Showing papers on "Avian Influenza A Virus published in 2015"

Avian Influenza A(H10N7) virus-associated mass deaths among harbor seals.

Abstract: To the Editor: Avian influenza A viruses occasionally cross the species barrier; influenza A(H5N1) virus and the recently emerged influenza A(H7N9) virus are prime examples of bird-to-human transmission (1,2). In addition, avian influenza A viruses can cross to various other mammalian species, including pinnipeds (e.g., seals) (3,4). Recently, mass deaths have occurred among harbor seals (Phoca vitulina); hundreds of carcasses washed up the shores of Sweden (March 2014), Denmark (July 2014), and Germany (October 2014). Approximately 1,400 dead harbor seals were seen in the coastal waters of Schleswig-Holstein in Germany alone, where the population is ≈12,000 animals. We report the investigation of the deaths of 17 seals from different age groups that were found dead on the islands of Helgoland and Sylt, Germany, during the second week of October 2014. Complete necropsies were performed on the carcasses, which were in variable nutritional conditions, ranging from very poor to good. Necropsy results showed consistently poorly retracted lungs with severe congestion, occasional diffuse consolidation, and multifocal firm nodular areas of gray-yellow discoloration with varying numbers of metazoic parasites. Histologic examinations showed acute necrotizing bronchitis and adenitis of bronchial glands with sloughing of epithelial cells (Figure, panel A). Occasionally, mild interstitial pneumonia was found. Multifocal pyogranulomatous to necrotizing pneumonia was associated with parasite infestation. A few animals had suppurative to necrotizing or nonsuppurative rhinitis and tracheitis. Figure Histopathologic and phylogenetic analyses of necropsy samples from harbor seals infected with avian influenza A(H10N7) virus, Germany, 2014. A) Lung of harbor seal showing marked necrosis and sloughing of epithelial cells in bronchial glands (arrows); ... Because mass deaths among seals were caused by phocine distemper virus in the same area in 1988 and 2002, we tested lung and throat swab samples for morbillivirus using reverse transcription PCR (RT-PCR) and immunohistochemical analysis (5). In addition, real-time RT-PCR targeting the influenza A virus matrix gene was performed (6). No indications for the morbillivirus were detected by RT-PCR and immunohistochemistry; however, in lung lesions and throat swab samples of 11 animals, a positive signal was observed by the influenza A matrix gene real-time RT-PCR (cycle threshold values 15.0–33.9). Influenza A virus (A/harbor seal/Germany/1/2014) was subsequently isolated from lung and throat swab samples; the virus replicated to high titers in 11-day-old embryonated chicken eggs and on MDCK cells. By PCR using specific primers and subsequent Sanger sequencing of the hemagglutinin and neuraminidase genes, this virus was characterized as an influenza A virus of the H10N7 subtype, commonly found in migratory waterfowl (6). In addition, genetic analyses of all other gene segments indicated that the influenza virus A/harbor seal/Germany/1/2014 was most closely related to various influenza A viruses detected in wild birds. Specifically, the hemagglutinin and neuraminidase genes were genetically most closely related to subtype H10N7 viruses recently found in migratory ducks in Georgia, Egypt, and the Netherlands (Figure, panel C) (7). Genetic analyses were based on BLAST analyses using public databases available as of October 17, 2014 (http://www.ncbi.nlm.nih.gov, http://www.gisaid.com) and supplemented with H10 and N7 sequences from the international wild bird surveillance program of Erasmus Medical Center (Technical Appendix Table). A maximum-likelihood phylogenetic tree of the hemagglutinin gene was generated by using PhyML version 3.1 (8) with the general time reversible +I+Γ model of nucleotide substitution; a full heuristic search and subtree pruning and regrafting searches were performed. The tree was visualized by using Figtree version 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree). To further elucidate the role of influenza A(H10N7) virus in the pathogenesis of the disease causing deaths among the seals, we conducted immunohistochemical analysis on the lungs using an influenza A virus nucleoprotein-specific monoclonal antibody (9). Evaluation of the lung tissues of the dead seals showed viral antigen in cytoplasm and nuclei of epithelial cells of bronchi and bronchial glands of affected lung areas (Figure, panel B), which suggests that this virus played a major role in the deaths. Immunohistochemical analysis performed on various organs (including brain and olfactory bulb) indicated that viral antigen was restricted to the respiratory tract. Although avian influenza A virus infections previously have caused mass deaths in seals (3,4,10), subtype H10N7 has not been associated with such events. We can speculate that the ongoing deaths could eventually affect all harbor seal populations of northwestern Europe and have consequences for wildlife management and seal rehabilitation activities. In addition, preliminary analysis of the hemagglutinin sequence of the influenza A(H10N7) virus suggests the presence of molecular determinants that indicate mammalian adaptation. Various analyses are ongoing to answer questions about the route of transmission among seals and possible transmissibility to humans. Note added in proof: Zohari et al. also recently reported the involvement of avian influenza A(H10N7) virus in mass deaths of harbor seals in Sweden (Euro Surveill. 2014;19:pii: 20967). Technical Appendix: Details of hemagglutinin sequences shown in the Figure. Click here to view.(22K, pdf)

Serological and virological surveillance of avian influenza A virus H9N2 subtype in humans and poultry in Shanghai, China, between 2008 and 2010.

Abstract: We report the serological evidence of low-pathogenic avian influenza (LPAI) H9N2 infection in an occupational poultry-exposed population and a general population. A serological survey of an occupational poultry-exposed population and a general population was conducted using a haemagglutinin-inhibiting (HI) assay in Shanghai, China, from January 2008 to December 2010. Evidence of higher anti-H9 antibodies was found in serum samples collected from poultry workers. During this period, 239 H9N2 avian influenza viruses (AIVs) were isolated from 9297 tracheal and cloacal paired specimens collected from the poultry in live poultry markets. In addition, a total of 733 influenza viruses were isolated from 1569 nasal and throat swabs collected from patients with influenza-like symptoms in a sentinel hospital, which include H3N2, H1N1, pandemic H1N1 and B, but no H9N2 virus was detected. These findings highlight the need for long-term surveillance of avian influenza viruses in occupational poultry-exposed workers.

Oral immunization of haemaggulutinin H5 expressed in plant endoplasmic reticulum with adjuvant saponin protects mice against highly pathogenic avian influenza A virus infection

Abstract: Pandemics in poultry caused by the highly pathogenic avian influenza (HPAI) A virus occur too frequently globally, and there is growing concern about the HPAI A virus due to the possibility of a pandemic among humans. Thus, it is important to develop a vaccine against HPAI suitable for both humans and animals. Various approaches are underway to develop such vaccines. In particular, an edible vaccine would be a convenient way to vaccinate poultry because of the behaviour of the animals. However, an edible vaccine is still not available. In this study, we developed a strategy of effective vaccination of mice by the oral administration of transgenic Arabidopsis plants (HA-TG) expressing haemagglutinin (HA) in the endoplasmic reticulum (ER). Expression of HA in the ER resulted in its high-level accumulation, N-glycosylation, protection from proteolytic degradation and long-term stability. Oral administration of HA-TG with saponin elicited high levels of HA-specific systemic IgG and mucosal IgA responses in mice, which resulted in protection against a lethal influenza virus infection with attenuated inflammatory symptoms. Based on these results, we propose that oral administration of freeze-dried leaf powders from transgenic plants expressing HA in the ER together with saponin is an attractive strategy for vaccination against influenza A virus.

Comprehensive analysis of antibody recognition in convalescent humans from highly pathogenic avian influenza H5N1 infection

Abstract: Understanding the mechanism of protective antibody recognition against highly pathogenic avian influenza A virus H5N1 in humans is critical for the development of effective therapies and vaccines. Here we report the crystal structure of three H5-specific human monoclonal antibodies bound to the globular head of hemagglutinin (HA) with distinct epitope specificities, neutralization potencies and breadth. A structural and functional analysis of these epitopes combined with those reported elsewhere identifies four major vulnerable sites on the globular head of H5N1 HA. Chimeric and vulnerable site-specific mutant pseudoviruses are generated to delineate broad neutralization specificities of convalescent sera from two individuals who recovered from the infection with H5N1 virus. Our results show that the four vulnerable sites on the globular head rather than the stem region are the major neutralizing targets, suggesting that during natural H5N1 infection neutralizing antibodies against the globular head work in concert to provide protective antibody-mediated immunity.

Comparative analysis of selected innate immune-related genes following infection of immortal DF-1 cells with highly pathogenic (H5N1) and low pathogenic (H9N2) avian influenza viruses.

Abstract: H5N1 and H9N2 viruses are important causes of avian influenza in China. H5N1 is typically associated with severe to fatal disease in poultry, while H9N2 is usually associated with mild disease. Differences in viral virulence prompted us to investigate whether innate immune responses would be differentially regulated following infection by H5N1 and H9N2 viruses. To address this hypothesis, expression of a panel of innate immune-related genes including IFN-α, IFN-β, Mx1, OASL, ISG12, IFIT5, IRF7, USP18, SST, and KHSRP in immortal DF-1 cells following H5N1 and H9N2 infection was analyzed and compared by real-time quantitative RT-PCR. Cells infected by either virus overall exhibited a similar expression profile for four ISGs (Mx1, OASL, ISG12, and IFIT5), IFN-α, IFN-β, and SST gene. However, two immune-regulatory genes (IRF7 and KHSRP) were not responsive to highly pathogenic H5N1 infection but were strongly up-regulated in DF-1 cells infected with low pathogenic H9N2 infection. The subtype-dependent host response observed in this study offers new insights into the potential roles of IRF7 and KHSRP in control and modulation of the replication and virulence of different subtypes or strains of avian influenza A virus.

Production of H5N1 Influenza Virus Matrix Protein 2 Ectodomain Protein Bodies in Tobacco Plants and in Insect Cells as a Candidate Universal Influenza Vaccine

Abstract: The spread of influenza A viruses is partially controlled and prevented by vaccination. The matrix protein 2 ectodomain (M2e) is the most conserved sequence in influenza A viruses, and is therefore a good potential target for a vaccine to protect against multiple virus subtypes. We explored the feasibility of an M2e-based universal influenza A vaccine candidate based on the highly pathogenic avian influenza A virus, H5N1. A synthetic M2e gene was human- and plant-codon optimized and fused in-frame with a sequence encoding the N-terminal proline-rich domain (Zera(®)) of the γ-zein protein of maize. Zera(®)M2e was expressed transiently in Nicotiana benthamiana and Sf21 baculovirus/insect cell expression systems, and Zera(®)M2e protein bodies (PBs) were successfully produced in both expression systems. The plant-produced Zera(®)M2e PBs were purified and injected into Balb/c mice. Western blot analysis using insect cell-produced Zera(®)M2e PBs and multiple tandem M2e sequences (5xM2e) fused with the avian influenza H5N1 transmembrane and cytosolic tail (5xM2e_tHA) confirmed the presence of M2e-specific antibodies in immunized mice sera. The immunogenicity of the Zera(®)M2e indicates that our plant-produced protein has potential as an inexpensive universal influenza A vaccine.

Structure and receptor binding preferences of recombinant hemagglutinins from avian and human h6 and h10 influenza a virus subtypes.

Abstract: During 2013, three new avian influenza A virus subtypes, A(H7N9), A(H6N1), and A(H10N8), resulted in human infections. While the A(H7N9) virus resulted in a significant epidemic in China across 19 provinces and municipalities, both A(H6N1) and A(H10N8) viruses resulted in only a few human infections. This study focuses on the major surface glycoprotein hemagglutinins from both of these novel human viruses. The detailed structural and glycan microarray analyses presented here highlight the idea that both A(H6N1) and A(H10N8) virus hemagglutinins retain a strong avian receptor binding preference and thus currently pose a low risk for sustained human infections. IMPORTANCE Human infections with zoonotic influenza virus subtypes continue to be a great public health concern. We report detailed structural analysis and glycan microarray data for recombinant hemagglutinins from A(H6N1) and A(H10N8) viruses, isolated from human infections in 2013, and compare them with hemagglutinins of avian origin. This is the first structural report of an H6 hemagglutinin, and our results should further the understanding of these viruses and provide useful information to aid in the continuous surveillance of these zoonotic influenza viruses.

When fur and feather occur together: interclass transmission of avian influenza A virus from mammals to birds through common resources.

Abstract: The potential role of wild mammals in avian influenza A virus (IAV) transmission cycles has received some attention in recent years and cases where birds have transmitted IAV to mammals have been documented. However, the contrasting cycle, wherein a mammal could transmit an avian IAV to birds, has been largely overlooked. We experimentally tested the abilities of two mammalian species to transmit avian IAV to mallards (Anas platyrhynchos) in simulated natural environments. Results suggested that striped skunks (Mephitis mephitis) can successfully transmit avian IAV to mallards through indirect contact with shared resources, as transmission was noted in 1 of 4 of the mallards tested. Cottontail rabbits (Sylvilagus sp.) exhibited a similar pattern, as one of five cottontail rabbits successfully transmitted IAV to a mallard, likely through environmental contamination. For each mammalian species tested, the mallards that became infected were those paired with the individual mammals with the lowest shedding levels but were anecdotally observed to be the most active animals. Mammals associated with and around poultry rearing facilities should be taken into consideration in biosecurity plans.

An overview of the characteristics of the novel avian influenza A H7N9 virus in humans

Abstract: The novel avian influenza A H7N9 virus which caused the first human infection in Shanghai, China; was reported on the 31st of March 2013 before spreading rapidly to other Chinese provinces and municipal cities. This is the first time the low pathogenic avian influenza A virus has caused human infections and deaths; with cases of severe respiratory disease with pneumonia being reported. There were 440 confirmed cases with 122 fatalities by 16 May 2014; with a fatality risk of ∼28%. The median age of patients was 61 years with a male-to-female ratio of 2.4:1. The main source of infection was identified as exposure to poultry and there is so far no definitive evidence of sustained person-to-person transmission. The neuraminidase inhibitors, namely oseltamivir, zanamivir, and peramivir; have shown good efficacy in the management of the novel H7N9 virus. Treatment is recommended for all hospitalized patients, and for confirmed and probable outpatient cases; and should ideally be initiated within 48 h of the onset of illness for the best outcome. Phylogenetic analysis found that the novel H7N9 virus is avian in origin and evolved from multiple reassortments of at least four origins. Indeed the novel H7N9 virus acquired human adaptation via mutations in its eight RNA gene segments. Enhanced surveillance and effective global control are essential to prevent pandemic outbreaks of the novel H7N9 virus.

Emerging Infections of CNS: Avian Influenza A Virus, Rift Valley Fever Virus and Human Parechovirus.

Abstract: History is replete with emergent pandemic infections that have decimated the human population. Given the shear mass of humans that now crowd the earth, there is every reason to suspect history will repeat itself. We describe three RNA viruses that have recently emerged in the human population to mediate severe neurological disease. These new diseases are results of new mutations in the infectious agents or new exposure pathways to the agents or both. To appreciate their pathogenesis, we summarize the essential virology and immune response to each agent. Infection is described in the context of known host defenses. Once the viruses evade immune defenses and enter central nervous system (CNS) cells, they rapidly co-opt host RNA processing to a cataclysmic extent. It is not clear why the brain is particularly susceptible to RNA viruses; but perhaps because of its tremendous dependence on RNA processing for physiological functioning, classical mechanisms of host defense (eg, interferon disruption of viral replication) are diminished or not available. Effectiveness of immunity, immunization and pharmacological therapies is reviewed to contextualize the scope of the public health challenge. Unfortunately, vaccines that confer protection from systemic disease do not necessarily confer protection for the brain after exposure through unconventional routes.

Pyrosequencing reveals an oseltamivir- resistant marker in the quasispecies of avian influenza A (H7N9) virus

Abstract: Prompt diagnosis of an oseltamivir-resistant marker is important for patient management, in particular to prevent the spread of resistant strains in the recent human H7N9 outbreak. We tailored a pyrosequencing assay to reveal neuraminidase R292K, a resistant marker found in one isolate from China, and demonstrated its performance in both sensitivity and specificity. In addition, a semi-nested polymerase chain reaction was applied, which enhanced the detection rate by at least 10-fold. We validated this assay by examining the marker in Taiwan's first imported human case and found R and K in quasispecies.

A comparative evaluation of feathers, oropharyngeal swabs, and cloacal swabs for the detection of H5N1 highly pathogenic avian influenza virus infection in experimentally infected chickens and ducks

Abstract: Oropharyngeal and cloacal swabs have been widely used for the detection of H5N1 highly pathogenic avian Influenza A virus (HPAI virus) in birds. Previous studies have shown that the feather calamus is a site of H5N1 virus replication and therefore has potential for diagnosis of avian influenza. However, studies characterizing the value of feathers for this purpose are not available, to our knowledge; herein we present a study investigating feathers for detection of H5N1 virus. Ducks and chickens were experimentally infected with H5N1 HPAI virus belonging to 1 of 3 clades (Indonesian clades 2.1.1 and 2.1.3, Vietnamese clade 1). Different types of feathers and oropharyngeal and cloacal swab samples were compared by virus isolation. In chickens, virus was detected from all sample types: oral and cloacal swabs, and immature pectorosternal, flight, and tail feathers. During clinical disease, the viral titers were higher in feathers than swabs. In ducks, the proportion of virus-positive samples was variable depending on viral strain and time from challenge; cloacal swabs and mature pectorosternal feathers were clearly inferior to oral swabs and immature pectorosternal, tail, and flight feathers. In ducks infected with Indonesian strains, in which most birds did not develop clinical signs, all sampling methods gave intermittent positive results; 3-23% of immature pectorosternal feathers were positive during the acute infection period; oropharyngeal swabs had slightly higher positivity during early infection, while feathers performed better during late infection. Our results indicate that immature feathers are an alternative sample for the diagnosis of HPAI in chickens and ducks.

Newly Emergent Highly Pathogenic H5N9 Subtype Avian Influenza A Virus

Abstract: The novel H7N9 avian influenza virus (AIV) was demonstrated to cause severe human respiratory infections in China. Here, we examined poultry specimens from live bird markets linked to human H7N9 infection in Hangzhou, China. Metagenomic sequencing revealed mixed subtypes (H5, H7, H9, N1, N2, and N9). Subsequently, AIV subtypes H5N9, H7N9, and H9N2 were isolated. Evolutionary analysis showed that the hemagglutinin gene of the novel H5N9 virus originated from A/Muscovy duck/Vietnam/LBM227/2012 (H5N1), which belongs to clade 2.3.2.1. The neuraminidase gene of the novel H5N9 virus originated from human-infective A/Hangzhou/1/2013 (H7N9). The six internal genes were similar to those of other H5N1, H7N9, and H9N2 virus strains. The virus harbored the PQRERRRKR/GL motif characteristic of highly pathogenic AIVs at the HA cleavage site. Receptor-binding experiments demonstrated that the virus binds α-2,3 sialic acid but not α-2,6 sialic acid. Identically, pathogenicity experiments also showed that the virus caused low mortality rates in mice. This newly isolated H5N9 virus is a highly pathogenic reassortant virus originating from H5N1, H7N9, and H9N2 subtypes. Live bird markets represent a potential transmission risk to public health and the poultry industry. IMPORTANCE This investigation confirms that the novel H5N9 subtype avian influenza A virus is a reassortant strain originating from H5N1, H7N9, and H9N2 subtypes and is totally different from the H5N9 viruses reported before. The novel H5N9 virus acquired a highly pathogenic H5 gene and an N9 gene from human-infecting subtype H7N9 but caused low mortality rates in mice. Whether this novel H5N9 virus will cause human infections from its avian host and become a pandemic subtype is not known yet. It is therefore imperative to assess the risk of emergence of this novel reassortant virus with potential transmissibility to public health.

Molecular characterization of mammalian-adapted Korean-type avian H9N2 virus and evaluation of its virulence in mice

Abstract: Avian influenza A virus (AIV) is commonly isolated from domestic poultry and wild migratory birds, and the H9N2 subtype is the most prevalent and the major cause of severe disease in poultry in Korea. In addition to the veterinary concerns regarding the H9N2 subtype, it is also considered to be the next potential human pandemic strain due to its rapid evolution and interspecies transmission. In this study, we utilize serial lung-to-lung passage of a low pathogenic avian influenza virus (LPAI) H9N2 (A/Ck/Korea/163/04, WT163) (Y439-lineage) in mice to increase pathogenicity and investigate the potential virulence marker. Mouse-adapted H9N2 virus obtained high virulence (100% mortality) in mice after 98 serial passages. Sequence results show that the mouse adaptation (ma163) possesses several mutations within seven gene segments (PB2, PA, HA, NP, NA, M, and NS) relative to the wild-type strain. The HA gene showed the most mutations (at least 11) with one resulting in the loss of an N-glycosylation site (at amino acid 166). Moreover, reverse genetic studies established that an E627K substitution in PB2 and the loss of the N-glycosylation site in the HA protein (aa166) are critical virulence markers in the mouse-adapted H9N2 virus. Thus, these results add to the increasing body of mutational analysis data defining the function of the viral polymerase and HA genes and their roles in mammalian host adaptation. To our knowledge, this is first report of the generation of a mammalian-adapted Korea H9N2 virus (Y493-lineages). Therefore, this study offers valuable insights into the molecular evolution of the LPAI Korean H9N2 in a new host and adds to the current knowledge of the molecular markers associated with increased virulence.

Identification of Hsp90 as a species independent H5N1 avian influenza A virus PB2 interacting protein.

Abstract: The avian influenza polymerase protein PB2 subunit is an important mediator of cross species adaptation and adaptation to mammalian cells is strongly but not exclusively associated with an adaptive mutation of the codon at position 627 of the PB2 protein which alters the glutamate normally found at this position to a lysine. This study sought to identify host cell factors in both mammalian and avian cells that interacted in a species specific or species independent manner. Two PB2 fusion proteins differing only in codon 627 were generated and transfected into mammalian and avian cells and interacting proteins identified through co-immunoprecipitation. A number of proteins including Hsp90 were identified and further investigation showed that Hsp90 interacted with both isoforms of PB2 in both mammalian and avian cells. Hsp90 is thus identified as a species independent interacting protein, further confirming that this protein may be a suitable target for anti-influenza drug development.

Gene expression signatures as a therapeutic target for severe H7N9 influenza - what do we know so far?

Abstract: A novel H7N9 avian influenza A virus (IAV) emerged in China in early 2013 causing > 450 cases of respiratory illness and 175 deaths within a 20-month period. Though avian viruses infect humans infrequently, the lack of human immunity to these viruses raises the possibility of a pandemic if they were to acquire the ability to transmit efficiently. Despite the fact that IAV pathogenicity results from the cytopathic effects and tissue damage caused by both viral replication and an overly robust immune response, current IAV therapeutics only target the viral proteins. This has led to the emergence of drug resistance due to the high mutation rates of viruses. The growing obsolescence of our current influenza therapeutics underscores the need for alternative treatment strategies. One promising area of research is the use of drugs that target the host response to IAV infection. This article describes how gene expression profiling can be used to predict drugs that reverse the destructive effects of the host response to H7N9 and other pathogenic influenza viruses.

Epidemiological and molecular characteristics of the PB1-F2 proteins in H7N9 influenza viruses, Jiangsu.

Abstract: The recent sporadic infections of humans in China with previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern. The aim is to find out the epidemiological and molecular analysis of the PB1-F2 proteins in H7N9 influenza viruses, in Jiangsu province. Sequences were obtained from GISAID database. Data were analyzed by using Molecular Evolutionary Genetics Analysis software and Bayesian Markov chain Monte Carlo method. From March 1, 2013, to May 31, 2014, 53 patients were confirmed to be infected with the H7N9 virus; one was a retrospective case in Jiangsu province. 38 sequences of PB1 in H7N9 of Jiangsu were obtained from the GISAID online and were then divided into three lineages. Of these sequences, 4 sequences and 3 sequences encode an N-terminally truncated PB1-F2 (52aa)polypeptide and C-terminally truncated PB1-F2 (76aa) polypeptide, respectively. The remaining sequences encode a full-length PB1-F2 (90aa). We estimated a mean evolutionary rate of 3.053 × 10−3 subs/site/year (95% HPD: 2.021 × 10−3–4.051 × 10−3). The site-by-site analysis of selection pressure analysis revealed positively and negatively (12, 3), respectively, selected sites. Influenza A (H7N9) virus adapting into new host, PB1-F2 of H7N9, might be faced with higher selection pressures.

Detection of avian influenza A virus in Malaysian birds

Abstract: A study was conducted to assess the presence of avian influenza A virus (AIV A) especially in wild forest birds of Malaysia. The annual north-south movements of migratory birds have been implicated as a factor that contributes to the spread of AIV A throughout the world. Mixing of local forest birds and migratory birds has been reported to occur during migrational stops of the migratory birds. It has been postulated that infected wild birds can pass on the virus to poultry, especially at areas that are considered as interface between wild birds and the domesticated poultry reared at the forest fringes. The possible widespread of AIV A in Malaysia and reports of outbreaks of H5N1 strain during the early 2003 to 2006, have led to the interest in finding support for this postulate. Occurrence of AIV A was postulated with possible implications with the ecological parameters of the areas. Transmission of viruses had been speculated to occur in certain seasons which give rise to the idea that some climatic factors such as temperature and rainfall plays their role in the transmission. Seventeen study areas from 12 geographical locations in Malaysia were studied and grouped into seven habitat types given as primary forest, secondary forest, urban, monoculture, mixed forest of lowland and limestone, beach forest and mixed forest of secondary growth and orchard to reflect a portion of the vast tropical habitat. The average temperature for all the habitats ranges from 23°C to 28°C while rainfall measure range from 0.7 mm to 9.6 mm. There are no significant differences in the temperature and rainfall measures that could be used to discriminate them into different habitats accordingly. The diversity indices were calculated based on Shannon's diversity index (H') and Simpson's diversity index (1-D) using Multivariate Statistical Package (MVSP) version 3.0. Both indices reflect the highest bird diversity in mixed forest of the limestone and lowland habitat (H'= 3.698, 1-D = 0.969). A total of 2199 throat washing and blood samples were collected from 1132 birds. The samples were screened for the presence of viral RNA genome. Out of the total samples, 1797 were analysed using reverse transcriptase polymerase chain reaction (RT-PCR) and 402 samples were analysed by the immunocapture RT-PCR. Detection by RT PCR was carried out using H5, H6, H7, H9 and nucleoprotein (NP) primers while immunocapture to the microplate was conducted using serowell plates pre-coated with H5, H6, H7 and H9 antigens. The results of the detection were negative for all isolates. This probably suggests that birds in Malaysia are free from avian influenza A virus. Optimisation of the detection based on the two techniques had been carried out using positive control. Amplification and detection of viral RNA were optimised at the temperature of 70°C with the use of H5 gene. However, the potential hosts, preferred habitats as well as climatic factor (rainfall and temperature) that were associated to the transmission and infection of the AIV could not be discriminated with reference to the negative detection. Although all isolates were tested negative, the infection of avian influenza virus is still a major concern because the fact that birds are free moving organisms suggests that higher chances of interaction between the birds and other fauna including human is plausible.

Could A Deletion in Neuraminidase Stalk Strengthen Human Tropism of the Novel Avian Influenza Virus H7N9 in China, 2013?

Abstract: Objective. A novel avian influenza A virus (AIV) H7N9 subtype which emerged in China in 2013 caused worldwide concern. Deletion of amino-acids 69 to 73 in the neuraminidase stalk was its most notable characteristic. This study is aimed to discuss the tropism and virulence effects of this deletion. Methods: Neuraminidase gene sequences of N9 subtype were collected from NCBI and GISAID. MEGA6.0, Stata12.0, and UCSF Chimera were employed for sequence aligning, significance testing, and protein tertiary structure homology modeling. Results: A total of 736 sequences were obtained; there were 81 human isolates of the novel AIV H7N9, of which 79 had the deletion. Among all the 654 avian origin sequences, only 43 had the deletion (p < 0.001). Tertiary structure displayed that the deletion obviously changed the spatial direction of neuraminidase. Conclusions: The deletion in neuraminidase stalk could have strengthened human tropism of the novel AIV H7N9, as well as its virulence.

Avian influenza a virus h5n1 vaccine for oral administration comprising hemagglutinin and saponin

Abstract: The present invention relates to an orally-administered influenza A virus subtype H5N1 vaccine composition comprising a plant body for expressing a recombinant protein containing hemagglutinin (HA) of influenza A virus subtype H5N1 and saponin, and an application thereof, more specifically, to a composition which can have a preventive effect for influenza A virus subtype H5N1 by orally administering a plant body transformed into a recombinant protein containing hemagglutinin (HA) of influenza A virus subtype H5N1 in combination with saponin, a feed composition comprising the same as an active component, and a method for preventing influenza A virus subtype H5N1 infection comprising a step of orally administering the feed composition to an entity requiring vaccination for influenza A virus subtype H5N1 infection. The present invention provides the orally-administered influenza A virus subtype H5N1 vaccine which can also be used as an edible vaccine.

[A preclinical trial of the reassortant influenza virus vaccine strain A/17/Quail/Hong Kong/97/84 (H9N2)].

Abstract: In this work, we examined the reassortant influenza virus strain A/17/Quail/Hong Kong/97/84 (H9N2) prepared at the Virology Department, Institute of Experimental Medicine, Russian Academy of Medical Sciences. The A/ Leningrad/134/17 (H2N2)-based vaccine candidate contained hemagglutinin and the neuraminidase from the nonpathogenic avian influenza A virus A(H9N2) of the G1 antigenic lineage. The vaccine candidate showed the ts-properties and cold adaptation. When administered intranasally to mice, the vaccine strain A(H9N2) was attenuated. It did not multiply in the lungs but was reproduced well in the nasal cavity, causing the production of the post-vaccination antibody. The A/17/Quail/Hong Kong/97/84(H9N2) virus was immunogenic when administered to mice as a LAIV intranasally or as a IIV intramuscularly. Intranasal A(H9N2) LAIV stimulated local production of the antibodies, which resulted in reduction in lung titers of the challenge virus G9.

Analysis of avian influenza virus surveillance in live poultry markets in Jining City, 2013-2014

Abstract: Objective To understand the distribution of avian influenza virus in live poultry market, and assess the risk of human infection caused by avian influenza. Methods Six live poultry markets were selected for the study, the environmental samples were collected by swab smear, Influenza A virus nucleic acid was detected by fluorescence quantitative PCR, the positive specimens were further subtypied. Results A total of 759 environmental samples were collected and tested, 37 samples were positive with avian influenza A virus nucleic acid, the overall positive rate was 4.87%.The positive rate in 2014 was higher than that in 2013(7.99% vs 1.63%,χ2=16.61,P0.01) The positive rate in the winter-spring season was higher than that in the summer-autumn season(7.98% vs2.54%, χ~2=11.85, P0.01). Positive specimens were detected in Yanzhou City and Weishan County, there were statistically differences among different counties(χ~2=29.72,P0.01) or among different types of specimens(χ~2=40.97,P0.01). Conclusion Influenza A virus in live poultry markets exists indeed, H5,H7 and H9 were the prevailing subtypes. It is necessary to conduct surveillance and health education among the people exposed to poultry in live poultry markets, We will effectively strengthen the market oversight, quarantine access, disinfection and suspended business.

Published sequences do not support transfer of oseltamivir resistance mutations from avian to human influenza A virus strains

Abstract: Tamiflu (oseltamivir phosphate ester, OE) is a widely used antiviral active against influenza A virus. Its active metabolite, oseltamivir carboxylate (OC), is chemically stable and secreted into wastewater treatment plants. OC contamination of natural habitats of waterfowl might induce OC resistance in influenza viruses persistently infecting waterfowl, and lead to transfer of OC-resistance from avian to human influenza. The aim of this study was to evaluate whether such has occurred. A genomics approach including phylogenetic analysis and probability calculations for homologous recombination was applied on altogether 19,755 neuraminidase (N1 and N2) genes from virus sampled in humans and birds, with and without resistance mutations. No evidence for transfer of OE resistance mutations from avian to human N genes was obtained, and events suggesting recombination between human and avian influenza virus variants could not be traced in the sequence material studied. The results indicate that resistance in influenza viruses infecting humans is due to the selection pressure posed by the global OE administration in humans rather than transfer from avian influenza A virus strains carrying mutations induced by environmental exposure to OC.

Role of Glycans in Viral Infection

Abstract: A variety of viruses show specific binding to glycans on the cellular surface, such as sialoglycoconjugates, glycosaminoglycans, and histo-blood group antigens. The viral surface proteins recognize terminal sugar chain moieties of glycan and select glycans for binding to specific tissues and hosts. For example, orthomyxoviruses (influenza viruses) and paramyxoviruses recognize terminal moieties of sialic acid linked to galactose for infecting target cells. In most cases, glycans are thought to be involved in cellular surface attachment and cell entry of viruses, as viral receptors and/or coreceptors. Expression of sugar chain moieties is generally dependent on specific tissues, cells, and hosts. Therefore, the specific interactions of viruses with glycans significantly affect tissue tropism and pathogenicity by selection of the viral replication site. For example, human influenza A virus preferentially binds to sialic acid α2,6 linkage to galactose, which is expressed in the human upper respiratory tract. On the other hand, avian influenza A virus preferentially binds to sialic acid α2,3 linkage to galactose, which is expressed in chicken eggs and trachea. The difference in recognition is believed to determine host specificity of influenza A virus. Platforms of the sugar chain are N-linked glycan, O-linked glycans (containing proteoglycans), and sphingolipid. Difference in these platforms also affects functions of viral receptors. This chapter presents a review about glycans bound and recognized by representative viruses including coronavirus, flavivirus, herpesvirus, norovirus, orthomyxovirus, paramyxovirus, parvovirus, polyomavirus, retrovirus, and reovirus.