Jianping Qin

ISOLATION AND GENETIC ANALYSIS REVEALED NO PREDOMINANT NEW STRAINS OF AVIAN INFECTIOUS BRONCHITIS VIRUS CIRCULATING IN SOUTH CHINA DURING 2004-2008

Abstract Twenty-seven strains of avian infectious bronchitis virus (IBV) were isolated from dead or diseased chickens at different chicken farms in South China during 2004–2008, of which the S1 gene was sequenced. Phylogenetic analysis of the S1 gene sequences of the isolated 27 strains together with 29 strains published in Genbank revealed that all IBV strains except for one isolated and one published were clustered into six distinct genotypes I-VI. 26 isolated strains belong to genotypes I, II, and III, forming a big phylogenetic branch without new predominant strains, whereas all five vaccine strains belong to genotype V that is evolutionarily distant from genotypes I, II, and III. The study of the protease cleavage motif within the S1 protein found 12 different cleavage motifs, of which 3 motifs are shared by both isolated and published strains, 2 motifs unique to isolated strains, and 7 motifs unique to published strains, further bolstering the notion of no new predominant strains. Alignment analysis of the S1 amino acid sequences indicated that the amino acid substitutions, insertions, and deletions are polymorphic and diverse, showing no sign of predominant genetic changes among the isolated strains. Taken together, there was no predominant new strain circulating in South China during 2004–2008. Nonetheless, circulating IBV strains have been continuously evolving with genetic compositions distant from vaccine strains; this explains why there have been constant but infrequent outbreaks in commercial flocks in South China during 2004–2008. Furthermore, in order to safe guard against the sudden emergence of new predominant strains, continuing surveillance of IBV strains circulating in the field is of extreme importance.

Phylogenetic distribution and predominant genotype of the avian infectious bronchitis virus in China during 2008-2009.

BackgroundThe nephropathogenic avian infectious bronchitis (IB) caused unprecedented economic losses to the commercial chicken industry of China in 2008-2009. To investigate the prevalence of nephropathogenic IB in China, eighty IBV isolates from different provinces during 2008-2009 were identified by dwarf embryo test and RT-PCR.ResultsThe strains were mostly isolated in winter and spring with a wide age range of IB outbreaks, from 4 to 69 days. By the virus recovery trials, 70/80 of the strains resulted in the deaths or distresses of birds from nephritis. To learn more about the molecular evolutionary characteristics of the circulating field strains, the coding region of major spike 1 (S1) protein gene of these strains was RT-PCR amplified and sequenced. Compared to the published representative strains, nucleotides and amino acids sequence analysis indicated that the S1 genes of these strains and the reference strains displayed homologies ranging from 75.1% to 99.8% and from 73.1% to 99.8% respectively. S1 protein of the major pandemic strains contained 540 or 542 amino acids with the cleavage site of HRRRR or RRFRR. Phylogenetic analysis revealed that recent field isolates of IBV in China were mostly belonged to A2-branch (QXIBV-branch) and HN08-branch, only one isolate was belonged to Gray-branch and M41-branch respectively. Most of the 80 strains showed evolutionarily distant from vaccine strains.ConclusionsThe results of this study suggested that nephropathogenic IBVs were mainly A2-like strains in China during 2008-2009.

Phylogenetic analysis of the S1 glycoprotein gene of infectious bronchitis viruses isolated in China during 2009–2010

As part of our ongoing surveillance program, 40 field strains of avian infectious bronchitis virus (IBV) were isolated from dead or diseased chicken flocks in different areas of China between 2009 and 2010. S1 glycoprotein genes of these strains were sequenced and analyzed with 38 strains published in GenBank. S1 genes of these isolated strains and the vaccine strains showed nucleotide homologies ranging from 65.2 to 82% and amino acid homologies ranging from 58.4 to 81.9%. Meanwhile, Chinese IBV strains isolated in this study, which were mainly nephropathogenic, could be separated into six variant lineages (CH I–CH VI), and current vaccine strains used in China formed Mass variant lineage that is evolutionarily distant from Chinese isolates. Moreover, CK/CH/GD/NC10, CK/CH/GD/KP10, and our previous isolates TC07-2 formed the CH VI lineage, showing larger evolutionary distances from other strains. Taken together, these findings suggested that various variant lineages were co-circulating in China now, and appeared to be continuously evolving, alternative indigenous vaccines indeed need for effective control of IB in China.

Preparation and immune activity analysis of H5N1 subtype avian influenza virus recombinant protein-based vaccine.

Avian influenza is a severe disease among farmed poultry and free-living birds and a constant threat to the commercial chicken industry around the world. Hemagglutinin (HA) is the major immunogen on the envelope of influenza A virus and is the predominant inducer of neutralizing antibody. To obtain the bioactive antigen proteins in large quantities, a new protein expression vector pBCX was constructed, which is based on the pET32a vector. The HA gene of the H5N1 subtype of avian influenza virus (AIV) was inserted into the pBCX vector and expressed efficiently in Escherichia coli BL21 (DE3). Fused expression of the exogenous gene and msyB produced a 97-kDa msyB-HA fusion protein. Sodium dodecyl sulfate-PAGE combined with scanning analysis demonstrated that the msyB-HA fusion protein accounted for 29.5% of the total bacterial protein, 90.5% being soluble. The msyB-HA fusion protein was purified with nondenaturing 50% Ni-NTA column chromatography, and the result showed that 24 mg of purified msyB-HA fusion protein could be obtained from 1 L of induced expression bacterial culture medium. The comparative results in the present study showed that pBCX was superior to pET32a as a protein expression vector. Western blotting showed the recombinant msyB-HA (rHA) to have better antigenic activity, which may be the result from the better posttranslation protein modification and folding in the pBCX expression system. With the rHA fusion protein as antigen, we successfully prepared and screened specific monoclonal antibodys against the H5N1 subtype AIV, which indicated that the rHA had antigen epitopes and biofunctions. The immune test confirmed that the rHA protein vaccine could also induce high neutralizing antibodies, and the AIV challenge test proved that the rHA protein-based vaccine could prevent the corresponding infection. This study demonstrates that the recombinant HA protein produced by the pBCX expression system could be used as a recombinant protein-based vaccine and has potential for further development for diagnosis.

Isolation and phylogenetic analysis of hemagglutinin gene of H9N2 influenza viruses from chickens in South China from 2012 to 2013.

As part of our ongoing influenza surveillance program in South China, 19 field strains of H9N2 subtype avian influenza viruses (AIVs) were isolated from dead or diseased chicken flocks in Guangdong province, South China, between 2012 and 2013. Hemagglutinin (HA) genes of these strains were sequenced and analyzed and phylogenic analysis showed that 12 of the 19 isolates belonged to the lineage h9.4.2.5, while the other seven belonged to h9.4.2.6. Specifically, we found that all of the viruses isolated in 2013 belonged to lineage h9.4.2.5. The lineage h9.4.2.5 viruses contained a PSRSSR↓GLF motif at HA cleavage site, while the lineage h9.4.2.6 viruses contained a PARSSR↓GLF at the same position. Most of the isolates in lineage h9.4.2.5 lost one potential glycosylation site at residues 200-202, and had an additional one at residues 295-297 in HA1. Notably, 19 isolates had an amino acid exchange (Q226L) in the receptor binding site, which indicated that the viruses had potential affinity of binding to human like receptor. The present study shows the importance of continuing surveillance of new H9N2 strains to better prepare for the next epidemic or pandemic outbreak of H9N2 AIV infections in chicken flocks.

Complete Genome Sequence of a Recombinant Nephropathogenic Infectious Bronchitis Virus Strain in China

ABSTRACT Recently, nephropathogenic infectious bronchitis virus (IBV) outbreaks have occurred in commercial broiler flocks and have been associated with a high incidence and morbidity in China. The CK/CH/Zhejiang/06/10 strain (IBV-YX10) was isolated from a 12-day-old broiler chicken in a flock of chickens with swollen speckled kidneys and distended ureters filled with uric acid in China in 2010. Here we reported the complete genomic sequence of the IBV-YX10 which was a natural recombinant nephropathogenic infectious bronchitis virus strain. These findings will contribute additional insights into the molecular characteristics of evolving IBV genomes and the need for effective control of IBV in China.

Complete Genome Sequence Analysis of a Natural Reassortant Infectious Bursal Disease Virus in China

ABSTRACT A novel isolate of infectious bursal disease virus (IBDV) was designated GX-NN-L. The GX-NN-L IBDV was a very virulent infectious bursal disease virus (vvIBDV) isolated from broiler flocks in Guangxi province, China, in 2011. The GX-NN-L IBDV caused high mortality, immunosuppression, low weight gain, and bursal atrophy in commercial broilers. Here, we report the complete genome sequence of the GX-NN-L IBDV, a reassortment strain with segments A and B derived from very virulent strains and attenuated IBDV, respectively. These findings from this study provide additional insights into the genetic exchange between attenuated and very virulent strains of IBDV and continuous monitoring of the spread of the virus in chicken.

Complete Genome Sequence of a Newcastle Disease Virus Strain Isolated from Broiler Breeder Flocks in China

ABSTRACT In 2010 and 2011, several devastating Newcastle disease (ND) outbreaks occurred in China, affecting broilers, layers, and breeders. The CK-JSX1-201005 virus was isolated from broiler breeder flocks vaccinated with the classical ND virus (NDV) vaccine program, but laying rate decreased from 80% to 30 to 40% in the clinic. Here, we report the complete genome sequence and molecular characteristic of the CK-JSX1-201005 NDV. These findings provide additional insights into the genetic variation of NDV circulating in China and are useful for vaccine development for NDV.

Pathogenicity and genetic characterization of a duck Tembusu virus associated with egg-dropping in Muscovy ducks.

Duck Tembusu virus (DTMUV) has spread to the major duck-farming region in China, causing acute egg-production drop in Chinese duck population. In this study, we characterized a DTMUV strain (named GD2014) isolated from an egg-production drop duck farm in Guangdong province, South China. The virus was pathogenic to Muscovy duck embryos and caused severe egg production drop for laying Muscovy ducks. The genome sequence of GD2014 shared 97-99% homologies with other waterfowl-origin Tembusu viruses, and shared 89% identities with MM1775 strain isolated from mosquito. Phylogenetic analysis of entire open reading frame (ORF), E gene and NS5 gene indicated that GD2014 belonged to Ntaya group. These results have implications for understanding the orgin, emergence and pathogenicity of DTMUV as well as for the development of vaccines and diagnostics based on epidemiological data.

Sequence and phylogenetic analysis of surface protein genes of emerging H9N2 influenza viruses isolated from poultry in two geographical regions of China

Subtype H9N2 avian influenza viruses (AIVs) circulating in China have aroused increasing concerns for their impact on poultry and risk to public health. The present study was an attempt to elucidate the phylogenetic relationship of H9N2 AIVs in two geographically distinct regions of China where vaccination is routinely practiced. A total of 18 emerging H9N2 isolates were identified and genetically characterized. Phylogenetic analysis of hemagglutinin (HA) and neuraminidase (NA) genes confirmed that the isolates belonged to the Y280 lineage. Based on the HA genes, the isolates were subdivided into two subgroups. The viruses from Zhejiang Province were clustered together in Group I, while the isolates from Guangdong Province were clustered together in Group II. Antigenic characterization showed that the tested viruses were antigenically different when compared to the current used vaccine strain. It was notable that 14 out of total 18 isolates had an amino acid exchange (Q→L) at position 216 (226 by H3 Numbering) in the receptor-binding site, which indicated that the virus had potential affinity of binding to human like receptor. These results suggest that the emerging viruses have potential risk to public health than previously thought. Therefore, continuous surveillance studies of H9N2 influenza virus are very important to the prognosis and control of future influenza pandemics.