Zilin Qiao

A novel genotype of hepatitis E virus prevalent among farmed rabbits in China.

In total, 335 serum samples were collected from rabbits from two farms in Gansu province, China, and tested for anti‐hepatitis E virus (HEV) antibody using EIA and for HEV RNA using nested RT‐ PCR with ORF2 primers. The overall prevalence of anti‐HEV antibody and HEV RNA was 57.0% (191/335) and 7.5% (25/335), respectively. The positivity rate of HEV RNA in the anti‐HEV antibody negative group (7.6% (11/144)) did not differ significantly from that in the positive group (7.3% (14/191)). The concordance between HEV RNA and anti‐HEV antibody was 43.3% with no significant correlation (P < 0.05). All 25 amplicons from the ORF2 region were cloned and sequenced. On the basis of nucleotide sequence comparison, they had 84–99% identity to each other and 73–77%, 70–76%, 75–82%, 71–77%, and 53–65% with the corresponding regions of genotypes 1, 2, 3, 4, and avian HEV, respectively. Samples that were positive with the ORF2 primers were amplified using ORF1 region primers; 17 were positive and shared 71–78%, 73–76%, 74–82%, 72–78%, and 39–58% identity with the corresponding regions of genotypes 1, 2, 3, 4, and avian HEV, respectively, at the nucleotide level. Two representative full‐length sequences were determined. These two sequences shared 85% identity with each other and had 74%, 73%, 78–79%, 74–75%, and 46–47% identity to full‐length genotypes 1, 2, 3, 4, and avian HEV, respectively. Thus, the sequences isolated from the rabbits represent a novel genotype of HEV. This study provides novel information about HEV genotypes infecting rabbits as well as evidence of a new mammalian genotype of HEV. J. Med. Virol. 81:1371–1379, 2009.

Infection dynamics of hepatitis E virus in naturally infected pigs in a Chinese farrow-to-finish farm

To analyze the changes that occur in pigs during hepatitis E virus (HEV) infection, 256 serial serum samples were obtained from 32 pigs from one pig farm at ages 0 (cord blood), 15, 30, 60, 75, 90, 120, and 150 days. All HEV markers were assayed in these samples and showed that total anti-HEV antibodies and IgG formed two peaks. The first peak occurred at 0-60 days and the second after 75 days. No markers of infection, such as HEV RNA, antigen and anti-HEV IgM, were detectable during the first peak. Most newborn piglets (< 24 h of age) were negative for total anti-HEV and IgG. However, colostrum from all of the sows had evidence of these antibodies. Thus, the anti-HEV in the first peak was assumed to be acquired from maternal milk. Some infectious markers were positive at the beginning of second peak. PCR products were cloned and sequenced and the results indicated those sequences belonged to HEV genotype 4. The antibody present during the second peak may be induced by natural infection with HEV. In conclusion, pigs are susceptible to HEV infection and may remain infectious after the first peak of anti-HEV antibody.

Seroprevalence and distribution of hepatitis E virus in various ethnic groups in Gansu province, China.

Gansu province is located in northwestern China and is home to 45 ethnic groups including Han, Hui, Zang and others. Different ethnic groups have varying involvement with livestock and meat consumption, especially pork. To investigate the prevalence of hepatitis E virus (HEV) infection and the distribution of HEV genotypes among the major ethnic groups in Gansu province, 2090 serum samples were collected from individuals from four regions and three ethnic groups, the Han, Hui and Zang. All serum samples were tested for anti-HEV IgG and IgM antibodies, as well as HEV antigen, and selected samples were then tested for HEV RNA. The data showed that the seroprevalence of anti-HEV IgG in the Hui, Han and Zang ethnic groups from the four regions was 8.9%, 18.7% and 32.9%, respectively, and these differed significantly (P<0.05). The seroprevalence of anti-HEV antibody for each ethnic group varied among the different regions. In general, within the same region, the three ethnic groups also show differences. Genomic analysis indicated that HEV isolated from humans belonged to genotype 4, and resembled closely swine HEV isolates from Gansu province. The seroprevalence of anti-HEV antibodies was in accordance with the amount of contact with pigs in the different regions. Pigs are the primary host for HEV, so people in frequent contact with pigs may be at risk of zoonotic infection. However, populations that have rare contact with pigs are more likely to be susceptible to HEV when exposed, suggesting that should be the target of vaccination.

Isolation, molecular and phylogenetic analysis of encephalomyocarditis virus strain GS01 in China

Encephalomyocarditis virus (EMCV) is a small non-enveloped, single-stranded RNA virus. It can infect many host species and cause acute myocarditis and respiratory failure in piglets, reproductive failure in pregnant sows. Diseases caused by EMCV currently affect the swine industry worldwide. In this study, an EMCV strain was isolated from an aborted fetus in western China. It was identified by reverse-transcription polymerase chain reaction (RT-PCR) and genome sequencing. The subsequent results showed that the virus could produce a specific cytopathic effect on BHK-21 cells and could cause severe clinical symptoms and pathological changes in mice. Complete genome sequencing and multiple sequence alignment indicated that the GS01 strain was 79.9-99.9% identical with other isolates worldwide. Phylogenetic analysis showed that EMCV isolates fell into five clusters: lineage 1, 2, 3, 4, and 5 based on the nucleotide sequences of the entire ORF and VP3/VP1 junction, as well as 3D gene. GS01 isolate was grouped into lineage 1. The results of this study confirmed that an EMCV strain GS01 isolated from an aborted pig fetus in western China was fatal to mice and provided new epidemiologic data on EMCV in China.

Genome sequence of foot-and-mouth disease virus outside the 3A region is also responsible for virus replication in bovine cells

The deletion of residues 93-102 in non-structure protein 3A of foot-and-mouth disease virus (FMDV) is associated with the inability of FMDV to grow in bovine cells and attenuated virulence in cattle.Whereas, a previously reported FMDV strain O/HKN/21/70 harboring 93-102 deletion in 3A protein grew equally well in bovine and swine cells. This suggests that changes inFMDV genome sequence, in addition to 93-102 deletion in 3A, may also affectthe viral growth phenotype in bovine cellsduring infection and replication.However, it is nuclear that changes in which region (inside or outside of 3A region) influences FMDV growth phenotype in bovine cells.In this study, to determine the region in FMDV genomeaffecting viral growth phenotype in bovine cells, we constructed chimeric FMDVs, rvGZSB-HKN3A and rvHN-HKN3A, by introducing the 3A coding region of O/HKN/21/70 into the context of O/SEA/Mya-98 strain O/GZSB/2011 and O Cathay topotype strain O/HN/CHA/93, respectively, since O/GZSB/2011 containing full-length 3A protein replicated well in bovine and swine cells, and O/HN/CHA/93 harboring 93-102 deletion in 3A protein grew poorly in bovine cells.The chimeric virusesrvGZSB-HKN3A and rvHN-HKN3A displayed growth properties and plaque phenotypes similar to those of the parental virus rvGZSB and rv-HN in BHK-21 and primary fetal porcine kidney (FPK) cells. However, rvHN-HKN3A and rv-HN replicated poorly in primary fetal bovine kidney (FBK) cells with no visible plaques, and rvGZSB-HKN3A exhibited lower growth rate and smaller plaque size phenotypes than those of the parental virus in FBK cells, but similar growth properties and plaque phenotypes to those of the recombinant viruses harboring 93-102 deletion in 3A. These results demonstrate that the difference present in FMDV genome sequence outside the 3A coding region also have influence on FMDV replication ability in bovine cells.

Construction and characterization of 3A-epitope-tagged foot-and-mouth disease virus

Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids (aa) in most FMDVs examined to date. Specific deletion in the FMDV 3A protein has been associated with the inability of FMDV to grow in primary bovine cells and cause disease in cattle. However, the aa residues playing key roles in these processes are poorly understood. In this study, we constructed epitope-tagged FMDVs containing an 8 aa FLAG epitope, a 9 aa haemagglutinin (HA) epitope, and a 10 aa c-Myc epitope to substitute residues 94-101, 93-101, and 93-102 of 3A protein, respectively, using a recently developed O/SEA/Mya-98 FMDV infectious cDNA clone. Immunofluorescence assay (IFA), Western blot and sequence analysis showed that the epitope-tagged viruses stably maintained and expressed the foreign epitopes even after 10 serial passages in BHK-21 cells. The epitope-tagged viruses displayed growth properties and plaque phenotypes similar to those of the parental virus in BHK-21 cells. However, the epitope-tagged viruses exhibited lower growth rates and smaller plaque size phenotypes than those of the parental virus in primary fetal bovine kidney (FBK) cells, but similar growth properties and plaque phenotypes to those of the recombinant viruses harboring 93-102 deletion in 3A. These results demonstrate that the decreased ability of FMDV to replicate in primary bovine cells was not associated with the length of 3A, and the genetic determinant thought to play key role in decreased ability to replicate in primary bovine cells could be reduced from 93-102 residues to 8 aa residues at positions 94-101 in 3A protein.