Xintian Wen

Comparative analysis of oncogenic genes revealed unique evolutionary features of field Marek's disease virus prevalent in recent years in China.

BackgroundMareks disease (MD) is an economically important viral disease of chickens caused by Mareks disease virus (MDV), an oncogenic herpesvirus. This disease was well controlled since the widespread use of commercial vaccines, but field MDVs have shown continuous increasing in virulence and acquired the ability to overcome the immune response induced by vaccines. Nowadays, MD continues to be a serious threat to poultry industry, isolation and characterization of MDVs are essential for monitoring changes of viruses and evaluating the effectiveness of existing vaccines.ResultsBetween 2008 and 2010, 18 field MDV strains were isolated from vaccinated chicken flocks in Sichuan province, China. Three oncogenic genes including Meq, pp38 and vIL-8 genes of the 18 isolates were amplified and sequenced. Homology analysis showed that the deduced amino acid sequences of these three genes exhibit 95.0-98.8%, 99.3-100% and 97.0-98.5% homology respectively with these of other reference strains published in GenBank. Alignment analysis of the nucleotide and deduced amino acid sequences showed that four amino acid mutations in Meq gene and two amino acid mutations in vIL-8 gene displayed perfect regularity in MDVs circulating in China, which could be considered as features of field MDVs prevalent in recent years in China. In addition, one amino acid mutation in pp38 gene can be considered as a feature of virulent MDVs from USA, and three amino acid mutations in Meq gene were identified and unique in very virulent plus (vv+) MDVs. Phylogenetic analysis based on Meq and vIL-8 protein sequences revealed that field MDVs in China evolved independently. Virulence studies showed that CVI988 could provide efficient protection against the field MDVs epidemic recently in China.ConclusionsThis study and other published data in the GenBank have demonstrated the features of Meq, pp38 and vIL-8 genes of MDVs circulating in recent years in Sichuan, China. Mutations, deletions or insertions were observed in these three genes, and some mutations could be considered as the unique marks of the MDVs circulating presently in China. The paper supplies some valuable information concerning the evolution of MDV which is useful for the vaccine development and control of MD in China.

Genetic analysis revealed LX4 genotype strains of avian infectious bronchitis virus became predominant in recent years in Sichuan area, China

Between 2008 and 2010, 19 strains of infectious bronchitis virus (IBV) were isolated from the vaccinated chicken flocks in Sichuan province, China. The S1 genes of the isolates were amplified and sequenced. Phylogenetic analysis revealed that the 19 isolates and 37 reference IBV strains can be grouped into eight genotypes. Although IBVs of Taiwan-I type, massachusetts type, and proventriculitis type were isolated, but most isolates were LX4 genotype. Homology analysis of the sequences of S1 genes of the 19 isolates and 37 reference IBV strains revealed that the identity of the nucleotides and amino acid sequences of the S1 genes between the 15 LX4-type isolates and other IBV strains were 71.9–99.3% and 72.1–99.1%, respectively, while those of the analyzed IBV of LX4 type were 96.0–99.9% and 94.3–99.8%, respectively. The results from this study and other published results in the GenBank database showed that isolates circulating in Sichuan province in recent years were mainly LX4 genotype, which is the predominant genotype circulated in China in recent years.

Intragastric administration of attenuated Salmonella typhimurium harbouring transmissible gastroenteritis virus (TGEV) DNA vaccine induced specific antibody production

n Abstractn n Attenuated Salmonella typhimurium was selected as a transgenic vehicle for the development of live mucosal vaccines against transmissible gastroenteritis virus (TGEV). A 2.2kb DNA fragment, encoding for N-terminal domain glycoprotein S of TGEV, was amplified by RT-PCR and cloned into eukaryotic expression vector pVAX1. The recombinant plasmid pVAX-S was transformed by electroporation into attenuated S. typhimurium SL7207, the expression and translation of the pVAX-S delivered by recombinant S. typhimurium SL7207 (pVAX-S) was detected in vitro and in vivo respectively. BALB/c mice were inoculated orally with SL7207 (pVAX-S) at different dosages, the bacterium was safe to mice at dosage of 2×109n CFU and eventually eliminated from the spleen and liver at week 4 post-immunization. Mice immunized with different dosages of SL7207 (pVAX-S) elicited specific anti-TGEV local mucosal and humoral responses as measured by indirect ELISA assay. Moreover, the immunogenicity of the DNA vaccine was highly dependent on the dosage of the attenuated bacteria used for oral administration, 109n CFU dosage group showed higher antibody response than 108n CFU and 107n CFU dosages groups during week 4–8 post-immunization. The results indicated that attenuated S. typhimurium could be used as a delivery vector for oral immunization of TGEV DNA vaccine.n n

Analysis of a QX-like avian infectious bronchitis virus genome identified recombination in the region containing the ORF 5a, ORF 5b, and nucleocapsid protein gene sequences

The complete genome of a QX-like infectious bronchitis virus (IBV) strain Sczy3 isolated recently in Sichuan was sequenced. The genome contains 27,695 nucleotides (nt), and possesses a genomic structure similar to other IBV strains. Sequence comparisons demonstrated that the Sczy3 genome had the highest nt sequence identity with QX-like IBVs and was most dissimilar to the Massachusetts type IBV. Differences in the sequences of genes present in the Sczy3 genome and other IBVs gene sequences were also identified. Phylogenic analysis showed that the entire genome and most of the Sczy3 genes were located in the same cluster as LX4. Recombination analysis showed that Sczy3 is a chimeric strain derived from LX4 (major parental sequence) and H120 (minor parental sequence) suggesting that recombination occurred in a region containing the 3′ terminal 5a sequence (83xa0nt), the 5′ terminal 5b sequence (222xa0nt), and the 5′ terminal nucleocapsid protein gene sequence (132xa0nt). Mutations and intergenic recombination may have played an important role in the evolution of IBVs.

Identification of canine parvovirus with the Q370R point mutation in the VP2 gene from a giant panda (Ailuropoda melanoleuca).

BackgroundIn this study, we sequenced and phylogenetic analyses of the VP2 genes from twelve canine parvovirus (CPV) strains obtained from eleven domestic dogs and a giant panda (Ailuropoda melanoleuca) in China. A novel canine parvovirus (CPV) was detected from the giant panda in China.ResultsNucleotide and phylogenetic analysis of the capsid protein VP2 gene classified the CPV as a new CPV-2a type. Substitution of Gln for Arg at the conserved 370 residue in CPV presents an unusual variation in the new CPV-2a amino acid sequence of the giant panda and is further evidence for the continuing evolution of the virus.ConclusionsThese findings extend the knowledge on CPV molecular epidemiology of particular relevance to wild carnivores.

The different expression of immune-related cytokine genes in response to velogenic and lentogenic Newcastle disease viruses infection in chicken peripheral blood

Newcastle disease virus (NDV) is an important pathogen hazardous to poultry industry, and the pathogenicity of NDV strains varies with different virulence. Peripheral blood serves as an important producer and carrier of viruses and cytokines in NDV infection. In order to explore the difference of cytokine expression in the peripheral blood between velogenic strain and lentogenic strain infection, NDV virulent strain F48E9 and vaccine strain Lasota were used to infect specific-pathogen-free (SPF) chickens separately, and peripheral blood was collected on 0, 3, 7, 10, 14, and 21xa0days post-infection (d.p.i.). Real-time PCR was then used to detect the expression of six kinds of immune-related cytokine genes. For the F48E9 group, a sharp increase of the expression of interferon-alpha (IFN-α), interferon-gamma (IFN-γ), interleukin-16 and IL-18 was observed on 3xa0d.p.i. before the NDV blood peak (7xa0d.p.i.), followed by a rapid decline to the level lower than that of control group, then the expression of IFN-α increased slowly and reached or exceeded the level of control group in the later phase of the infection, while the expression of IFN-γ, IL-16, and IL-18 fluctuated at the level of control group for the rest of study period. The increase of IL-2 expression was not obvious, and no increase of IL-15 expression was noted. For the Lasota (vaccine) group, the picture was quite different, a sharp increase of IFN-γ (but not IFN-α), IL-2 was observed on 7xa0d.p.i. before the NDV blood peak (10xa0d.p.i.). On the contrary, there was no dramatic increase of IL-16 and IL-18. Interestingly, in contrast to the F48E9 group, there was an increase of IL-15 on day 10xa0d.p.i., but it remained modest. There was also an increase of IFN-α on day 21xa0d.p.i. Our results revealed that infection with NDV strains of different virulence was associated with distinct cytokine expression patterns in peripheral blood, modulation of cytokine responses may play a key role in mediation of NDV pathogenesis.

HtrA Is Important for Stress Resistance and Virulence in Haemophilus parasuis

ABSTRACT Haemophilus parasuis is an opportunistic pathogen that causes Glässers disease in swine, with polyserositis, meningitis, and arthritis. The high-temperature requirement A (HtrA)-like protease, which is involved in protein quality control, has been reported to be a virulence factor in many pathogens. In this study, we showed that HtrA of H. parasuis (HpHtrA) exhibited both chaperone and protease activities. Finally, nickel import ATP-binding protein (NikE), periplasmic dipeptide transport protein (DppA), and outer membrane protein A (OmpA) were identified as proteolytic substrates for HpHtrA. The protease activity reached its maximum at 40°C in a time-dependent manner. Disruption of the htrA gene from strain SC1401 affected tolerance to temperature stress and resistance to complement-mediated killing. Furthermore, increased autoagglutination and biofilm formation were detected in the htrA mutant. In addition, the htrA mutant was significantly attenuated in virulence in the murine model of infection. Together, these data demonstrate that HpHtrA plays an important role in the virulence of H. parasuis.

A TolC-Like Protein of Actinobacillus pleuropneumoniae Is Involved in Antibiotic Resistance and Biofilm Formation

Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, a significant disease that causes serious economic losses to the swine industry worldwide. Persistent infections caused by bacterial biofilms are recalcitrant to treat because of the particular drug resistance of biofilm-dwelling cells. TolC, a key component of multidrug efflux pumps, are responsible for multidrug resistance (MDR) in many Gram-negative bacteria. In this study, we identified two TolC-like proteins, TolC1 and TolC2, in A. pleuropneumoniae. Deletion of tolC1, but not tolC2, caused a significant reduction in biofilm formation, as well as increased drug sensitivity of both planktonic and biofilm cells. The genetic-complementation of the tolC1 mutation restored the competent biofilm and drug resistance. Besides, biofilm formation was inhibited and drug sensitivity was increased by the addition of phenylalanine-arginine beta-naphthylamide (PAβN), a well-known efflux pump inhibitor (EPI), suggesting a role for EPI in antibacterial strategies toward drug tolerance of A. pleuropneumoniae. Taken together, TolC1 is required for biofilm formation and is a part of the MDR machinery of both planktonic and biofilm cells, which could supplement therapeutic strategies for resistant bacteria and biofilm-related infections of A. pleuropneumoniae clinical isolate SC1516.

Comparative proteome analysis of the extracellular proteins of two Haemophilus parasuis strains Nagasaki and SW114

This study used a comparative proteomics approach to distinguish between the two-dimensional electrophoresis profiles of extracellular proteins in Nagasaki and SW114. Protein spots were identified using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. The ten proteins unique to Nagasaki were putative adhesin AidA protein, putative extracellular serine protease (autotransporter) (771aa), putative extracellular serine protease (autotransporter) (780aa), protective surface antigen D15, 30S ribosomal protein S2, periplasmic serine protease do/hhoA-like protein, acid phosphatase, membrane protein, protein-disulfide isomerase, and iron ABC transporter substrate-binding protein. Meanwhile, the two proteins unique to SW114 were C4-dicarboxylate ABC transporter substrate-binding protein and peptide ABC transporter substrate-binding protein. Quantitative PCR was used to analyze the mRNA transcript levels of three randomly selected proteins. The afuA, AidA, and ompD15 genes encoding iron ABC transporter substrate-binding protein, putative adhesin AidA protein and protective surface antigen D15 respectively demonstrated significantly higher mRNA transcript levels (39.606, 3.924, and 36.668, respectively) in Nagasaki than in SW114. These observations suggest the levels of differentially expressed proteins were directly proportional to their cellular mRNA levels. Three virulence-related proteins, namely, putative adhesin AidA protein, putative extracellular serine protease (autotransporter) (771aa) and putative extracellular serine protease (autotransporter) (780aa) were identified in Nagasaki.

Phaeohyphomycotic dermatitis in a giant panda (Ailuropoda melanoleuca) caused by Cladosporium cladosporioides

We report here a clinical case of phaeohyphomycosis in an 18-year-old male giant panda (Ailuropoda melanoleuca). Skin lesions on the giant panda disappeared following 2 months of treatment with ketoconazole. Three months after discontinuing the treatment, there was a clinical and mycological relapse. The disease progression was no longer responsive to ketoconazole. Microscopy and polymerase chain reaction (PCR) analysis revealed that the infection was caused by Cladosporium cladosporioides. A 4-month treatment regime with Itraconazole oral solution (700 mg per day) successfully terminated the infection.