Optimal expression and purification of sapelovirus A structural protein VP1, and its immunogenicity in mice.

Sapelovirus A (SV-A) is a positive-sense single-stranded RNA virus which is associated with acute diarrhea, pneumonia and reproductive disorders. The virus capsid is composed of four proteins, and the functions of the structural proteins are unclear. In this study, we expressed SV-A structural protein VP1 and studied its antigenicity and immunogenicity. SDS-PAGE analysis revealed that the target gene was expressed at high levels at 0.6 mM concentration of IPTG for 24 h. The mouse polyclonal antibody against SV-A VP1 protein was produced and reached a high antiserum titer (1: 2,048,000). Immunized mice sera with the recombinant SV-A VP1 protein showed specific recognition of purified VP1 protein by western blot assay and could recognize native SV-A VP1 protein in PK-15 cells infected with SV-A by indirect immunofluorescence assay. The successfully purified recombinant protein was able to preserve its antigenic determinants and the generated mouse anti-SV-A VP1 antibodies could recognize native SV-A, which may have the potential to be used to detect SV-A infection in pigs.

Serological investigations on West Nile virus in birds and horses in Shanghai, China.

West Nile virus (WNV) infection is an emerging zoonosis that threatens global public health. In this study, a total of 95 bird serum samples from 14 species and 341 horse serum samples were collected from 2008 to 2010 in Shanghai, China. All serum samples were screened initially for WNV-reactive antibodies using a competitive ELISA. The positive samples detected by ELISA were further confirmed using a plaque-reduction neutralization test (PRNT) for WNV and its most closely related flaviviruses in the area to avoid false positives due to cross-reactivity. Five (5·3%) of the bird serum samples and none (0·0%) of the horse serum samples tested positive for WNV antibodies. The findings strongly suggest that some of the birds, specifically the resident birds in China, had been exposed to WNV.

Molecular detection of Torque teno sus virus from tissues samples of sick pigs in China.

In the present study, Torque teno sus virus (TTSuV) was detected from different tissues, stool and serum samples of 25 sick pigs. The total prevalence of TTSuV1 and TTSuV2 were 64% (16/25) and 28% (7/25), 24% (6/25) were co-infected with both TTSuV1 and TTSuV2. The prevalence of TTSuV infection in spleen is a slightly higher, with positive rates of 52% (13/25) for TTSuV1 and 24% (6/25) for TTSuV2. Phylogenetic analysis of TTSuV1 showed that 21 isolates were distributed into two clusters (genotype TTSuV1a and TTSuV1b), with genotype TTSuV1b was the dominant genotype. Phylogenetic analysis of TTSuV2 showed that the nine isolates shared 80.9-99.2% nucleotide homology with each other, and were distributed in different genotypes (TTSuV2a-TTSuV2f). TTSuV2d was the most prevalent genotype in this study, which contained five Spanish strains and nine Chinese strains, and shared 94.2-96.8% homology.

Segmentation expression of capsid protein as an antigen for the detection of avian nephritis virus infection in chicken flocks.

Subclinical pathological changes in the kidneys of broiler chickens and suppression of growth caused by the avian nephritis virus (ANV) affect poultry flocks worldwide. A test for detection of virus-specific antibodies in serum would be useful for epidemiological investigations, however the poor propagation in cell cultures has restricted the development of serological tests based on the use of ANV particles as antigens. An enzyme-linked immunosorbent assay (ELISA) was developed for detection of ANV-specific antibodies in chicken serum, using a recombinant protein antigen prepared by segmentation expression of the capsid protein antigen epitope of ANV (HM029238) transfected into Escherichia coli. The expressed fusion protein was detected by Western blotting with ANV-positive serum, and the optimal immunoreactive fusion P1 protein was determined. Using the optimized P1-ELISA, ANV-specific antibodies were detected in commercial chicken flocks aged 10-25 days obtained from the Liaoning Province, China. Out of 960 serum samples, 459 (47.8%) were positive for infection with ANV. These results indicate that the P1-ELISA is helpful for preliminary serological diagnosis of ANV infection, and could be used to for screening in ANV infection and for determining antibodies against ANV.

Detection of astrovirus infection in pigeons (Columbia livia) during an outbreak of diarrhoea.

Avian astrovirus infections are widespread in many countries, and infections have been linked to enteritis and increased mortality in young poultry. Although pigeons are treated as an important poultry product in some countries, their diseases are often poorly understood and astrovirus infection in pigeons has not been reported. In the present study, faecal samples were collected during an outbreak of gastrointestinal illness in a population of Shanghai pigeons. The samples were examined for astroviruses by reverse transcription-polymerase chain reaction. Eighty-nine per cent (40/45) and 4% (2/45) were found to be positive for avian nephritis virus (ANV) and chicken astrovirus, respectively. One positive sample indicated a co-infection with both ANV and chicken astrovirus. Phylogenetic analysis based on the partial polymerase gene sequence and full-length capsid protein from published avian astrovirus sequences in GenBank revealed that the pigeon viruses detected in this study were evolutionarily closely related to chicken ANV. The present study provided evidence for the presence of astrovirus in pigeons and suggests that cross-infection between pigeons and commercial chickens was likely. Whether the astroviruses in pigeons were responsible for the diarrhoea remains to be determined.

Complete sequence and genetic characterization of pigeon avian nephritis virus, a member of the family Astroviridae.

In the current study, the complete genome sequence of a member of the family Astroviridae isolated from pigeons was determined through genetic characterization and phylogeny analysis. The isolated genome sequence was proposed to be that of pigeon avian nephritis virus (ANV), whose genome structure and characteristics were similar to previously reported avian astroviruses. The sequenced ssRNA genome comprises 6928 nucleotides, excluding the poly(A) tail, and contains three open reading frames. Phylogenetic analysis using a partial nucleotide sequence of the polymerase gene and the entire amino acid sequence of the full-length capsid protein revealed that pigeon avian nephritis virus is closely related to the previously published ANV, especially to the Japanese G-4260 and Chinese strains. This investigation provides information on the sequence and genetic characteristics of this virus and contributes to a better understanding of pigeon ANV and the possible occurrence of astrovirus transmission between chickens and pigeons.

Isolation and characterization of canine astrovirus in China.

In this study, we first investigated the prevalence of astrovirus in stools of dogs with and without diarrhea in Shanghai, China. Of all the specimens, 22 (12.02%) from the 183 dogs with diarrhea and none (0%) from the 138 healthy controls were positive for astrovirus. Furthermore, we cloned partial sequences of ORF1b (442 bp) and the entire sequences of ORF2 (2475 bp). Phylogenetic analysis showed that the new isolates were belonged to genus Mamastrovirus and most closely clustered with the Italy strain, based on the ORF2 sequences available. However, the new isolates and the Italy strain were divided into two different clusters. The new isolates may be a new strain of canine astrovirus.

Sequence analyses of the representative Chinese-prevalent strain of avian nephritis virus in healthy chicken flocks.

Avian nephritis virus (ANV), which belongs to the Astroviridae family, has been associated with acute nephritis in chickens. Cases of ANV infection have been recorded in Japan and in several European countries. However, related studies have never been performed in China. Thus, this study isolated ANV in Chinese chicken flocks. ANV RNA was detected by reverse transcription-PCR in stool samples collected from healthy layer chickens in the Sichuan Province of China in 2009. Of the 192 stool specimens collected, 32.3% (62/192) were positive for ANV infection. The whole genome of ANV-Sichuan54, the first representative Chinese strain, was 6941 nucleotides in length, including the 5' untranslated region, three open reading frames (ORFs), a 3' UTR, and a poly-(A) tail. Comparative and phylogenetic analyses based on partial RNA-dependent RNA polymerase (ORF1b) demonstrated that the majority of ANV investigations were more closely related to the U.S. ANV strain (DQ324827-324836) than to the G-4260 (AB033998).

Molecular detection and sequence analysis of feline Torque teno virus (TTV) in China.

In the present study, two isolates (SH-F1 and SH-F2) of Torque teno felis virus (feline TTV) were detected in 16 (12.5%) serum samples collected from cats in China. Their full length genomes were cloned and sequenced. The results showed that they were 2063bp in length and contained three open reading frames (ORFs) (ORF1: nt438-1748, ORF2: nt268-585 and ORF3: nt268-581, 1461-1842). Phylogenetic analysis showed that they were clustered with the strain of Japan (Fc-TTV4, AB076003) and the strain of France (PRA4, EF538878). Sequence analysis indicated that SH-F1 had high (97.5% and 93.3%) identity with the strain of Japan and the strain of France, and SH-F2 shared 94.5% and 92.1% homology with them, respectively. In conclusion, we demonstrated that feline TTV is present in China.

Human parechovirus infections in monkeys with diarrhea, China.

Information about human parechovirus (HPeV) infection in animals is scant. Using 5' untranslated region reverse transcription-PCR, we detected HPeV in feces of monkeys with diarrhea and sequenced the complete genome of 1 isolate (SH6). Monkeys may serve as reservoirs for zoonotic HPeV transmissions and as models for studies of HPeV pathogenesis.

Sequence analysis of an isolate of minute virus of canines in China reveals the closed association with bocavirus.

In the present study, we have cloned and sequenced the nearly-full-length genome of minute virus of canines (MVC), SH26, in China. The genome of MVC, 5,132 nucleotides (nts) in length, contains three open reading frames (ORFs), which are 2,325-bp of NS1, 561-bp of NP1 and 2,112-bp of VP1/VP2 encoding three proteins of 774, 186 and 703 residues, respectively. Predicted amino acids sequence of NS1 of MVC has 44% identity with human bocavirus (HBoV) and human boacvirus 2 (HBoV2), NP1 has 48 and 45% identity with HBoV and HBoV2, VP1/VP2 has 45 and 46% identity with HBoV and HBoV2, respectively. Phylogenetic analysis showed that the present Chinese MVC strain was also closely clustered with the previous American and Japanese MVC isolates, and MVCs formed a different branch together with bovine parvovirus and HBoVs from other parvoviruses classified into Parvovirinae.

Cross-species infection of hepatitis E virus in a zoo-like location, including birds.

Hepatitis E virus (HEV) is a zoonotic pathogen of which several species of animals are considered to be reservoirs. Thirty-eight faecal samples, obtained from 22 species of animals including birds in a wildlife first-aid centre in Eastern China, were tested for HEV RNA. Our survey revealed that in total 28.9% (95% confidence interval 14.5-43.4) of the faecal samples from various mammals and birds were HEV RNA positive. Sequence and phylogenetic analyses of the 11 isolates demonstrated that all sequences clustered in genotype 4 with 96-100% identity to each other. In addition, serum samples from seven animal handlers have shown that five (71.4%) were seropositive. The findings imply that cross-species infection of HEV had probably occurred in this zoo-like location, and moreover, birds can be infected naturally with mammalian HEV.

Sequence and analysis of genomic segment A and B of very virulent infectious bursal disease virus isolated from China.

A very virulent infectious bursal disease virus (vvIBDV) field strain, named SH95, was identified and characterized from flocks with vaccination failure in Shanghai. The use of random primer and a reverse transcriptase lacking RNase-H activity produced full-length cDNA copies of the viral genomic A and B segments of SH95. The 3259 base pairs (bp) of segment A and 2827 bp of segment B were amplified by long and accurate PCR in a single step, then successfully cloned and sequenced. There were five to 27 amino acid substitutions compared with other IBDV strains within the segment A polyprotein (of these, three are unique) and about nine to 38 amino acid substitutions within VP1 (of which, four are unique). The comparison of sequences encoding the polyprotein showed that vvIBDV SH95 was most closely related to Asiatic vvIBDVs, which formed a closely related group clearly distinguishable from other classical strains of IBDV. Phylogenetic analysis suggested that vvIBDV SH95 and some other Asiatic vvIBDVs were derived from similar origin. But the topology tree performed on segment B was quite different from that performed on segment A, indicating that a genetic reassortment had played an important role in the emergence of vvIBDV SH95.