Avian Influenza Virus H5 Subtype Research Articles

N-glycosylation on hemagglutinin head reveals inter-branch antigenic variability of avian influenza virus H5-subtypes

H5-subtype avian influenza virus (AIV) is globally prevalent and undergoes frequent antigenic drift, necessitating regular updates to vaccines. One of the many influencing elements that cause incompatibility between vaccinations and epidemic strains is the dynamic alteration of glycosylation sites. However, the biological significance of N-glycosylation in the viral evolution and antigenic changes is unclear. Here, we performed a systematic analysis of glycosylation sites on the HA1 subunit of H5N1, providing insights into the changes of primary glycosylation sites, including 140 N, 156 N, and 170 N within the antigenic epitopes of HA1 protein. Multiple recombinant viruses were then generated based on HA genes of historical vaccine strains and deactivated for immunizing SPF chickens. Inactivated recombinant strains showed relatively closer antigenicity compared to which has identical N-glycosylation patterns. The N-glycosylation modification discrepancy highlights the inter-branch antigenic diversity of H5-subtype viruses in avian influenza and serves as a vital foundation for improving vaccination tactics.

Rapid detection of H5 subtype avian influenza virus using CRISPR Cas13a based-lateral flow dipstick

Due to its high mortality rate, highly pathogenic avian influenza (HPAI), a notifiable animal illness designated by the World Organisation for Animal Health (WOAH), has caused enormous financial losses to the poultry sector. The H5 subtype of avian influenza virus (H5-AIV) is regarded as the most common highly pathogenic avian influenza virus (HPAIV) that threatens public health and safety. Virus isolation and reverse transcription quantitative PCR (RT-qPCR) are usually used to detect H5-AIV and are important for the timely diagnosis and control of H5-AIV. However, these methods are time-consuming and require a significant amount of effort. In this study, we established a recombinase-aided amplification (RAA) combined with CRISPR-Cas13a and lateral flow dipstick (LFD) assay for the detection of H5-AIV. The results showed that the process can be completed within 40 min at 37°C. The method had a detection limit of 0.1 copy/μL, which was comparable to the RT-qPCR. There was no cross-reactivity with H3-AIV, H7-AIV, H9-AIV, H10-AIV, IBV, NDV, RVA and DAstV. The kappa value of RT-RAA-Cas13a-LFD and RT-qPCR in 380 clinical samples was 0.89 (κ>0.75). In conclusion, we established a convenient, efficient and accurate method to detect H5-AIV, and the results can be visualized and interpreted using LFD, which can be adapted to the needs of grassroots laboratories and field-deployable assays. This approach provides a new perspective for clinical H5-AIV diagnosis and has great potential for application in clinical quarantine of the poultry farming.

Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development

BackgroundHighly pathogenic emerging and re-emerging viruses continuously threaten lives worldwide. In order to provide prophylactic prevention from the emerging and re-emerging viruses, vaccine is suggested as the most efficient way to prevent individuals from the threat of viral infection. Nonetheless, the highly pathogenic viruses need to be handled in a high level of biosafety containment, which hinders vaccine development. To shorten the timeframe of vaccine development, the pseudovirus system has been widely applied to examine vaccine efficacy or immunogenicity in the emerging and re-emerging viruses. MethodsWe developed pseudovirus systems for emerging SARS coronavirus 2 (SARS-CoV-2) and re-emerging avian influenza virus H5 subtypes which can be handled in the biosafety level 2 facility. Through the generated pseudovirus of SARS-CoV-2 and avian influenza virus H5 subtypes, we successfully established a neutralization assay to quantify the neutralizing activity of antisera against the viruses. ResultsThe result of re-emerging avian influenza virus H5Nx pseudoviruses provided valuable information for antigenic evolution and immunogenicity analysis in vaccine candidate selection. Together, our study assessed the potency of pseudovirus systems in vaccine efficacy, antigenic analysis, and immunogenicity in the vaccine development of emerging and re-emerging viruses. ConclusionInstead of handling live highly pathogenic viruses in a high biosafety level facility, using pseudovirus systems would speed up the process of vaccine development to provide community protection against emerging and re-emerging viral diseases with high pathogenicity.

A study of the relationship between human infection with avian influenza a (H5N6) and environmental avian influenza viruses in Fujian, China

BackgroundAvian influenza A (H5N6) virus poses a great threat to the human health since it is capable to cross the species barrier and infect humans. Although human infections are believed to largely originate from poultry contaminations, the transmissibility is unclear and only limited information was available on poultry environment contaminations, especially in Fujian Province.MethodsA total of 4901 environmental samples were collected and tested for Avian Influenza Virus (AIV) from six cities in Fujian Province through the Fujian Influenza Surveillance System from 2013 to 2017. Two patient-related samples were taken from Fujian’s first confirmed H5N6 human case and his backyard chicken feces in 2017. Chi-square test or Fisher’s exact probability test was used to compare the AIV and the viral subtype positive rates among samples from different Surveillance cities, surveillance sites, sample types, and seasons. Phylogenetic tree analysis and molecular analysis were conducted to track the viral transmission route of the human infection and to map out the evolutions of H5N6 in Fujian.ResultsThe overall positive rate of the H5 subtype AIVs was 4.24% (208/4903). There were distinctive differences (p < 0.05) in the positive rates in samples from different cities, sample sites, sample types and seasons. The viruses from the patient and his backyard chicken feces shared high homologies (99.9–100%) in all the eight gene segments. Phylogenetic trees also showed that these two H5N6 viruses were closely related to each other, and were classified into the same genetic clade 2.3.4.4 with another six H5N6 isolates from the environmental samples. The patient’s H5N6 virus carried genes from H6N6, H5N8 and H5N6 viruses originated from different areas. The R294K or N294S substitution was not detected in the neuraminidase (NA). The S31 N substitution in the matrix2 (M2) gene was detected but only in one strain from the environmental samples.ConclusionsThe H5 subtype of AIVs has started circulating in the poultry environments in Fujian Province. The patient’s viral strain originated from the chicken feces in his backyard. Genetic reassortment in H5N6 viruses in Fujian Province was indicated. The H5N6 viruses currently circulating in Fujian Province were still commonly sensitive to Oseltamivir and Zanamivir, but the resistance against Amantadine has emerged.

Genetic characteristics of hemagglutinin genes of nine H5 subtype avian influenza viruses in Weining, Guizhou Province during 2015—2017

Objective To investigate the molecular characteristics of H5 subtype avian influenza viruses (AIV) in Weining, Guizhou Province. Methods Nine representative strains were randomly selected from H5 subtype AIV that were identified by real-time PCR in Weining, Guizhou Province from 2015 to 2017. Nucleic acid was extracted from each sample and hemagglutinin (HA) genes were amplified and then sequenced. Homology, genetic evolution and the sites related to pathogenicity, receptor binding regions as well as potential glycosylation of H5 AIV were analyzed by bioinformation software. Results Homology analysis revealed that there was 96.1%-99.9% and 95.7%-100% similarity among the nine strains in nucleotide and amino acid of HA gene, respectively. These strains belonged to two branches, H5-1 and H5-2. The cleavage site motifs were PLREKRRKR↓GLF for five strains in H5-1 branch and PQRERRRKR↓GLF for four strains in H5-2 branch, which made them high pathogenic. QSG and QRG at the key receptor binding sites were found in H5-1 and H5-2 branch strains, respectively. They were responsible for receptor binding specificity of AIV. Mutations of 138Q, 139G and 53K were all detected in the nine strains. 129K, 189T, 140K and 282V mutations were discovered in the five strains of H5-1 branch, while 189N, 140M and 282I mutations were found in the four strains of H5-2 branch. Results of the glycosylation motif analysis showed that six sites were conservative, but there was an addition of 124NHT site in two strains of H5-2 branch isolated in 2017. Conclusion Two high pathogenic H5 subtypes of AIV could be epidemic in Weining, Guizhou Province during 2015 to 2017. Although H5 subtype AIV did not possess specific receptor binding regions like human influenza viruses, they were in continuous variation with an increase in glycosylation motifs, which might enhance their virulence and pathogenicity to human beings. Hence, surveillance and study on the molecular properties of H5 subtype AIV should be strengthened. Key words: H5 subtype avian influenza virus; Hemagglutinin gene; Molecular characteristic

Expression and purification of recombinant H5HA1 protein of H5N1 avian influenza virus in E. coli and its application in indirect ELISA

ABSTRACTThe PCR amplified HA1 fragment of H5N1 (H5HA1) avian influenza virus (AIV) hemagglutinin gene was cloned into pET28a (+) expression vector and expressed in Rosetta Blue (DE3) pLysS cells. The recombinant H5HA1 (rH5HA1) protein purified by passive gel elution after SDS-PAGE of the inclusion bodies reacted specifically with H5N1 serum in Western blot analysis. A subtype specific indirect enzyme linked immunosorbent assay (iELISA) using the rH5HA1 protein as the coating antigen was developed for detecting antibodies to H5 subtype of AIV. The assay had 89.04% sensitivity and 95.95% specificity when compared with haemagglutination inhibition test. The Kappa value of 0.842 indicated a perfect agreement between the tests. The iELISA developed can be used for serosurveillance of avian influenza in chickens.

Serological Evaluation of Turkey Herpesvirus Vector Vaccines Expressing the Hemagglutinin Gene of Avian Influenza Virus H5 Subtype under Three Different Promoters

Marek’s disease virus, including turkey herpesvirus (HVT), have been utilized as vectors to express foreign antigen genes and induce immunity against the antigens in chickens. Selection of promoters in developing such vector vaccines is one of the most important factors influencing efficacy of vector vaccines. In this study, in order to find a suitable promoter for expressing the hemagglutinin gene of avian influenza virus H5 subtype in HVT vector vaccines, three HVT vector avian influenza virus H5 subtype (HVT-AI) viruses expressing the hemagglutinin gene were constructed using three promoters; the cytomegalovirus (CMV) promoter, the chicken β-actin (Bac) promoter, and CMV/Bac chimera (Pec) promoter. Of those three vector vaccines, HVT-AI with the CMV promoter induced significantly higher avian influenza virus (AIV) hemagglutinin inhibition titers than the other HVT-AI vaccines with the Bac or the Pec promoters, after inoculation into chickens at one day old. When evaluated with two of commercially available AIV enzyme-linked immunosorbent assay kits, the HVT-AI vaccines did not induce positive titers, indicating that these HVT-AI vaccines may be utilized for easy differentiation of vaccinated chickens from ones infected with field AIV.

Ultrasensitive electrochemical immunoassay for avian influenza subtype H5 using nanocomposite.

We report a novel electrochemical immunosensor that can sensitively detect avian influenza virus H5 subtype (AIV H5) captured by graphene oxide-H5-polychonal antibodies-bovine serum albumin (GO-PAb-BSA) nanocomposite. The graphene oxide (GO) carried H5-polychonal antibody (PAb) were used as signal amplification materials. Upon signal amplification, the immunosensor showed a 256-fold increase in detection sensitivity compared to the immunosensor without GO-PAb-BSA. We designed a PAb labeling GO strategy and signal amplification procedure that allow ultrasensitive and selective detection of AIV H5. The established method responded to 2−15 HA unit/50 µL H5, with a linear calibration range from 2−15 to 2−8 HA unit/50 µL. In summary, we demonstrated that the immunosenser has a high specificity and sensitivity for AIV H5, and the established assay could be potentially applied in the rapid detection of other pathogenic microorganisms.

Avian influenza in village chickens, its awareness and presence of potential risk practices among rural dwellers

ObjectiveTo assess the presence of antibodies to the avian influenza virus H5 subtype in village chickens. MethodsA total of 480 sera samples were obtained from apparently healthy local chickens in four local government areas (LGAs). The sera samples were subjected to the haemagglutination inhibition (HI) test using the H5N2 avian influenza antigen. ResultsAn overall prevalence of 2.92% with a mean antibody titre of (7.07±0.73) log2 was obtained. There was a low level of awareness of avian influenza among the rural dwellers, the electronic and print media were the most common source of awareness. ConclusionsThis result highlights the important role apparently healthy village chickens may play in virus perpetuation (reservoir) and in the spread of avian influenza to other animals and humans.

Inactivation of high and low pathogenic avian influenza virus H5 subtypes by copper ions incorporated in zeolite-textile materials

The effect of cotton textiles containing Cu2+ held by zeolites (CuZeo-textile) on the inactivation of H5 subtype viruses was examined. Allantoic fluid (AF) containing a virus (AF virus) (0.1ml) was applied to the textile (3×3-cm), and incubated for a specific period at ambient temperature. After each incubation, 0.9ml of culture medium was added followed by squeezing to recover the virus into the medium. The recovered virus was titrated using Madin–Darby canine kidney (MDCK) cells or 10-day-old embryonated chicken eggs.The highly pathogenic H5N1 and the low pathogenic H5N3 viruses were inactivated on the CuZeo-textile, even after short incubation. The titer of A/chicken/Yamaguchi/7/04 (H5N1) in MDCK cells and in eggs declined by >5.0 log10 and 5.0log10, respectively, in 30s. The titer of A/whooper swan/Hokkaido/1/08 (H5N1) in MDCK cells declined by 2.3 and 3.5 in 1 and 5min, respectively. When A/whistling swan/Shimane/499/83 (H5N3) was treated on the CuZeo-textile for 10min, the titer declined by >5.0log10 in MDCK cells and by >3.5log10 in eggs. In contrast, no decrease in the titers was observed on cotton textiles containing zeolites alone (Zeo-textile). Neither cytopathic effects nor NP antigens were detected in MDCK cells inoculated with the H5N1 virus treated on the CuZeo-textile. The viral genes (H5, N1, M, and NP) were amplified from the virus treated on the CuZeo-textile by RT-PCR. The hemagglutinating activity of the CuZeo-textile treated virus was unaffected, indicating that virus–receptor interactions were maintained. Electron microscopic analysis revealed a small number of particles with morphological abnormalities in the H5N3 virus samples recovered immediately from the CuZeo-textile, while no particles were detectable in the 10-min treated sample, suggesting the rapid destruction of virions by the Cu2+ in the CuZeo-textile. The loss of infectivity of H5 viruses could, therefore, be due to the destruction of virions by Cu2+. Interestingly, CuCl2 treatment (500 and 5000μM) did not have an antiviral effect on the AF viruses (H5N1 and H5N3) even after 48h of incubation, although the titer of the purified H5N3 virus treated with CuCl2 declined greatly. The antiviral effect was inhibited by adding the AF to the purified H5N3 virus prior to the CuCl2 treatment. The known antibacterial/antifungal activities of copper suggest that the CuZeo-textile can be applied at a high level of hygiene in both animals and humans.