Conservation and distribution of the DRACH motif for potential m6A sites in avian leukosis virus subgroup J.

N6-methyladenosine (m6A) methylation is an internal post-transcriptional modification that has been linked to viral multiplication and pathogenicity. To elucidate the conservation patterns of potential 5'-DRACH-3' motifs in avian leukosis virus subgroup J (ALV-J), 149 ALV-J strains (139 isolates from China; ALV-J prototype HPRS-103 from the UK; and 9 strains from the USA, Russia, India, and Pakistan) available in GenBank before December 2023 were retrieved. According to the prediction results of the SRAMP web-server, these ALV-J genomes contained potential DRACH motifs, with the total number ranging from 43 to 64, which were not determined based on the isolation region and time. Conservative analysis suggested that 37 motifs exhibited a conservation of >80%, including 17 motifs with a grading above "high confidence." Although these motifs were distributed in the U5 region of LTRs and major coding regions, they were enriched in the coding regions of p27, p68, p32, and gp85. The most common m6A-motif sequence of the DRACH motif in the ALV-J genome was GGACU. The RNA secondary structure of each conserved motif predicted by SRAMP and RNAstructure web-server was mainly of two types-A-U pair (21/37) and hairpin loop (16/37)-based on the core adenosine. Considering the systematic comparative analysis performed in this study, future thorough biochemical research is warranted to determine the role of m6A modification during the replication and infection of ALV-J. These conservation and distribution analysis of the DRACH motif for potential m6A sites in ALV-J would provide a foundation for the future intervention of ALV-J infection and m6A modification.

Molecular analysis of Gyrovirus galga1 variants identified from the sera of dogs and cats in China.

Gyrovirus galga1 (GyVg1), a member of the Anelloviridae family and Gyrovirus genus, has been detected in chicken and human tissue samples. In this study, the DNA of GyVg1-related gyroviruses in the sera of six dogs and three cats from Central and Eastern China was identified using PCR. Alignment analysis between the nine obtained and reference GyVg1 strains revealed that the genome identity ranged from 99.20% (DOG03 and DOG04 strains) to 96.17% (DOG01 and DOG06 strains). Six recombination events were predicted in multiple strains, including DOG01, DOG05, DOG06, CAT01, CAT02, and CAT03. The predicted major and minor parents of DOG05 came from Brazil. The DOG06 strain is potentially recombined from strains originating from humans and cats, whereas DOG01 is potentially recombined from G17 (ferret-originated) and Ave3 (chicken-originated), indicating that transmissions across species and regions may occur. Sixteen representative amino acid mutation sites were identified: nine in VP1 (12 R/H, 114S/N, 123I/M, 167 L/P, 231 P/S, 237 P/L, 243 R/W, 335 T/A, and 444S/N), four in VP2 (81 A/P, 103 R/H, 223 R/G, and 228 A/T), and three in VP3 (38 M/I, 61 A/T, and 65 V/A). These mutations were only harbored in strains identified in dogs and cats in this study. Whether this is related to host tropism needs further investigation. In this study, GyVg1 was identified in the sera of dogs and cats, and the molecular characteristics prompted the attention of public health.

Transcriptome-wide N6-methyladenosine modification and microRNA jointly regulate the infection of avian leukosis virus subgroup J in vitro.

N6-methyladenosine (m6A) methylation in transcripts has been suggested to influence tumorigenesis in liver tumors caused by the avian leukosis virus subgroup J (ALV-J). However, m6A modifications during ALV-J infection in vitro remain unclear. Herein, we performed m6A and RNA sequencing in ALV-J-infected chicken fibroblasts (DF-1). A total of 51 differentially expressed genes containing differentially methylated peaks were identified, which were markedly enriched in microRNAs (miRNAs) in cancer cells as well as apoptosis, mitophagy and autophagy, RNA degradation, and Hippo and MAPK signaling pathways. Correlation analysis indicated that YTHDC1 (m6A-reader gene) plays a key role in m6A modulation during ALV-J infection. The env gene of ALV-J harbored the strongest peak, and untranslated regions and long terminal repeats also contained peaks of different degrees. To the best of our knowledge, this is the first thorough analysis of m6A patterns in ALV-J-infected DF-1 cells. Combined with miRNA profiles, this study provides a useful basis for future research into the key pathways of ALV-J infection associated with m6A alteration.

Multienzyme isothermal rapid amplification and lateral flow dipstick combination assay for visible detection of chicken chaphamaparvovirus.

Chicken chaphamaparvovirus (CkChpV) is a newly emerging pathogen that is currently prevalent in chickens with diarrhea symptoms. To diagnose CkChpV more conveniently and rapidly, this study established a multienzyme isothermal rapid amplification (MIRA) assay, with a reaction time of only 15 min and optimal reaction temperature of 38°C. In combination with the lateral flow dipstick assay, the CkChpV-MIRA assay can be completed within 20 min. We revealed that the detection limit of the MIRA assay using standard plasmids as templates was as low as 21.3 copies, and its sensitivity was 100 times higher than that of nested PCR. Moreover, the designed primer set and probe could only detect CkChpV specifically, and there was no cross reaction with avian nephritis virus, rotavirus, chicken parvovirus virus, Newcastle disease virus, and infectious bronchitis virus, which may cause diarrhea. These findings demonstrated that the CkChpV-MIRA assay established in this study is convenient, sensitive, and specific and does not require sophisticated equipment. It is more suitable for the detection of CkChpV in clinical samples.

Molecular identification of carnivore chaphamaparvovirus 2 (feline chaphamaparvovirus) in cats with diarrhea from China.

Chaphamaparvovirus carnivoran2 (feline chaphamaparvovirus, FeChPV) is a novel feline parvovirus originally detected in Canadian cats in 2019, and it has also been identified in domestic cats in other nations. To evaluate the prevalence and genetic diversity of FeChPV in China, rectal swabs of pet cats from Henan, Guangdong, Anhui, Zhejiang, and Inner Mongolia provinces were collected. Of the 230 samples subjected to nested polymerase chain reaction, 6 (2.6%) tested positive for FeChPV. Although all positive samples were from cats with diarrhea, statistical analyses revealed no correlation between the presence of the virus and clinical symptoms (p > 0.05). Phylogenetic trees of nonstructural protein 1 (NS1) and capsid protein (VP1) demonstrated that these six new strains formed a major branch with other reference FeChPV strains and considerably differed from Chaphamaparvoviru carnivoran1. Moreover, recombination analysis revealed that the FeChPV strain CHN20201025, previously detected in a dog, was a recombinant and strains CHN200228 and CHN180917, identified in this study, were the closest relatives to the parental strains. The findings of this study and a previous study wherein FeChPV was detected in dogs suggest that FeChPV can propagate between species. Additionally, these findings indicate that the genetic diversity of FeChPV can provide an insight into the epidemiological status of FeChPV in China.

Molecular Characteristics of Chicken Infectious Anemia Virus in Central and Eastern China from 2020 to 2022.

To evaluate the recent evolution of CIAV in China, 43 flocks of chickens from the provinces of Henan, Jiangsu, Hubei, and Anhui were screened via polymerase chain reaction during 2020-2022. Of these, 27 flocks tested positive for CIAV nucleic acids, including 12 which were positive for other immunosuppression viruses. Additionally, 27 CIAV strains were isolated, and their whole genomes were sequenced. The AH2001 and JS2002 strains shared the highest identity at 99.56%, and the HB2102 and HB2101 strains shared the lowest identity at 95.34%. Based on the genome sequences of these strains and reference strains, a phylogenetic tree was constructed and divided into eight main branches. Most of the strains were grouped with the East Asian strains, whereas the HB2101 strain belonged to the Brazil and Argentina cluster. A recombination event was detected in multiple strains, in which AH2002 recombined from KJ728827/China/2014 (from Taiwan Province) and HN2203, and AH2202 recombined from KX811526/China/2017 (from Shandong Province) and HN2203. All the obtained strains had a highly pathogenic Gln amino acid site at position 394 of the VP1. Overall, our findings demonstrate the importance of CIAV monitoring and provide data that aid in understanding the evolution of CIAV.

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.

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.

Genome analysis and recombination characterization of duck hepatitis B virus isolated from ducks and geese in central China, 2017 to 2019.

Owing to its high similarity to human hepatitis B virus (HBV), duck HBV (DHBV) is often used as an essential model for HBV research. Although intergenotypic recombination of HBV is common, it remains unclear whether the intergenotypic recombination of human HBV is exactly the same as that of DHBV. In this study, 119 serum samples of duck and goose were collected from 51 farms (29 duck and 22 goose farms) in the central and eastern regions of China. A total of 22 strains isolated from the 22 DHBV positive flock were sequenced. Genome sequence alignment revealed that the duck- and goose-origin strains shared the highest and lowest similarities (99.7 and 90.52%, respectively). The complete genomes of these DHBV and 31 reference strains were analyzed using phylogenetic methods and classified into 3 clusters, which corresponded to the previously identified DHBV-I, DHBV-II, and DHBV-III branches. Recombination analyses of the 53 DHBV genomes indicated 2 major intergenotypic recombination events with high confidence values. These recombination events occurred between the genotypes of the Chinese isolates Y180813HB (Chinese branch [DHBV-Ⅰ]) and E170101AH (Chinese branch [DHBV-Ⅱ]) and the Western isolate DHBV-XY (Western branch [DHBV-Ⅲ]), resulting in the emergence of 2 Chinese recombinant isolates Y190303HN and Y170101HB. In addition, 40% (2/5) goose-origin and 58.8% (10/17) duck-origin DHBV in this study harbored the mutation site of G133E in preS, which promote the pathogenicity of DHBV. This is the first study to report on the genome analysis and recombination characterization of DHBV isolated from Chinese geese. Further, continuous investigation and molecular identification of DHBV should be conducted to attract researchers' attention.

Molecular characteristics of novel chaphamaparvovirus identified in chickens.

Chicken chaphamaparvovirus (CkChpV) is a novel parvovirus species that belongs to the Chaphamaparvovirus genus and is frequently detected in different vertebrates exhibiting diarrhea symptoms. In this study, screening tests were performed on samples from 478 chickens, including 357 with diarrhea and 121 healthy, collected from 25 farms in China to investigate CkChpV infection in China. CkChpV, avian nephritis virus, rotavirus, chicken parvovirus, Newcastle disease virus, infectious bronchitis virus, chicken proventricular necrosis virus, and chicken circovirus were all detected in the samples at a positivity rate of 32%, 9%, 6%, 2%, 2%, 1%, 0%, and 0%, respectively. Statistical analyses suggested a correlation between the infection by the virus and diarrhea (P < 0.05). The genome of 9 strains from the CkChpV-positive samples, whose length was 4,432 nucleotides, have been completely sequenced. The strains shared 97.2 to 98.7% genomic similarity, 98.1 to 99.1%, and 98.2 to 99.2% amino acid similarity, respectively, for NS1 and VP1 compared with CkChpV strain RS/BR/15/2S in GenBank. The genetic relationship between these strains and CkChpV was established through phylogenetic analysis. These findings indicated the infection existence of CkChpV in China, which enriches our understanding of the diversity of the chaphamaparvoviruses and its host spectrum.

Extensive genetic heterogeneity and molecular characteristics of emerging astroviruses causing fatal gout in goslings.

Since 2017, outbreaks of gosling astroviruses (GoAstV) causing the major symptoms related to gout in geese have posed a threat to China's poultry industry and caused huge economic losses. In this study, tissue samples from goslings with gout and urate deposition as the main symptoms were taken from 14 goose farms in different regions of China and screened for pathogen infection. The infection rate of GoAstV was 100%, whereas the infection rates of goose parvovirus, reovirus, Tembusu virus, and goose hemorrhagic polyomavirus were 2, 4, 0, and 0%, respectively. In total, 14 GoAstV strains were isolated and their complete genomes were sequenced. Based on the phylogenetic trees, the 14 isolated strains were classified as GoAstV (G-I) and were considered distant from strains belonging to GoAstV (G-II). The multiple sequence alignments indicated a tremendous amount of amino acid mutations in some parts of the encoding proteins of these strains; the main mutations were located in open reading frames (ORFs)-ORF1a and ORF2, such as M533V and F568S in ORF1a and A614T in ORF2. On the other hand, Further, 2 of the 14 GoAstV strains were possibly derived through inter-GoAstV-I recombination. Taken together, these findings indicate that GoAstVs are evolving in a more complex manner and have diverse transmission routes.

Molecular characterization of the Gyrovirus galga 1 strain detected in various zoo animals: the first report from China.

Since 2011, the Gyrovirus galga 1 (GyVg1, previously recognized as avian gyrovirus 2) strain has extensively been detected worldwide. The virus has been identified in several species, including chickens, humans, domestic cats, and snakes, especially in China. Therefore, in this study, the presence of GyVg1 was investigated in various zoo animals to determine whether it exists in various species in Nanyang, China. A total of 63 whole blood samples (1 sample from each animal) from 24 animal species were collected from the Nanyang Zoo. Eight different GyVg1 strains were identified in eight types of animals using polymerase chain reaction, and the full genome of each strain was sequenced. The whole genome of four GyVg1 strains, namely, HN2019-H1, HN2019-T1, HN2019-SD1, and HN2019-L1 identified in hippopotamus (Hippopotamus amphibius), tiger (Panthera tigris), sika deer (Cervus nippon), and lion (Panthera leo), respectively, comprised 2375 nucleotides (nt). The whole genome of the other strains, namely, HN2019-E1, HN2019-S1, HN2019-PF1, and HN2019-P1 identified in egret (Egretta garzetta), silver pheasant (Lophura nycthemera), peafowl (Pavonini), and common pheasants (Phasianus colchicus), respectively, comprised 2376 nt. Subsequently, a phylogenetic tree was constructed based on the 8 whole-genome sequence strains and 29 reference strains. These 37 strains were grouped into two major branches, group A and group B, and the 8 strains identified in this study were placed in group A. An analysis of the amino acids encoded by three open reading frames revealed some mutation sites unique to these eight strains. The substitution occurred at site 110 of viral protein 2 of HN2019-PF1, which is located in the highly conserved phosphatase motif WX7HX3CXCX5H (95-115aa). Recombination analysis revealed that, all these viral sequences were obtained as a result of recombination among the three GyVg1 strains (JL1511 and GS1512 from chickens and 17CC0810 from cat) from China and two strains (G17 from ferret of Hungary and RS-BR-15-2S from chicken of Brazil) from other countries. These findings indicate the complex evolution of GyVg1. Nevertheless, its transmission across the hosts is worth exploring.

Epidemiological investigations and locally determined genotype diversity of Mycoplasma synoviae in Central China from 2017 to 2019.

Mycoplasma synoviae (M. synoviae) has been identified worldwide to cause respiratory diseases, infectious synovitis, airsacculitis, and eggshell apex abnormalities (EAA) in commercial chickens, which results in substantial economic losses to the poultry industry. Therefore, in this study, 258 flocks were investigated between 2017 and 2019 for M. synoviae by screening samples from Central China. Subsequently, 129 M. synoviae strains were isolated, with a positive rate of 50%. Moreover, a higher incidence of M. Synoviae infections was in layers (74.1%) than in broilers (20%) in this study. The 5'-end conserved segment of the variable lipoprotein hemagglutinin A (vlhA) gene of these isolates was then cloned and sequenced because it is a common genomic target identified so far for M. synoviae genotyping. Genotyping of all isolates was based on the phylogenetic analysis and length analysis of the proline-rich-repeat (PRR) regions, respectively. Phylogenetic analysis based on 5'-end conserved segment of the vlhA gene (76-421 nt) assigned the majority of the occurring strains as being from group 6, and others from groups 2 and 3. Results identified that these isolates were of 6 types: A (38aa), D (23aa), E (19aa), I (28aa), J (20aa), and L (35aa), based on the size of the PRR region analysis. Furthermore, most of the isolates (81.4% were identified as type L. Additionally, the epidemic types included only I and L in 2017; however, the types rose to 5 (A, D, E, I, L) in 2018 and rose to 6 (A, D, E, I, J, L) in 2019. These data showed the genotype diversity of M. synoviae in Central China. The high rate of positive flocks suggests the urgent need to take real-time supervisory controls of this Mycoplasma species in avian flocks.

First report of a novel goose astrovirus outbreak in Muscovy ducklings in China.

A highly acute disease characterized as visceral gout broke out in Muscovy ducklings in Henan province (China) in June 2020, with a mortality rate of up to 61%. In this study, common pathogenic agents were screened using reverse-transcription polymerase chain reaction or polymerase chain reaction. The results found the novel goose astrovirus (GoAstV) to be the pathogenic agent. We isolated the GoAstV, which has been designated as HNNY0620, using the Leghorn male chicken hepatocellular carcinoma (LMH) cell line and sequenced the complete genome. The phylogenetic tree showed that the amino acid (aa) sequences of ORF1a and ORF2 and the completed nucleotide sequences of the HNNY0620 strain were clustered in the GoAstV-I clade. ORF1a aa and whole-genome sequences were genetically close to TAstV-2 and DHV-3, whereas the ORF2 aa sequences were clustered with TAstV-2 and DHV2. Both the duck-origin GoAstVs and HNNY0620 harbored some special mutations, but ORF1a in 700 (I/T), ORF1b in 288 (F/L), and ORF2 in 306 (A/T) were only found in HNNY0620. These results suggest that the host range of GoAstV is diffusing, which can potentially affect other waterfowl.

Genetic Characterisation and Local Genotypes of Canine Parvovirus Strains Collected from Pet Dogs in Central and Eastern China During 2018-2019.

INTRODUCTION: 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. MATERIAL AND METHODS: A total of 118 faecal samples from dogs identified as CPV-positive were collected from veterinary clinics in central and eastern China. Overall, 16 strains collected from Anhui, 29 from Henan, and 16 from Zhejiang Province were sequenced to determine the genotypic composition of CPV-2 and mutational complexity of CPV-VP2. RESULTS: The CPV-2a, CPV-2b, and CPV-2c genotypes were detected in Anhui and Henan Provinces, while CPV-2c alone was detected in Zhejiang Province. Sequence analysis of all strains showed 98.5%-99.8%, 98.3%-99.9%, and 98.7%-99.8% identity among the 16 Anhui, 29 Henan, and 16 Zhejiang strains, respectively. Strains collected from Anhui and Henan Provinces showed lower identity (97.0%), suggesting greater genetic divergence in central China. The mutation rates of Henan and Anhui strains were lower than that of Zhejiang strains. Major amino acid mutations occurred at sites 5, 370, 426, and 440. Epitope and entropy analyses implied these sites' likely conformance to the principles of mutation tendency, complexity, and diversity. CONCLUSION: The findings for the evolutionary structure of CPV-2 strains collected from three provinces in central and eastern China advance trend monitoring of the genetic variation in canine parvovirus and point to its implications in the development of novel vaccines.

Simple and Visible Detection of Novel Astroviruses Causing Fatal Gout in Goslings Using One-Step Reverse Transcription Polymerase Spiral Reaction Method.

In this study, a one-step isothermal method combining polymerase spiral reaction (PSR) with reverse transcription (RT-PSR) was established for rapid and specific detection of novel astroviruses causing fatal gout in goslings (N-GoAstV). The one-step RT-PSR was accomplished at the optimal temperature of 62°C and time of 40 min and used primers simply designed as conventional PCR primers, and the results of detection were visible to the naked eye. The detection limit of PSR was above 34.7 copies/µL at a 95% probability level according to probit regression analysis. The assay specifically detected N-GoAstV, and no other reference viruses were detected. These results suggest that the newly established RT-PSR assay could, in one step, accomplish reverse-transcription, amplification, and result determination providing a visible, convenient, rapid, and cost-effective test that can be carried out onsite, in order to ensure timely quarantine of N-GoAstV-infected birds, leading to effective disease control.

An Improved Polymerase Cross-Linking Spiral Reaction Assay for Rapid Diagnostic of Canine Parvovirus 2 Infection.

With increasing complications of canine parvovirus infection cases, disease diagnosis and treatment have become more difficult. In this study, specificity primers for the conserved region of the VP2 gene of canine parvovirus 2 (CPV-2) were synthesized and evaluated. An improved polymerase cross-linking spiral reaction (PCLSR) method for early and rapid diagnosis of CPV-2 was established. The results showed that the amplification reaction was optimal when run at 62°C for 50 min and could be used to detect CPV-2 without any cross-reactions with other pathogens of canine infectious diseases. Reaction results were directly judged by the naked eyes, with the positive amplification tube shown as luminous yellow and the negative tube as bright purple. Compared with the previously reported polymerase spiral reaction (PSR) method for CPV-2 detection, this reaction was performed using improved primer pairs and a better dye identification method (using an indicator comprising phenol red and cresol red). The detection limit of PCLSR was 3.9 × 101 copies using gel electrophoresis or a visible dye. The positive rate of 132 clinical samples was 42.42%, which was identically the same as that of the PSR method and slightly higher than that of the colloidal gold strip method (39.39%). The newly developed CPV-PCLSR assay shows the advantage of rapid visualization of results and offers a convenient and rapid method for early CPV-2 diagnosis with higher sensitivity and specificity than the established methods.

Molecular Characterisation and Genetic Diversity of Canine Parvovirus Type 2 Prevalent in Central China.

INTRODUCTION: Canine parvovirus (CPV) disease is one of the most threatening to domestic and wild dogs. MATERIAL AND METHODS: A total of 132 clinical samples were isolated from domestic dogs with diarrhoea from Henan, Hubei, Jiangsu, and Anhui provinces from 2016 to 2017, and 56 were positive for CPV-2 by PCR. A phylogenetic tree was constructed for the isolate sequences incorporating 53 non-Chinese reference strains. RESULTS: VP2 sequences showed the strains mainly to be new CPV-2a/2b and CPV-2c genotypes. The Ala5Gly, Phe267Tyr, Ser297Ala, Tyr324Ile, Gln370Arg, Asn426Asp or Asn426Glu, and Thr440Ala sites in the VP2 protein antigenic region were found to have high mutation rates. The VP2 tertiary structural model shows that the change at these mutation points is a factor for the changes in the protein structure. Significant differences between the Central Chinese strains and others were found, indicating that evolution is geographically related and extended in major regions. The homology between the identified strains confirmed their relationship. Phylogenetic analysis indicated that the common genotypes in the same clusters differ slightly in homology and evolutionary history. CONCLUSION: This epidemiological study enriches the available data and serves as an important reference for studies on the evolution of CPV and selection of vaccines in China.

Novel Genotype Definition and the First Epidemiological Investigation of Canine Adenovirus Type 2 in Dogs in Central China.

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.

Novel genotype definition and genome characteristics of duck circovirus in central and Eastern China.

To explore genetic variations in duck circovirus (DuCV) and the molecular epidemiology of its infection, tissue samples were collected from 219 dead ducks from 20 farms in the central and eastern regions of China. All farms tested positive for DuCV, with duck-origin goose parvovirus, reovirus and Tembusu virus having co-infection rates of 100%, 0% and 0%, respectively. A total of 20 strains from the DuCV-positive flock were sequenced. The total sequence length was 1987-1996 nt, and the sequences shared 82% (JX499186, DuCV2 from Sichuan province, China) to 99.7% (KY328304, DuCV1 from Shandong Province, China) sequence identity with DuCV sequences available in GenBank. Hyper-variable regions were mainly located in open reading frame (ORF)2, ORF3 and intergenic regions. The tertiary structure of ORF2 from four provinces (Henan, Anhui, Zhejiang and Fujian) in China showed a canonical viral jelly roll and the antigenic epitope of ORF2 located in the bulge of the protein surface. Overall, 15 of the 20 DuCV strains are possibly derived through inter-genotypic and intragenotypic recombination. Based on sequence and phylogenetic analyses, six strains from Fujian Province clustered into a novel genotype-DuCV-1d. These findings may enrich our understanding of DuCV evolution and circulation and lay the foundation for vaccine strain selection.