reaction (PCR)-positive FeMV-1 from only 14/113 (12.39%) dogs with respiratory disease (p = .001). Of these 14 dogs, six were co-infected with other canine respiratory viruses (6/14; 42.86%). Two independent immunohistochemistry procedures, using antibodies against matrix and phosphoprotein of FeMV-1, confirmed the presence of FeMV-1 in lung tissues of two necropsied dogs (out of a total of 22 dogs, 9.09%) that died from respiratory disease. This finding corresponded to transmission electron microscopy findings that paramyxoviral particles exist in lung epithelia. FeMV-1 antigen localization was also evident in the kidney, lymphoid and brain tissues of two deceased dogs. FeMV-1 was successfully isolated from a necropsied dog and from two living dogs, all with respiratory illnesses, which supports FeMV infection in dogs. The detection of FeMV-1 in dog tissues expands the known tropism of this virus to a non-felid host. Our findings indicate that FeMV-1, alone or in co-infection with other viral pathogens, might contribute to respiratory illness and death in dogs.

Canine bocavirus-2 infection and its possible association with encephalopathy in domestic dogs.

Canine bocaviruses (CBoVs) have been recognized as pathogens associated with intestinal diseases. Hematogenous spreading caused by CBoV has been documented and may potentiate the virus entry across the blood-brain barrier to initiate a brain infection. This study focused attention on CBoV detection in cases of encepahlopathy and attempted to determine its viral localization. A total of 107 dog brains that histologically exhibited encephalopathy (ED) were investigated for the presence of CBoVs using polymerase chain reaction (PCR). Thirty-three histologically normal brain samples from dogs were used as a control group (CD). CBoV-2 was detected in 15 ED dogs (14.02%) but not in CD dogs (p = 0.02), while no CBoV-1 and -3 were detected. Among the CBoV-2 positive dogs, brain histological changes were characterized by nonsuppurative encephalitis, with inclusion body-like materials in some brains. In situ hybridization (ISH) and transmission electron microscopy (TEM) confirmed the presence of CBoV-2 viral particles in glial cells, supporting neurotropism of this virus. ISH signals were also detected in the intestines, lymphoid organs, and the heart, suggesting both enteral and parenteral infections of this virus. Whole genome characterization and evolutionary analysis revealed genetic diversity of CBoV-2 sequences and it was varying among the different countries where the virus was detected. This study points to a possible association of CBoV-2 with encephalopathy in dogs. It also highlights the genetic diversity and cellular tropism of this virus.