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Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute watery diarrhea and vomiting in unweaned piglets, and is associated with high mortality, thus causing severe economic losses in the pig industry. Currently, although attenuated vaccines are commonly used in commercial pig farms in China, they do not completely protect against all mutated wild-type strains. Existing nucleic acid assays have high sensitivity and specificity, but the complexity of the assay process and expensive instrumentation hinder disease detection. Here, reverse transcription–enzymatic recombinase amplification (RT-ERA) was combined with the CRISPR-Cas12a system to develop a rapid diagnostic method to distinguish PEDV wild-type strains from attenuated vaccine strains. The protocol used crRNA and RT-ERA amplification primers against open reading frame 3 (ORF3), followed by Cas12a/crRNA complex detection of predefined target sequences at 37 °C for 30 min, thus producing results visible to the naked eye under LED blue light. The assay is highly sensitive and specific, detecting as few as two copies of the target gene per test and showing no cross-reactivity with other porcine pathogens. Overall, this integrated RT-ERA pre-amplification and Cas12a/crRNA cleavage assay is a practical tool for reliable and rapid detection of PEDV for diagnostic differentiation. [ABSTRACT FROM AUTHOR]

Canine kobuvirus (CaKoV), a newly described virus, is the causative agent of gastroenteritis in dogs. In this study, 57 fecal samples from dogs with diarrhea in Anhui Province, eastern China, were collected. Among these, five samples were identified to be infected with CaKoV, by polymerase chain reaction targeting the CaKoV 3D gene. The five CaKoV strains were subjected to phylogenetic analysis. The sequences of VP1 from the five CaKoV strains were 93.6%–96.1% identical to each other and 91.75%–97.95% identical to other reported CaKoV VP1 sequences. In addition, the complete genome of one strain was successfully amplified and sequenced. The genome consisted of 8223 nucleotides and shared 94.6%–97.0% nucleotide and 93.1%–94.0% amino acid sequence identity with other CaKoV isolates. Phylogenetic analysis revealed that the CaKoV strain from Anhui Province was similar to other Chinese strains, and it was more closely related to feline and mouse kobuviruses than to sheep and bovine kobuviruses. Interestingly, all of the CaKoV-positive samples were coinfected with canine parvovirus. The finding of CaKoV infection in dogs with diarrhea and coinfection with canine parvovirus are a cause for concern and highlight the need for management and preventive measures. [ABSTRACT FROM AUTHOR]

This study aimed to explore the phylogenetic and molecular characteristics of feline astrovirus. A total of 33 fecal samples of domestic cats with or without diarrhea were collected from the Anhui province, and two positive samples were detected. The complete genome and ORF2 of the two strains were sequenced and phylogenetically analyzed. AH-1-2020 and AH-2-2020 displayed 83.4% homology, and their homologies with other reference strains were 75.3%–83.4% and 83.4%–95.0%, respectively. Phylogenetic tree analysis revealed that all strains could be classified into three different clusters; therefore, the mean amino acid genetic distances (p-dist) among the three clusters were estimated. The results suggested that the two strains and other FeAstV strains were grouped into three genotypes, with AH-1-2020 belonging to a novel genotype. High similarity was observed (65.9%–66.5% nucleotide identity and 63.8%–64.8% amino acid identity) in ORF2 between porcine astrovirus type 1 and AH-1-2020. Furthermore, inter-specific recombination between porcine astrovirus type 1 and FeAstV was observed. We, therefore, inferred that inter-specific transmission may exist between pigs and cats; however, further studies are required to verify this. This is the first report on the genetic characterization and phylogenetic analysis of FeAstVs in the Anhui province and would further the current understanding of the genetic diversity and epidemiology of FeAstVs. [ABSTRACT FROM AUTHOR]

In this study, we develop a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid and easy detection of goose astroviruses (GAstVs) in clinical samples. The specific LAMP primer sets were designed targeting the ORF2 gene of GAstV. The conditions of LAMP amplification were optimized in terms of reaction time and temperature. The optimal conditions are 60 min in a 60 °C water bath. No cross-reactivity was noted with fowl adenovirus serotype 4 (FAdV-4), duck tembusu virus (DTMUV), goose parvovirus (GPV), avian infectious bronchitis virus (IBV), or chicken anemia virus (CAV). The proposed RT-LAMP method was compared with conventional RT polymerase chain reaction (RT-PCR) and with nested RT-PCR. The results showed that the sensitivity of the proposed method was comparable to that of nested RT-PCR and tenfold higher than that of the conventional RT-PCR. Clinical samples (N = 129) of the liver and kidney from sick geese collected from six commercial goose farms were tested. The positive rate was 39.5% (51/129), 38.8% (50/129), and 34.9% (45/129) using RT-LAMP, nested RT-PCR and conventional RT-PCR, respectively. The developed RT-LAMP diagnostic method is not only simple, rapid, and highly specific, but also portable for use on the field. It may be used in epidemiological investigation to detect GAstVs. [ABSTRACT FROM AUTHOR]

Orf is a non-systemic, ubiquitous disease of sheep and goats caused by the orf virus (ORFV). ORFV occasionally causes cutaneous lesions in humans in contact with infected animals. In the present study, a real-time PCR method was established for detection of ORFV using the fluorescent chimeric dye SYBR Green I. Specific primers were designed to target a highly conserved region of the ORFV B2L gene. This method was able to detect a minimum of 20 copies of ORFV genomic DNA. The results showed no cross-reactions with other common DNA viruses. The time required for the test was approximately 1.5 h. Clinical test samples showed that this method was faster and had a higher sensitivity than traditional PCR. In conclusion, this novel, real-time PCR-based assay provides a rapid, sensitive, and specific method for ORFV detection. This test provides improved technical support for studies regarding the clinical diagnosis and epidemiology of ORFV. [ABSTRACT FROM AUTHOR]