A Single Vaccination of Chimeric Bivalent Virus-Like Particle Vaccine Confers Protection Against H9N2 and H3N2 Avian Influenza in Commercial Broilers and Allows a Strategy of Differentiating Infected from Vaccinated Animals.

H9N2 and H3N2 are the two most important subtypes of low pathogenic avian influenza viruses (LPAIV) because of their ongoing threat to the global poultry industry and public health. Although commercially available inactivated H9N2 vaccines are widely used in the affected countries, endemic H9N2 avian influenza remains uncontrolled. In addition, there is no available avian H3N2 vaccine. Influenza virus-like particles (VLPs) are one of the most promising vaccine alternatives to traditional egg-based vaccines. In this study, to increase the immunogenic content of VLPs to reduce production costs, we developed chimeric bivalent VLPs (cbVLPs) co-displaying hemagglutinin (HA) and neuraminidase (NA) of H9N2 and H3N2 viruses with the Gag protein of bovine immunodeficiency virus (BIV) as the inner core using the Bac-to-Bac baculovirus expression system. The results showed that a single immunization of chickens with 40µg/0.3mL cbVLPs elicited an effective immune response and provided complete protection against H9N2 and H3N2 viruses. More importantly, cbVLPs with accompanying serological assays can successfully accomplish the strategy of differentiating infected animals from vaccinated animals (DIVA), making virus surveillance easier. Therefore, this cbVLP vaccine candidate would be a promising alternative to conventional vaccines, showing great potential for commercial development.

Immunoprotection against influenza virus H9N2 by the oral administration of recombinant Lactobacillus plantarumNC8 expressing hemagglutinin in BALB/c mice.

The H9N2 avian influenza virus (AIV) has become increasingly concerning due to its role in severe economic losses in the poultry industry. Transmission of AIV to mammals, including pigs and humans, has accelerated efforts to devise preventive strategies. To develop an effective oral vaccine against H9N2 AIV, a recombinant Lactobacillus plantarum NC8 strain expressing the hemagglutinin (HA) gene of H9N2 AIV was constructed in this study. Mice were orally immunized with the recombinant NC8-pSIP409-HA strain, and sIgA, IgG and HI antibodies were produced by the NC8-pSIP409-HA strain, which also induced CD8(+) T cell immune responses. Most importantly, oral administration produced complete protection against challenge with mouse-adapted H9N2 virus. These results indicate that the recombinant NC8-pSIP409-HA was more effective at inducing the mucosal, humoral and cellular immune responses. Therefore, L. plantarum NC8-pSIP409-HA could become a promising oral vaccine candidate against H9N2 AIV.

Schisandrin A and B affect subventricular zone neurogenesis in mouse.

Schisandrin A and B (Sch A and B) are the main effective components extracted from the oriental medicine Schisandra chinensis which is traditionally used to enhance mental and intellectual function. Although their neuroprotective effects have been demonstrated, their influences on neurogenesis are still unknown. In the brain, new neural cells born in the subventricular zone (SVZ) next to the lateral ventricles migrate along the rostral migratory stream (RMS) to the olfactory bulb (OB). To investigate the effects of Sch A and B on neurogenesis in the SVZ-RMS-OB system, Sch A and B were intragastrically administrated at dosages of 1, 10 and 20 mg/kg d respectively. The dose of 10 mg/kg d was selected for further analysis based on the preliminary analysis. In the SVZ, significant increases of phosphohistone H3 positive proliferating cells and the intensity of glial fibrillary acidic protein (GFAP+) cells were noticed in Sch B group. In the RMS, Sch A treatment augmented the intensity of doublecortin positive neuroblasts. In the OB, Sch A decreased tyrosine hydroxylase cells and Calbindin (CalB+) cells, while Sch B increased CalB+ cells and Calretinin (CalR+) cells. These results suggest that Sch B stimulates SVZ proliferation by enhancing GFAP+ cells and improves the survival of OB interneurons, while Sch A promotes neuroblast formation in the RMS but impairs the survival of OB interneurons. The present study provides the first evidence that Sch B exerts neuroprotective functions by enhancing neurogenesis, but Sch A mainly negatively regulates neurogenesis, in the adult SVZ-RMS-OB system.

Protective immunity conferred by porcine circovirus 2 ORF2-based DNA vaccine in mice.

Post-weaning multisystemic wasting syndrome (PMWS) associated with porcine circovirus type 2 (PCV2) has caused the swine industry significant health challenges and economic damage. Although inactivated and subunit vaccines against PMWS have been used widely, so far no DNA vaccine is available. In this study, with the aim of exploring a new route for developing a vaccine against PCV2, the immunogenicity of a DNA vaccine was evaluated in mice. The pEGFP-N1 vector was used to construct a PCV2 Cap gene recombinant vaccine. To assess the immunogenicity of pEGFP-Cap, 80 BALB/c mice were immunized three times at 2 weekly intervals with pEGFP-Cap, LG-strain vaccine, pEGFP-N1 vector or PBS and then challenged with PCV2. IgG and cytokines were assessed by indirect ELISA and ELISA, respectively. Specimens stained with hematoxylin and eosin (HE) and immunohistochemistry (IHC) techniques were examined histopathologically. It was found that vaccination of the mice with the pEGFP-Cap induced solid protection against PCV2 infection through induction of highly specific serum IgG antibodies and cytokines (IFN-γ and IL-10), and a small PCV2 viral load. The mice treated with the pEGFP-Cap and LG-strain developed no histopathologically detectable lesions (HE stain) and IHC techniques revealed only a few positive cells. Thus, this study demonstrated that recombinant pEGFP-Cap substantially alleviates PCV2 infection in mice and provides evidence that a DNA vaccine could be an alternative to PCV2 vaccines against PMWS.

Genetic analysis of avian paramyxovirus-1 (Newcastle disease virus) isolates obtained from swine populations in China related to commonly utilized commercial vaccine strains.

Newcastle disease virus (NDV) has been thought to only infect avian species. However, at least eight NDV strains were isolated from swine populations in China during 1999-2006, four of which were characterized genetically and phylogenetically. Genetic analysis revealed that JL106 and SP13 had a (112)G-R-Q-G-R-L(117) motif at the cleavage site of F protein, while JL01 and MP01 possessed a (112)G-K-Q-G-R-L(117) motif, which indicated that all of them were typical of low-virulence viruses. Phylogenetic analysis based on the full-length F gene sequences showed that JL106 and SP13 belonged to genotype II, similar to the commonly utilized commercial La Sota vaccine strain in China. While JL01 and MP01 clustered within genotype I, genetically identical to the V4 vaccine strain. The animal trials showed that JL106 can effectively infect chickens. The present results indicated that the use of live La Sota and V4 vaccines and close contact between avian and pigs maybe resulted in cross-species infection, therefore, it is necessary to further carry out swine NDV epidemiology surveillance.

Antigenic variation between Newcastle disease viruses of goose and chicken origin.

Newcastle disease virus (NDV) is believed to infect only domestic avian species, with waterfowl such as geese either not being infected, even by virulent strains, or developing only inapparent infection. Since the late 1990s, a new infectious disease producing high morbidity and mortality among geese broke out in many provinces of China. This disease was caused by a serotype I avian paramyxovirus known as (APMV-1)-NDV. To investigate how NDV spreads between chickens and geese, the serological similarities of NDV strains (goose-origin NDV/NA-1 and chicken-origin NDV/F48E9, F48E8) were assessed by cross-neutralization assays both in vivo and in vitro. The results indicated that antigenic variation had occurred between NDV/NA-1 strains and NDV/F48E9, F48E8 strains. Notably, NDV/NA-1 effectively protected vaccinated birds from morbidity and mortality against NDV/NA-1 strain challenge and significantly reduced virus shedding from the vaccinated birds when compared with F48E9-vaccinated birds. This might provide clues to the evolution of the goose NDV.

Swine infection with H9N2 influenza viruses in China in 2004.

In 2004, H9N2 influenza A viruses were isolated from pigs with respiratory syndrome in commercial swine farms in Henan province, China. Antigenic and genetic characterization were performed for seven swine H9N2 influenza viruses. The hemagglutinin antigenicity of swine H9N2 viruses was similar to those of avian H9N2 viruses of A/duck/Hong Kong/Y280/1997 (Dk/HK/Y280/97)-like sublineage prevalent in China. It is noteworthy that the neuraminidase of these isolates had no deletions in the stalk, which was seldom observed in those viruses of Dk/HK/Y280/97-like sublineage. Genetic analysis revealed that all seven isolates had an -R-S-S-R- motif at the HA cleavage site, which was the same as those of Dk/HK/Y280/97-like viruses established in avian population in China. Phylogenetic analyses showed that the seven swine H9N2 viruses were completely derived from avian influenza viruses of Dk/HK/Y280/97-like sublineage. The present results indicated that avian-to-pig interspecies transmission of H9N2 viruses continued to exist in China through 2004; therefore, surveillance of swine influenza should be given a high priority.

Rapid detection of avian influenza virus a and subtype H5N1 by single step multiplex reverse transcription-polymerase chain reaction.

Outbreaks of H5N1 highly pathogenic avian influenza (HPAI) virus caused great economic losses to the poultry industry and resulted in human deaths in Thailand and Viet Nam in 2004. Rapid typing and subtyping of H5N1 viruses, especially from clinical specimens, are desirable for taking prompt control measures to prevent the spread of the disease. Here, we developed a set of oligonucleotide primers able to detect, type and subtype H5 and N1 influenza viruses in a single step multiplex reverse transcription-polymerase chain reaction (RT-PCR). RNA was extracted from allantoic fluid or from specimens with guanidinium isothiocyanate reagent. Reverse transcription and PCR were carried out with a mixture of primers specific for influenza viruses of type A, subtype H5 and N1 in a single reaction system under identical conditions. The amplified DNA fragments were analyzed by agarose gel electrophoresis. All the H5N1 viruses tested in the study and the experimental specimens presented three specific bands by the method established here. The results presented here suggest that the method described below is rapid and specific and, therefore, could be valuable in the rapid detection of H5N1 influenza viruses in clinics.

[Expression of recombinant Vp6 gene of porcine rotavirus A with non-antibiotic Lactobacillus vector in Escherichia coli].

The antigenic determinants of Vp6 gene of porcine rotavirus A was amplified from infected MA 104 cell by the reverse transcription-polymerase chain reaction (RT-PCR), the product of which was a 1194bp cDNA segment. Using T-A cloning technique, the PCR product was cloned into pGEM-T vector. Cloning plasmid pGEM-T-Vp6 and the prokaryotic shuttle expression vector pW425t between E. coli and Lactobacillus, were digested by SacI and KpnI double enzymes, respectively. The purified Vp6 gene was subcloned into the expression vector pW425t. Thus, the recombinant pW425t-Vp6 was constructed, which then was transformed into the competence thyA gene-mutant E. coli X13. Treated lysates of bacterium were loaded directly onto SDS-PAGE, on which approximately 44.88 kD fusion protein was observed. The protein was further analyzed using Western blot, which indicated that the protein was reactive with the antibody of rotavirus A. The results lay foundation for further studies on the Lactobacillus subunit vaccine and DNA vaccine of Vp6 gene for prevention and control of porcine rotavirus.