Molecular characterization and genetic diversity of parvoviruses prevalent in cats in Central and Eastern China from 2018 to 2022

Cats are a potential source of genetic diversity for parvoviruses. Herein, 134 samples were collected from cats with clinical gastroenteritis and analyzed for the presence of viral DNA via polymerase chain reaction, which revealed 48 positive samples. Identity analysis of VP2 nucleotide sequences indicated that these 48 strains, belonging to feline panleukopenia virus (FPV) and canine parvovirus type-2 (CPV-2; including new CPV-2a and CPV-2c genotypes), shared 94.59–99.94% nucleotide identity with the reference strains. The FPV strain F8 (isolated from Vietnam) appeared to be a recombinant of strains HB2003 and JS1901, whereas the Chinese CPV-2b strain BM-(11) isolated in 2011 was believed to be a recombinant of strains AH2008 and JS1901. In phylogenetic tree analysis based on VP2 nucleotide sequences, all obtained FPV strains and most reference FPV strains were clustered together, except strain BJ-22, which originated from monkeys. Further, two new CPV-2a strains (AH2005 and AH2008) were close to the newly reported Chinese CPV-2a strains but were distant from the other CPV-2a strains, namely CPV-339 (from the United States) and K022 (from South Korea). Additionally, the FPV and CPV-2 strains had high mutation rates in the antigenic regions of the VP2 protein. According to model prediction of the CPV–VP2 protein, these mutations may cause changes in the tertiary structure of VP2. The findings of this study can be used to improve the pre-evaluation of vaccination efficacy against diseases caused by FPV and CPV-2 in domestic cats and understand their genotypic transmission and mutation trends

Insect-Specific microRNA Involved in the Development of the Silkworm Bombyx mori

MicroRNAs (miRNAs) are endogenous non-coding genes that participate in post-transcription regulation by either degrading mRNA or blocking its translation. It is considered to be very important in regulating insect development and metamorphosis. We conducted a large-scale screening for miRNA genes in the silkworm Bombyx mori using sequence-by-synthesis (SBS) deep sequencing of mixed RNAs from egg, larval, pupal, and adult stages. Of 2,227,930 SBS tags, 1,144,485 ranged from 17 to 25 nt, corresponding to 256,604 unique tags. Among these non-redundant tags, 95,184 were matched to the silkworm genome. We identified 3,750 miRNA candidate genes using a computational pipeline combining RNAfold and TripletSVM algorithms. We confirmed 354 miRNA genes using miRNA microarrays and then performed expression profile analysis on these miRNAs for all developmental stages. While 106 miRNAs were expressed in all stages, 248 miRNAs were egg- and pupa-specific, suggesting that insect miRNAs play a significant role in embryogenesis and metamorphosis. We selected eight miRNAs for quantitative RT-PCR analysis; six of these were consistent with our microarray results. In addition, we searched for orthologous miRNA genes in mammals, a nematode, and other insects and found that most silkworm miRNAs are conserved in insects, whereas only a small number of silkworm miRNAs has orthologs in mammals and the nematode. These results suggest that there are many miRNAs unique to insects

Differential Expression of miRNAs in Response to Topping in Flue-Cured Tobacco (Nicotiana tabacum) Roots

Topping is an important cultivating measure for flue-cured tobacco, and many genes had been found to be differentially expressed in response to topping. But it is still unclear how these genes are regulated. MiRNAs play a critical role in post-transcriptional gene regulation, so we sequenced two sRNA libraries from tobacco roots before and after topping, with a view to exploring transcriptional differences in miRNAs.Two sRNA libraries were generated from tobacco roots before and after topping. Solexa high-throughput sequencing of tobacco small RNAs revealed a total of 12,104,207 and 11,292,018 reads representing 3,633,398 and 3,084,102 distinct sequences before and after topping. The expressions of 136 conserved miRNAs (belonging to 32 families) and 126 new miRNAs (belonging to 77 families) were determined. There were three major conserved miRNAs families (nta-miR156, nta-miR172 and nta-miR171) and two major new miRNAs families (nta-miRn2 and nta-miRn26). All of these identified miRNAs can be folded into characteristic miRNA stem-loop secondary hairpin structures, and qRT-PCR was adopted to validate and measure the expression of miRNAs. Putative targets were identified for 133 out of 136 conserved miRNAs and 126 new miRNAs. Of these miRNAs whose targets had been identified, the miRNAs which change markedly (>2 folds) belong to 53 families and their targets have different biological functions including development, response to stress, response to hormone, N metabolism, C metabolism, signal transduction, nucleic acid metabolism and other metabolism. Some interesting targets for miRNAs had been determined.The differential expression profiles of miRNAs were shown in flue-cured tobacco roots before and after topping, which can be expected to regulate transcripts distinctly involved in response to topping. Further identification of these differentially expressed miRNAs and their targets would allow better understanding of the regulatory mechanisms for flue-cured tobacco response to topping

Biochemical Characterization of a Novel Alkaline-Tolerant Xaa-Pro Dipeptidase from <i>Aspergillus phoenicis</i>

Xaa-Pro dipeptidase (XPD, EC 3.4.13.9; also known as prolidase) catalyzes the hydrolysis of the iminopeptide bond in the trans-Xaa-Pro dipeptides (Xaa represents any amino acid except proline), which makes it find wide applications in food, medical and environmental protection fields. In the present study, a novel Xaa-Pro dipeptidase from Aspergillus phoenicis ATCC 14332 (ApXPD) was heterologously expressed and biochemically characterized. Reclassification based on phylogenetic analysis and the version 12.5 MEROPS database showed that this enzyme was the only fungal XPD in the unassigned subfamily that shared the highest sequence identity with Xanthomonas campestris prolidase but not with that from the more related fungal species A. niudulans. As compared with other prolidases, ApXPD also contained a long N-terminal tail (residues 1–63) and an additional region (PAPARLREKL) and used a different arginine residue for dipeptide selectivity. After heterologous expression and partial purification, recombinant ApXPD was highly active and stable over the alkaline range from 8.5 to 10.0, with maximum activity at pH 9.0 and more than 80% activity retained after 1 h incubation at pHs of 8.5–10.0 (55 °C). It also had an apparent optimum temperature of 55 °C and remained stable at 20–30 °C. Moreover, this enzyme was a cobalt-dependent prolidase that only cleaved dipeptides Lys-Pro, Gly-Pro, and Ala-Pro rather than other dipeptides, tripeptides, and tetrapeptides. All these distinct features make A. phoenicis ATCC 14332 XPD unique among currently known prolidases, thus defining a novel Xaa-Pro dipeptidase subfamily

Genetic Analysis of Avian Gyrovirus 2 Variant-Related Gyrovirus Detected in Farmed King Ratsnake (Elaphe carinata): The First Report from China

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

Genetic characterisation and local genotypes of canine parvovirus strains collected from pet dogs in central and eastern China during 2018–2019

Canine parvovirus type-2 (CPV-2) causes acute infectious diseases in puppies, which show high morbidity and mortality. Better effect of vaccination against these diseases could be achieved with deeper knowledge of CPV-2 genotype dissemination and mutation history. This study investigated CPV-2–positive samples collected recently over a wide region of China

Molecular characterisation and genetic diversity of canine parvovirus type 2 prevalent in Central China

Canine parvovirus (CPV) disease is one of the most threatening to domestic and wild dogs

Visual detection of porcine epidemic diarrhea virus using a novel reverse transcription polymerase spiral reaction method

Abstract Background Porcine epidemic diarrhea virus (PEDV) is a major etiological agent of porcine epidemic diarrhea around the world. Point-of-care testing in the field is lacking owing to the requirement for a simple, robust field applicable test that does not require professional laboratory equipment. The aim of this study was to establish a novel reverse transcription polymerase spiral reaction (RT-PSR) assay for the rapid detection of porcine epidemic diarrhea virus (PEDV). For the assay, a specific RT-PSR primer pair was designed against a conserved region in PEDV ORF3. Results The RT-PSR was optimized, and PEDV could be detected after a 50 min incubation at 62 °C, in addition to the 15 min required for reverse transcription. No cross-reaction with other porcine infectious viruses was observed. This new method for PEDV detection was 10 times more sensitive than the conventional reverse transcription-polymerase chain reaction (RT-PCR) assay. The positive rates for 65 clinical samples using the new RT-PSR assay and the conventional RT-PCR assay were 58.46% (38/65) and 53.84% (35/65), respectively. In the RT-PSR assay, the addition of a mixture of dyes allowed a positive reaction to be directly observed by the naked eye. Conclusions These results indicate that this RT-PSR assay is capable of accurately detecting PEDV, and has the advantages of high specificity and sensitivity for the detection of PEDV

Genetic Heterogeneity and Mutated PreS Analysis of Duck Hepatitis B Virus Recently Isolated from Ducks and Geese in China

In this study, we detected 12 duck and 11 goose flocks that were positive for duck hepatitis B virus (DHBV) using polymerase chain reaction and isolated 23 strains between 2020 and 2022 in China. The complete genomes of goose strains E200801 and E210501 shared the highest identity (99.9%), whereas those of strains Y220217 and E210526 shared the lowest identity (91.39%). The phylogenetic tree constructed based on the genome sequences of these strains and reference strains was classified into three major clusters: the Chinese branch DHBV-I, the Chinese branch DHBV-II, and the Western branch DHBV-III. Furthermore, the duck-origin strain Y200122 was clustered into a separate branch and was predicted to be a recombinant strain derived from DHBV-M32990 (belonging to the Chinese branch DHBV-I) and Y220201 (belonging to the Chinese branch DHBV-II). Additionally, preS protein analysis of the 23 DHBV strains revealed extensive mutation sites, almost half of which were of duck origin. All goose-origin DHBV contained the mutation site G133E, which is related to increased viral pathogenicity. These data are expected to promote further research on the epidemiology and evolution of DHBV. Continuing DHBV surveillance in poultry will enhance the understanding of the evolution of HBV

RNA Interference-Mediated Knockdown of <i>Bombyx mori</i> Haemocyte-Specific Cathepsin L (<i>Cat L</i>)-Like Cysteine Protease Gene Increases <i>Bacillus thuringiensis kurstaki</i> Toxicity and Reproduction in Insect Cadavers

The silkworm’s Cat L-like gene, which encodes a lysosomal cathepsin L-like cysteine protease, is thought to be part of the insect’s innate immunity via an as-yet-undetermined mechanism. Assuming that the primary function of Cat L-like is microbial degradation in mature phagosomes, we hypothesise that the suppression of the Cat L-like gene expression would increase Bacillus thuringiensis (Bt) bacteraemia and toxicity in knockdown insects. Here, we performed a functional analysis of Cat L-like in larvae that were fed mulberry leaves contaminated with a commercial biopesticide formulation based on Bt kurstaki (Btk) (i.e., Dipel) to investigate its role in insect defence against a known entomopathogen. Exposure to sublethal doses of Dipel resulted in overexpression of the Cat L-like gene in insect haemolymph 24 and 48 h after exposure. RNA interference (RNAi)-mediated suppression of Cat L-like expression significantly increased the toxicity of Dipel to exposed larvae. Moreover, Btk replication was higher in RNAi insects, suggesting that Cat L-like cathepsin may be involved in a bacterial killing mechanism of haemocytes. Finally, our results confirm that Cat L-like protease is part of the antimicrobial defence of insects and suggest that it could be used as a target to increase the insecticidal efficacy of Bt-based biopesticides