Hemagglutinin Gene Of Influenza Research Articles

P1614: MOLECULAR EPIDEMIOLOGY OF INFLUENZA VIRUS IN PATIENTS WITH HEMATOLOGIC DISORDERS

Background: Whereas influenza virus infections usually present as self-limiting upper respiratory tract infections, they are also one of the most common causes of viral lower respiratory tract infections. Especially immunocompromised patients, such as ones suffering from malignant diseases, are at a higher risk of severe influenza virus infection, thus leading to increased morbidity and mortality. With influenza viruses possessing the ability of antigenic shift and antigenic drift, consequently altering the viral genome frequently and avoiding long-term immunity of the host, the necessity for regular data collection is clear. Aims: With this study we want to retrospectively assess influenza virus epidemiology in patients with hematological malignancies and investigate clinical and virological risk factors of severe infection to be able to apply information about improvement of hygiene and care. Methods: We included clinical data from 159 influenza positive patients from the Department of Haematology of the University Hospital Heidelberg from the seasons of 2014 to 2019 to analyse occurrence of nosocomial transmission, prolonged viral shedding and associated risk factors for severe infection. Virological data was obtained by nasopharyngeal swabs, which were collected routinely, then Sanger sequencing of the hemagglutinin gene was performed for phylogenetic and mutational analysis. Combination of clinical and virological data was assessed to evaluate specific susceptibility to influenza subtype, subgroup or amino acid mutation. Results: 42 patients (26%) developed LRTI. Associated risk factors with development of lower respiratory tract infection (LRTI) (p<0.05) were nosocomial infection (p=0.008), IgG levels lower than 6g/dl (p=0.046), bacterial (p=0.001), fungal (p<0.001) and bacterial and fungal (p=0.017) coinfection. 9 of 31 (29%) of bacterial copathogens belonged to multi-resistant strains. LRTI associated mortality rate in our study population was noticeably high with 38%. 67% of patients with consecutive testing (n=54) presented with prolonged viral shedding (median 9.5 days). Epidemiological comparison with national surveillance data mostly showed no divergence of our patient group regarding influenza type and subtype. In 115 patients, sequencing of influenza hemagglutinin gene was successful, and 13 virus isolates revealed significant substitutions: In A/H1N1 samples P137S, D187N and D222V; in A/H3N2 samples D7N, T10M, D101E, I140M, Q197K, Q197R and I214T; in B/Yamagata samples Y165H, P171Q and N202R and none in B/Victoria samples. Most of these substitutions could be assigned to an antigenic site or receptor binding site. Summary/Conclusion: While no specific susceptibility to influenza subtypes or subgroups could be observed, high LRTI associated mortality rate and occurrence of nosocomial transmission as well as multi-resistant copathogens illustrate the need for better hospital hygiene and early evaluation of disease severity. To understand detected mutations and their impact on the course of infection as well as virus evolution and vaccination efficiency, further collection of data is needed.

Genetic Diversity of the Hemagglutinin Genes of Influenza a Virus in Asian Swine Populations.

Swine influenza (SI) is a major respiratory disease of swine; SI is due to the influenza A virus of swine (IAV-S), a highly contagious virus with zoonotic potential. The intensity of IAV-S surveillance varies among countries because it is not a reportable disease and causes limited mortality in swine. Although Asia accounts for half of all pig production worldwide, SI is not well managed in those countries. Rigorously managing SI on pig farms could markedly reduce the economic losses, the likelihood of novel reassortants among IAV-S, and the zoonotic IAV-S infections in humans. Vaccination of pigs is a key control measure for SI, but its efficacy relies on the optimal antigenic matching of vaccine strains with the viral strains circulating in the field. Here, we phylogenetically reviewed the genetic diversity of the hemagglutinin gene among IAVs-S that have circulated in Asia during the last decade. This analysis revealed the existence of country-specific clades in both the H1 and H3 subtypes and cross-border transmission of IAVs-S. Our findings underscore the importance of choosing vaccine antigens for each geographic region according to both genetic and antigenic analyses of the circulating IAV-S to effectively manage SI in Asia.

Construction of the vaccine strain of the influenza B virus with chimeric hemagglutinin to induce a cross-protective immune response

BACKGROUND: Influenza viruses cause worldwide epidemics, and the most effective method to prevent influenza disease is regular vaccinations. The development of new generation vaccines is aimed primarily at the formation of an immune response against a wide range of influenza viruses. One of the promising approaches is sequential vaccination with chimeric influenza viruses with identical stem domains of the hemagglutinin surface protein.&#x0D; AIM: The development of an experimental vaccine strain of influenza B virus with chimeric hemagglutinin consisting of head and stem domains of influenza B viruses belonging to different genetic lineages.&#x0D; MATERIALS AND METHODS: A chimeric influenza hemagglutinin gene was obtained by genetic engineering from the genetic material of B/Victoria and B/Yamagata influenza strains. The gene was inserted into the vector for the reverse genetics of the influenza virus. The influenza B virus strain with chimeric hemagglutinin was obtained by transfection of Vero cells using an 8-plasmid system. The rest of the genes were obtained from the attenuated influenza B virus with cold-adapted and temperature-sensitive phenotypes. The biological properties of the obtained recombinant strain, its infectious titer in developing chicken embryos and MDCK cell culture were evaluated.&#x0D; RESULTS: A recombinant vaccine strain has been successfully rescued. The head domain of the hemagglutinin of the virus is inherited from the B/Victoria influenza virus, and the stem domain from the B/Yamagata virus. The virus actively replicated in eggs and MDCK cells, with temperature-sensitive and cold-adapted phenotypes identical to classical live attenuated influenza vaccine viruses. The thermal stability of the chimeric hemagglutinin did not differ significantly from the thermal stability of the hemagglutinins of the donor viruses.&#x0D; CONCLUSIONS: The results obtained indicate the possibility of creating a strain with chimeric hemagglutinin, fragments of which are inherited from different genetic lineages. The growth characteristics and biological properties of the strain make it a promising candidate for the experimental evaluation of the possibility of inducing a cross-protective immune response by sequential vaccination with vaccine strains with identical stem hemagglutinin domains.

Spatial and temporal coevolution of N2 neuraminidase and H1 and H3 hemagglutinin genes of influenza A virus in US swine.

The neuraminidase (NA) and hemagglutinin (HA) are essential surface glycoproteins of influenza A virus (IAV). In this study, the evolution of subtype N2 NA paired with H1 and H3 subtype HA in swine was evaluated to understand if the genetic diversity of HA and NA were linked. Using time-scaled Bayesian phylodynamic analyses, the relationships of paired swine N2 with H1 or H3 from 2009 to 2018 were evaluated. These data demonstrated increased relative genetic diversity within the major N2 clades circulating in swine in the USA (N2.1998 between 2014 and 2017 and N2.2002 between 2010 and 2016). Preferential pairing was observed among specific NA and HA genetic clades. Gene reassortment between cocirculating influenza A strains resulted in novel pairings that persisted. The changes in genetic diversity in the NA gene were quantified using Bayesian phylodynamic analyses, and increases in diversity were observed subsequent to novel NA–HA reassortment events. The rate of evolution among NA–N2 clades and HA–H1 and HA–H3 clades were similar. Bayesian phylodynamic analyses demonstrated strong spatial patterns in N2 genetic diversity, but frequent interstate movement of rare N2 clades provided opportunity for reassortment and emergence of new N2–HA pairings. The frequent regional movement of pigs and their influenza viruses is an explanation for the documented patterns of reassortment and subsequent changes in gene diversity. The reassortment and evolution of NA and linked HA evolution may result in antigenic drift of both major surface glycoproteins, reducing vaccine efficacy, with subsequent impact on animal health.

In-depth phylogenetic analysis of the hemagglutinin gene of influenza A(H3N2) viruses circulating during the 2016-2017 season revealed egg-adaptive mutations of vaccine strains

ABSTRACTObjectives: The authors conducted an in-depth phylogenetic analysis of the hemagglutinin (HA) gene of A(H3N2) identified during the 2016–2017 season to compare the circulating strains with both the egg-derived and cell-derived 2016–2017 candidate vaccine virus (CVV).Methods: 162 HA1 sequences of H3N2 viruses identified during the 2016–2017 season were phylogenetically analyzed and compared with egg-/cell-derived CVV. The predicted vaccine efficacy (pVE) of egg-/cell-derived CVV against field strains was quantified by Pepitope model.Results: All H3N2 belonged to 3C.2a genetic clade, most (80.2%) to 3C.2a1 sub-clade. Several H3N2 variants circulated in the 2016–2017 season. HA sequences of field H3N2 strains displayed greater identity with cell-derived CVV than with egg-derived CVV. The amino acid substitutions in positions 160 and 194 identified in egg-derived CVV affected the pVE, which was lower for egg-derived CVV than for cell-derived CVV.Conclusions: The results suggested that reduced vaccine effectiveness observed in 2016–2017 season was probably due to changes in the HA of vaccine strains acquired upon adaptation in eggs, which are the basis of most manufacturing systems currently used globally. Egg-free vaccine manufacturing systems would be advisable to improve the effectiveness of influenza vaccines.

Respiratory syncytial, parainfluenza and influenza virus infection in young children with acute lower respiratory infection in rural Gambia

Respiratory viral infections contribute significantly to morbidity and mortality worldwide, but representative data from sub-Saharan Africa are needed to inform vaccination strategies. We conducted population-based surveillance in rural Gambia using standardized criteria to identify and investigate children with acute lower respiratory infection (ALRI). Naso- and oropharyngeal swabs were collected. Each month from February through December 2015, specimens from 50 children aged 2–23 months were randomly selected to test for respiratory syncytial (RSV), parainfluenza (PIV) and influenza viruses. The expected number of viral-associated ALRI cases in the population was estimated using statistical simulation that accounted for the sampling design. RSV G and F proteins and influenza hemagglutinin genes were sequenced. 2385 children with ALRI were enrolled, 519 were randomly selected for viral testing. One or more viruses were detected in 303/519 children (58.4%). RSV-A was detected in 237 and RSV-B in seven. The expected incidence of ALRI associated with RSV, PIV or influenza was 140 cases (95% CI, 131–149) per 1000 person-years; RSV incidence was 112 cases (95% CI, 102–122) per 1000 person-years. Multiple strains of RSV and influenza circulated during the year. RSV circulated throughout most of the year and was associated with eight times the number of ALRI cases compared to PIV or IV. Gambian RSV viruses were closely related to viruses detected in other continents. An effective RSV vaccination strategy could have a major impact on the burden of ALRI in this setting.

Genetic characterization of hemagglutinin (HA) gene of influenza A viruses circulating in Southwest India during 2017 season.

Molecular surveillance of influenza viruses is essential for early detection of novel variants. The aim of the present study was to analyze the hemagglutinin gene of influenza A(H1N1)pdm09 and A(H3N2) viruses circulating during the 2017 season. To investigate the genetic diversity of hemagglutinin gene of influenza A(H1N1)pdm09 and A(H3N2) viruses from 2017 season, ten samples from each subtype were sequenced and analyzed. The season was predominated by influenza A(H1N1)pdm09 viruses. Ten samples were sequenced from each subtype and all sequenced influenza A(H1N1)pdm09 and A(H3N2) viruses belonged to clades 6B.1 and 3C.2a, respectively. Sequence analysis of H1 gene in comparison to 2010-2016 vaccine strain showed mutations K166Q and S188T (K180Q and S202T here) that most likely resulted in antigenic drift and emergence of variant viruses. H3 gene substitutions N137K, N187K, I422V, and G500E that define clade 3C.2a1 were detected during analysis of sequences in comparison to 2017-2018 vaccine strain of northern hemisphere. These substitutions contributed to the change of WHO's recommendation of the 2018-2019 vaccine strain for northern hemisphere. The results of this study provide insights about the continuous genetic variability of the HA gene.

Significant circulation of influenza B viruses mismatching the recommended vaccine-lineage in South Korea, 2007–2014

We aimed to characterize the lineages of influenza B viruses obtained from clinical specimens during the 2007–2014 seasons in South Korea. RT-PCR for the partial hemagglutinin gene of influenza B virus was performed on laboratory-confirmed influenza B samples from the 2007–2008 season to 2013–2014 season. A phylogenetic tree was generated, and current influenza vaccine strains for the Northern Hemisphere were used as representative strains of Victoria and Yamagata lineages.A total of 571 influenza B virus sequences were analyzed. During the 2009–2010 season, most of the circulating influenza B viruses matched the vaccine strain; 91.0% (91/100) of viruses belonged to the Victoria lineage. In the 2007–2008, 2011–2012, and 2013–2014 seasons, co-circulation of each influenza B lineage was found with a match ratio to the vaccine strain of 53.2% (42/79), 40.9% (63/154), and 58.3% (134/230), respectively. Overall, 41.7% (238/571) of the circulating influenza B viruses belonged to the lineage mismatching the vaccine strain.During the seven influenza seasons, influenza B epidemics were substantial in four seasons in South Korea. Significant mismatches of the vaccine and lineage of the circulating influenza B viruses were found. The current trivalent influenza vaccine may not be fully suitable for effective protection against influenza B.

Molecular analysis of influenza A H1N1pdm09 virus circulating in Madhya Pradesh, India in the year 2017.

Influenza A (H1N1) pdm09 emerged as a major public health problem in 2009. The virus has evolved since then and has acquired several mutations that are epidemiologically important. Viral hemagglutinin (HA) protein is crucial determinant for virus entry, antigenicity, and virulence. The information regarding amino acid substitutions in HA protein of viruses circulating in India during 2017 is lacking. We sequenced HA gene of Influenza A (H1N1) pdm09 detected in Central India and compared the amino acid sequences with present vaccine component and contemporary Influenza A (H1N1) pdm09 sequences. We observed 7 amino acid changes of which two (T508A and I510T) were novel to Central Indian strain. Further, the substitution of Aspartate by Glycine at 222, which is known to have clinical implications, was detected in sequences from western India. The phylogenetic analysis revealed that the circulating virus belonged to clade 6B.1. The appreciable acquired mutations by the virus are not important antigenically and the present vaccine provides effective protection. On the other hand, the amino acid substitutions may play important role in epidemiology; we suggest rigorous molecular monitoring and documentation for timely interventions.

Influenza DNA vaccine purification using pHEMA cryogel support

Influenza virus is a huge financial and social burden for health care systems over the world. Currently, traditional approaches are not effective in the fight of the epidemy and new alternatives like DNA vaccines have been developed. However, the downstream process of DNA vaccines is a constant challenge in the biotechnology industry. Cryogels has several advantages over traditional supports and have been tested as stationary phase in chromatographic separations. In this work, a method based on poly(2-hydroxyethyl methacrylate) cryogel was used to purify the plasmid NTC7482-41H-VA2 HA, which express the Influenza hemagglutinin gene. For this purpose, the cryogel was synthesized by cryo-polymerization of 2-hydroxyethyl methacrylate and characterized by scanning electron microscopy. The purification of supercoiled isoform of the plasmid NTC7482-41H-VA2 HA from a clarified lysate sample was achieved in a two-step experiment using NaCl and the dynamic binding capacity of pHEMA cryogel was determined. The assessment of DNA vaccine allowed to conclude that the level of contaminants such as proteins, genomic DNA, RNA and endotoxins are in accordance with FDA agency.

Swine Influenza Virus (H1N2) Characterization and Transmission in Ferrets, Chile.

Phylogenetic analysis of the influenza hemagglutinin gene (HA) has suggested that commercial pigs in Chile harbor unique human seasonal H1-like influenza viruses, but further information, including characterization of these viruses, was unavailable. We isolated influenza virus (H1N2) from a swine in a backyard production farm in Central Chile and demonstrated that the HA gene was identical to that in a previous report. Its HA and neuraminidase genes were most similar to human H1 and N2 viruses from the early 1990s and internal segments were similar to influenza A(H1N1)pdm09 virus. The virus replicated efficiently in vitro and in vivo and transmitted in ferrets by respiratory droplet. Antigenically, it was distinct from other swine viruses. Hemagglutination inhibition analysis suggested that antibody titers to the swine Chilean H1N2 virus were decreased in persons born after 1990. Further studies are needed to characterize the potential risk to humans, as well as the ecology of influenza in swine in South America.

Molecular epidemiology and phylogenetic analysis of HA gene of influenza A(H1N1)pdm09 strain during 2010-2014 in Dalian, North China.

The objective of the present study was to evaluate the epidemiology of influenza A(H1N1)pdm09 and its hemagglutinin (HA) molecular and phylogenetic analysis during 2010-2014 in Dalian, North China. A total of 3717 influenza-like illness (ILI) cases were tested by real-time PCR and 493 were found to be positive. Out of these 493 cases, 121 were subtype influenza A(H1N1)pdm09, of which 14 cases were reported in 2010-2011, 29 in 2012-2013, and 78 in 2013-2014. HA coding regions of 45 isolates were compared to that of the vaccine strain A/California/7/09(H1N1), and a number of variations were detected. P83S, S185T, S203T, R223Q, and I321V mutations were observed in all of the Dalian isolates. Furthermore, a high proportion >71% of the strains possessed the variation D97N and K283E. Phylogenetic analysis confirmed the close match of the majority of circulating strains with the vaccine strains. However, it also reveals a trend of strains to accumulate amino acid variations and form new phylogenetic groups.

Detection of influenza A virus by reverse transcription-loop mediated isothermal amplification

Objective To detect influenza A virus by reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay to established a rapid, simple and visualization nucleic acid detection method. Methods The RT-LAMP primers were designed in accordance with the hemagglutinin gene of influenza A virus. Then, the specificity of the primers was evaluated by detection of different influenza viruses, and the sensitivity was confirmed by testing multiple diluted RNA samples. Hydroxynaphthol blue (HNB) was used for visually evaluation and gel electrophoresis was used for validation. Clinical samples were detected by RT-LAMP assay. Its consistency with fluorescent quantitative polymerase chain reaction (PCR) methods was compared. Results The primers of RT-LAMP assay had high specificity. This technique could amplify influenza A virus accurately. The detection limit of RT-LAMP assay was 2.5×103 copies/mL by detection of multiple diluted RNA samples. In addition, the results of RT-LAMP assay could be visually inspected using HNB by color change, and the results was in accordance with that of gel electrophoresis. RT-LAMP assay was in consistence with fluorescent quantitative PCR when clinically applied. Conclusions RT-LAMP assay is a rapid, specific, sensitive and simple method to detect influenza A virus. Key words: Influenza A virus; Reverse transcription-loop mediated isothermal amplification; Rapid detection

Bioinformatics studies of Influenza A hemagglutinin sequence data indicate recombination-like events leading to segment exchanges.

BackgroundThe influenza genome is highly variable due primarily to two mechanisms: antigenic drift and antigenic shift. A third mechanism for genetic change, known as copy choice or template switching, can arise during replication when, if two viral strains infect a cell, a part of a gene from the second viral strain can be copied into the growing progeny of a gene of the first viral strain as replacement leading to a new variety of the virus. This template switching between the same genes of the two strains is known as homologous recombination. While genetic drift and shift are well-understood, the presence or absence of intra-segment homologous recombination in influenza genomes is controversial.Context and purpose of studyWe are interested to study the possibility of subunit-wise homologous recombination. The idea is that where well-defined subunits are separated by consensus sequences, it might be possible for template switching to take place at such junctions. The influenza hemagglutinin gene has basically two subunits, HA1 and HA2, with HA1 being mostly surface exposed and containing the active site for binding to cells, while HA2 secures the hemagglutinin to the viral coat. We undertook a thorough search of the major human infecting influenza hemagglutinin gene sequences, viz., the H1N1, H5N1, H3N2 and H7N9 subtypes, over the period 2010–2014 in Asia to determine if certain sequences could be identified that had HA1 from a previous strain and HA2 from another.ResultsOur search yielded several instances where sequence identities between segments of various strains could be interpreted as indicating possibilities of segment exchange. In some cases, on closer examination they turn out to differ by a few mutations in each segment, due perhaps to the short time span of our database.Conclusions and potential implicationsThe study reported here, and in combination with our earlier observations on the neuraminidase, shows that subunit-wise recombination-like events in the influenza genes may be occurring more often than have been accounted for and merits further detailed studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-2017-3) contains supplementary material, which is available to authorized users.

Evaluation of the Universal Viral Transport system for long-term storage of virus specimens for microbial forensics

Forensic microbial specimens, including bacteria and viruses, are collected at biocrime and bioterrorism scenes. Although it is preferable that the pathogens in these samples are alive and kept in a steady state, the samples may be stored for prolonged periods before analysis. Therefore, it is important to understand the effects of storage conditions on the pathogens contained within such samples. To evaluate the capacity to preserve viable virus and the viral genome, influenza virus was added to the transport medium of the Universal Viral Transport system and stored for over 3 months at various temperatures, after which virus titrations and quantitative analysis of the influenza hemagglutinin gene were performed. Although viable viruses became undetectable 29 days after the medium was stored at room temperature, viruses in the medium stored at 4 °C were viable even after 99 days. A quantitative PCR analysis indicated that the hemagglutinin gene was maintained for 99 days at both 4 °C and room temperature. Therefore, long-term storage at 4 °C has little effect on viable virus and viral genes, so the Universal Viral Transport system can be useful for microbial forensics. This study provides important information for the handling of forensic virus specimens.

Vaccine protection of chickens against antigenically diverse H5 highly pathogenic avian influenza isolates with a live HVT vector vaccine expressing the influenza hemagglutinin gene derived from a clade 2.2 avian influenza virus

Vaccination is an important tool in the protection of poultry against avian influenza (AI). For field use, the overwhelming majority of AI vaccines produced are inactivated whole virus formulated into an oil emulsion. However, recombinant vectored vaccines are gaining use for their ability to induce protection against heterologous isolates and ability to overcome maternal antibody interference. In these studies, we compared protection of chickens provided by a turkey herpesvirus (HVT) vector vaccine expressing the hemagglutinin (HA) gene from a clade 2.2 H5N1 strain (A/swan/Hungary/4999/2006) against homologous H5N1 as well as heterologous H5N1 and H5N2 highly pathogenic (HP) AI challenge. The results demonstrated all vaccinated birds were protected from clinical signs of disease and mortality following homologous challenge. In addition, oral and cloacal swabs taken from challenged birds demonstrated that vaccinated birds had lower incidence and titers of viral shedding compared to sham-vaccinated birds. Following heterologous H5N1 or H5N2 HPAI challenge, 80–95% of birds receiving the HVT vector AI vaccine at day of age survived challenge with fewer birds shedding virus after challenge than sham vaccinated birds. In vitro cytotoxicity analysis demonstrated that splenic T lymphocytes from HVT-vector-AI vaccinated chickens recognized MHC-matched target cells infected with H5, as well as H6, H7, or H9 AI virus. Taken together, these studies provide support for the use of HVT vector vaccines expressing HA to protect poultry against multiple lineages of HPAI, and that both humoral and cellular immunity induced by live vaccines likely contributes to protection.

Safety and immunogenicity of a modified-vaccinia-virus-Ankara-based influenza A H5N1 vaccine: a randomised, double-blind phase 1/2a clinical trial

Modified vaccinia virus Ankara (MVA) is a promising viral vector platform for the development of an H5N1 influenza vaccine. Preclinical assessment of MVA-based H5N1 vaccines showed their immunogenicity and safety in different animal models. We aimed to assess the safety and immunogenicity of the MVA-haemagglutinin-based H5N1 vaccine MVA-H5-sfMR in healthy individuals. In a single-centre, double-blind phase 1/2a study, young volunteers (aged 18-28 years) were randomly assigned with a computer-generated list in equal numbers to one of eight groups and were given one injection or two injections intramuscularly at an interval of 4 weeks of a standard dose (10(8) plaque forming units [pfu]) or a ten times lower dose (10(7) pfu) of the MVA-H5-sfMR (vector encoding the haemagglutinin gene of influenza A/Vietnam/1194/2004 virus [H5N1 subtype]) or MVA-F6-sfMR (empty vector) vaccine. Volunteers and physicians who examined and administered the vaccine were masked to vaccine assignment. Individuals who received the MVA-H5-sfMR vaccine were eligible for a booster immunisation 1 year after the first immunisation. Primary endpoint was safety. Secondary outcome was immunogenicity. The trial is registered with the Dutch Trial Register, number NTR3401. 79 of 80 individuals who were enrolled completed the study. No serious adverse events were identified. 11 individuals reported severe headache and lightheadedness, erythema nodosum, respiratory illness (accompanied by influenza-like symptoms), sore throat, or injection-site reaction. Most of the volunteers had one or more local (itch, pain, redness, and swelling) and systemic reactions (rise in body temperature, headache, myalgia, arthralgia, chills, malaise, and fatigue) after the first, second, and booster immunisations. Individuals who received the 10(7) dose had fewer systemic reactions. The MVA-H5-sfMR vaccine at 10(8) pfu induced significantly higher antibody responses after one and two immunisations than did 10(7) pfu when assessed with haemagglutination inhibition geometric mean titre at 8 weeks against H5N1 A/Vietnam/1194/2004 (30·2 [SD 3·8] vs 9·2 [2·3] and 108·1 [2·4] vs 15·8 [3·2]). 27 of 39 eligible individuals were enrolled in the booster immunisation study. A single shot of MVA-H5-sfMR 10(8) pfu prime immunisation resulted in higher antibody responses after the booster immunisation than did two shots of MVA-H5-sfMR at the ten times lower dose. The MVA-based H5N1 vaccine was well tolerated and immunogenic and therefore the vaccine candidates arising from the MVA platform hold great promise for rapid development in response to a future influenza pandemic threat. However, the immunogenicity of this vaccine needs to be compared with conventional H5N1 inactivated non-adjuvanted vaccine candidates in head-to-head clinical trials. European Research Council.

Application of single-strand conformational polymorphism for detection of amino acid changes in neutralizing epitope of hemagglutinin gene of influenza A strains

Background: Chronic hepatitis B (CHB) is a serious health problemworldwidewitha substantialminorityofpatients experiencing premature death due to end-stage liver disease and/or hepatocelluar carcinoma. The purpose of the present study is to clarify whether the changes of liver histology in patients with mild CHB, whose alanine aminotransferase (ALT) is more than and less than two times up limit of normal (ULN), are different. The study will provide clue to solve vital problems in CHB treatment. It is meaning for the cases which even thoughmeet the requirements of both the “Chronic Hepatitis B Prophylaxis and Treatment Guide 2010” and HBV DNA detective value demond for normative anti-virus treatment, but ALT 0.05). However, for staging liver fibrosis, it shows an obvious difference as group ALT≥80U/L holds a higher S1 ratio (P <0.05). It means group ALT≥80U/L bears lower hepatic fibrosis. Conclusion: Since the present study shows that the comparison of hepatic inflammation activity is statistically meaningless comparing across groups of different ALT, and comparing to group ALT≥80U/L, group ALT<80U/L bears more serious hepatic fibrosis. It suggest that for mild CHB patients, includes both ALT≥80u and ALT<80u patients, as long as patients meet HBV DNA viral load demond for normative anti-virus treatment, normative antivirus treatment should be conducted. This study supplements and improves “Chronic Hepatitis B Prophylaxis and Treatment Guide” regarding to antiviral treatment for mild CHB patients with serum ALT abnormal (ALT<2×ULN).

Cross-protective immunity against influenza A/H1N1 virus challenge in mice immunized with recombinant vaccine expressing HA gene of influenza A/H5N1 virus

BackgroundInfluenza virus undergoes constant antigenic evolution, and therefore influenza vaccines must be reformulated each year. Time is necessary to produce a vaccine that is antigenically matched to a pandemic strain. A goal of many research works is to produce universal vaccines that can induce protective immunity to influenza A viruses of various subtypes. Despite intensive studies, the precise mechanisms of heterosubtypic immunity (HSI) remain ambiguous.MethodIn this study, mice were vaccinated with recombinant virus vaccine (rL H5), in which the hemagglutinin (HA) gene of influenza A/H5N1 virus was inserted into the LaSota Newcastle disease virus (NDV) vaccine strain. Following a challenge with influenza A/H1N1 virus, survival rates and lung index of mice were observed. The antibodies to influenza virus were detected using hemagglutination inhibition (HI). The lung viral loads, lung cytokine levels and the percentages of both IFN-γ+CD4+ and IFN-γ+CD8+ T cells in spleen were detected using real-time RT-PCR, ELISA and flow cytometry respectively.ResultsIn comparison with the group of mice given phosphate-buffered saline (PBS), the mice vaccinated with rL H5 showed reductions in lung index and viral replication in the lungs after a challenge with influenza A/H1N1 virus. The antibody titer in group 3 (H1N1-H1N1) was significantly higher than that in other groups which only low levels of antibody were detected. IFN-γ levels increased in both group 1 (rL H5-H1N1) and group 2 (rL H5 + IL-2-H1N1). And the IFN-γ level of group 2 was significantly higher than that of group 1. The percentages of both IFN-γ+CD4+ and IFN-γ+CD8+ T cells in group 1 (rL H5-H1N1) and group 2 (rL H5 + IL-2-H1N1) increased significantly, as measured by flow cytometry.ConclusionAfter the mice were vaccinated with rL H5, cross-protective immune response was induced, which was against heterosubtypic influenza A/H1N1 virus. To some extent, cross-protective immune response can be enhanced by IL-2 as an adjuvant. Cellular immune responses may play an important role in HSI against influenza virus.

Application of three duplex real-time PCR assays for simultaneous detection of human seasonal and avian influenza viruses

This study was performed to develop real-time PCR (qPCR) for detection of human seasonal and avian influenza viruses in duplex format. First duplex qPCR detects haemagglutinin (HA) gene of influenza virus A(H1N1)pdm09 and HA gene of influenza virus A(H3N2), the second reaction detects neuraminidase (NA) gene of influenza virus A(H3N2) and NA gene of influenza virus A(H1N1)pdm09 and A(H5N1), and the third reaction detects HA gene of influenza A(H5N1) and nonstructural protein gene of influenza B virus. Primers and probes were designed using multiple alignments of target gene sequences of different reference strains. Assays were optimised for identical thermocycling conditions. Their specificity was confirmed by conventional PCR and monoplex qPCR with nucleic acids isolated from different influenza viruses and other respiratory pathogens. Plasmid constructs with a fragment of specific gene were used to assess sensitivity of the assay. The limit of detection ranged from 27 to 96 cDNA copies/reaction. Clinical specimens (n = 107) have been tested using new assays, immunofluorescence and monoplex qRT-PCR. It has been shown that developed assays have been capable of rapid and accurate simultaneous detection and differentiation of influenza viruses. They are more sensitive than immunofluorescence and at least as sensitive as monoplex qRT-PCR.