Goose astrovirus (GAstV), an agent of fatal visceral gout in goslings, has been widely circulating in eastern China since 2017, but little is known about its genetic diversity and systematic evolution. In this study, we isolated and sequenced nine nearly full-length GAstV genomes and conducted comprehensive genetic diversity and evolutionary analysis and compared them with other reported GAstV sequences. Our results indicated that two genotypic species of GAstV were circulating in China, and GAstV-2 subgenotype II-c had arisen as the dominant genotype in Shandong province and across the whole country. Multiple alignments of GAstV amino acid sequences revealed several characteristic mutations in GAstV-2 II-c strains, as well as additional residues in the nine new isolates which varied over time. Phylogenetic analysis of three open reading frames demonstrated different evolutionary histories. Evidence of natural recombination was also detected in GAstV, with most of the recombination occurring in the GAstV-2 II-c subgenotype. Molecular adaptation analyses revealed that the evolution of GAstV was shaped by strong negative selection, although a number of amino acids, which potentially affect host infection and cell entry, were subjected to positive pressure. Overall, these findings improve our understanding of the epidemiology and evolution of GAstV and may help in the development of vaccines and diagnostics.

The clinical outcome of porcine circovirus 3 (PCV3) infection is still controversial. Herein, a novel PCV3 isolate (PCV3-China/DB-1/2017) with the molecular characterization of 24A and 27K in the Cap protein was used to inoculate three-week-old cesarean-derived, colostrum-deprived piglets. The nine PCV3 DB-1 inoculated piglets exhibited no obvious clinical symptoms or macroscopic lesions. PCV3 displayed a broad histotropism, including the heart, liver, spleen, lung, kidney, brain, lymph nodes, and tonsil, and the lungs and lymph nodes contained a higher quantity of viral genomes compared to that of the other organs. From 7 days after PCV3 DB-1 inoculation, the piglets showed obvious IgG antibody responses against PCV3 rCap-VLPs. The cumulative results demonstrated that PCV3 trend to low pathogenicity.

Canine circovirus (CanineCV), a new pathogen, was found to be associated with canine hemorrhagic diarrhea, vasculitis, granulomatous lymphadenitis, and acute gastroenteritis. Although CanineCV was highly positive rate in diarrhea cases, its pathogenicity remains controversial. In this study, the seroprevalence and associated risk factors of CanineCV infection among domestic dogs in northeastern China was investigated by an indirect enzyme-linked immunosorbent assay (iELISA) based on recombinant capsid protein. Results revealed the proposed iELISA had no cross-reactivity with other related pathogens, and yielded good diagnostic values. Then, to evaluate the rCap iELISA, this study applied it to detect antibodies against CanineCV in 1,047 clinical serum samples obtained from northeastern China in 2016-2017. Results showed the positive rates in the five cities of Jilin, Liaoning, and Heilongjiang provinces ranged from 22.22 to 42.29%. Statistical analysis shows a significant difference in age between dogs <3 months old with respect to the >1-year-old dogs (p = 0.005), that is, the CanineCV infection was more frequently identified from older dogs. In the artificially infected experiment, the dogs developed seroconversion after 9 or 12 days and the main way of virus excretion was through feces. More interestingly, among the 32 ELISA-positive serum samples, 34.75% samples tested positive for the CanineCV DNA by qPCR, far higher than that in ELISA-negative serum samples (5.26%, 2/38). This report is the first to demonstrate that CanineCV infection is common in the dog population in northeastern China. The results showed obvious differences in the positive rate associated with diarrhea, age, but not with different cities. This study also provide basis for evaluating the pathogenic potential of CanineCV. But, the pathogenicity, the relationship between antibody level and immune protection, and the harmful effects of this virus remain to be established.

Canine circovirus (canine CV) is an etiological agent associated with diarrhea, hemorrhagic gastroenteritis and vasculitis. Although canine CV has been identified and characterized in southern China in recent years, its epidemiology in other regions of China and its precise molecular characteristics have not been examined. In this study, we examined 141 fecal specimens collected from domestic dogs with or without diarrhea in Heilongjiang province, Northeastern China, during 2014 to 2016. A total of 18 out of 141 samples were found to be positive for canine CV by real-time quantitative PCR. In the diarrhea samples, canine CV was detected in coinfections with canine parvovirus 2. More importantly, two different canine CV strains were detected in one sample. Five canine CV genomes were successfully amplified. Sequence analysis showed that there were two unique amino acid changes in the Rep protein (N39S in the K1 strain, and T71A in the XF16 strain). Phylogenetic analysis indicated that canine CV could be divided into four genotypes, and specific nucleotide mutations could be used for confirming the four genotypes. Moreover, recombination analysis revealed that a total of eight recombination events were found in five genomic sequences. Molecular evolution analysis showed that the canine CV has been under purifying selection. This study provides evidence that at least three genotypes of canine CV are co-circulating in China. Continuous epidemiological surveillance is therefore necessary to understand their importance for the evolution of canine CV.

Autophagy is a tightly regulated catabolic process and is activated in cells in response to stress signals. Despite extensive study, the interplay between duck hepatitis A virus type 1 (DHAV-1) and the autophagy of host cells is not clear. In this study, we applied proteomics analysis to investigate the interaction mechanism between DHAV-1 and duck embryo fibroblast (DEF) cells. In total, 507 differentially expressed proteins (DEPs) were identified, with 171 upregulated proteins and 336 downregulated proteins. The protein expression level of heat shock proteins (Hsps) and their response to stimulus proteins and zinc finger proteins (ZFPs) were significantly increased while the same aspects of ribosome proteins declined. Bioinformatics analysis indicated that DEPs were mainly involved in the "response to stimulus", the "defense response to virus", and the "phagosome pathway". Furthermore, Western blot results showed that the conversion of microtubule-associated protein 1 light chain 3-I (LC3-I) to the lipidation form of LC3-II increased, and the conversion rate decreased when DEF cells were processed with 4-phenylbutyrate (4-PBA). These findings indicated that DHAV-1 infection could cause endoplasmic reticulum (ER) stress-induced autophagy in DEF cells, and that ER stress was an important regulatory factor in the activation of autophagy. Our data provide a new clue regarding the host cell response to DHAV-1 and identify proteins involved in the DHAV-1 infection process or the ER stress-induced autophagy process.

As a disease characterized by severe liver necrosis and hemorrhage, duck viral hepatitis (DVH) is mainly caused by duck hepatitis A virus (DHAV). The positive-strand RNA genome of DHAV type 1 (DHAV-1) contains an internal ribosome entry site (IRES) element within the 5' untranslated region (UTR), structured sequence elements within the 3' UTR, and a poly(A) tail at the 3' terminus. In this study, we first examined that insulin-like growth factor-2 mRNA-binding protein-1 (IGF2BP1) specifically interacted with the DHAV-1 3' UTR by RNA pull-down assay. The interaction between IGF2BP1 and DHAV-1 3' UTR strongly enhanced IRES-mediated translation efficiency but failed to regulate DHAV-1 replication in a duck embryo epithelial (DEE) cell line. The viral propagation of DHAV-1 strongly enhanced IGF2BP1 expression level, and viral protein accumulation was identified as the key point to this increment. Collectively, our data demonstrated the positive role of IGF2BP1 in DHAV-1 viral proteins translation and provided data support for the replication mechanism of DHAV-1.

The duck hepatitis A virus type 1 (DHAV-1) is a member of Picornaviridae family, the genome of the virus contains a 5′ untranslated region (5′ UTR), a large open reading frame that encodes a polyprotein precursor and a 3′ UTR followed by a poly(A) tail. The translation initiation of virus proteins depends on the internal ribosome-entry site (IRES) element within the 5′ UTR. So far, little information is known about the role of the 3′ UTR and poly(A) tail during the virus proliferation. In this study, the function of the 3′ UTR and poly(A) tail of DHAV-1 in viral replication and IRES-mediated translation was investigated. The results showed that both 3′ UTR and poly(A) tail are important for maintaining viral genome RNA stability and viral genome replication. During DHAV-1 proliferation, at least 20 adenines were required for the optimal genome replication and the virus replication could be severely impaired when the poly (A) tail was curtailed to 10 adenines. In addition to facilitating viral genome replication, the presence of 3′ UTR and poly(A) tail significantly enhance IRES-mediated translation efficiency. Furthermore, 3′ UTR or poly(A) tail could function as an individual element to enhance the DHAV-1 IRES-mediated translation, during which process, the 3′ UTR exerts a greater initiation efficiency than the poly(A)25 tail.

DNA-launched infectious system is a useful tool with high rescue efficiency that allows the introduction of mutations in specific positions to investigate the function of an individual viral element. Rescued virus particles could be harvested by directly transfecting the DNA-launched recombinant plasmid to the host cells, which will reduce labor and experimental cost by skipping the in vitro transcription assay.

The circulation of duck hepatitis A virus types 1 (DHAV-1) and 3 (DHAV-3) in Southeast Asia has resulted in a continuously changing epidemiological scenario. In this study, a duplex real-time PCR assay for simultaneous quantitative detection of DHAV-1 and DHAV-3 was established, and 200 liver samples from dead ducklings collected from 31 different flocks in Shandong province, China, were tested. Fifty-eight (29.0 %) samples from 13 flocks were positive for DHAV-1 single infection, 113 (56.5 %) samples from 13 other flocks were positive for DHAV-3 single infection, and 24 samples (12.0 %) from four flocks were positive for both viruses. DHAV-1 and DHAV-3 were detected with high viral loads in all of the organs tested (liver, spleen, pancreas, kidney, heart, thymus, bursa of Fabricius and brain). No significant difference in DHAV-1 and DHAV-3 viral loads was found between singly infected and coinfected samples, and there was no correlation between the viral loads of the two viruses and the age of dead ducklings. To the best of our knowledge, this is the first report about the in vivo distribution of DHAV-1 and DHAV-3 in clinically infected ducklings.

Dear Editor,Duck virus hepatitis（DVH）is caused by at least threedifferent RNA viruses,including duck hepatitis A virus（DHAV）,duck astrovirus type 1（DAstV-1）,and duckastrovirus type 2（DAstV-2）.The first of these,DHAV,has been classified into three serotypes by neutralization

We report here the complete genome sequence of a duck astrovirus (DAstV) isolated from a dead duckling in eastern China. Sequence analyses indicated that the genome of the astrovirus possessed a typical astrovirus organization. Comparison of the partial polymerase gene sequences of DAstV-1 and DAstV-2 showed that the astrovirus shared 94.4% and 64.2% nucleotide identity, respectively. The whole nucleotide sequence of the astrovirus had the highest homology with the sequence of DAstV-1 strain C-NGB (98.7%). Therefore, the strain we describe here is a DAstV-1 isolate.

The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1-17 and 18-36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNA binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome.

The duck circovirus (DuCV) infection in sick ducks from Fujian Province was investigated. The liver samples of 43 sick Muscovy ducks with infectious serositis were collected from 12 duck farms in Fujian Province. Based on the published sequences of DuCV, two primers were designed for the detection of DuCV and four pairs of primers were designed to amplify four overlapping fragments that cover the complete genome of DuCV. The specific PCR products were amplified from positive samples. The fragments were then cloned into pMD18-T vector and sequenced, and the full length genomic sequence of the FJ0601 isolate of DuCV was obtained. PCR analysis showed that the proportion of ducks which were positive for circovirus was 79% and 10 out of the 12 farms were positive. Sequence analysis showed that the complete genome of DuCV-FJ0601 was 1988 bp and possessed features common to the family Circoviridae which included a stem-loop structure and the Rep protein motifs. Homology analysis showed that FJ0601 isolate of DuCV had 97.3%∼97.5% nucleotide sequence identity to all the four Taiwan isolates (TC1/2002, TC2/2002, TC3/2002, TC4/2002), 82.9% identity to the America (33753-52) isolate and 82.3% identity to the Germany isolate. Phylogenetic analysis with Clustal W, however, showed that FJ0601 isolate of DuCV was on a common branch with Taiwan isolates, and Germany and America isolates belonged to the other branch.

White spot syndrome virus(WSSV) from shrimp was first found in Taiwan in 1992.Since 1993,white spot syndrome disease of shrimp caused by this virus was widely spread throughout China,Asia and Pan-Pacific Ocean.It can cause 100% cumulative mortality of cultured shrimps in 3—4 days.The virus particles contain five major proteins:VP28,VP26,VP24,VP19 and VP15.The mode of entry and systemic infection of WSSV in shrimp and the funotions of these virion proteins are not clear.VP60B is a kind of minor structure proteins in WSSV particles.Using interproscan to analysis on homology to VP60B,a piece of sequence of amino acid in VP60B was found homology with adenovirus type 5 fiber protein knob domain.In this study,the gene of VP60B had been cloned into a prokaryotic expression system,and induced to express at low temperature.The result showed that the expressed protein was existed mainly as inclusion body in this expression system. Prokaryotic expressed VP60B could not recognize by polyclone antiserum against WSSV,which suggested that this protein might be a weak immunogen.According to the analysis of the sequence of amino acid,a trans-membrane domain was found,which suggested that this protein might locate on the envelop membrane of WSSV.

The genomic DNA extracted from chicken embryo fibroblast (CEF) infected with a Chinese field isolate HA9901 of reticuloendotheliosis virus (REV) was used as the template to amplify the REV proviral genomic cDNA by PCR with 6 pairs of primers according to published sequences. Six overlapping fragments were amplified, cloned into the TA vector and sequenced, including a fragment which was amplified from the circular proviral cDNA and covering both 5′-and 3′-ends. The complete sequence of the whole genome was established and analyzed with a DNAstar software. Comparisons of the sequence with two other strains demonstrated that the genomes of REV were relatively conservative, the homogenecity for all genes or LTR fragments of the 3 strains was over 92%, no matter whether they were isolated from different species and regions in different years. But, the homology of Chinese strain HA9901 to a fowl pox virus-associated strain from Chickens was higher than that to strain SNV isolated from ducks.