To visually and rapidly detect a novel goose astrovirus (N-GoAstV) causing fatal gout in goslings, an isothermal detection method based on one-step reverse transcription loop-mediated isothermal amplification (one-step RT-LAMP) was established. The one-step RT-LAMP assay for N-GoAstV detection, using Bst 3.0 DNA polymerase with strong reverse transcription activity and primer sets targeting the opening reading frame 1b (ORF1b) of N-GoAstV, could be completed in 30 min using a water bath at 61°C; the detection results could be visually observed by adding a pH-sensitive dye containing phenol red and cresol red. The detection limit of the one-step RT-LAMP assay was 57.8 copies, which was similar to that of reverse transcription–quantitative polymerase chain reaction. The assay specifically detected N-GoAstV without any cross-reaction with other reference viruses, and this was further confirmed using enzyme digestion. These results indicated that the newly established RT-LAMP assay could accomplish reverse transcription, amplification, and visual result determination in one step, and the results obtained via this rapid and cost-effective method could be used to support disease control on farms in terms of N-GoAstV infection.

Infectious bronchitis (IB), caused by avian IB virus (IBV), is an acute and highly contagious disease of chickens. From 2016 to 2018, 56 IBV strains were isolated and identified from clinical samples obtained from various chicken farms located in central China. The S1 sequencing of these strains revealed nucleotide and amino acid identities of 70.2 to 100% and 62.6 to 100%, respectively, compared with those of reference strains. Phylogenetic analysis indicated that the genotypes of the isolates included GI-13 (4/91), GI-7 (TW-I), GI-24 (Mass), GI-19 (QX), and GI-18 (LDT3-A), with GI-19 (QX) being the predominant genotype. Meanwhile, GI-13 (4/91) was the second most dominant genotype in Henan Province, whereas it was GI-7 (TW-I) in Hunan and Hubei provinces. Recombination analysis of 3 variant strains showed that CK/CH/HeN/20160113 might be a recombination of LDT3-A- and QX-type strains and that CK/CH/HeN/20160316 might be a recombination of Italy-02-type strain and CK-CH-LJS08II. The predicted tertiary structure between CK/CH/HeN/20160113 and LDT3-A-type strain revealed that the novel 336 (L-P) and 455 (S-A) mutations changed the structure from an alpha helix to a random crimp. In addition, the 275 (Y-F) site reduced the length of the β-sheet, whereas the site 353 (A-T) extended the β-sheet. These findings suggested that GI-19 (QX) remains the predominant genotype in central China, and a locally determined complex genotype associated with variable clinical symptoms exists related to gene recombination and mutations.

Porcine circovirus type 3 (PCV3) is a newly emerging circovirus that might be associated with porcine dermatitis and nephropathy syndrome, reproductive failure, and cardiac and multisystemic inflammation. To aid the prevention and control of the infectious disease caused by PCV3, we developed a novel isothermal amplification assay using polymerase spiral reaction (PSR), which allows the visual detection of preserved strains and clinical samples. This assay precisely amplified the PCV3 genome with the use of a water bath at 62 °C for 50 min. The detection limit was found to be 1.13 × 102 copies/μL by gel electrophoresis or with the use of a visible dye (an indicator comprising phenol red and cresol red). No cross-reaction with other porcine infectious viruses was observed. The detection results for 23 PCV3-positive samples by PSR were in accordance with loop-mediated isothermal amplification (LAMP) assay. The detection rate of the PSR assay for PCV3 positivity of clinical samples was 68/97, which was higher than LAMP assay (67/97). These results indicated that the PSR assay provides an accurate and rapid method for the detection of PCV3 with high sensitivity and specificity. It is particularly suited for use in a simple laboratory setting without a thermal cycler or gel electrophoresis equipment.

Avian gyrovirus 2 (AGV2), which is similar to chicken infectious anemia virus, is a new member of the genus Gyrovirus. AGV2 has been detected not only in chicken but also in human tissues and feces. This study analyzed 91 samples (8 from liver tissue and 83 from fecal samples) collected from king ratsnakes (Elaphe carinata) from 7 separate farms in Hubei and Henan, China, for AGV2 DNA using PCR. The results demonstrated a low positive rate of AGV2 (6.59%, 6/91) in the snakes, and all the positive samples were collected from the same farm. The AGV2 strain HB2018S1 was sequenced, and its 2376 nt genome comprised three partially overlapping open reading frames: VP1, VP2, and VP3. Phylogenetic analysis revealed that the HB2018S1 and NX1506-1 strains from chickens in China belong to the same clade and that they have a nucleotide identity as high as 99.5%. Additionally, recombination analysis showed that HB2018S1 might originate from the recombination of viruses similar to those detected in chickens and a ferret. A total of 10 amino acid mutation sites (44(R/K), 74(T/A), 256 (C/R), 279(L/Q), and 373(V/A) in AGV2 VP1; 60(I/T), 125(T/I), 213(D/N), and 215(L/S) in AGV2 VP2; and 83(H/Y) in AGV2 VP3) different from those observed in most reference strains were found in the genome of HB2018S1, indicating that the differences may be related to a transboundary movement among hosts, which needs further elucidation. To the best of our knowledge, this study is the first report on an AGV2-infected poikilotherm, suggesting that cross-host transmission of viruses with circular single-stranded DNA genomes would be a public health concern.

In this study, loop-mediated isothermal amplification (LAMP) was used to establish a rapid, specific, and visual detection method for duck hepatitis B virus (DHBV). The design and synthesis of 4 specific LAMP primers were based on the conserved gene region of the DHBV genome, and the optimum temperature and time of the LAMP reaction were 63°C and 50 min, respectively. The LAMP assay was confirmed to be specific for DHBV detection and had the same sensitivity as the quantitative PCR assay. A visual detection method for rapid determination of results was developed using a color indicator containing phenol red and cresol red. A color change was produced based on a pH change in the reaction system, indicating a positive reaction. For the detection of samples from ducks and geese, the LAMP method has the advantages of simplicity, high sensitivity and specificity, good visibility, and low cost. Moreover, it is more practical and convenient than PCR-related assays for the clinical detection of DHBV.

Infections caused by canine adenovirus (CAdV) type 1 have been reported worldwide in the past two decades. However, only few studies have specifically reported the prevalence of CAdV type 2 (CAdV-2). The present study investigated the persistent circulation of CAdV-2 in dogs with diarrhea in the Henan, Hubei, and Jiangsu provinces in central China from 2017 to 2019. We conducted polymerase chain reaction for detecting CAdV-2 and other related pathogens in 224 rectal swabs of pet dogs and the co-infection of canine diseases was also analyzed. In addition, the structural protein genes—Fiber, Hexon, and Penton—of the isolated CAdV-2 strains were sequenced and analyzed. The similarity between Hexon and Penton among the 19 strains was 97.4%, as revealed by sequence alignment. Multiple sequence alignment results showed that the Fiber gene sequences of these CAdV-2 strains shared 97.4%–99.8% nucleotide and 94.1%–99.3% amino acid identity with reference sequences and shared only 79.0%–80.5% nucleotide and 77.3%–80.5% amino acid identity with the vaccine strain CLL, indicating that Fiber harbored most of the variant sites. Furthermore, pairwise sequence comparisons of Hexon of CH-JS-1901 and CH-HN-1801 with that of India2006 revealed a novel genotype. Furthermore, protein model prediction showed that the amino acid mutation of fiber protein in 19 strains was located in the head region, that may cause structural changes on the surface of the fiber protein. These findings are of significance for monitoring the epidemiology of CAdV-2 infection and developing a novel vaccine which contribute to understanding genetic evolution of CAdV-2 in China.