Hemagglutinin Gene Of Influenza Virus Research Articles

Features of influenza virus hemagglutinin genes and their recoding possibilities

The world has already entered the stage of increasing odds for a new pandemic, which prompts to seek out for new flu vaccines, because existing vaccines demonstrate only suboptimal effectiveness. With the Covid-19 pandemic, the possibility of using mRNA vaccines has been opened up, and a prospect of finding hemagglutinin (HA) gene mRNA-based new influenza vaccines seems very attractive. As a rule, the mRNA vaccine is a product of recoding, which ensures the mRNA stability. However, the results of mRNA recoding can be ambiguous. The purpose of this report is to analyze the features of genes and proteins and to consider opportunities and limitations in their recoding. Primary structures of NA proteins and relevant genes were retrieved from Internet publicly available databases. The amino acid composition and frequency of dipeptides, nucleotide and dinucleotide compositions, %GC, translational code and compositions of neighboring di- and tricodones, distribution along primary structure for explicit and synonymous mutations were determined. H1N1 and H3N2 subtypes have both specific and general features (limitations) in their genes, differing not only in the number of protein substitutions, but also in the number and distribution of gene synonymous codons, which do not manifest in the protein primary structure, but appear, apparently, as a hidden factor, which causes the low effectiveness of classical influenza vaccines. The identification of several limitations in gene structure suggests that its any modification (in any gene) must not contradict each of the restrictions established by nature. The frequency of CpG dinucleotides in all studied strains is low, but a potential for optimizing it in H1N1 strains due to the prohibition of the quartet in the gene for arginine-encoding codons is especially limited and can be implemented through synonymous codons of other amino acids (alanine, proline, threonine or serine). Compared to the H1N1 subtype, the H3N2 subtype can be expected to have more possibilities in constructing stable NA gene mRNA.

Co-production of hemagglutinin H9N2 influenza virus and fusion protein Newcastle virus in insect cell using baculovirus expression system.

Influenza and Newcastle disease are the most important poultry diseases that cause high annual damage to poultry farms worldwide. Newcastle virus fusion (F) gene and Influenza Virus Hemagglutinin (HA) gene are capable of encoding F and HA proteins that are the main factors in creating immunity, so this study aimed to clone and express these genes in Spodoptera frugiperda (Sf9) cells using baculovirus expression system. After isolating the Newcastle and Influenza virus genome, the HA gene of influenza virus and the F gene of Newcastle virus were amplified by reverse transcriptase PCR and specific primers and then cloned into pFastBacTM Dual plasmid. A recombinant sucker with these genes was produced in the DH10Bac host cell. By transfecting Sf9 cells with recombinant bacmid, expression was assessed by SDS-PAGE, western blotting, and Bradford methods. Cloning of genes into the bacmid was successful. By transfecting the recombinant bacmid into Spodoptera frugiperda cells, 218 µg/ml of the recombinant protein was obtained in the supernatant. In addition, the presence of protein was confirmed by western blotting. The PCR products of HA and F genes showed one band of 1.7 kb size using specific primers. The pFastHA1 vector was about 7 kb in size. Two bands of about 7 kb and 1.7 kb were created by ligation of the F gene and pFastHA1 vector based on enzymatic digestion, indicating the correct ligation of F gene under the P10 promoter. This is the first report on the cloning and Co-expression of two HA and F genes using baculovirus expression system and can be a candidate for dual influenza and Newcastle vaccine. Mixtures of these recombinant proteins can be used as vaccine candidates against both avian influenza and Newcastle disease.

Recombinant duck enteritis virus bearing the hemagglutinin genes of H5 and H7 influenza viruses is an ideal multivalent live vaccine in ducks

ABSTRACT Due to the fact that many avian influenza viruses that kill chickens are not lethal to ducks, farmers are reluctant to use avian influenza inactivated vaccines on ducks. Large numbers of unvaccinated ducks play an important role in the transmission of avian influenza viruses from wild birds to domestic poultry, creating a substantial challenge to vaccination strategies for avian influenza control. To solve this problem, we constructed a recombinant duck enteritis virus (DEV), rDEV-dH5/H7, using a live attenuated DEV vaccine strain (vDEV) as a vector. rDEV-dH5/H7 carries the hemagglutinin gene of two H5 viruses [GZ/S4184/17 (H5N6) (clade 2.3.4.4 h) and LN/SD007/17 (H5N1) (clade 2.3.2.1d)] and an H7 virus [GX/SD098/17 (H7N9)]. These three hemagglutinin genes were stably inherited in rDEV-dH5/H7 and expressed in rDEV-dH5/H7-infected cells. Animal studies revealed that rDEV-dH5/H7 and vDEV induced similar neutralizing antibody responses and protection against lethal DEV challenge. Importantly, rDEV-dH5/H7 induced strong and long-lasting hemagglutinin inhibition antibodies against different H5 and H7 viruses and provided complete protection against challenges with homologous and heterologous highly pathogenic H5 and H7 influenza viruses in ducks. Our study shows that rDEV-dH5/H7 could serve as an ideal live attenuated vaccine to protect ducks against infection with lethal DEV and highly pathogenic avian influenza viruses.

Emergence of a new designated clade 16 with significant antigenic drift in hemagglutinin gene of H9N2 subtype avian influenza virus in eastern China

ABSTRACT H9N2 avian influenza viruses (AIVs) pose an increasing threat to the poultry industry worldwide and have pandemic potential. Vaccination has been principal prevention strategy to control H9N2 in China since 1998, but vaccine effectiveness is persistently challenged by the emergence of the genetic and/or antigenic variants. Here, we analysed the genetic and antigenic characteristics of H9N2 viruses in China, including 70 HA sequences of H9N2 isolates from poultry, 7358 from online databases during 2010–2020, and 15 from the early reference strains. Bayesian analyses based on hemagglutinin (HA) gene revealed that a new designated clade16 emerged in April 2012, and was prevalent and co-circulated with clade 15 since 2013 in China. Clade 16 viruses exhibited decreased cross-reactivity with those from clade 15. Antigenic Cartography analyses showed represent strains were classified into three antigenic groups named as Group1, Group2 and Group3, and most of the strains in Group 3 (15/17, 88.2%) were from Clade 16 while most of the strains in Group2 (26/29, 89.7%) were from Clade 15. The mean distance between Group 3 and Group 2 was 4.079 (95%CI 3.605–4.554), revealing that major switches to antigenic properties were observed over the emergence of clade 16. Genetic analysis indicated that 11 coevolving amino acid substitutions primarily at antigenic sites were associated with the antigenic differences between clade 15 and clade 16. These data highlight complexities of the genetic evolution and provide a framework for the genetic basis and antigenic characterization of emerging clade 16 of H9N2 subtype avian influenza virus.

The genetic characterization of hemagglutinin (HA), neuraminidase (NA) and polymerase acidic (PA) genes of H3N2 influenza viruses circulated in Guangdong Province of China during 2019-2020.

The evolution of seasonal influenza viruses, which can cause virus antigenic drift to escape human herd immunity, is a significant public health problem. Here, we obtained hemagglutinin (HA), neuraminidase (NA), and polymerase acidic protein (PA) the gene sequences of 84 influenza virus isolates collected in Guangdong Province during the 2019-2020 influenza season. Phylogenetic analyses revealed all these isolates were genetically similar to the viruses of clade 3C2a A1b, specifically those within subclades of A1b 137F (59 cases), A1b 186D (19 cases), and A1b 94N (6 cases). The influenza virus isolates were distinct from the World Health Organization recommended influenza A vaccine virus for the 2019-2020 Northern Hemisphere season (A/Kansas/14/2017; H3N2). Phylogenies inferred from the individual gene segment sequences revealed that one reassortment event occurred among these clades. The genetic variation involved mutations within viral antigenic epitopes and two N-glycosylation site alterations. The novel mutation sites of G202D and D206N in the HA gene, E344K in the NA gene, and K626R in the PA gene which may affect the spread of the virus were observed. We investigated the evolution of these genes and found that the HA and NA genes were under greater pressure than PA gene. Mutations associated with conferring resistance to NA inhibitors or baloxavir acid were not found. Our results suggest that a rapid evolution of the H3N2 influenza virus occurred, thus continuous monitoring is critical for establishing appropriate vaccine formulations or drug delivery for targeting influenza.

Isolation and molecular characterization of the hemagglutinin gene of H9N2 avian influenza viruses from poultry in Java, Indonesia.

Objective:The avian influenza virus (AIV) subtype H9N2 circulating in Indonesia has raised increasing concern about its impact on poultry and its public health risks. In this study, the H9N2 virus from chicken poultry farms in Java was isolated and characterized molecularly.Materials and Methods:Thirty-three pooled samples of chicken brain, cloacal swab, trachea, and oviduct were taken from multiple chickens infected with AIV in five regions of Java, Indonesia. The samples were isolated from specific pathogenic-free embryonated eggs that were 9 days old. Reverse transcription polymerase chain reaction and sequencing were used to identify H9N2 viruses.Results:This study was successful in detecting and characterizing 13 H9N2 isolates. The sequencing analysis of hemagglutinin genes revealed a 96.9%–98.8% similarity to the H9N2 AIV isolated from Vietnam in 2014 (A/muscovy duck/Vietnam/LBM719/2014). According to the phylogenetic analysis, all recent H9N2 viruses were members of the lineage Y280 and clade h9.4.2.5. Nine of the H9N2 isolates studied showed PSKSSR↓GLF motifs at the cleavage site, while four had PSKSSR↓GLF. Notably, all contemporary viruses have leucine (L) at position 216 in the receptor-binding region, indicating that the virus can interact with a human-like receptor.Conclusion:This study described the features of recent H9N2 viruses spreading in Java’s poultry industry. Additionally, H9N2 infection prevention and management must be implemented to avoid the occurrence of virus mutations in the Indonesian poultry industry.

Minoxidil nanoparticles as potential intradermal carrier for H5 DNA vaccine delivery

Background: DNA formulations provide the basis for safe and cost-effective vaccine. Low efficiency is often observed in the delivery of DNA vaccines and to induce a strong and long-lasting immune response, effective DNA delivery technologies are required that can induce high and continued levels of antigen production and stimulation at appropriate target sites. Methods and materials: In order to develop a new strategy for intradermal delivery of DNA vaccine, a recombinant plasmid encoding hemagglutinin (HA) gene of avian influenza virus, was formulated using green synthesis of Minoxidil nanoparticles (Min/NP) to develop nanoencapsulated H5 DNA vaccine (Min/NP/H5). Min/NP was successfully synthesized and uniformly dispersed with sizes in the range of 22–50 nm with an average size of 32 nm and positive charge of 16 mV. Ex vivo intradermal delivery of Min/NP/H5 were analysed by using Parallel Artificial Membrane Permeability Assay (PAMPA) and qPCR. Results: The results showed 100% of nanoencapsulated H5 DNA vaccine passed the artificial membrane. BALB/c mice (10 weeks old-female and male) immunized once intradermally with 150 μl of Min/NP/H5 behind the ear showed no clinical manifestations. H5 plasmid DNA was successfully detected from the dermis of the vaccinated mice as early as 1 h post-vaccination. In addition, sera collected from mice immunized with Min/NP/H5 showed rapidly increasing antibody titres against H5 on day 14 after immunization. The highest average antibody titres were detected on day 35 post immunization at 89.6 ± 35.05. Conclusion: Hence, single intradermal administrations of Min/NP/H5 to mice was able to efficiently deliver DNA vaccine and induced high levels of the antibody.

Molecular characterization of the hemagglutinin gene of H9N2 avian influenza viruses isolated from broiler flocks in Morocco from 2016 to 2018

Avian influenza viruses of the H9N2 subtype continue to spread in wild birds and poultry worldwide. Infection with H9N2 avian influenza virus was detected for the first time in Morocco in January 2016. In this study, a total of 105 organ and tracheal swab samples from 21 broiler farms in Morocco were collected from July 2016 to October 2018 for H9N2 screening. The suspicion of disease was based on severe respiratory signs such as sneezing, coughing, rales and gasping, while H9N2 virus infection was confirmed by real-time RT-PCR. Hemagglutinin (HA) genes of four isolates were amplified by conventional RT-PCR, sequenced, and aligned for phylogenetic analyses. Among the 21 flocks, 48% (10/21) were qRT-PCR positive for H9, with the cycle threshold values ranging from18.6 to 34.8. The maximum similarity in nucleotide and protein sequences (96-98%) was observed between the Moroccan viruses and an H9 virus isolated from broiler chickens in 2017 in Burkina Faso (A/chicken/BurkinaFaso/17RS93-19/2017) and from a layer chicken in the United Arab Emirates in 2015 (A/chicken/Dubai/D2506/2015). The HA genes revealed the close relationship between the four Moroccan viruses, with 97.9%-99.9% nucleotide identity. Phylogenetic analysis showed that the Moroccan viruses belonged to the G1 lineage, and likely originated from the Middle East, as previously reported in 2016.

Conserved structural RNA domains in regions coding for cleavage site motifs in hemagglutinin genes of influenza viruses.

The acquisition of a multibasic cleavage site (MBCS) in the hemagglutinin (HA) glycoprotein is the main determinant of the conversion of low pathogenic avian influenza viruses into highly pathogenic strains, facilitating HA cleavage and virus replication in a broader range of host cells. In nature, substitutions or insertions in HA RNA genomic segments that code for multiple basic amino acids have been observed only in the HA genes of two out of sixteen subtypes circulating in birds, H5 and H7. Given the compatibility of MBCS motifs with HA proteins of numerous subtypes, this selectivity was hypothesized to be determined by the existence of specific motifs in HA RNA, in particular structured domains. In H5 and H7 HA RNAs, predictions of such domains have yielded alternative conserved stem-loop structures with the cleavage site codons in the hairpin loops. Here, potential RNA secondary structures were analyzed in the cleavage site regions of HA segments of influenza viruses of different types and subtypes. H5- and H7-like stem-loop structures were found in all known influenza A virus subtypes and in influenza B and C viruses with homology modeling. Nucleotide covariations supported this conservation to be determined by RNA structural constraints that are stronger in the domain-closing bottom stems as compared to apical parts. The structured character of this region in (sub-)types other than H5 and H7 indicates its functional importance beyond the ability to evolve toward an MBCS responsible for a highly pathogenic phenotype.

Innovation in Newcastle Disease Virus Vectored Avian Influenza Vaccines.

Highly pathogenic avian influenza (HPAI) and Newcastle disease are economically important avian diseases worldwide. Effective vaccination is critical to control these diseases in poultry. Live attenuated Newcastle disease virus (NDV) vectored vaccines have been developed for bivalent vaccination against HPAI viruses and NDV. These vaccines have been generated by inserting the hemagglutinin (HA) gene of avian influenza virus into NDV genomes. In laboratory settings, several experimental NDV-vectored vaccines have protected specific pathogen-free chickens from mortality, clinical signs, and virus shedding against H5 and H7 HPAI viruses and NDV challenges. NDV-vectored H5 vaccines have been licensed for poultry vaccination in China and Mexico. Recently, an antigenically chimeric NDV vector has been generated to overcome pre-existing immunity to NDV in poultry and to provide early protection of poultry in the field. Prime immunization of one-day-old poults with a chimeric NDV vector followed by boosting with a conventional NDV vector has shown to protect broiler chickens against H5 HPAI viruses and a highly virulent NDV. This novel vaccination approach can provide efficient control of HPAI viruses in the field and facilitate poultry vaccination.

Generation of highly productive and mammalian nonpathogenic recombinant H9N2 avian influenza viruses by optimization of 3’end promoter and NS genome

We developed A/PR/8/34 (PR8) virus-based reverse genetics system in which six internal genes of PR8 and attenuated hemagglutinin and intact neuraminidase genes of field avian influenza viruses (AIVs) have been used for the generation of highly productive recombinant vaccine strains. The 6 + 2 recombinant vaccine strains can induce protective humoral immunity against intended field AIVs; however, the epitopes of B and T cells encoded by internal genes may be important for heterosubtypic protection. Therefore, it is advantageous to use homologous internal genes of field AIVs for recombinant vaccine strains. However, the rescue of recombinant viruses having whole internal genes of field AIVs by the PR8-based reverse genetics system was unsuccessful in some cases. Although partial replacement of an internal gene has been successful for generation of highly productive and mammalian nonpathogenic recombinant viruses, complete replacement of internal genes may be more favorable. In this study, we successfully generated complete recombinant H9N2 AIVs possessing 8 genomes of H9N2 AIVs by optimal combinations of 3’ end promoter sequences of polymerase genomes, and a NS genome. All the generated recombinant viruses showed highly productive and mammalian nonpathogenic traits but some of them showed much higher virus titers in embryonated chicken eggs. Additionally, we found the same mutations of NS1 gene determined pathogenicity of AIVs in chicken embryos as well as mammals. Thus, the 3’ end promoter optimization, and highly productive and mammalian nonpathogenic internal genes may be useful to develop vaccines against AIVs.

Development of a reverse transcription recombinase polymerase amplification combined with lateral-flow dipstick assay for avian influenza H9N2 HA gene detection.

H9N2 avian influenza viruses (AIVs) have been detected from wild birds and domestic poultry worldwide. Serious diseases combined with secondary infection have caused high mortality and great economic losses to poultry industry. Therefore, simple, rapid, sensitive and accurate methods suitable for field detection of H9N2 AIVs are crucial to efficiently control virus infection and spread in time. In this study, an isothermal reverse transcription recombinase polymerase amplification with lateral-flow dipstick (RT-RPA-LFD) assay for detection of hemagglutinin (HA) gene of H9 subtype influenza viruses was developed. The optimal forward and reverse primers targeting HA gene of H9 subtype influenza viruses were labeled with fluorescein isothiocyanate (FITC) and biotin at the 5'-end, respectively. The amplification reaction could be finished in 20min at a wide temperature range of 30-42°C, and then the products could be visualized with naked eyes. The developed H9 RT-RPA-LFD was able to detect 0.15pg of H9N2 AIV RNA, which was 10 times more sensitive than that of conventional RT-PCR. The H9 RT-RPA-LFD assay did not detect nucleic acids extracted from H9 negative samples or from other poultry respiratory pathogens. The clinical performance of H9 RT-RPA-LFD was determined by testing 120 cloacal samples collected from chickens with respiratory syndromes. The coincidence rate of the detection results between RT-RPA-LFD and conventional RT-PCR was 95.8%. Therefore, the developed RT-RPA-LFD assay provides a rapid, reliable and sensitive method for field diagnosis of H9 subtype AIVs.

Fluorometric detection of influenza viral RNA using graphene oxide

Simple and reliable detection of influenza viruses is important for timely prescription of antiviral therapy. Here, we developed a facile fluorometric system for detection of influenza subtype viral genes using graphene oxide (GO). A fluorescent DNA probe complementary to hemagglutinin gene of influenza virus is degraded by the 5′ to 3′ exonuclease activity of Taq polymerase during PCR. Upon addition of GO, the released fluorophore retains fluorescence without adsorption onto GO, whereas the intact fluorescent DNA probe is adsorbed onto GO with fluorescence quenching. Our multi well plate system can detect as low as 3.8 pg of influenza viral RNA.

Phylogenetic classification of hemagglutinin gene of H9N2 avian influenza viruses isolated in China during 2012-2016 and evaluation of selected candidate vaccine strains.

H9N2 subtype avian influenza virus (AIV) was the highly contagious pathogen which has caused severe losses in the poultry industry throughout China in recent years. Using current epidemic viruses as vaccine was an effective way to prevent infection of H9N2 subtype AIV. In this study, a total of 23 H9N2 subtype AIV strains were isolated in 200 samples from 13 provinces of China during 2012-2016. The sequencing and phylogenetic analysis of the hemagglutinin gene sequence of the isolation strains showed that 22 isolation strains were clustered to h9.4.2.5 lineage, while only 1 belonged to h9.4.2.6. The data of cross-HI, neutralization and cross-immune protection shown that the A/chicken/Hunan/HN/2015 (HN) and A/chicken/Shandong/SD/2014 (SD) strains as vaccine could effectively protect present viruses infection compared with other strains. These results indicated that current epidemic viruses were mainly belong to h9.4.2.5 lineage and HN and SD strains as candidate vaccine strains were potentiality for the protection of present H9N2 subtype AIV infection.

Subpopulation Primers Essential for Exhaustive Detection of Diverse Hemagglutinin Genes of H5 Subtype Avian Influenza Viruses by Loop-Mediated Isothermal Amplification Method.

Loop-mediated isothermal amplification (LAMP) is a potential screening test for avian influenza (AI), but its narrow detection spectrum limits its applications. To improve this narrow detection spectrum, 3 types of primers were compared for detection of diverse H5 subtype hemagglutinin (HA) genes. Four and 6 genes, of 10 genetically different H5 HA genes tested, were detected with S primers specific for A/duck/Tsukuba/9/2005 (H5N2) and with M primers (which contained mixed bases), respectively. In contrast, all 10 HA genes became positive with population primers (P primers) (a mixture of primers designed for each subpopulation of 2,202 HA genes). Our study indicated that the P primers for the forward inner primer (FIP) and backward inner primer (BIP) sites were essential for exhaustive detection, whereas those for the F3, forward loop (FL), backward loop (BL), and B3 sites were exchangeable with M primers. A base mismatch experiment demonstrated that HA genes with ≤2 base mismatches per primer site and ≤10 base mismatches per HA gene were amplifiable. Reverse transcription-LAMP was broadly reactive, specific for H5 subtype HA genes, and applicable to field samples, with the sensitivity of real-time PCR. The in silico analysis suggested that most H5 HA genes (2,586 positive genes/2,588 genes tested) registered in the GenBank database might be amplifiable. These results indicate that the use of subpopulation primers in LAMP allows exhaustive detection of diverse HA genes and H5 LAMP can be used as a reliable AI screening test in general diagnostic laboratories.

DNA assembly with error correction on a droplet digital microfluidics platform

BackgroundCustom synthesized DNA is in high demand for synthetic biology applications. However, current technologies to produce these sequences using assembly from DNA oligonucleotides are costly and labor-intensive. The automation and reduced sample volumes afforded by microfluidic technologies could significantly decrease materials and labor costs associated with DNA synthesis. The purpose of this study was to develop a gene assembly protocol utilizing a digital microfluidic device. Toward this goal, we adapted bench-scale oligonucleotide assembly methods followed by enzymatic error correction to the Mondrian™ digital microfluidic platform.ResultsWe optimized Gibson assembly, polymerase chain reaction (PCR), and enzymatic error correction reactions in a single protocol to assemble 12 oligonucleotides into a 339-bp double- stranded DNA sequence encoding part of the human influenza virus hemagglutinin (HA) gene. The reactions were scaled down to 0.6-1.2 μL. Initial microfluidic assembly methods were successful and had an error frequency of approximately 4 errors/kb with errors originating from the original oligonucleotide synthesis. Relative to conventional benchtop procedures, PCR optimization required additional amounts of MgCl2, Phusion polymerase, and PEG 8000 to achieve amplification of the assembly and error correction products. After one round of error correction, error frequency was reduced to an average of 1.8 errors kb− 1.ConclusionWe demonstrated that DNA assembly from oligonucleotides and error correction could be completely automated on a digital microfluidic (DMF) platform. The results demonstrate that enzymatic reactions in droplets show a strong dependence on surface interactions, and successful on-chip implementation required supplementation with surfactants, molecular crowding agents, and an excess of enzyme. Enzymatic error correction of assembled fragments improved sequence fidelity by 2-fold, which was a significant improvement but somewhat lower than expected compared to bench-top assays, suggesting an additional capacity for optimization.

Sequence and phylogenetic analysis of hemagglutinin genes of H9N2 influenza viruses isolated from chicken in China from 2013 to 2015

H9N2 avian influenza virus (AIV) infection is a major problem in poultry industry worldwide. In this study, molecular characterizations and phylogenetic relationships of hemagglutinin (HA) gene sequences of H9N2 AIV of 5 Chinese isolates in 2014 recently available in GenBank, 3 widely used vaccine strains, and 52 novel isolates in China from 2013 to 2015 were analyzed. The homology analysis showed that the nucleotide sequences of HA gene of these recent Chinese H9N2 AIV isolates shared homologies from 94.1 to 99.9%. Phylogenetic analysis showed that all isolates belonged to AIV lineage h9.4.2.5. Fifty-six out of the 57 recent Chinese H9N2 AIV isolates had the motifs PSRSSR↓GLF at the cleavage sites within the HA protein, while one isolate PWH01 harbored LSRSSR↓GLF. Remarkably, all of the recent Chinese H9N2 AIV strains had the Q216L substitution in the receptor binding site, which indicated that they had potential to infect humans. Most of recent Chinese H9N2 AIV isolates lost the potential N-linked glycosylation site at residues 200–202 compared with vaccine strains. This present study demonstrated that AIV lineage h9.4.2.5 was more predominant in China than other lineages as it harbored all the H9N2 AIV isolated between 2013 and 2015. Also we showed the importance of continuous surveillance of emerging H9N2 AIV in China and update of vaccine formulation accordingly in order to prevent and control H9N2 AIV.

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.

Development of reverse transcription recombinase polymerase amplification assay for avian influenza H5N1 HA gene detection

The 2006 outbreaks of H5N1 avian influenza in Egypt interrupted poultry production and caused staggering economic damage. In addition, H5N1 avian influenza viruses represent a significant threat to public health. Therefore, the rapid detection of H5 viruses is very important in order to control the disease. In this study, a qualitative reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of hemagglutinin gene of H5 subtype influenza viruses was developed. The results were compared to the real-time reverse transcription polymerase chain reaction (RT-PCR). An in vitro transcribed RNA standard of 970 nucleotides of the hemagglutinin gene was developed and used to determine the assay sensitivity. The developed H5 RT-RPA assay was able to detect one RNA molecule within 7min, while in real-time RT-PCR, at least 90min was required. H5 RT-RPA assay did not detect nucleic acid extracted from H5 negative samples or from other pathogens producing respiratory manifestation in poultry. The clinical performance of the H5 RT-RPA assay was tested in 30 samples collected between 2014 and 2015; the sensitivity of H5 RT-RPA and real-time RT-PCR was 100%. In conclusion, H5 RT-RPA was faster than real-time RT-PCR and easily operable in a portable device. Moreover, it had an equivalent sensitivity and specificity.

Sequencing and genetic analysis of hemagglutinin gene of H9 avian influenza viruses from Iran (2008 to 2011)

ObjectiveTo survey on molecular characterization of Hemagglutinin(HA) gene in H9N2 viruses and determine genetic relationship between Iranian H9N2 viruses and other Asian viruses. MethodsTen H9N2 viruses were isolated from commercial broiler chickens in Iran during 2008-2011 and their HA genes were analyzed by reverse transcription-polymerase chain reaction and sequencing. Meanwhile, nucleotide sequences (open reading frame) of the HA genes were used for phylogenetic tree construction. ResultsAll the isolates possessed the same amino acid motif -R-S-S-R/G-L- at the cleavage site of HA and possessed H at amino acid position 183, A and T at position 190, S at position 138, whereas the representative strains of the other sublineage (except CK/HK/G1/97-like) of H9N2 viruses had N, E, and A at these positions respectively. Phylogenetic analysis of HA genes showed that it shared a common ancestor Qa/HK/G1/97 isolate which had contributed internal genes of human H5N1 viruses. ConclusionsThe present results indicated the HA genes of H9N2 influenza viruses circulating in Iran were well conserved in the past years and the earliest Iranian isolates may be considered to represent such a progenitor.