Monoclonal antibodies (mAbs) are widely used in virus research and disease diagnosis.  The nucleoprotein (NP) of influenza A virus (IAV) plays important roles in multiple stages of the virus life cycle.  Therefore, generating conserved mAbs against NP and characterizing their properties will provide useful tools for IAV research.  In this study, two mAbs against the NP protein, 10E9 and 3F3, were generated with recombinant truncated NP proteins (NP-1 and NP-2) as immunogens.  The heavy-chain subclass of both 10E9 and 3F3 was determined to be IgG2α, and the light-chain type was κ.  Truncation and site-specific mutation analyses showed that the epitopes of mAbs 10E9 and 3F3 were located in the N terminal 84–89 amino acids and the C terminal 320–324 amino acids of the NP protein, respectively.  We found that mAbs 10E9 and 3F3 reacted well with the NP protein of H1–H15 subtypes of IAV.  Both 10E9 and 3F3 can be used in immunoprecipitation assay, and 10E9 was also successfully applied in confocal microscopy.  Furthermore, we found that the 10E9-recognized ₈₄SAGKDP₈₉ epitope and 3F3-recognized ₃₂₀ENPAH₃₂₄ epitope were highly conserved in NP among all avian and human IAVs.  Thus, the two mAbs we developed could be used as powerful tools in the development of diagnostic methods of IAV, and also surely promote the basic research in understanding the replication mechanisms of IAV.

H7 avian influenza viruses (AIVs) normally circulated among birds before.  From 1996 to 2012, human infections with H7 AIVs (H7N2, H7N3, and H7N7) were reported in Canada, Italy, Mexico, the Netherlands, the United Kingdom and the USA.  Until March 2013, human infections with H7N9 AIVs were reported in China.  Since then, H7N9 AIVs have continued to circulate in both humans and birds.  Therefore, the detection of antibodies against the H7 subtype of AIVs has become an important topic.  In this study, a competitive enzyme-linked immunosorbent assay (cELISA) method for the detection of antibody against H7 AIVs was established.  The optimal concentration of antigen coating was 5 μg mL^–1, serum dilution was 1/10, and enzyme-labeled antibody was 1/3 000.  To determine the cut-off value of cELISA, percent inhibition (PI) was determined by using receiver operating characteristic (ROC) curve analysis in 178 AIVs negative samples and 368 AIVs positive serum samples (n=546).  When PI was set at 40%, the specificity and sensitivity of cELISA were 99.4 and 98.9%, respectively.  This method could detect the antibodies against H7Nx (N1–N4, N7–N9) AIVs, and showed no reaction with AIVs of H1–H6 and H8–H15 subtypes or common avian viruses such as Newcastle disease virus (NDV), Infectious bronchitis virus (IBV) and Infectious bursal disease virus (IBDV), exhibiting good specificity.  This method showed a coincidence rate of 98.56% with hemagglutinin inhibition (HI) test.  And the repeatability experiment revealed that the coefficients of variation (CV) of intra- and inter-batch repetition were all less than 12%.  The data indicated that the cELISA antibody-detection method established in this study provided a simple and accurate technical support for the detection of a large number of antibody samples of H7-AIV.

   In 2013, a human influenza outbreak caused by a novel H7N9 virus occurred in China.  Recently, the H7N9 virus acquired multiple basic amino acids at its hemagglutinin (HA) cleavage site, leading to the emergence of a highly pathogenic virus.  The development of an effective diagnostic method is imperative for the prevention and control of highly pathogenic H7N9 influenza.  Here, we designed and synthesized three pairs of primers based on the nucleotide sequence at the HA cleavage site of the newly emerged highly pathogenic H7N9 influenza virus.  One of the primer pairs and the corresponding probe displayed a high level of amplification efficiency on which a real-time RT-PCR method was established.  Amplification using this method resulted in a fluorescent signal for only the highly pathogenic H7N9 virus, and not for any of the H1–H15 subtype reference strains, thus demonstrating high specificity.  The method detected as low as 39.1 copies of HA-positive plasmid and exhibited similar sensitivity to the virus isolation method using embryonated chicken eggs.  Importantly, the real-time RT-PCR method exhibited 100% consistency with the virus isolation method in the diagnosis of field samples.  Collectively, our data demonstrate that this real-time RT-PCR assay is a rapid, sensitive and specific method, and the application will greatly aid the surveillance, prevention, and control of highly pathogenic H7N9 influenza viruses.

To develop a modified live vaccine (MLV) against porcine reproductive and respiratory syndrome virus (PRRSV), virulent CH-1a strain was attenuated by serial passages up to 130 passage (P130) in Marc-145 cells. The virulence and immune efficacy of the attenuated CH-1a were evaluated in pigs. The results showed that animals inoculated with P130 did not develop any clinical sign of the disease, but produced rapid and effective humoral immune responses against PRRSV challenge, indicating that attenuated CH-1a P130 is the candidate as the effective vaccine against PRRSV. To define the potential mutations in the attenuated CH-1a genome, we sequenced and analyzed the ORF5 gene of CH-1a strain of different passages (P39, P55, P65, P70, P85, P100, P115, P120, P125, and P130) and found that three mutations (C5Y, H38Q and L146Q) which may be related with the attenuation of CH-1a. In addition, we also found a unique restriction enzyme site (TspEI) in the ORF5 gene of attenuated CH-1a, which can be used as a genetic marker to distinguish original and attenuated CH-1a.