Detection of intestinal intraepithelial lymphocytes, goblet cells and secretory IgA in the intestinal mucosa during Newcastle disease virus infection.

Newcastle disease, which is caused by Newcastle disease virus (NDV), is a highly contagious viral disease of poultry and other bird species. The mucosa is the first line of defence to invading pathogens, including NDV, and it has been confirmed that the mucosa can contribute to host protection. This study was conducted to evaluate the intestinal mucosal immunology in NDV infection. Forty specific-pathogen-free chickens were divided into two groups, 20 birds in each group. Group 1 was inoculated with NDV by the intravenous route. Group 2 was used as the control group and was given sterile phosphate-buffered saline by the same route. At 24, 48, 72, and 96 h post infection (h.p.i.), five chickens from each treatment were killed. Samples of the duodenum, jejunum, and ileum were collected to quantify intestinal intraepithelial lymphocytes (IEL), goblet cells and secretory IgA (sIgA) by cytochemistry and immunohistochemistry analysis. The results indicated that IEL were increased from 24 to 72 h.p.i. in the infected tissues, and were significantly higher than in the control group at 48 h.p.i. (P < 0.01). In contrast to IEL, goblet cell numbers were reduced dramatically from 24 to 96 h.p.i. in the infected birds (P < 0.01) Furthermore, the content of sIgA was significantly higher at 48 and 72 h.p.i. in the infected tissues (P < 0.01). sIgA positivity was observed in the epithelial lining of the intestinal mucosa. These data suggest that IEL, goblet cells, and sIgA were involved in the intestinal mucosal immunity against NDV infection.

The Interaction between B87 Vaccine Strain and BC6/85 of Infectious Bursa Disease Virus in SPF Chickens.

This study was initiated to determine the interaction between two infectious bursal disease virus (IBDV) strains in the early stages of infection by detection and quantification of IBDV RNA in lymphoid and non-lymphoid tissues. SPF chickens were inoculated with single infection or dual infection by the mild strain B87 followed by the pathogenic strain BC6/85 at 0, 1, 2, and 3 days post-inoculation (dpi) with B87. Real-time RT-PCR assays were developed to examine the viral loads of the tissues collected at various time intervals. The results reveal that B87 could delay the time point of positive detection of the BC6/85 strain in the bursa of Fabricius from 1 dpi to 3 dpi, indicating that B87 interfered with the replication of BC6/85. The interference occurred when BC6/85 was inoculated at 2 dpi and 3 dpi with the B87 strain. Moreover, BC6/85 could affect the proliferation and duration of B87 in SPF chickens. The rates of positive detection for B87 decreased significantly during dual infection. The investigation of the interaction between the two strains is important for the implementation of appropriate control measures.

Avian leukosis virus contamination in live vaccines: A retrospective investigation in China.

Avian leukosis (AL) is one of the most pandemic immunosuppressive diseases and has been widely spread between 2006 and 2009 in China. The contamination of avian leukosis virus (ALV) in attenuated vaccine is considered as one of the possible transmission routes of this disease. Based on a retrospective survey of 918 batches of attenuated vaccine produced before 2010, three of them were identified as ALV-positive and corresponding ALV strains were successfully isolated from a live Fowlpox virus vaccine, a live Newcastle disease virus vaccine and a live Infectious Bursal Disease virus vaccine, respectively, and whole-genome sequencing showed that these three isolates shared the highest homology with ALV-A wild strains isolated in China (97.7%) over the same period, and the phylogenetic analysis based on their gp85 genes further confirmed that they belong to subgroup A. Meanwhile, although these three ALV-A strains isolated from contaminated vaccines shared a close genetic relationship, their U3 region of genome have a relatively low identity, suggesting that these three strains may have different sources. This study reminds us once again that the possibility of ALV infecting chickens through contaminated live vaccines, requiring us to carry out stricter exogenous virus monitoring in vaccines.

Identification, Genetic Analysis, and Pathogenicity of Classical Swine H1N1 and Human-Swine Reassortant H1N1 Influenza Viruses from Pigs in China.

Swine influenza virus causes a substantial disease burden to swine populations worldwide and poses an imminent threat to the swine industry and humans. Given its importance, we characterized two swine influenza viruses isolated from Shandong, China. The homology and phylogenetic analyses showed that all eight gene segments of A/swine/Shandong/AV1522/2011(H1N1) were closely related to A/Maryland/12/1991(H1N1) circulating in North America. The HA, NA, M, and NS genes of the isolate were also confirmed to have a high homology to A/swine/Hubei/02/2008(H1N1) which appeared in China in 2008, and the virus was clustered into the classical swine lineage. The gene segments of A/swine/Shandong/AV1523/2011(H1N1) were highly homologous to the early human H1N1 and H2N2 influenza viruses, except for the HA gene, and the virus was a reassortant H1N1 virus containing genes from the classical swine (HA) and human (NA, PB2, PB1, PA, NP, M, and NS) lineages. Both the viruses could cause lethal infection and replicate efficiently in the lungs, brains, spleens, and kidneys of mice. Histopathological examinations showed that AV1522 and AV1523 viruses caused a spectrum of marked pneumonia and meningoencephalitis according to the duration of infection, demonstrating a progression of respiratory disease and neurological disease over the course of infection that ultimately resulted in lethality for the infected mice. The changes in the pathogenicity of swine influenza viruses to mammals, accompanied with the continuous reassortment and evolution of the viruses, highlights the importance of ongoing epidemiological investigation.

Isolation and molecular characterization of equine H3N8 influenza viruses from pigs in China.

During 2004-2006 swine influenza virus surveillance, two strains of H3N8 influenza viruses were isolated from pigs in central China. Sequence and phylogenetic analyses of eight gene segments revealed that the two swine isolates were of equine origin and most closely related to European equine H3N8 influenza viruses from the early 1990s. Comparison of hemagglutinin (HA) amino acid sequences showed several important substitutions. One substitution caused the loss of a potential glycosylation site, and two substitutions, located at the cleavage site and adjacent to the receptor-binding pocket, respectively, had been reported previously in canine H3 HAs. This expansion of host range of equine H3N8 influenza viruses with mutations in the HA protein might raise the possibility of transmission of these viruses to humans.

Genetic Characteristics and Pathogenicity Analysis in Chickens and Mice of Three H9N2 Avian Influenza Viruses.

H9N2 avian influenza is a remarkable disease that has circulated in domestic poultry in large regions of China and posed a serious threat to the poultry industry. The H9N2 virus can not only infect mammals directly, but also provide gene segments to generate novel, but lethal human reassortants. Therefore, it is important to study the evolution, pathogenicity, and transmission of the H9N2 virus. In this study, three H9N2 viruses isolated from chickens in different layer farms were identified. Phylogenetic analysis revealed that these H9N2 viruses were all multiple genotype reassortants, with genes originating from Y280-like, F/98-like, and G1-like viruses. Animal studies indicated that the AV1535 and AV1548 viruses replicated efficiently in the lungs, tracheas, spleens, kidneys, and brains of chickens; the viruses shed for at least 11 days post-inoculation (DPI) and were transmitted efficiently among contact chickens. The AV1534 virus replicated poorly in chickens, shed for 7 DPI, and were not transmitted efficiently among contact chickens. The AV1534 virus replicated well in mice lungs and caused about 2% weight loss. The AV1535 and AV1548 viruses were not able to replicate in the lungs of mice. Our results indicate that we should pay attention to H9N2 avian influenza virus surveillance in poultry and changes in the pathogenicity of them to mammals.

Rapid detection of Burkholderia pseudomallei with a lateral flow recombinase polymerase amplification assay.

Melioidosis is a severe infectious disease caused by gram-negative, facultative intracellular pathogen Burkholderia pseudomallei (B. pseudomallei). Although cases are increasing reported from other parts of the world, it is an illness of tropical and subtropical climates primarily found in southeast Asia and northern Australia. Because of a 40% mortality rate, this life-threatening disease poses a public health risk in endemic area. Early detection of B. pseudomallei infection is vital for prognosis of a melioidosis patient. In this study, a novel isothermal recombinase polymerase amplification combined with lateral flow dipstick (LF-RPA) assay was established for rapid detection of B. pseudomallei. A set of primer-probe targeting orf2 gene within the putative type III secretion system (T3SS) cluster genes was generated and parameters for the LF-RPA assay were optimized. Result can be easy visualized in 30 minutes with the limit of detection (LOD) as low as 20 femtogram (fg) (ca. 25.6 copies) of B. pseudomallei genomic DNA without a specific equipment. The assay is highly specific as no cross amplification was observed with Burkholderia mallei, members of the Burkholderia cepacia-complex and 35 non-B. pseudomallei bacteria species. Moreover, isolates from patients in Hainan (N = 19), Guangdong (N = 1), Guangxi (N = 3) province of China as well as in Australia (N = 3) and Thailand (N = 1) were retrospectively confirmed by the newly developed method. LODs for B. pseudomallei-spiked soil and blood samples were 2.1×103 CFU/g and 4.2×103 CFU/ml respectively. The sensitivity of the LF-RPA assay was comparable to TaqMan Real-Time PCR (TaqMan PCR). In addition, the LF-RPA assay exhibited a better tolerance to inhibitors in blood than TaqMan PCR. Our results showed that the LF-RPA assay is an alternative to existing PCR-based methods for detection of B. pseudomallei with a potentiality of early accurate diagnosis of melioidosis at point of care or in-field use.

A new gene from Xenorhabdus bovienii and its encoded protease inhibitor protein against Acyrthosiphon pisum.

BACKGROUND: Aphids are insect pests with significant importance worldwide for agricultural and horticultural crops. The chemical pesticides used to control aphids could result in pesticide residues in agricultural and horticultural products as well as in negative effects on the environment. Therefore, alternative control methods are urgently needed. This study identified a new gene from strain BJFS526 of the symbiotic bacterium Xenorhabdus bovienii and expressed the protease inhibitor protein encoded by the gene. The effects of the protein on the pea aphids, Acyrthosiphon pisum, were also investigated. RESULTS: The gene PIN1 encoding the protease inhibitor protein against aphids was successfully cloned from BJFS526. The study demonstrated that the protein had adverse effects on pea aphid survival, and that the activity of aphid aminopeptidase was significantly inhibited by the protein. CONCLUSION: The results from this study suggest that this gene and the protease inhibitor protein encoded may offer an alternative method to control aphids in the future.

Reduction of infectious bursal disease virus replication in cultured cells by proteasome inhibitors.

Infectious bursal disease virus (IBDV) is the etiological agent of a highly contagious disease in chickens. In a recent report, proteasome inhibitor MG132 has been shown to completely inhibit IBDV-induced apoptosis. This raises the possibility that the ubiquitin-proteasome pathway may be used by the virus to promote viral replication. In this study, we examined the interplay between IBDV replication and the ubiquitin-proteasome pathway in cultured cells. Treatment of DF-1 cells with the proteasome inhibitors MG132 or lactacystin significantly decreased virus release in the supernatant and prevented virus-induced cytopathic effect. Inhibition of the ubiquitin-proteasome pathway did reduce markedly viral RNA transcription and protein translation but not affect virus internalization. We also demonstrated that IBDV activates caspase pathway via triggering the efflux of cytochrome c in mitochondria into cytosol of infected cells. This activity was dose-dependently reduced by proteasome inhibitor treatment. Taken together, our data suggest that proteasome inhibitor reduces IBDV replication through inhibition of viral RNA transcription and protein synthesis, and thus preventing IBDV-induced apoptosis.

Large-scale manufacture and use of recombinant VP2 vaccine against infectious bursal disease in chickens.

A new subunit vaccine against infectious bursal disease (IBD) was developed; the antigen used in the vaccine was expressed by a new engineering strain, E. coli BL21/pBV220-VP2. The study on the production and use of the vaccine was performed. The results showed that the recombinant VP2 was water-soluble and demonstrated natural antigen activity in vitro. The antibody induced by rVP2 subunit vaccine could protect chickens from challenges of IBDV strains, both BC 6/85 and JZ 3/02. The vaccine, in which the VP2 AGP titre is 1:4, would be enough to protect SPF chickens of 19-day-old, but seemed to be relatively lower to protect commercial Avian Broilers under 10-day-age. In field study, Avian Broilers were vaccinated with rVP2 subunit vaccine of 1:16 AGP titre at the age of 7 days. The protection rate was varied from 72% to 95% in different chicken farms. To study the method of serological evaluation, antibody respond to vaccination had been detected with three kinds of tests. The correct ratio of detection decreased in the order of VP2-based ELISA, AGP test, and virions-based ELISA, if the result of IBDV detection was used as standard of judging. Correlation coefficient between the OD values of VP2-based ELISA and the virions-based ELISA was 0.782. The results will make it possible for the vaccine to be produced commercially and used in poultry industry in large scale.

Development of recombinant VP2 vaccine for the prevention of infectious bursal disease of chickens.

The objective of the present study was to investigate the feasibility of a subunit vaccine and a live bacteria vaccine to protect chickens against infectious bursal disease virus (IBDV) infection. The gene for VP2 of a new wild-type very virulent IBDV (vvIBDV) strain was cloned into an Escherichia coli expression system. Following expression, the recombinant VP2 and the induced expression bacteria were used to vaccinate chickens against virulent IBDV (vIBDV). Three weeks after the vaccination, chickens were inoculated with IBDV strain BC 6/85 by intranasal route or eyedrop route, prior to challenge anti-IBDV serum antibody was detected by AGP. All chickens vaccinated with recombinant VP2 could be detected anti-IBDV antibody. The subunit vaccine of recombinant VP2 conferred protection for 90--100% chickens, live bacteria vaccine of recombinant VP2 conferred protection for 85.7% chickens. The results indicate that E. coli BL 21/pET 28 a-VP2 could be used to develop recombinant VP2 vaccine against infectious bursal disease in chickens.