Hongmei Bao

Development of a reverse transcription loop-mediated isothermal amplification method for the rapid detection of avian influenza virus subtype H7

A rapid and sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of the H7 avian influenza virus (H7 AIV) isotype was developed. The minimum detection limit of the RT-LAMP assay was 0.1-0.01 PFU per reaction for H7 AIV RNA, making this assay 100-fold more sensitive than the conventional RT-PCR method. This RT-LAMP assay also has the capacity to detect both high- and low-pathogenic H7 AIV strains. Using a pool of RNAs extracted from influenza viruses corresponding to all 15 HA subtypes (in addition to other avian pathogenic viruses), the RT-LAMP system was confirmed to amplify only H7 AIV RNA. Furthermore, specific pathogen free (SPF) chickens were infected artificially with H7 AIV, throat and cloacal swabs were collected, and viral shedding was examined using viral isolation, RT-PCR and RT-LAMP. Shedding was detected following viral isolation and RT-LAMP one day after infection, whereas viral detection using RT-PCR was effective only on day 3 post-infection. These results indicate that the RT-LAMP method could facilitate epidemiological surveillance and the rapid diagnosis of the avian influenza subtype H7.

H7N9 virulent mutants detected in chickens in China pose an increased threat to humans

Certain low pathogenic avian influenza viruses can mutate to highly pathogenic viruses when they circulate in domestic poultry, at which point they can cause devastating poultry diseases and severe economic damage. The H7N9 influenza viruses that emerged in 2013 in China had caused severe human infections and deaths. However, these viruses were nonlethal in poultry. It is unknown whether the H7N9 viruses can acquire additional mutations during their circulation in nature and become lethal to poultry and more dangerous for humans. Here, we evaluated the evolution of H7N9 viruses isolated from avian species between 2013 and 2017 in China and found 23 different genotypes, 7 of which were detected only in ducks and were genetically distinct from the other 16 genotypes that evolved from the 2013 H7N9 viruses. Importantly, some H7N9 viruses obtained an insertion of four amino acids in their hemagglutinin (HA) cleavage site and were lethal in chickens. The index strain was not lethal in mice or ferrets, but readily obtained the 627K or 701N mutation in its PB2 segment upon replication in ferrets, causing it to become highly lethal in mice and ferrets and to be transmitted efficiently in ferrets by respiratory droplet. H7N9 viruses bearing the HA insertion and PB2 627K mutation have been detected in humans in China. Our study indicates that the new H7N9 mutants are lethal to chickens and pose an increased threat to human health, and thus highlights the need to control and eradicate the H7N9 viruses to prevent a possible pandemic.

Development of a reverse transcription loop-mediated isothermal amplification method for the rapid detection of subtype H7N9 avian influenza virus.

A novel influenza A (H7N9) virus has emerged in China. To rapidly detect this virus from clinical samples, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of the H7N9 virus. The minimum detection limit of the RT-LAMP assay was 0.01 PFU H7N9 virus, making this method 100-fold more sensitive to the detection of the H7N9 virus than conventional RT-PCR. The H7N9 virus RT-LAMP assays can efficiently detect different sources of H7N9 influenza virus RNA (from chickens, pigeons, the environment, and humans). No cross-reactive amplification with the RNA of other subtype influenza viruses or of other avian respiratory viruses was observed. The assays can effectively detect H7N9 influenza virus RNA in drinking water, soil, cloacal swab, and tracheal swab samples that were collected from live poultry markets, as well as human H7N9 virus, in less than 30 min. These results suggest that the H7N9 virus RT-LAMP assays were efficient, practical, and rapid diagnostic methods for the epidemiological surveillance and diagnosis of influenza A (H7N9) virus from different resource samples.

Detection and Differentiation of Four Poultry Diseases Using Asymmetric Reverse Transcription Polymerase Chain Reaction in Combination with Oligonucleotide Microarrays

Asymmetric reverse transcription polymerase chain reaction (RT-PCR) and microarrays were combined to distinguish 4 viruses, including Avian influenza virus (AIV), Newcastle disease virus (NDV), Infectious bronchitis virus (IBV), and Infectious bursal disease virus (IBDV), and hemagglutinin (HA) subtypes H5, H7, and H9, and neuraminidase (NA) subtypes N1 and N2 of AIV. The AIV matrix protein (M), and HA and NA genes, IBV nucleoprotein (NP) gene, NDV NP gene, and IBDV A fragment gene were cloned into plasmids. These genes were amplified from these positive recombinant plasmids, which included the inserted target genes by PCR. The PCR products were purified and printed on the amino-modified slides as the probes. RNA was extracted from samples and labeled by asymmetric RT-PCR using a cyanine (Cy)3–labeled primers. The labeled complementary (c)DNA was hybridized to the probes immobilized on the glass slides. After hybridization, the microarrays were scanned, and the hybridization pattern agreed perfectly with the known location of each probe. No cross-hybridization could be detected. Results demonstrated that microarray based on asymmetric RT-PCR is an effective way to distinguish AIV, IBV, NDV, and IBDV simultaneously.

Novel influenza A(H7N2) virus in chickens, Jilin province, China, 2014.

In February 2014, while investigating the source of a human infection with influenza A(H7N9) virus in northern China, we isolated subtypes H7N2 and H9N2 viruses from chickens on the patient’s farm. Sequence analysis revealed that the H7N2 virus is a novel reassortant of H7N9 and H9N2 viruses. Continued surveillance is needed.

A protein chip designed to differentiate visually antibodies in chickens which were infected by four different viruses.

This study aimed to develop a visual protein chip that can differentiate the antibodies induced by avian influenza virus, Newcastle disease virus, infectious bronchitis virus and infectious bursal disease virus, simultaneously. Proteins from the four viruses were purified and spotted onto an aldehyde group-modified glass slide at 2mg/ml. After that, the protein chip was reacted with the corresponding positive serum against these viruses, hybridized with a colloidal gold-labeled secondary antibody and visualized by silver staining. A diagnostic protein chip was constructed to differentiate antibodies of four poultry diseases This protein chip showed good sensitivity compared with traditional methods, and it was more than 400 times as sensitive as the agar gel precipitin methods used to detect avian influenza and infectious bursal disease. The protein chip was used to test known serum samples of the four poultry diseases and field serum samples. The results showed that this method could hybridize specifically with the corresponding antibodies with strong signals and without cross-hybridization. In conclusion, this protein chip can be used to differentiate the antibodies induced by the four avian viruses.

Simultaneous detection of novel H7N9 and other influenza A viruses in poultry by multiplex real-time RT-PCR

BackgroundA novel reassortant H7N9 influenza A virus has crossed the species barrier from poultry to cause human infections in China in 2013 and 2014. Rapid detection of the novel H7N9 virus is important to detect this virus in poultry and reduce the risk of an epidemic in birds or humans.FindingsIn this study, a multiplex real-time RT-PCR (rRT-PCR) assay for rapid detection of H7N9 and other influenza A viruses was developed. To evaluate the assay, various influenza A viruses, other avian respiratory viruses, and 1,070 samples from poultry were tested. Fluorescence signals corresponding to H7 and N9 subtypes were detected only when H7 and N9 subtypes were present, while the fluorescence signal for the influenza A M gene was detected in all specimens with influenza A strains. The fluorescent signal can be detected in dilutions as low as 56 copies per reaction for the H7, N9 and M genes. Intra- and inter-assay variability tests showed a reliable assay. In poultry samples, a comparison of rRT-PCR with virus isolation showed a high level of agreement.ConclusionsThe multiplex rRT-PCR assay in this study has good specificity, sensitivity and reproducibility, and will be useful for laboratory surveillance and rapid diagnosis of H7N9 and other influenza A viruses.

Identification of a novel linear epitope on the NS1 protein of avian influenza virus.

BackgroundThe NS1 protein of avian influenza virus (AIV) is an important virulent factor of AIV. It has been shown to counteract host type I interferon response, to mediate host cell apoptosis, and to regulate the process of protein synthesis. The identification of AIV epitopes on NS1 protein is important for understanding influenza virus pathogenesis.ResultsIn this paper, we describe the generation, identification, and epitope mapping of a NS1 protein-specific monoclonal antibody (MAb) D9. First, to induce the production of MAbs, BALB/c mice were immunized with a purified recombinant NS1 expressed in E. coli. The spleen cells from the immunized mice were fused with myeloma cells SP2/0, and through screening via indirect ELISAs, a MAb, named D9, was identified. Western blot assay results showed that MAb D9 reacted strongly with the recombinant NS1. Confocal laser scanning microscopy showed that this MAb also reacts with NS1 expressed in 293T cells that had been transfected with eukaryotic recombinant plasmids. Results from screening a phage display random 7-mer peptide library with MAb D9 demonstrated that it recognizes phages displaying peptides with the consensus peptide WNLNTV--VS, which closely matches the 182WNDNTVRVS190 of AIV NS1. Further identification of the displayed epitope was performed with a set of truncated polypeptides expressed as glutathione S-transferase fusion proteins, and the motif 182WNDNT186 was defined as the minimal unit of the linear B cell epitope recognized by MAb D9 in western blot assays. Moreover, homology analysis showed that this epitope is a conserved motif among AIV.ConclusionsWe identified a conserved linear epitope, WNDNT, on the AIV NS1 protein that is recognized by MAb D9. This MAb and its epitope may facilitate future studies on NS1 function and aid the development of new diagnostic methods for AIV detection.

Rapid Evolution of H7N9 Highly Pathogenic Viruses that Emerged in China in 2017

H7N9 low pathogenic influenza viruses emerged in China in 2013 and mutated to highly pathogenic strains in 2017, resulting in human infections and disease in chickens. To control spread, a bivalent H5/H7 inactivated vaccine was introduced in poultry in September 2017. To monitor virus evolution and vaccine efficacy, we collected 53,884 poultry samples across China from February 2017 to January 2018. We isolated 252 H7N9 low pathogenic viruses, 69 H7N9 highly pathogenic viruses, and one H7N2 highly pathogenic virus, of which two low pathogenic and 14 highly pathogenic strains were collected after vaccine introduction. Genetic analysis of highly pathogenic strains revealed nine genotypes, one of which is predominant and widespread and contains strains exhibiting high virulence in mice. Additionally, some H7N9 and H7N2 viruses carrying duck virus genes are lethal in ducks. Thus, although vaccination reduced H7N9 infections, the increased virulence and expanded host range to ducks pose new challenges.

Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray.

Avian influenza virus (AIV) non-structural protein 1 (NS1) is a multifunctional protein. It is present at high levels in infected cells and can be used for AIV detection and diagnosis. In this study, we generated monoclonal antibody (MAb) D7 against AIV NS1 protein by immunization of BALB/c mice with purified recombinant NS1 protein expressed in Escherichia coli. Isotype determination revealed that the MAb was IgG1/κ-type subclass. To identify the epitope of the MAb D7, the NS1 protein was truncated into a total of 225 15-mer peptides with 14 amino acid overlaps, which were spotted for a peptide microarray. The results revealed that the MAb D7 recognized the consensus DAPF motif. Furthermore, the AIV NS1 protein with the DAPF motif deletion was transiently expressed in 293T cells and failed to react with MAb D7. Subsequently, the DAPF motif was synthesized with an elongated GSGS linker at both the C- and N-termini. The MAb D7 reacted with the synthesized peptide both in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays. From these results, we concluded that DAPF motif is the epitope of MAb D7. To our knowledge, this is the first report of a 4-mer epitope on the NS1 protein of AIV that can be recognized by MAb using a peptide microarray, which is able to simplify epitope identification, and that could serve as the basis for immune responses against avian influenza.