Yumin Zhu

Identification of a recombinant Muscovy Duck parvovirus (MDPV) in Shanghai, China.

The full-length genome of strain SAAS-SHNH, a MDPV isolated from Muscovy Duck in Shanghai, has been sequenced and shown to share 93.7% nucleotide identity with MDPV strain FM (NC_006147). Two putative genetic recombination events were identified as occurring within the 419-610 nt and 3113-4241 nt regions of the SAAS-SHNH genome which, for the first time, provide evidence of recombination between MDPVs and GPVs.

Retinoid protects rats against neutrophil-induced oxidative stress in acute experimental mastitis.

Activated polymorphonuclear neutrophilic leukocytes (PMN) are able to produce large quantities of bactericidal molecules such as reactive oxygen species (ROS) that are associated with tissue damage in models of inflammatory mastitis. In this study, the putative protective effect of retinoid was evaluated in a lipopolysaccharide (LPS) induced mastitis model in rats. Commencing at 10 d of gestation, retinoid (dissolved in olive oil) or an equal volume of olive oil were administered daily by gavage to pregnant rats until parturition. LPS or pyrogen-free physiological saline were infused into the mammary gland 72 h after parturition. At pre-infusion (defined as 0 h) and at 2, 4, 8, 16 and 24 h post-infusion, six rats from each group were euthanized. Retinoid administration decreased PMN accumulation in mammary alveoli, significantly decreased the level of TNF-alpha in mammary tissues and IL-8 in serum at the different time points. ROS release was significantly increased after LPS infusion and was reduced by retinoid at 16 h PI. Retinoid reduced N-acetyl-beta-D-glucosaminidase (NAGase) activity in both serum and mammary tissue at 8 h PI. Intercellular adhesion molecule 1 (ICAM-1) mRNA expression reached its peak value earlier in retinoid treated rats than in the control group. Overall, the results suggest that activated PMN play an important role in the pathogenesis of acute mastitis and retinoid administration may be an effective tool for protecting mammary tissue against PMN-induced oxidative stress during LPS-induced acute mastitis.

Reduced prevalence of genotype 3 HEV in Shanghai pig farms and hypothetical homeostasis of porcine HEV reservoir

A total of 493 fecal samples collected from local Shanghai pig farms were examined for Hepatitis E virus (HEV) after the introduction of stricter sanitary measures following outbreaks of a high fever-associated pig disease during 2006 and 2007. Our investigation revealed that, while the overall occurrence of HEV RNA positives decreased by only 3.7%, the incidence of HEV genotype 4 increased from 9.8% to 20.6% whereas the incidence of HEV genotype 3 decreased from 16.2% to 1.6%. As well as demonstrating that HEV genotype 3 was more sensitive than genotype 4 to the stricter sanitation procedures, our data also suggested that a homeostasis mechanism, whereby the overall incidence of HEV is maintained at a specific population level, might exist in the porcine HEV reservoir. Furthermore, in one case, we encountered the coexistence of HEV genotypes 3 and 4 within the same sample, indicating the possibility of future HEV infections of increased severity and even the occurrence of a HEV pandemic due to genetic recombination and species evolution.

Polysaccharide Nucleic Acid of Bacillus Calmette Guerin Modulates Th1/Th2 Cytokine Gene Expression in Lipopolysaccharide-Induced Mastitis in Rats

The aim of this study was to evaluate, in rats, the changes in the T helper type 1 (Th1)/Th2 radio in mammary glands after an intramammary infusion of lipopolysaccharide (LPS) and to characterize the moderating effects of the polysaccharide nucleic acid of Bacillus Calmette Guerin (BCG-PSN) on the mammary gland. In the control group, the levels of IL-2 and INF-γmRNA expression increased, whereas IL-4 mRNA expression decreased after LPS challenge. As a consequence, the INF-[.gamma]/IL-4 mRNA ratio was significantly higher at 3, 6, and 9 h post-infusion (PI) compared to the control value (0 h; P< 0.01). BCG-PSN increased mRNA expression of both INF-γ and IL-4 before infusion of LPS. LPS challenge significantly the reduced Th1/Th2 cytokine ratio due to Th1 cytokine IFN-γ suppression and Th2 cytokine IL-4 upregulation compared with the control group. A significant reduction of N-acetyl-[.beta]-D-glucosaminidase (NAGase) was observed at 24 h PI in the BCG-PSN treatment group compared to the control group (P < 0.05). Thus, it was demonstrated that level of BCG-PSN might change the Th1/Th2 ratio mainly by enhancing the Th2 immune response. This is the first report of a Th1/Th2 change induced by coliform mastitis and characterization of the effect of BCG-PSN on mammary gland inflammation. This study makes a better understanding of the mechanisms of coliform mastitis and provides a putative novel strategy for the prevention and/or treatment of mastitis.

Fine mapping and conservation analysis of linear B-cell epitopes of peste des petits ruminants virus nucleoprotein

Nucleoprotein (NP) is the most abundant and highly immunogenic protein of morbillivirus, and is presently the basis of most diagnostic assays for peste des petits ruminants virus (PPRV). In this study, fine epitope mapping and conservation analysis of linear B-cell epitopes on the PPRV NP has been undertaken using biosynthetic peptides. Nineteen linear B-cell epitopes were identified and their corresponding minimal motifs were located on the NP of PPRV China/Tibet/Geg/07-30. Conservation analysis indicated that ten of the 19 minimal motifs were conserved among 46 PPRV strains. Peptides containing the minimal motifs were recognized using anti-PPRV serum from a goat immunized with PPRV vaccine strain Nigeria 75/1. Identified epitopes and their motifs improve our understanding of the antigenic characteristics of PPRV NP and provide a basis for the development of epitope-based diagnostic assays.

Molecular epidemiology of hepatitis E virus infections in Shanghai, China

BackgroundHepatitis E virus (HEV) causes acute or fulminant hepatitis in humans and is an important public health concern in many developing countries. China has a high incidence of HEV epidemics, with at least three genotypes (1, 3 and 4) and nine subtypes (1b, 1c, 3b, 4a, 4b, 4d, 4g, 4h and 4i) so far identified. Since genotype 3 and the newly identified subtype 4i have been exclusively limited geographically to Shanghai and its neighboring provinces, the epidemiology of HEV infections within the municipality, a major industrial and commercial center, deserves closer attention.FindingsA total of 65 sequences, 60 located within the HEV SH-SW-zs1 genome [GenBank:EF570133], together with five full-length swine and human HEV genomic sequences, all emanating from Shanghai, were retrieved from GenBank. Consistent with the primary role of genotype 4 in China overall, analysis of the sequences revealed this to have been the dominant genotype (58/65) in Shanghai. Six HEV subtypes (3b, 4a, 4b, 4d, 4h and 4i) were also represented. However, although subtype 4a is the dominant subtype throughout China, subtype 4i (29/65) was the most prevalent subtype among the Shanghai sequences, followed by subtypes 4d (10/65) and 4h (9/65). Subtypes 4h, 4i and 4d were found in both swine and humans, whereas 4b was found only in swine and subtype 4a only in humans.ConclusionsSix different swine and human HEV subtypes have so far been documented in Shanghai. More molecular epidemiological investigations of HEV in swine, and particularly among the human population, should be undertaken.

Development of an epitope-based competitive ELISA for the detection of antibodies against Tibetan peste des petits ruminants virus.

Aims: To develop an effective diagnostic kit, based on a competitive ELISA-based system (cELISA), for detecting serum antibody against peste des petits ruminants virus (PPRV). Methods: Epitope peptides of the nucleocapsid (N) protein of Tibetan PPRV were synthesized chemically and injected into rabbits to prepare hyperimmune antisera. Test sera were incubated simultaneously with hyperimmune antisera and added to the wells of ELISA plates coated previously with recombinant N protein. Horseradish peroxidase-conjugated goat anti-rabbit antibody was employed to detect the quantity of hyperimmune antisera combined with recombinant N protein. Results: A cELISA has been developed for monitoring PPRV infections with a cutoff value of 35. Relative sensitivity and specificity values of the epitope-based cELISA were 96.18 and 91.29%, respectively, when compared with a commercial cELISA kit in a test involving 1,039 serum samples. Conclusion: We report an efficient method for preparing antibody suitable for incorporation into a cELISA that can be used routinely for the detection of PPRV antibodies in serum samples. The method eliminated the requirement for virus culture and monoclonal antibody preparation, reduced the biorisk posed by virus-dependent manipulations, and the performance of the resultant cELISA compared favorably with a commercially available cELISA kit.

Development of an Indirect ELISA with Artificially Synthesized N Protein of PPR Virus

The full-length gene encoding the nucleocapsid (N) protein of the virus (PPRV) responsible for an outbreak of peste des petits ruminants in Tibet in 2007 was synthesized in two stages using overlapping PCR without the need for viral genomic cDNA as template. The full-length N gene was successfully expressed in Escherichia coli, and the purified gene product bound to monoclonal antibody raised against PPRV N protein. Furthermore, it was able to replace recombinant B-N antigen as the coating antigen in a commercial ELISA kit prepared with another PPRV strain. Recombinant protein was employed as the coating antigen to develop an indirect ELISA for PPRV antibody detection in the sera of infected small ruminants. Antibody detection was optimal at a 1:200 serum dilution and an antigen concentration of 3.2 µg/ml, and the positive threshold (cutoff) value of the assay was 2.18. Analysis of 697 serum samples revealed the sensitivity and specificity of the indirect ELISA to be 96.7 and 96.1%, respectively, compared with a commercially available ELISA test.

Infectivity of a genotype 4 hepatitis E virus cDNA clone by intrahepatic inoculation of laboratory rats.

A cDNA clone of a genotype 4 swine hepatitis E virus (HEV) strain (SAAS-FX17), identified in Shanghai, has been constructed. Capped RNA transcripts were prepared in vitro and shown to be replication-competent in Huh7 cells. Sprague-Dawley (SD) rats administered the RNA transcripts by intrahepatic inoculation developed active infections as evidenced by fecal virus shedding and sero-conversion to anti-HEV. The former was first detected between 23 and 30 days post-inoculation (dpi) and persisted until 45 dpi. Sera of rats inoculated with RNA transcripts became anti-HEV positive between 30 and 40 dpi, and reverted to anti-HEV negative at 52 dpi. Our data indicate for the first time that intrahepatic inoculation of rats with RNA transcripts of an HEV cDNA clone may serve as an alternative animal model for HEV research.

Inhibitory effects of recombinant porcine interferon-α on high- and low-virulence porcine reproductive and respiratory syndrome viruses.

The inhibitory effects of recombinant porcine interferon alpha (rPoIFN-α) on the propagation of low-virulence PRRSV (lvPRRSV) in MARC-145 cells, and on the progress and severity of high virulence PRRSV (hvPRRSV)-induced infections in pigs, were determined. Pre-treatment of MARC-145 cells with increasing concentrations of rPoIFN-α prior to infection with lvPRRSV decreased the observed cytopathic effects (CPEs) in a concentration-dependent manner. Viral propagation and antibody response were temporarily delayed in swine treated with rPoIFN-α either at the same time as the hvPRRSV challenge was administered or post-challenge. Exposure of challenged animals to rPoIFN-α after the onset of disease symptoms alleviated associated hyperthermia. Variations in lymphocyte subsets indicated that rPoIFN-α treatment might alleviate damage to the immune system or enhance propagation of host cytotoxic T-lymphocytes when the treatment was applied simultaneously with the virus or 1dpc, respectively.