Acute-onset respiratory signs in a Labrador Retriever with a positive SARS-CoV-2 rapid antigen test and infection confirmed by RT-PCR analysis: a case report.

A 10-year-old male neutered Labrador Retriever presented with a history of acute-onset tachypnoea, lethargy and anorexia. The dog was pyrexic, tachypnoeic and dyspnoeic on examination. A rapid antigen test for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was performed on an oropharyngeal swab and yielded a positive result. SARS-CoV-2 infection was subsequently confirmed by reverse transcription polymerase chain reaction (RT-PCR) analysis. Both of the dog's owners had positive rapid antigen test and RT-PCR analysis results for SARS-CoV-2. Additional diagnostics included computed tomography. Resolution of the dog's clinical signs was achieved with symptomatic treatment.

Molecular survey of infectious bronchitis virus on poultry farms in Gifu Prefecture, Japan from 2021 to 2022 by RT-PCR with an enhanced level of detection sensitivity for the S1 gene.

Infectious bronchitis virus (IBV) is the causative agent of infectious bronchitis (IB) in chickens. There is a correlation between cross-protection and percentage of similarity between nucleotide sequences encoding the S1 subunit, which is responsible for generating neutralizing and serotype-specific antibodies. Therefore, RT-PCR is commonly used to amplify the IBV-S1 gene following DNA sequencing in order to predict the efficacy of vaccines against IBV strains. We successfully enhanced the sensitivity for detection of the IBV-S1 gene by second PCR after purification of the 1st RT-PCR product. Using that method, we obtained detailed information on the prevalence of IBV on poultry farms in Gifu Prefecture, Japan. The IBV-S1 gene detection method used in the current study will enable accurate information on the prevalence of IBV in Japan to be obtained.

Different dynamics of mean SARS-CoV-2 RT-PCR Ct values between the first and second COVID-19 waves in the Madrid population.

SARS-CoV-2 RT-PCR cycle threshold values from 18,803 cases (2 March-4 October) in Madrid define three stages: (i) initial ten weeks with sustained reduction in viral load (Ct: 23.4-32.3), (ii) stability with low viral loads (Ct: 31.9-35.5) in the next nine weeks and (iii) sudden increase with progressive higher viral loads until reaching stability at high levels in the next twelve weeks, coinciding with an increased percentage of positive cases and reduced median age. These data indicate differential virological/epidemiological patterns between the first and second COVID-19 waves in Madrid.

Molecular detection of bat coronaviruses in three bat species in Indonesia.

Bats are an important reservoir of several zoonotic diseases. However, the circulation of bat coronaviruses (BatCoV) in live animal markets in Indonesia has not been reported. Genetic characterization of BatCoV was performed by sequencing partial RdRp genes. Real-time polymerase chain reaction based on nucleocapsid protein (N) gene and Enzyme-linked immunosorbent assay against the N protein were conducted to detect the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA and antibody, respectively. We identified the presence of BatCoV on Cynopterus brachyotis, Macroglossus minimus, and Rousettus amplexicaudatus. The results showed that the BatCoV included in this study are from an unclassified coronavirus group. Notably, SARS-CoV-2 viral RNA and antibodies were not detected in the sampled bats.

Development and application of a real-time RT-PCR assay to rapidly detect H2 subtype avian influenza A viruses.

The H2 subtypes of avian influenza A viruses (avian IAVs) have been circulating in poultry, and they have the potential to infect humans. Therefore, establishing a method to quickly detect this subtype is pivotal. We developed a TaqMan minor groove binder real-time RT-PCR assay that involved probes and primers based on conserved sequences of the matrix and hemagglutinin genes. The detection limit of this assay was as low as one 50% egg infectious dose (EID50)/mL per reaction. This assay is specific, sensitive, and rapid for detecting avian IAV H2 subtypes.

Molecular evidence and hematological alterations associated with the occurrence of coronavirus in domestic dogs in Pakistan.

Canine Enteric Coronavirus (CCoV) is one of the major enteric pathogen affecting dogs. This study aims to investigate the molecular prevalence, phylogenetic analysis, associated risk factors, and haemato-biochemical alterations in Canine Coronavirus in dogs in district Lahore, Pakistan. 450 fecal samples were collected from symptomatic dogs originating from various pet-clinics and kennels during 2018-2019. Samples were initially analyzed by sandwich lateral flow immunochromatographic assay and then further processed by RT-PCR (reverse transcriptase polymerase chain reaction) targeting the M gene followed by sequencing. RT-PCR based positive (n=20) and negative (n=20) dogs were samples for their blood for the haemato-biochemical analysis. A questionnaire was used to collect data from pet owners, in order to analyze the data for risk factors analysis by chi square test on SPSS. The prevalence of CCoV was 35.1%, and 23.8 % through Sandwich lateral flow immunochromatographic and RT-PCR respectively. Various risk factors like breed, age, sex, vomiting, diarrhea, sample source, body size, cohabitation with other animals, living environment, food, deworming history, contact with other animals or birds feces, and season were significantly associated with CCoV. The CCoV identified in Pakistan were 98% similar with the isolates from China (KT 192675, 1), South Korea (HM 130573, 1), Brazil (GU 300134, 1), Colombia (MH 717721, 1), United Kingdom (JX 082356, 1) and Tunisia (KX156806). Haematobiochemical alterations in CCoV affected dogs revealed anaemia, leucopenia, lymphopenia, neutrophilia, and decreased packed cell volume, and a significant increase in alkaline phosphate and alanine transaminase. It is concluded that infection with canine coronavirus appears widespread among dog populations in district Lahore, Pakistan. This study is the first report regarding the molecular detection and sequence analysis of CCoV in Pakistan.

Molecular-based cross-species evaluation of bovine coronavirus infection in cattle, sheep and goats in Ghana.

BACKGROUND: Apart from the huge worldwide economic losses often occasioned by bovine coronavirus (BCoV) to the livestock industry, particularly with respect to cattle rearing, continuous surveillance of the virus in cattle and small ruminants is essential in monitoring variations in the virus that could enhance host switching. In this study, we collected rectal swabs from a total of 1,498 cattle, sheep and goats. BCoV detection was based on reverse transcriptase polymerase chain reaction. Sanger sequencing of the partial RNA-dependent RNA polymerase (RdRp) region for postive samples were done and nucleotide sequences were compared with homologous sequences from the GenBank. RESULTS: The study reports a BCoV prevalence of 0.3%, consisting of 4 positive cases; 3 goats and 1 cattle. Less than 10% of all the animals sampled showed clinical signs such as diarrhea and respiratory distress except for high temperature which occurred in > 1000 of the animals. However, none of the 4 BCoV positive animals manifested any clinical signs of the infection at the time of sample collection. Bayesian majority-rule cladogram comparing partial and full length BCoV RdRp genes obtained in the study to data from the GenBank revealed that the sequences obtained from this study formed one large monophyletic group with those from different species and countries. The goat sequences were similar to each other and clustered within the same clade. No major variations were thus observed between our isolates and those from elsewhere. CONCLUSIONS: Given that Ghana predominantly practices the extensive and semi-intensive systems of animal rearing, our study highlights the potential for spillover of BCoV to small ruminants in settings with mixed husbandry and limited separation between species.

Establishment of method for dual simultaneous detection of PEDV and TGEV by combination of magnetic micro-particles and nanoparticles.

Transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) are the main pathogens causing viral diarrhea in pig, mixed infections of these two viruses are very common in intensive pig rearing. However, there is a lack of a method to simultaneously detect and distinguish PEDV and TGEV in preclinical levels. In this study, we aimed to establish a dual ultrasensitive nanoparticle DNA probe-based PCR assay (dual UNDP-PCR) based on functionalized magnetic bead enrichment and specific nano-technology amplification for simultaneous detection and distinguish diagnosis of PEDV and TGEV. The detection limit of dual UNDP-PCR for single or multiple infections of PEDV and TGEV is 25 copies/g, which is 400 times more sensitive than the currently known duplex RT-PCR, showing better specificity and sensitivity without cross-reaction with other viruses. For pre-clinical fecal samples, the dual UNDP-PCR showed a markedly higher positive detection rate (52.08%) than conventional duplex RT-PCR (13.21%), can rapidly and accurately identify targeted pathogens whenever simple virus infection or co-infection. In summary, this study provides a technique for detecting and distinguishing PEDV and TGEV in preclinical levels, which is high sensitivity, specificity, repeatability, low cost and broad application prospect.

Susceptibility of swine cells and domestic pigs to SARS-CoV-2.

The emergence of SARS-CoV-2 has resulted in an ongoing global pandemic with significant morbidity, mortality, and economic consequences. The susceptibility of different animal species to SARS-CoV-2 is of concern due to the potential for interspecies transmission, and the requirement for pre-clinical animal models to develop effective countermeasures. In the current study, we determined the ability of SARS-CoV-2 to (i) replicate in porcine cell lines, (ii) establish infection in domestic pigs via experimental oral/intranasal/intratracheal inoculation, and (iii) transmit to co-housed naïve sentinel pigs. SARS-CoV-2 was able to replicate in two different porcine cell lines with cytopathic effects. Interestingly, none of the SARS-CoV-2-inoculated pigs showed evidence of clinical signs, viral replication or SARS-CoV-2-specific antibody responses. Moreover, none of the sentinel pigs displayed markers of SARS-CoV-2 infection. These data indicate that although different porcine cell lines are permissive to SARS-CoV-2, five-week old pigs are not susceptible to infection via oral/intranasal/intratracheal challenge. Pigs are therefore unlikely to be significant carriers of SARS-CoV-2 and are not a suitable pre-clinical animal model to study SARS-CoV-2 pathogenesis or efficacy of respective vaccines or therapeutics.

Epidemiological investigation of feline infectious peritonitis in cats living in Harbin, Northeast China from 2017 to 2019 using a combination of an EvaGreen-based real-time RT-PCR and serum chemistry assays.

Feline infectious peritonitis (FIP) is caused by the FIP virus (FIPV), a highly virulent mutant form of feline coronavirus (FCoV). This disease is one of the most important infectious diseases in cats, and it is associated with high mortality, particularly among younger cats. In this study, we isolated a wild-type FIPV HRB-17 epidemic strain from the blood sample of household pet cat exhibiting the characteristic wet-form FIP symptoms, which has been confirmed further by animal infection. Further, we developed an EvaGreen-based real-time RT-PCR assay for the accurate detection of FCoV based on the amplification of the highly conserved FIPV N gene. Then, using a combination of the real-time RT-PCR approach and a serum chemistry assay, we performed an epidemiological survey of FIPV infection in cats living in Harbin City, Northeast China. The results indicated that the EvaGreen-based real-time RT-PCR assay can be used for screening FCoV infection in the affected cats at an analytical detection limit of 8.2 × 101 viral genome copies/µL, but could not effectively distinguish FIPVs from FECVs. Additionally, the results of the epidemiological survey investigating feline blood samples (n = 1523) collected between July 2017 to July 2019 revealed an FIPV prevalence of approximately 12% (189/1523). Maybe, the prevalence would be less than 12% due to the real-time RT-PCR assay could not accurately differentiate FIPV and FECV. Nevertheless, it still highlighted the severity of the FIP epidemic in cats and reiterated the urgent need to develop effective anti-FIP therapeutic agents and anti-FIPV vaccines. As pet cats are household animals, risk communication and continuous region-extended surveillance cat programs are recommended.

Detection of bovine viral diarrhea virus genotype 1 in aerosol by a real time RT-PCR assay.

BACKGROUND: As a pestivirus of the Flaviviridae family, bovine viral diarrhea virus (BVDV), has imposed a large burden on animal husbandry worldwide, and such virus can be transmitted mainly through direct contact with other infected animals and probably via aerosols. In the present study, we aimed to develop a real-time RT-PCR method for detection of BVDV-1 in aerosol samples. METHODS: A pair of primers specific for highly conserved regions of the BVDV-1 5'-UTR was designed. The standard curve and sensitivity of the developed assay were assessed based on 10-fold serial dilutions of RNA molecular standard. The specificity of the assay was evaluated with other pestiviruses and infectious bovine viruses. The clinical performance was examined by testing 169 aerosol samples. RESULTS: The results showed that a good linear relationship existed between the standard curve and the concentration of template. The lowest detection limit was 5.2 RNA molecules per reaction. This assay was specific for detection of BVDV-1, and no amplification was found for other pestiviruses such as classical swine fever virus (CSFV), border disease virus (BDV), and common infectious bovine viruses, including BVDV-2, infectious bovine rhinotracheitis virus (IBRV), bovine parainfluenza virus type 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), bovine ephemeral fever virus (BEFV) and bovine coronavirus (BcoV). The assay was highly reproducible with low variation coefficient values (CVs) for intra-assay and inter-assay. A total of 169 aerosol samples collected from six dairy herds were tested using this method. The results showed that the positive detection rate of BVDV-1 was 17.2% (29/169), which was significantly higher compared with the conventional RT-PCR. Additionally, the positive samples (n = 29) detected by real-time RT-PCR were verified by BVDV RPA-LFD, and a concordance rate of 100% was obtained between them. CONCLUSIONS: Taken together, we developed a real-time RT-PCR assay for quantitative analysis of BVDV-1 in aerosol samples, and our finding provided valuable insights into the risk on aerosol transmission of BVDV-1.