Influenza H7N9 Virus Hemagglutinin with T169A Mutation Possesses Enhanced Thermostability and Provides Effective Immune Protection against Lethal H7N9 Virus Challenge in Chickens.

H7N9 avian influenza virus (AIV) has caused huge losses in the poultry industry and impacted human public health security, and still poses a potential threat. Currently, immune prevention and control of avian influenza relies on traditional inactivated vaccines; however, they have some limitations and genetically engineered avian influenza subunit vaccines may be potential candidate vaccines. In this study, a T169A mutation in the HA protein derived from H7N9 AIV A/Chicken/Guangdong/16876 (H7N9-16876) was generated using the baculovirus expression system (BVES). The results showed that the mutant (HAm) had significantly increased thermostability compared with the wild-type HA protein (HA-WT). Importantly, immunizing chickens with HAm combined with ISA 71VG elicited higher cross-reactive hemagglutination inhibition (HI) antibody responses and cytokine (IFN-γ and IL-4) secretion. After a lethal challenge with heterologous H7N9 AIV, the vaccine conferred chickens with 100% (10/10) clinical protection and effectively inhibited viral shedding, with 90% (9/10) of the chickens showing no virus shedding. The thermostability of HAm may represent an advantage in practical vaccine manufacture and application. In general, the HAm generated in this study represents a promising subunit vaccine candidate for the prevention and control of H7N9 avian influenza.

Supplementation of H7N9 Virus-Like Particle Vaccine With Recombinant Epitope Antigen Confers Full Protection Against Antigenically Divergent H7N9 Virus in Chickens.

The continuous evolution of the H7N9 avian influenza virus suggests a potential outbreak of an H7N9 pandemic. Therefore, to prevent a potential epidemic of the H7N9 influenza virus, it is necessary to develop an effective crossprotective influenza vaccine. In this study, we developed H7N9 virus-like particles (VLPs) containing HA, NA, and M1 proteins derived from H7N9/16876 virus and a helper antigen HMN based on influenza conserved epitopes using a baculovirus expression vector system (BEVS). The results showed that the influenza VLP vaccine induced a strong HI antibody response and provided effective protection comparable with the effects of commercial inactivated H7N9 vaccines against homologous H7N9 virus challenge in chickens. Meanwhile, the H7N9 VLP vaccine induced robust crossreactive HI and neutralizing antibody titers against antigenically divergent H7N9 viruses isolated in wave 5 and conferred on chickens complete clinical protection against heterologous H7N9 virus challenge, significantly inhibiting virus shedding in chickens. Importantly, supplemented vaccination with HMN antigen can enhance Th1 immune responses; virus shedding was completely abolished in the vaccinated chickens. Our study also demonstrated that viral receptor-binding avidity should be taken into consideration in evaluating an H7N9 candidate vaccine. These studies suggested that supplementing influenza VLP vaccine with recombinant epitope antigen will be a promising strategy for the development of broad-spectrum influenza vaccines.

Detection and Genetic Characterization of Atypical Porcine Pestivirus in Piglets With Congenital Tremors in Southern China.

Atypical porcine pestivirus (APPV) is an RNA virus newly discovered from swine in Asia, Europe, and the Americas. This novel virus has been confirmed as the cause of congenital tremor (CT) in piglets, which causes extensive economic losses to the swine industry. To investigate the genetic diversity and evolutionary relationship of APPV in China, 83 piglet samples with severe CT clinical signs were obtained from 12 commercial swine farms in 3 provinces of Southern China. RT-PCR revealed that the positive rates of APPV were as high as 100% (12/12) for the swine farms and 90.4% (75/83) for the samples. Subsequently, 21 positive samples and 3 positive samples were selected for partial E2 gene and complete polyprotein gene sequencing, respectively. Phylogenetic analysis showed that 62.5% of the sequences belonged to a novel APPV clade provisionally named genotype 3, which showed 81.0-82.1% sequence identity to genotypes 1 and 2. Amino acid sequence alignment showed that E2 protein of genotype 3 has three specific mutation sites, namely I19V, Y82F, and N107G. The results of the present study demonstrate that a novel APPV subgenotype, which is widely distributed in severe CT clinical samples in Southern China, was genetically diverse. We advocate for the inclusion of genotype 3 during revision of the APPV typing method.

Genomic comparison of foot-and-mouth disease virus R strain and its chick-passaged attenuated strain.

The present study examined the genomic differences between foot-and-mouth disease virus (FMDV) R strain and its attenuated, chick-passaged (R(304)) strain. Eleven pairs of primers were used to amplify the complete genome of FMDV R and R(304) by RT-PCR. Each fragment was cloned into pMD18-T vector and sequenced. Nucleotide analyses showed that the genome encoding regions of R and R(304) strains open reading frame (ORF) were both 6966 nucleotides (nt) in length, encoding 2322 amino acids. One hundred and ten nucleotides or 32 amino acids were found to be mutated most frequently were in the 3A gene. The next highest rates of mutation were observed in the LP and 1D genes. No mutations were found in either the 2A or 2C genes. The length of 5'IRES region and 3'UTR were 450 nt and 94 nt, respectively. The 5'IRES region and 3'UTR had only 4 nt and 3 nt mutation, respectively after attenuation. The R(304) poly(A) tail length of 18 nt, while that of the R strain was 30 nt. This result demonstrated the primary genomic changes of a FMDV and its attenuated strain, which has important implications in understanding the molecular epidemiology and functional genomics of FMDV.