Investigating comparative polymerase chain reaction for antigen receptor rearrangement analysis in different types of feline lymphoma samples.

Cats have the highest incidence of lymphoma among all animal species. Lymphoma accounts for 41% of all malignant tumors in cats and is responsible for 90% of hematopoietic tumors in felines. Biopsies are considered the gold standard for diagnosis. Polymerase chain reaction (PCR)-based clonality assessment of antigen receptor gene rearrangements can be a valuable complementary tool for identifying infiltrating B-and T-lymphocyte clones. Many studies have focused on intestinal cases but few have addressed mediastinal lymphoma. This study aims to: (1) investigate the clonality patterns of lymphoma samples from various anatomical sites, with a particular focus on mediastinal lymphoma, and (2) evaluate the sensitivity and specificity of the clonality analysis of pleural effusion samples in comparison with cytology, histology, immunohistochemistry, and immunocytochemistry for diagnosing mediastinal lymphoma. There were 82 cases, divided into 49 formalin-fixed and paraffin-embedded biopsy specimens (FFPE), 22 cell pellets, and 11 fresh tissue. This study examined the sensitivity and specificity of PCR for antigen receptor rearrangement (PARR) compared to immunohistochemistry (IHC) and immunocytochemistry. For T-cell receptor gamma chain genes, PARR demonstrated a sensitivity of 58.33% for both fresh tissue and FFPE samples, with a specificity of 100%. Cell pellet analysis exhibited a sensitivity of 64.71% and maintained 100% specificity. A combined analysis of fresh tissue and FFPE with cell pellets showed a sensitivity of 62.07%. For IGH, the sensitivity for fresh tissue and FFPE samples was 56.25%, while cell pellet analysis showed a sensitivity of 62.50%. When considering fresh tissue and FFPE samples, the sensitivity was 57.14%. In conclusion, molecular techniques have emerged as valuable tools for detecting lymphoma, especially in cases where traditional diagnostic methods yield inconclusive results, such as mediastinal lymphoma. While biopsy may not always be feasible, cytology and cell pellets obtained from pleural effusion offer alternative immunocytochemistry and molecular analysis samples, provided they are of sufficient quality and quantity. All sample types considered in this study were suitable for PARR to aid in cases with inconclusive results. Therefore, the sample selection should be tailored to the clinical situation.

Feline bocaviruses found in Thailand have undergone genetic recombination for their evolutions.

Feline bocaviruses (FBoVs) have been discovered for a decade and are often detected in feces, possibly associated with diarrhea in cats. Studies on FBoV evolution remain limited and have mainly focused on prevalence and genetic characterization. Although genetic recombination serves as a potential mechanism in bocavirus evolution, research on this process for FBoVs has been scarce. In this study, we characterized 19 complete coding sequences of FBoVs obtained from Thai cats, revealing that FBoV-1, -2, and -3 were endemic in Thailand. Genetic characterizations showed that most Thai FBoVs were closely related to previously detected strains in Thailand and China. Recombination analyses indicated intragenic, intraspecies recombination in all FBoV species, with recombination breakpoints commonly found in the NP1 and VP1/2 genes, highlighting these genes may be hotspots for FBoV recombination. However, no interspecies recombination was detected. Selective pressure analysis of various FBoV genes revealed that these viruses underwent purifying selection. Although the VP1/2 gene of all FBoV species was under the strongest negative selection pressure, positive selection sites were only found in FBoV-1 and FBoV-3. This study is the first to identify natural recombination in FBoV-2 and FBoV-3 and provides evidence that genetic recombination is a potential driver of FBoV evolutions. Additionally, this study offers up-to-date information on the genetic characteristics, evolutionary dynamics, and selective pressure status of FBoVs, which should be continuously monitored.

Divergent DNA methylation patterns and gene expression in MYC and CDKN2B in canine transmissible venereal tumors.

Background and Aim: Canine transmissible venereal tumor (CTVT), a unique transmissible cancer in dogs, affects the external genitalia and potentially spreads to other parts of the body. While somatic mutations in oncogenic and tumor-suppressing genes are linked to CTVT development, the impact of DNA methylation, which affects gene expression, remains unclear. This study explored whether DNA methylation in the promoter regions of the MYC oncogene and CDKN2B tumor suppressor genes in CTVTs is associated with their expression, both at the gene and protein levels. Materials and Methods: To investigate promoter DNA methylation of MYC and CDKN2B in CTVTs, we analyzed frozen tissue samples from genital CTVT (GTVTs) and extragenital CTVT (ETVTs). Genomic DNA was extracted, bisulfite-treated, and analyzed using bisulfite polymerase chain reaction (PCR) and sequencing. The messenger RNA and protein of MYC and CDKN2B were also extracted and assessed by real-time PCR and Western blotting. Matching formalin-fixed, paraffin-embedded blocks were used for immunohistochemical staining to visualize protein distribution in GTVT and ETVT tissues. Results: Although both GTVT and ETVT samples showed MYC promoter methylation, the extent of methylation differed significantly. GTVTs displayed a much higher degree of methylation, potentially explaining the more pronounced downregulation of MYC gene expression and reduction in c-MYC protein levels observed in GTVTs compared with ETVTs. Our data revealed a prevalent hypermethylation pattern in the CDKN2B promoter across both sample types. However, DNA methylation, which was expected to have a suppressive effect, did not correlate with gene/protein expression. GTVTs displayed high protein levels despite significantly reduced CDKN2B expression. Conversely, ETVTs maintained regular CDKN2B expression but exhibited reduced protein production, suggesting a complex interplay between methylation and expression in these tumors. Conclusion: MYC demonstrated a clear association between its promoter methylation status, gene expression, and protein levels; however, CDKN2B lacked this correlation, implying the involvement of methylation-independent regulatory mechanisms and highlighting the need for further investigation.

Single nucleotide polymorphism profiles of canine T-cell and null-cell lymphomas.

Background: The histopathological classification of T-cell lymphoma (TCL) in humans has distinctive mutational genotyping that suggests different lymphomagenesis. A similar concept is assumed to be observed in dogs with different TCL phenotypes. Objective: This study aimed to identify the previously reported single-nucleotide polymorphisms (SNPs) in both human beings and dogs in canine TCLs and null-cell lymphomas (NCLs) and to design compatible oligonucleotides from each variant based on the multiplex polymerase chain reaction. Methods: Genomic DNA was extracted from 68 tumor specimens (62 TCLs and 6 NCLs) and 5 buffy coat samples from dogs with TCL. Four TCL subtypes and NCL were analyzed in 44 SNPs from 21 genes using the MassARRAY. Results: The greatest incidences of SNPs observed in all TCL subtypes and NCL ware SATB1 c.1259A > C, KIT c.1275A > G, SEL1L c.2040 + 200C > G, and TP53 c.1024C > T, respectively. Some SNP locations were statistically significant associated with NCL, including MYC p.S75F (p = 0.0003), TP53 p.I149N (p = 0.030), PDCD1 p.F37LX (p = 0.012), and POT1 p.R583* (p = 0.012). Conclusion: Each TCL histological subtype and NCL are likely to contain distinctive mutational genetic profiles, which might play a role in lymphoma gene-risk factors and might be useful for selecting therapeutic target drugs for each canine patient.

Detection of Antibodies against Feline Morbillivirus by Recombinant Matrix Enzyme-Linked Immunosorbent Assay.

Feline morbillivirus (FeMV) has been associated with feline health, although its exact role in pathogenesis is still debated. In this study, an indirect enzyme-linked immunosorbent assay (i-ELISA) targeting a recombinant matrix protein of FeMV (rFeMV-M) was developed and assessed in comparison to a Western blotting (WB) assay. The i-ELISA was evaluated using blood samples from 136 cats that were additionally tested with real-time reverse-transcription PCR (RT-qPCR). The i-ELISA exhibited a sensitivity of 90.1%, specificity of 75.6%, positive predictive value of 88.2%, and negative predictive value of 79.1%. The agreement between i-ELISA and WB analyses was substantial (a κ coefficient of 0.664 with a 95% confidence interval of 0.529 to 0.799). Within the study group, 68.4% (93/136) of the cats were serologically positive in the i-ELISA and 66.9% (91/136) in the WB assay, with 11.8% (11/93) of false positivity with the i-ELISA. However, only 8.1% (11/136) of the cats tested positive for FeMV using RT-qPCR (p < 0.001). The developed i-ELISA proved effective in identifying FeMV-infected cats and indicated the prevalence of FeMV exposure. Combining FeMV antibody detection through i-ELISA with FeMV RT-qPCR could offer a comprehensive method to determine and monitor FeMV infection status. Nevertheless, this assay still requires refinement due to a significant number of false positive results, which can lead to the misdiagnosis of cats without antibodies as having antibodies. This study also provided the first evidence of seroprevalence against FeMV among cat populations in Thailand, contributing valuable insights into the geographic distribution and prevalence of this virus.

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.

Molecular epidemiology and strain diversity of circulating feline Calicivirus in Thai cats.

Feline calicivirus (FCV) is a significant viral pathogen causing upper respiratory tract and oral diseases in cats. The emergence of the virulent systemic FCV variant (VS-FCV) has raised global concern in the past decade. This study aims to explore the epidemiology, genetic characterization, and diversity of FCV strains circulating among Thai cats. Various sample types, including nasal, oral, and oropharyngeal swabs and fresh tissues, were collected from 184 cats across different regions of Thailand from 2016 to 2021. Using reverse transcription real-time polymerase chain reaction (RT-qPCR), FCV infection was investigated, with additional screening for feline herpesvirus-1 (FHV-1) by qPCR. The detection rates for FCV, FHV-1, and co-infection were 46.7, 65.8, and 31.5%, respectively. Significantly, the odds ratio (OR) revealed a strong association between the detection of a single FCV and the presence of gingivostomatitis lesions (OR: 7.15, 95% CI: 1.89-26.99, p = 0.004). In addition, FCV detection is notably less likely in vaccinated cats (OR: 0.22, 95% CI: 0.07-0.75, p = 0.015). Amino acid sequence analysis based on the VP1 major capsid protein gene of the 14 FCV-Thai (FCV-TH) strains revealed genetic diversity compared to the other 43 global strains (0 to 86.6%). Intriguingly, a vaccine-like FCV variant was detected in one cat. In summary, this study provides insights into the epidemiology and molecular characteristics of FCV diversity within the Thai cat population for the first time. The identification of unique physicochemical characteristics in the capsid hypervariable region of some FCV-TH strains challenges previous hypotheses. Therefore, further exploration of vaccine-like FCV variants is crucial for a comprehensive understanding and to improve viral prevention and control strategies.

Evolutionary dynamics of canine kobuvirus in Vietnam and Thailand reveal the evidence of viral ability to evade host immunity.

Canine kobuvirus (CaKoV) is a pathogen associated with canine gastrointestinal disease (GID). This study examined 327 rectal swabs (RS), including 113 from Vietnam (46 healthy, 67 with GID) and 214 from Thailand (107 healthy and 107 with GID). CaKoV was detected in both countries, with prevalences of 28.3% (33/113) in Vietnam and 7.9% (17/214) in Thailand. Additionally, CaKoV was found in both dogs with diarrhea and healthy dogs. CaKoV was mainly found in puppies under six months of age (30.8%). Co-detection with other canine viruses were also observed. The complete coding sequence (CDS) of nine Vietnamese and four Thai CaKoV strains were characterized. Phylogenetic analysis revealed a close genetic relationship between Vietnamese and Thai CaKoV strains, which were related to the Chinese strains. CDS analysis indicated a distinct lineage for two Vietnamese CaKoV strains. Selective pressure analysis on the viral capsid (VP1) region showed negative selection, with potential positive selection sites on B-cell epitopes. This study, the first of its kind in Vietnam, provides insights into CaKoV prevalence in dogs of different ages and healthy statuses, updates CaKoV occurrence in Thailand, and sheds light on its molecular characteristics and immune evasion strategies.

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.

Diagnosis, isolation and description of a novel amnoonvirus recovered from diseased fancy guppies, Poecilia reticulata.

The guppy, Poecilia reticulata, is one of the most common cultured ornamental fish species, and a popular pet fish highly desired by hobbyists worldwide due to its availability of many brilliantly coloured fish of many varieties. The susceptibility of guppies to diseases presents a remarkable concern for both breeders and hobbyists. In this study, we report the emergence of disease in fancy guppies caused by a previously uncharacterized virus in the USA. This virus was isolated from moribund guppies in two separate outbreaks in California and Alabama, from December 2021 to June 2023. The infected guppies presented with acute morbidity and mortality shortly after shipping, displaying nonspecific clinical signs and gross changes including lethargy, anorexia, swimming at the water surface, gill pallor, mild to moderate coelomic distension and occasional skin lesions including protruding scales, skin ulcers and hyperaemia. Histological changes in affected fish were mild and nonspecific; however, liver and testes from moribund fish were positive for Tilapia lake virus (TiLV), the single described member in the family Amnoonviridae, using immunohistochemistry and in situ hybridization, although the latter was weak. A virus was successfully recovered following tissue inoculation on epithelioma papulosum cyprini and snakehead fish cell lines. Whole genome sequencing and phylogenetic analyses revealed nucleotide and amino acid homologies from 78.3%-91.2%, and 78.2%-97.7%, respectively, when comparing the guppy virus genomes to TiLV isolates. Based on the criteria outlined herein, we propose the classification of this new virus, fancy tailed guppy virus (FTGV), as a member of the family Amnoonviridae, with the name Tilapinevirus poikilos (from the Greek 'poikilos', meaning of many colours; various sorts, akin to 'poecilia').

Anticoagulant rodenticide poisoning in farmed Patagonian mara (Dolichotis patagonum).

BACKGROUND: Anticoagulant rodenticide (AR) poisoning was diagnosed in 3 Patagonian maras (Dolichotis patagonum) raised in the mara farm in Thailand. To date, there have been no reports of maras with diagnosed AR poisoning. CASE PRESENTATION: The first clinical sign of the sickening maras was anorexia. Fifteen from 50 maras were dead over a 3-5 day period after the clinical signs had occurred. Positive results to AR were detected in all of the maras' liver specimens by screening test using thin layer chromatography and spectrophotometry methods. Supportive therapy was selected for the treatment of the 35 surviving maras. During the follow - up observation period of 12 months, all of the surviving maras were healthy and no reproductive loss. CONCLUSIONS: This is the first report on suspected AR poisoning in maras in Thailand based on history taking, clinical signs, gross pathology lesions and chemical analysis. AR poisoning in the present report is possibly from contaminated animal food. Therefore, quality control of food should be fastidious when feeding maras.

Preparation of Decellularized Tissue as Dual Cell Carrier Systems: A Step Towards Facilitating Re-epithelization and Cell Encapsulation for Tracheal Reconstruction.

Surgical treatment of tracheal diseases, trauma, and congenital stenosis has shown success through tracheal reconstruction coupled with palliative care. However, challenges in surgical-based tracheal repairs have prompted the exploration of alternative approaches for tracheal replacement. Tissue-based treatments, involving the cultivation of patient cells on a network of extracellular matrix (ECM) from donor tissue, hold promise for restoring tracheal structure and function without eliciting an immune reaction. In this study, we utilized decellularized canine tracheas as tissue models to develop two types of cell carriers: a decellularized scaffold and a hydrogel. Our hypothesis posits that both carriers, containing essential biochemical niches provided by ECM components, facilitate cell attachment without inducing cytotoxicity. Canine tracheas underwent vacuum-assisted decellularization (VAD), and the ECM-rich hydrogel was prepared through peptic digestion of the decellularized trachea. The decellularized canine trachea exhibited a significant reduction in DNA content and major histocompatibility complex class II, while preserving crucial ECM components such as collagen, glycosaminoglycan, laminin, and fibronectin. Scanning electron microscope and fluorescent microscope images revealed a fibrous ECM network on the luminal side of the cell-free trachea, supporting epithelial cell attachment. Moreover, the ECM-rich hydrogel exhibited excellent viability for human mesenchymal stem cells encapsulated for 3 days, indicating the potential of cell-laden hydrogel in promoting the development of cartilage rings of the trachea. This study underscores the versatility of the trachea in producing two distinct cell carriers-decellularized scaffold and hydrogel-both containing the native biochemical niche essential for tracheal tissue engineering applications.

Domestic Cat Hepadnavirus Antigens in Lymphoma Tissues. Comment on Beatty et al. Domestic Cat Hepadnavirus and Lymphoma. Viruses 2023, 15, 2294.

We are addressing the comments made by Beatty et al [...].

Canine bufavirus (Carnivore protoparvovirus-3) infection in dogs with respiratory disease.

Canine bufavirus (CBuV) or Carnivore protoparvovirus-3, a nonenveloped DNA virus belonging to the genus Protoparvovirus, family Parvoviridae, has been identified in dogs with respiratory and enteric diseases. Although CBuV detection has been reported in multiple countries, descriptions of pathologic findings associated with infection have not yet been provided. In this study, the authors necropsied 14 dogs (12 puppies and 2 adult dogs) from a breeding colony that died during multiple outbreaks of respiratory diseases. Postmortem investigations revealed extensive bronchointerstitial pneumonia with segmental type II pneumocyte hyperplasia in all necropsied puppies but less severe lesions in adults. With negative results of common pathogen detection by ancillary testing, CBuV DNA was identified in all investigated dogs using a polymerase chain reaction (PCR). Quantitative PCR demonstrated CBuV DNA in several tissues, and in situ hybridization (ISH) indicated CBuV tissue localization in the lung, tracheobronchial lymph node, and spinal cord, suggesting hematogenous spread. Dual CBuV ISH and cellular-specific immunohistochemistry were used to determine the cellular tropism of the virus in the lung and tracheobronchial lymph node, demonstrating viral localization in various cell types, including B-cells, macrophages, and type II pneumocytes, but not T-cells. Three complete CBuV sequences were successfully characterized and revealed that they clustered with the CBuV sequences obtained from dogs with respiratory disease in Hungary. No additional cases were identified in small numbers of healthy dogs. Although association of the bufavirus with enteric disease remains to be determined, a contributory role of CBuV in canine respiratory disease is possible.

Natural SARS-CoV-2 infection in dogs: Determination of viral loads, distributions, localizations, and pathology.

Instances of reverse zoonosis involving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been documented in both controlled experiments and spontaneous cases. Although dogs are susceptible to infection, clinical significance is limited to mild or asymptomatic. Here, we investigate the fatal cases of natural SARS-CoV-2 infection in dogs in Thailand. Pathological findings of SARS-CoV-2-infected dogs reveal severe diffuse alveolar damage, pulmonary hyalinization and fibrosis, and syncytial formation, together with minor lesions in brain and kidney. Employing reverse transcription-digital PCR, substantial viral loads of SARS-CoV-2 were detected in lung, kidney, brain, trachea, tonsil, tracheobronchial lymph node, liver, and intestine, respectively. Localization of SARS-CoV-2 within various tissues was examined through immunohistochemistry (IHC), where the co-localization of the viral spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor was illustrated using double IHC. SARS-CoV-2 localization was markedly identified in the epithelial cells of the lung, trachea, intestine and kidneys, and moderately presented in the salivary gland and gall bladder, where the co-localization with the ACE2 was also evident. Neurons in the brainstem where exhibited lymphocytic perivascular cuffing were also found to be positive for SARS-CoV-2 in IHC testing, despite lacking ACE2 receptor expression. In addition, SARS-CoV-2 replication within the lungs of infected dogs was confirmed by transmission electron microscopy, visualizing free viral particles within the cytosol or the endoplasmic reticulum of syncytial cells within the lung. This study considerably expanded on the knowledge of the pathology associated with natural SARS-CoV-2 infection in dogs, a scenario that is relatively infrequent but occasionally leads to fatal outcome. Furthermore, these findings suggest the potential utility of dogs as a model for studying SARS-CoV-2 infection in humans, warranting further investigation.

Genetic characterization of canine astrovirus in non-diarrhea dogs and diarrhea dogs in Vietnam and Thailand reveals the presence of a unique lineage.

The role of canine astrovirus (CaAstV) in canine gastrointestinal disease (GID) is unknown. In this study, a total of 327 fecal swab (FS) samples were collected, including 113 FSs in Vietnam (46 samples from healthy dogs and 67 samples from GID dogs) and 214 FSs in Thailand (107 samples from healthy dogs and 107 samples from GID dogs). Overall, the prevalence of CaAstV in Vietnam and Thailand was 25.7% (29/113) and 8.9% (19/214), respectively. CaAstV was detected in both non-diarrhea dogs (21.7 and 7.5%) and diarrhea dogs (28.4% and 10.3%), respectively, in Vietnam and Thailand. In both countries, CaAstV was frequently detected in puppies under 6 months of age (23.3%) (p = 0.02). CaAstV-positive samples in Vietnam and Thailand were identified as co-infected with canine parvovirus, canine enteric coronavirus, canine distemper virus, and canine kobuvirus. The complete coding sequence of seven Vietnamese CaAstV and two Thai CaAstV strains were successfully characterized. Phylogenetic analyses showed that Vietnamese and Thai CaAstV strains were genetically close to each other and related to the Chinese strains. Furthermore, analysis of complete coding sequences indicated that the OR220030_G21/Thailand/2021 strain formed a unique lineage, whereas no recombination event was found in this study, suggesting that this strain might be an original lineage. In summary, this is the first study to report the presence of CaAstV in dogs with and without diarrhea in Vietnam and Thailand, and it was most often found in puppies with diarrhea. Our results highlight the importance of the CaAstV in dog populations and the need for continued surveillance of these emerging pathogens.

High-resolution melting analysis for simultaneous detection and discrimination between wild-type and vaccine strains of feline calicivirus.

High-resolution melting (HRM) analysis, a post-polymerase chain reaction (PCR) application in a single closed tube, is the straightforward method for simultaneous detection, genotyping, and mutation scanning, enabling more significant dynamic detection and sequencing-free turnaround time. This study aimed to establish a combined reverse-transcription quantitative PCR and HRM (RT-qPCR-HRM) assay for diagnosing and genotyping feline calicivirus (FCV). This developed method was validated with constructed FCV plasmids, clinical swab samples from living cats, fresh-frozen lung tissues from necropsied cats, and four available FCV vaccines. We performed RT-qPCR to amplify a 99-base pair sequence, targeting a segment between open reading frame (ORF) 1 and ORF2. Subsequently, the HRM assay was promptly applied using Rotor-Gene Q® Software. The results significantly revealed simultaneous detection and genetic discrimination between commercially available FCV vaccine strains, wild-type Thai FCV strains, and VS-FCV strains within a single PCR reaction. There was no cross-reactivity with other feline common viruses, including feline herpesvirus-1, feline coronavirus, feline leukemia virus, feline immunodeficiency virus, and feline morbillivirus. The detection limit of the assay was 6.18 × 101 copies/µl. This study, therefore, is the first demonstration of the uses and benefits of the RT-qPCR-HRM assay for FCV detection and strain differentiation in naturally infected cats.

Domestic cat hepadnavirus detection in blood and tissue samples of cats with lymphoma.

Domestic cat hepadnavirus (DCH), a relative hepatitis B virus (HBV) in human, has been recently identified in cats; however, association of DCH infection with lymphoma in cats is not investigated. To determine the association between DCH infection and feline lymphoma, seven hundred and seventeen cats included 131 cats with lymphoma (68 blood and 63 tumor samples) and 586 (526 blood and 60 lymph node samples) cats without lymphoma. DCH DNA was investigated in blood and formalin-fixed paraffin-embedded (FFPE) tissues by quantitative polymerase chain reaction (qPCR). The FFPE lymphoma tissues were immunohistochemically subtyped, and the localization of DCH in lymphoma sections was investigated using in situ hybridization (ISH). Feline retroviral infection was investigated in the DCH-positive cases. DCH DNA was detected in 16.18% (11/68) (p = 0.002; odds ratio [OR], 5.15; 95% confidence interval [CI], 2.33-11.36) of blood and 9.52% (6/63) (p = 0.028; OR, 13.68; 95% CI, 0.75-248.36) of neoplastic samples obtained from lymphoma cats, whereas only 3.61% (19/526) of blood obtained from non-lymphoma cats was positive for DCH detection. Within the DCH-positive lymphoma, in 3/6 cats, feline leukemia virus was co-detected, and in 6/6 were B-cell lymphoma (p > 0.9; OR, 1.93; 95% CI, 0.09-37.89) and were multicentric form (p = 0.008; OR, 1.327; 95% CI, 0.06-31.18). DCH was found in the CD79-positive pleomorphic cells. Cats with lymphoma were more likely to be positive for DCH than cats without lymphoma, and infection associated with lymphoma development needs further investigations.

Canine respiratory coronavirus in Thailand undergoes mutation and evidences a potential putative parent for genetic recombination.

Canine respiratory coronavirus (CRCoV) is associated with canine infectious respiratory disease complex. Although its detection has been reported worldwide, the genomic characteristics and evolutionary patterns of this virus remain poorly defined. In this study, 21 CRCoV sequences obtained from dogs in Thailand during two episodes (2013-2015, group A; 2021-2022, group B) were characterized and analyzed. The genomic characteristics of Thai CRCoVs changed from 2013 to 2022 and showed a distinct phylogenetic cluster. Phylogenetic analysis of the spike (S) genes divided the analyzed CRCoV strains into five clades. The full-length genome characterization revealed that all Thai CRCoVs possessed a nonsense mutation within the nonstructural gene located between the S and envelope genes, leading to a truncated putative nonstructural protein. Group B Thai CRCoV strains represented the signature nonsynonymous mutations in the S gene that was not identified in group A Thai CRCoVs, suggesting the ongoing evolutionary process of Thai CRCoVs. Although no evidence of recombination of Thai CRCoV strains was found, our analysis identified one Thai CRCoV strain as a potential parent virus for a CRCoV strain found in the United States. Selective pressure analysis of the hypervariable S region indicated that the CRCoV had undergone purifying selection during evolution. Evolutionary analysis suggested that the CRCoV was emerged in 1992 and was first introduced in Thailand in 2004, sharing a common ancestor with Korean CRCoV strains. These findings regarding the genetic characterization and evolutionary analysis of CRCoVs add to the understanding of CRCoVs. IMPORTANCE Knowledge of genomic characterization of the CRCoV is still limited and its evolution remains poorly investigated. We, therefore, investigated the full-length genome of CRCoV in Thailand for the first time and analyzed the evolutionary dynamic of CRCoV. Genomic characterization of Thai CRCoV strains revealed that they possess unique genome structures and have undergone nonsynonymous mutations, which have not been reported in previously described CRCoV strains. Our work suggests that the Thai CRCoVs were not undergone mutation through genetic recombination for their evolution. However, one Thai CRCoV strain PP158_THA_2015 was found to be a potential parent virus for the CRCoV strains found in the United States. This study provides an understanding of the genomic characterization and highlights the signature mutations and ongoing evolutionary process of CRCoV that could be crucial for monitoring in the future.

Quantification of domestic cat hepadnavirus DNA in various body fluid specimens of cats: the potential viral shedding routes.

Domestic cat hepadnavirus (DCH) belongs to the Hepadnaviridae family together with human hepatitis B virus (HBV) that remains to be a major health problem worldwide. The transmission of HBV infectious virion has been one of the essential factors that contribute to high number of HBV infection in humans. It has been long known that various body fluid specimens of human with chronic HBV infection contain HBV DNA and demonstrated to be infectious. In contrast to this knowledge, the detection of DCH in various body fluid specimens of cats, has not been reported. This study explored the detection of DCH DNA in various body fluid specimens of cats by quantitative polymerase chain reaction (qPCR) and investigated whether the detection of DCH DNA from broader routes was correlated with any genomic diversity by phylogenetic analysis. A total of 1,209 body fluid specimens were included, and DCH DNA was detected not only in 4.70% (25/532) of blood samples; but also in 12.5% (1/8), 1.14% (1/88), 2.54% (10/394), and 1.65% (3/182) of auricular swab (AS), nasal swab (NS), oral swab (OS), and rectal swab (RS) specimens, respectively. Furthermore, the level of DCH DNA detected in the blood was significantly correlated with DCH DNA detection in OS (P = 0.02) and RS (P = 0.04) specimens. Genomic analysis revealed that there was no notable genomic diversity within the complete genome sequences obtained in this study. In conclusion, this study highlighted the presence of DCH DNA in various body fluid specimens of cats, and the potential role of these specimens in DCH horizontal transmission within the cat population warrants further studies.