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').

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 [...].

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.

Tilapia lake virus (TiLV) causes severe anaemia and systemic disease in tilapia.

Tilapia lake virus disease (TiLVD) is an emerging disease in tilapia that is associated with mass mortality affecting global tilapia aquaculture. In this study, red hybrid tilapias (Oreochromis spp.) were experimentally infected by intracoelomic injection with Tilapia lake virus (TiLV) to gain a better understanding of the clinicopathological changes during infection. Pale bodies and gill were observed in infected fish after 7 days of post-challenge (dpc) associated with severe anaemia. Further haematological analysis in TiLV-infected fish revealed decreased levels of haemoglobin and haematocrit at 3 dpc. Common pathological findings included pale and friable liver, pale intestine with catarrhal content, and dark and shrunken spleen in TiLV-infected fish at 7 dpc and 14 dpc. Histologically, reduced numbers of red blood cells and accumulation of melano-macrophage centre in the spleen were found in infected fish at 3 dpc, and severe lesions were more commonly observed at 7 and 14 dpc. Lymphocyte infiltration, syncytial cell formation and multifocal necrotic hepatitis were the prominent pathological findings in the liver of infected fish. The severity of pathological changes was associated with TiLV-infection with higher viral loads and with the expression pattern of pro-inflammatory cytokines and antiviral genes, including interferon regulatory factor 1 (irf1), interleukin (il-8), radical s-adenosyl methionine domain containing 2 (rsad2) and mx. Our study provides a comprehensive analysis of the haematological profile and pathological changes in tilapia during TiLV infection. Overall, lesions present in various organs, together with alteration of host immune response in TiLV-infected fish, indicate the systemic infection of this virus. The knowledge gained from this study improves our understanding of how TiLV causes pathological and haematological changes in tilapia.

Detection and molecular characterization of two canine circovirus genotypes co-circulating in Vietnam.

BACKGROUND: Canine circovirus is reported in dogs in many countries, including the USA, China and Thailand. It has been detected in healthy dogs and dogs with diarrhea, hemorrhagic gastroenteritis, and vasculitis. It comprises five genotypes and is frequently found as a coinfection with canine parvovirus-2 (CPV-2). AIM: To characterize canine circovirus genotypes co-circulating with CPV-2 in Vietnam. METHOD: PCR assessment of 81 CPV-2-positive fecal samples from Vietnamese diarrheic dogs up to seven months of age for other viral enteric pathogens, including canine bocavirus, canine adenovirus, paramyxovirus, canine coronavirus, porcine circovirus-3 and canine circovirus. In addition, eight selected full genome sequences of Vietnamese canine circovirus were analyzed and used for phylogeny. RESULTS: In total 19.8% of samples were found to be positive for canine circovirus. Phylogeny revealed that the Vietnamese canine circovirus strains were clustered in two different genotypes (genotype-1 and -3). The genetic diversity among Vietnamese canine circovirus was 86.0-87.2%. The nucleotide discrepancy among both genotypes altered the deduced amino acid sequence in 14 and ten residues of the replicase and capsid proteins, respectively. Genetic recombination analysis revealed that the Vietnamese canine circovirus-6 strain has the American and Chinese canine circovirus as its major and minor parents, respectively. Only a single dog revealed triple detections of CPV-2c, Canine circovirus and canine adenovirus (1.2%). CONCLUSION: The co-circulation of two different genotypes of canine circovirus and CPV-2c in dogs in Vietnam has been illustrated. CLINICAL RELEVANCE: The mortality rate with CPV-2 only (22%) doubled in dogs with canine circovirus and CPV-2 co-infection.

Infection of Tilapia tilapinevirus in Mozambique Tilapia (Oreochromis mossambicus), a Globally Vulnerable Fish Species.

Tilapia tilapinevirus, or tilapia lake virus (TiLV), is a highly contagious virus found in tilapia and its hybrid species that has been reported worldwide, including in Asia, the Americas, and Africa. In this study, we experimentally challenged Mozambique tilapia (Oreochromis mossambicus) with a virulent TiLV strain, VETKU-TV01, at both low (1 × 103 TCID50/mL) and high (1 × 105 TCID50/mL) concentration. After the challenge, the Mozambique tilapia showed pale skin with some hemorrhage and erosion, lethargy, abdominal swelling, congestion around the eye, and exophthalmos; there was a cumulative mortality rate at 48.89% and 77.78% in the groups that received the low and high concentration, respectively. Quantitative PCR and in situ hybridization confirmed the presence of TiLV in the internal organs of moribund fish. Notably, severe histopathological changes, including glycogen depletion, syncytial hepatic cells containing multiple nuclei and intracytoplasmic inclusion bodies, and infiltration of melanomacrophage into the spleen, were frequently found in the Mozambique tilapia challenged with high TiLV concentration. Comparatively, the infectivity and pathology of the TiLV infection in Mozambique tilapia and red hybrid tilapia (Oreochromis spp.) were found to be similar. Our results confirmed the susceptibility of Mozambique tilapia, which has recently been determined to be a vulnerable species, to TiLV infection, expanding knowledge that the virus can cause disease in this fish species.

Cerebral cysticercosis in a wild Bengal tiger (Panthera tigris tigris) in Bhutan: A first report in non-domestic felids.

The endangered Bengal tiger (Panthera tigris tigris) is a keystone species playing an essential role in ecology as well as in the social and spiritual lives of the Himalayan people. The latest estimate of the Bengal tiger population in Bhutan accounts for 103 individuals. Infectious organisms, including zoonotic parasites causing high burden in human health, have received little attention as a cause of mortality in tigers. Taeniosis/cysticercosis, caused by the cestode Taenia solium, is considered one of the major neglected tropical diseases in Southeast Asia. We present here a case of neurocysticercosis in a Bengal tiger showing advanced neurological disease outside Thimphu, the capital city of Bhutan. After palliative care, the animal died, and necropsy revealed multiple small cysts in the brain. Here we show the presence of two genetic variants of T. solium in the parasite material collected based on PCR and sequencing of the complete cox1 and cytB genes. The sequences form a discrete branch within the Asia plus Madagascar cluster of the parasite. On other hand, tests for feline morbillivirus, feline calicivirus, canine distemper virus, Nipah, rabies, Japanese encephalitis, feline leukaemia and feline immunodeficiency virus were negative. In contrast, PCR for feline herpesvirus was positive and a latex agglutination test revealed an elevated antibody titer against Toxoplasma gondii (titer 1:256). The molecular examination of taeniid eggs isolated from the tiger faeces produced sequences for which the highest homology in GenBank is between 92% and 94% with T. regis and T. hydatigena. This fatal case of T. solium neurocysticercosis, a disease previously unrecorded in tigers or other non-domestic felids, demonstrates an anthropogenically driven transmission of a deadly pathogen which could become a serious threat to the tiger population.

Insights into the genetic diversity, recombination, and systemic infections with evidence of intracellular maturation of hepadnavirus in cats.

Hepatitis B virus (HBV) is a human pathogen of global concern, while a high diversity of viruses related to HBV have been discovered in other animals during the last decade. Recently, the novel mammalian hepadnavirus, tentatively named domestic cat hepadnavirus (DCH), was detected in an immunocompromised cat. Herein, a collection of 209 cat sera and 15 hepato-diseased cats were screened for DCH using PCR, resulting in 12.4% and 20% positivity in the tested sera and necropsied cats, respectively. Among the DCH-positive sera, a significantly high level of co-detection with retroviral infection was found, with the highest proportion being co-detection with feline immunodeficiency virus (FIV). Full-length genome characterization of DCH revealed the genetic diversity between the nine Thai DCH sequences obtained, and that they phylogenetically formed three distinct monophyletic clades. A putative DCH recombinant strain was found, suggesting a possible role of recombination in DCH evolution. Additionally, quantitative PCR was used to determine the viral copy number in various organs of the DCH-moribund cats, while the pathological findings were compared to the viral localization in hepatocytes, adjacent to areas of hepatic fibrosis, by immunohistochemical (IHC) and western blot analysis. In addition to the liver, positive-DCH immunoreactivity was found in various other organs, including kidneys, lung, heart, intestine, brain, and lymph nodes, providing evidence of systemic infection. Ultrastructure of infected cells revealed electron-dense particles in the nucleus and cytoplasm of hepatocytes, bronchial epithelial cells, and fibroblasts. We propose the intracellular development mechanism of this virus. Although the definitive roles of pathogenicity of DCH remains undetermined, a contributory role of the virus associated with systemic diseases is possible.

Genetic Adaptations, Biases, and Evolutionary Analysis of Canine Distemper Virus Asia-4 Lineage in a Fatal Outbreak of Wild-Caught Civets in Thailand.

Canine morbillivirus (CDV) is a serious pathogen that can cause fatal systemic disease in a wide range of domestic and wildlife carnivores. Outbreaks of CDV in wildlife species lead to questions regarding the dispersal of the CDV origin. In the present study, we identified a fatal CDV outbreak in caged wild-caught civets in Thailand. Full-length genetic analysis revealed that CDV from the Asia-4 lineage served as the likely causative agent, which was supported by the viral localization in tissues. Evolutionary analysis based on the CDV hemagglutinin (H) gene revealed that the present civet CDV has co-evolved with CDV strains in dogs in Thailand since about 2014. The codon usage pattern of the CDV H gene revealed that the CDV genome has a selective bias of an A/U-ended codon preference. Furthermore, the codon usage pattern of the CDV Asia-4 strain from potential hosts revealed that the usage pattern was related more to the codon usage of civets than of dogs. This finding may indicate the possibility that the discovered CDV had initially adapted its virulence to infect civets. Therefore, the CDV Asia-4 strain might pose a potential risk to civets. Further epidemiological, evolutionary, and codon usage pattern analyses of other CDV-susceptible hosts are required.

Cross-sectional investigation and risk factor analysis of community-acquired and hospital-associated canine viral infectious respiratory disease complex.

Canine infectious respiratory disease complex (CIRDC) is associated with multiple factors. The possible transmission source can be via community-acquired infection (CAI) or hospital-associated infection (HAI), but the variable factors within these two routes are not well described. This study aimed to (i) investigate a cross-sectional incidence of canine respiratory viruses, including influenza (CIV), parainfluenza, distemper (CDV), respiratory coronavirus (CRCoV), adenovirus-2, and herpesvirus, in respiratory-diseased dogs, and (ii) analyze the possibly related risk factors. In total 209 dogs with respiratory illness, consisting of 133 CAI and 76 HAI dogs, were studied. Both nasal and oropharyngeal swabs were sampled from each dog and subjected for CIRDC virus detection using multiplex PCRs. Common six viruses associated with CIRDC were detected in both groups with CIV and CRCoV being predominantly found. Only CDV was significantly more prevalent in CAI than HAI dogs. Multiple virus detections were found in 81.2% and 78.9% of CAI and HAI dogs, respectively. Co-detection of CIV and CRCoV was represented the highest proportion and most often found with other CIRD viruses. Moreover, the clinical severity level was notably related to the age of infected dogs, but not to the vaccination status, sex and transmission route. Since healthy or control dogs were not included in this study, the prevalence of the CIRD virus infections could not be assessed.

Epizootic of multi-centric, squamous cell carcinomas in populations of Indo-Pacific humpbacked dolphins Sousa chinensis in Thai waters.

Over the span of several years, 3 Indo-Pacific humpbacked dolphins died and were necropsied in Thailand. These 3 animals were all captive-bred at Oasis Sea World (Chanthaburi, Thailand), and displayed similar macroscopic progressive cutaneous lesions diagnosed as squamous cell carcinomas. In 2 of the 3 animals, necropsy revealed a severe fibrinosuppurative tracheitis and pneumonia secondary to metastasis of a cutaneous squamous cell carcinoma which extended from the head throughout the trunk and flippers. The tumors were characterized by coalescing botryoid masses with severe areas of cutaneous erosion, ulceration and necrohemorrhagic dermatitis. There was evidence of metastasis to the lungs and hilar lymph nodes. Necropsy of the third animal revealed similar progressive cutaneous squamous cell carcinomas but without evidence of metastasis. DNA molecular analysis of homogenized neoplastic tissue was conducted using polymerase chain reaction for both herpesvirus and papillomavirus in 2 of the 3 cases. In the first case, the tissues were positive for a herpesvirus alone, and this was phylogenetically classified as an alphaherpesvirus. This new herpesvirus has been tentatively named Sousa chinensis alphaherpesvirus. The second animal was negative for this novel herpesvirus and the third was not analyzed. In addition to the captive population, there is photographic evidence from 2 separate wild populations of Indo-Pacific humpbacked dolphins in the Gulf of Thailand, of a macroscopically identical proliferative and ulcerative process suspected to be squamous cell carcinomas.

Development and application of multiplex PCR assays for detection of virus-induced respiratory disease complex in dogs.

Canine infectious respiratory disease complex (CIRDC) viruses have been detected in dogs with respiratory illness. Canine influenza virus (CIV), canine parainfluenza virus (CPIV), canine distemper virus (CDV), canine respiratory coronavirus (CRCoV), canine adenovirus type 2 (CAdV-2) and canine herpesvirus 1 (CaHV-1), are all associated with the CIRDC. To allow diagnosis, two conventional multiplex polymerase chain reactions (PCR) were developed to simultaneously identify four RNA and two DNA viruses associated with CIRDC. The two multiplex PCR assays were then validated on 102 respiratory samples collected from 51 dogs with respiratory illness by sensitivity and specificity determination in comparison to conventional simplex PCR and a rapid three-antigen test kit. All six viruses were detected in either individual or multiple infections. The developed multiplex PCR assays had a >87% sensitivity and 100% specificity compared to their simplex counterpart. Compared to the three-antigen test kit, the multiplex PCR assays yielded 100% sensitivity and more than 83% specificity for detection of CAdV-2 and CDV, but not for CIV. Therefore, the developed multiplex PCR modalities were able to simultaneously diagnose a panel of CIRDC viruses and facilitated specimen collection through being suitable for use of nasal or oral samples.

Anti-diabetic and anti-oxidative activity of fixed oil extracted from Ocimum sanctum L. leaves in diabetic rats.

Ocimum sanctum L. (OS) leaves have been shown to exert diverse potential benefits in a variety of stress conditions. The present study was conducted to elucidate the effects of the fixed oil extracted from OS leaves on the blood glucose levels and serum lipid profile of streptozotocin-induced diabetic rats. In addition, the anti-oxidative activity of OS leaves to protect various organs including the liver, kidney and heart was investigated. The fixed oil of the OS leaves was extracted using hexane, and the various fatty acid contents of the oil were determined using gas chromatography-mass spectrometry. Male Wistar rats were allocated into three groups (n=7 per group): Normal control rats, diabetic rats and diabetic rats fed daily with the fixed oil for three weeks. The results showed that α-linolenic acid was the primary fatty acid contained in the fixed oil of OS. After 3 weeks of diabetic induction, the rats exhibited increased blood glucose levels and serum lipid profile, in addition to elevated serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), creatine kinase MB subunit (CK-MB), creatinine and blood urea nitrogen (BUN). The fixed oil significantly decreased the elevated levels of blood glucose, the serum lipid profile and the levels of serum creatinine and BUN (P<0.001), without exerting significant effects on the elevated serum levels of AST, ALT, LDH and CK-MB. Furthermore, the fixed oil increased the diabetically-reduced levels of serum insulin and decreased the rat kidney weight. Fixed oil suppressed the elevated thiobarbituric acid reactive substances (TBARS) level and increased the activity of various antioxidative enzymes in the rat renal tissue. By contrast, the fixed oil had no effect on the elevated TBARS level and the inhibited activity of the antioxidative enzymes in the rat liver and cardiac tissues. Histopathological results indicated that the fixed oil preserved the renal tissue against oxidative stress in diabetes. In summary, the results of the present study suggest that the fixed oil extracted from OS leaves exerted anti-hyperglycemic, anti-hyperlipidemic and free radical scavenging effects in diabetic rats, thus providing renal protection against diabetes. The α-linolenic acid contained in the fixed oil may be responsible for these effects.