Whole-genome analysis of porcine epidemic diarrhea virus (PEDV) from eastern China.

The complete genome sequence of a porcine epidemic diarrhea virus variant, strain SHQP/YM/2013, from China was determined and compared with those of other porcine epidemic diarrhea viruses. The full-length genome was 28,038 nucleotides (nt) in length without the poly (A) tail, and it was similar to that of other reported PEDV strains, with the characteristic gene order 5'-replicase (1a/1b) -S-ORF3-E-M-N-3'. Nucleotide sequence analysis based on individual virus genes indicated a close relationship between the S gene of SHQP/YM/2013 and those of the four Korean field strains from 2008-2009. Its ORF3 gene, however, fell into three groups. Recent prevalent Chinese PEDV field isolates were divided between group 1 and group 3, which suggests that the recent prevalent Chinese PEDV field isolates represent a new genotype that differs from the genotype that includes the vaccine strains. Based on phylogenetic analysis of the M gene, ORF3 gene and S gene, our study demonstrated that prevalent PEDV isolates in China may have originated from Korean strains. This report describes the complete genome sequence of SHQP/YM/2013, and the data will promote a better understanding of the molecular epidemiology and genetic diversity of PEDV field isolates in eastern China.

Genetic evolution of H9 subtype influenza viruses from live poultry markets in Shanghai, China.

H9N2 influenza viruses have become established and maintain long-term endemicity in poultry. The complete genomes of seven avian H9N2 influenza viruses were characterized. These seven influenza virus isolates were obtained from live poultry markets in Shanghai, China, in 2002 and from 2006 to 2008. Genetic analysis revealed that all seven isolates had an RSSR motif at the cleavage site of hemagglutinin (HA), indicating low pathogenicity in chickens. Phylogenetic analyses indicated that the seven avian H9N2 viruses belonged to the lineage represented by Duck/Hong Kong/Y280/97 (H9N2), a virus belonging to the Chicken/Beijing/1/94-like (H9N2) lineage, and that they are all quadruple reassortants consisting of genes from different lineages. The six internal genes of the isolates possessed H5N1-like sequences, indicating that they were reassortants of H9 and H5 viruses. All of the viruses had nonstructural (as well as HA and neuraminidase) genes derived from the Duck/Hong Kong/Y280/97-like virus lineage but also had other genes of mixed avian virus origin, including genes similar to those of H5N1 viruses (Gs/GD-like). The infected chickens showed no signs of disease. These results show the genetic and biological diversity of H9N2 viruses in Shanghai and support their potential role as pandemic influenza agents.

[Development and characterization of monoclonal antibodies against H7 hemagglutinin of avian influenza virus].

AIM: To prepare monoclonal antibodies (mAbs) against the hemagglutinin protein of H7 subtype of avian influenza virus (AIV). METHODS: (6-8 weeks old) BALB/c mice of were immunized endermicly with H7 subtype of AIV. The splenocytes from the immunized mice were fused with Sp2/0-Ag-14 myeloma cells after the last immunization. Hybridoma cells were screened by hemagglutination (HA) and hemagglutination inhibition (HI) tests. The reactivity and specificity of mAbs were evaluated by HI test and Western blot assay. RESULTS: Four hybridoma cell lines secreting specific mAbs named 2E2, 2A4, 5F5 and 7G5 were developed. The HI titers of these mAbs were 5 x 2(7)-5 x 2(11), and the immunoglobulin subclass of 2E2 was IgM, that of 2A4 was IgG1, and that of 5F5 and 7G5 was IgG2a. Western blot analysis confirmed that the mAbs only reacted with M(r) 75 000 HA protein of H7 subtype of AIV but did not react with the proteins of Newcastle disease virus (NDV). The results of HI reactivity assay suggested that 2E2, 5F5 and 7G5 only reacted with H7 subtype of AIV but did not react with other subtypes of AIV, NDV and infectious bronchitis virus (IBV). However 2A4 reacted not only with H7 subtype of AIV but also with H15N8 reference strain of AIV at low HI level. CONCLUSION: These mAbs can be used as a useful tool to analyze the HA structure of AIV. They also provide the effective reagents for the rapid detection of H7 subtype of AIV.

[Preparation and characterization of monoclonal antibodies against the hemagglutinin of H9 subtype of avian influenza virus].

AIM: To prepare monoclonal antibodies (mAb) against the hemagglutinin(HA) of H9 subtype of avian influenza virus (AIV). METHODS: 8 week-old female BALB/c mice were immunized with the inactivated vaccine of H9 subtype of AIV. Splenocytes from the immunized mice were fused with Sp2/0 myeloma cells, and positive hybridoma clones were screened by indirect ELISA and hemagglutination inhibition test (HI). The specificity of the mAb was characterized by ELISA, HI test, indirect immunofluorescence (IF) staining and Western blot. RESULTS: Three hybridoma cell lines named 2H1, 2A3 and 1C8 against HA of AIV H9 were obtained. The HI titers of 3 mAbs were 1 x 2(8)-1 x 2(13), and the ELISA titers were 1 x 10(-7), 1 x 10(-5) and 5 x 10(-6), respectively. The immunoglobulin subclass of all 3 mAbs was IgG1. Western blot analysis confirmed that mAb 2H1 could recognize HA and reacted to 31 out of 32 isolates of H9 subtype of AIV. CONCLUSION: Three mAbs recognizing HA of H9 subtype of AIV were obtained, which may provide an useful tool for the antigenic analysis, the serological diagnosis, the epidemiological survey and the evaluation of AIV vaccine.