Erythrocyte binding preference of avian influenza H5N1 viruses

Five erythrocyte species (horse, goose, chicken, guinea pig, and human) were used to agglutinate avian influenza H5N1 viruses by hemagglutination assay and to detect specific antibody by hemagglutination inhibition test. We found that goose erythrocytes confer a greater advantage over other erythrocyte species in both assays.     

The emergence and diversification of panzootic H5N1 influenza viruses

The Asian highly pathogenic avian influenza H5N1 virus was first detected in the goose population of Guangdong, China in 1996. The viruses in this lineage are unique in their ecological success, demonstrating an extremely broad host range and becoming established in poultry over much of Asia and in Africa. H5N1 viruses have also diverged into multiple clades and subclades that generally do not cross neutralize, which has greatly confounded control measures in poultry and pre-pandemic vaccine strain selection. Although H5N1 viruses currently cannot transmit efficiently between mammals they exhibit high mortality in humans and recent experimental studies have shown that it is possible to generate an H5N1 virus that is transmissible in mammals. In addition to causing unprecedented economic losses, the long-term presence of the H5N1 virus in poultry and its frequent introductions to humans continue to pose a significant pandemic threat. Here we provide a summary of the genesis, molecular epidemiology and evolution of this H5N1 lineage, particularly the factors that have contributed to the continued diversification and ecological success of H5N1 viruses, with particular reference to the poultry production systems they have emerged from.     

Protection of chickens against highly lethal H5N1 and H7N1 avian influenza viruses with a recombinant fowlpox virus co-expressing H5 haemagglutinin and N1 neuraminidase genes

Inactivated whole avian influenza virus (AIV) vaccine provides protection against homologous haemagglutinin (HA) subtype virus, but poor protection against a heterologous HA virus. Moreover, it induces chickens to produce antibodies to cross-reactive antigens, especially nucleoprotein, which is limits AIV serological surveillance. In this study, a recombinant fowlpox virus co-expressing HA (H5 subtype) and NA (N1 subtype) genes of AIV was evaluated for its ability to protect chickens against intramuscular challenge with a lethal dose of highly pathogenic (HP) AIV. Susceptible chickens were also vaccinated by wing-web puncture with the parent fowlpox vaccine virus. Following challenge 4 weeks later with HPAIV, all chickens vaccinated with recombinant virus were protected, while the chickens vaccinated with either the unaltered parent fowlpox vaccine virus or unvaccinated controls experienced 100% mortality following challenge. This protection was accompanied by the high levels of specific antibody to the respective components of the recombinant vaccine. The above results showed that rFPV-HA-NA could be a potential vaccine to replace current inactivated vaccines for preventing AI.     

Transmission of influenza A/H5N1 viruses in mammals

Highly pathogenic avian H5N1 influenza A viruses occasionally infect humans and cause severe respiratory disease and fatalities. Currently, these viruses are not efficiently transmitted from person to person, although limited human-to-human transmission may have occurred. Nevertheless, further adaptation of avian H5N1 influenza A viruses to humans and/or reassortment with human influenza A viruses may result in aerosol transmissible viruses with pandemic potential. Although the full range of factors that modulate the transmission and replication of influenza A viruses in humans are not yet known, we are beginning to understand some of the molecular changes that may allow H5N1 influenza A viruses to transmit via aerosols or respiratory droplets among mammals. A better understanding of the biological basis and genetic determinants that confer transmissibility to H5N1 influenza A viruses in mammals is important to enhance our pandemic preparedness.     

Enhanced growth of seed viruses for H5N1 influenza vaccines

Seed viruses used to produce inactivated H5N1 influenza vaccines are recombinant viruses with modified avirulent-type hemagglutinin (HA) and intact neuraminidase (NA) genes, both derived from an H5N1 isolate, and all remaining genes from the PR8 strain, which grows well in eggs. However, some reassortants grow suboptimally in eggs, imposing obstacles to timely, cost-efficient vaccine production. Here, we demonstrate that our PR8 strain supports better in ovo growth than the PR8 strain used for the WHO-recommended seed virus, NIBRG-14. Moreover, inclusion of an alternative NA protein further enhanced viral growth in eggs. These findings suggest that our H5N1 vaccine candidates would increase the availability of H5N1 vaccine doses at the onset of a new pandemic.     

Differential susceptibility of different cell lines to swine-origin influenza A H1N1, seasonal human influenza A H1N1, and avian influenza A H5N1 viruses

BackgroundThe novel swine-origin influenza A H1N1 virus (S-OIV) causes the current pandemic. Its tissue tropism and replication in different cell lines are not well understood.ObjectiveCompare the growth characteristics of cell lines infected by S-OIV, seasonal influenza A H1N1 (sH1N1) and avian influenza A H5N1 (H5N1) viruses and the effect of temperature on viral replication.Study designCytopathic effect (CPE), antigen expression by immunofluorescence (IF) and viral load profile by quantitative RT-PCR in 17 cell lines infected by S-OIV, sH1N1 and H5N1 were examined. Comparison of their replication efficiency in chick embryo was performed. The effect of temperature on viral replication in Madin–Darby canine kidney (MDCK) cells was determined by TCID₅₀ at 33 °C, 37 °C and 39 °C for 5 consecutive days.ResultsS-OIV replicated in cell lines derived from different tissues or organs and host species with comparable viral load to sH1N1. Among 13 human cell lines tested, Caco-2 has the highest viral load for S-OIV. S-OIV showed a low viral load with no CPE or antigen expression in pig kidney cell PK-15, H5N1 demonstrated the most diverse cell tropism by CPE and antigen expression, and the highest viral replication efficiency in both cell lines and allantoic fluid. All three viruses demonstrated best growth at 37 °C in MDCK cells.ConclusionCell line growth characteristics of S-OIV, sH1N1 and H5N1 appear to correlate clinically and pathologically with involved anatomical sites and severity. Low replication of S-OIV in PK-15 suggests that this virus is more adapted to human than swine.     

Classical swine H1N1 influenza viruses confer cross protection from swine-origin 2009 pandemic H1N1 influenza virus infection in mice and ferrets

The hemagglutinin of the 2009 pandemic H1N1 influenza virus is a derivative of and is antigenically related to classical swine but not to seasonal human H1N1 viruses. We compared the A/California/7/2009 (CA/7/09) virus recommended by the WHO as the reference virus for vaccine development, with two classical swine influenza viruses A/swine/Iowa/31 (sw/IA/31) and A/New Jersey/8/1976 (NJ/76) to establish the extent of immunologic cross-reactivity and cross-protection in animal models. Primary infection with 2009 pandemic or NJ/76 viruses elicited antibodies against the CA/7/09 virus and provided complete protection from challenge with this virus in ferrets; the response in mice was variable and conferred partial protection. Although ferrets infected with sw/IA/31 virus developed low titers of cross-neutralizing antibody, they were protected from pulmonary replication of the CA/7/09 virus. The data suggest that prior exposure to antigenically related H1N1 viruses of swine-origin provide some protective immunity against the 2009 pandemic H1N1 virus.     

Extent of antigenic cross-reactivity among highly pathogenic H5N1 influenza viruses

Highly pathogenic H5N1 avian influenza viruses emerged in 1996 and have since evolved so extensively that a single strain can no longer be used as a prepandemic vaccine or diagnostic reagent. We therefore sought to identify the H5N1 strains that may best serve as cross-reactive diagnostic reagents. We compared the cross-reactivity of 27 viruses of clades 0, 1, 2.1, 2.2, 2.3, and 4 and of four computationally designed ancestral H5N1 strains by hemagglutination inhibition (HI) and microneutralization (MN) assays. Antigenic cartography was used to analyze the large quantity of resulting data. Cartographs of HI titers with chicken red blood cells were similar to those of MN titers, but HI with horse red blood cells decreased antigenic distances among the H5N1 strains studied. Thus, HI with horse red blood cells seems to be the assay of choice for H5N1 diagnostics. Whereas clade 2.2 antigens were able to detect antibodies raised to most of the tested H5N1 viruses (and clade 2.2-specific antisera detected most of the H5N1 antigens), ancestral strain A exhibited the widest reactivity pattern and hence was the best candidate diagnostic reagent for broad detection of H5N1 strains.     

H5N1型禽流感病毒动物模型的研究现状

H5N1亚型禽流感病毒在全球多个国家肆虐,已严重威胁人类健康。禽流感病毒动物模型的建立可以为研究病毒的突变、传染性和发病机制提供良好的技术平台。本综述概括了H5N1对几种哺乳动物:食蟹猴、雪貂、小鼠、大鼠、沙鼠、家猫等的致病性,为以后理想动物模型的建立和研究提供一定的帮助。     

Asia: avian influenza H5N1

The emergence of the first human cases of avian influenza in Hong Kong in 1997 has raised fears of a new pandemic originating from the Asian continent. Despite unprecedented international mobilization, first to stop then to limit its diffusion, the highly pathogenic avian virus A/H5N1 has successfully spread in Asia, Europe and Africa by successive epizootic outbreaks affecting migratory birds and poultry, Transmission from animals to humans is uncommon but severe with a fatality rate exceeding 60% in confirmed cases. Nearly half of the countries struck by the disease is in Asia. With 87% of confirmed cases and 91% of deaths, Asia is also one of the continent most heavily affected. Like the neighbouring countries in South-East Asia, Cambodia has been repeatedly hit by avian flu in the recent years. The measures implemented to prevent the spread of A/H5N1 virus in farms, to improve the behaviour of farmers and to clean up the poultry markets emphasize the multiple difficulties to control this zoonosis. Meanwhile, influenza surveillance is being carried out in humans, based on a reporting system and a network of sentinel hospitals connected to the Institut Pasteur du Cambodge. Thus, Cambodia takes an active part in the wide international network which should quickly detect any mutational event among avian flu viruses that might lead to the emergence of a pandemic.     

Immune responses to infection with H5N1 influenza virus

Influenza A H5N1 viruses remain a substantial threat to global public health. In particular, the expanding genetic diversity of H5N1 viruses and the associated risk for human adaptation underscore the importance of better understanding host immune responses that may protect against disease or infection. Although much emphasis has been placed on investigating early virus–host interactions and the induction of innate immune responses, little is known of the consequent adaptive immune response to H5N1 virus infection. In this review, we describe the H5N1 virus-specific and cross-reactive antibody and T cell responses in humans and animal models. Data from limited studies suggest that although initially robust, there is substantial waning of the serum antibody responses in survivors of H5N1 virus infection. Characterization of monoclonal antibodies generated from memory B cells of survivors of H5N1 virus infection has provided an understanding of the fine specificity of the human antibody response to H5N1 virus infection and identified strategies for immunotherapy. Human T cell responses induced by infection with seasonal influenza viruses are directed to relatively conserved internal proteins and cross-react with the H5N1 subtype. A role for T cell-based heterosubtypic immunity against H5N1 viruses is suggested in animal studies. Further studies on adaptive immune responses to H5N1 virus infection in both humans and animals are needed to inform the design of optimal immunological treatment and prevention modalities.     

Antiviral etiotropic chemicals: efficacy against influenza A viruses A subtype H5N1

The paper analyzes data of an experimental study of the efficacy of antiviral agents (amantadine, remantadine, ozeltamivir, zanamivir, arbidol, ribavirin) in the cultured cells and on a model of murine influenza pneumonia against influenza A viruses subtype H5N1. It also gives data on their use in the treatment of human beings during avian influenza outbreak. The mechanism of action of the agents, pharmacokinetics, adverse reactions, and their potential resistance are considered.     

Pathogenicity of Chinese H5N1 highly pathogenic avian influenza viruses in pigeons

It has long been thought that pigeons are resistant against H5 highly pathogenic avian influenza (HPAI) viruses. Recently, however, highly pathogenic H5N1 avian influenza viruses have demonstrated distinct biological properties that may be capable of causing disease in pigeons. To examine the susceptibility of domestic pigeons to recent H5N1 viruses, we inoculated pigeons using H5N1 viruses isolated in China from 2002 to 2004. Within 21 days following inoculation, all pigeons had survived and fully recovered from temporary clinical signs. However, seroconversion assays demonstrated that several viruses did in fact establish infection in pigeons and caused a certain amount of viral shedding in the oropharynx and cloaca. There was not, however, a definitive relationship between viral shedding and viral origin. Viruses were also inconsistently isolated from various organs of pigeons in infected groups. Pathological examination revealed that the infection had started as respiratory inflammation and caused the most severe lesions in the brain in later stages. These results indicate that pigeons are susceptible to the more recent Asian H5N1 HPAI and could be a source of infection to other animals, including humans.     

H5N1禽流感病毒HA假病毒的构建及特性研究

目的 构建包膜蛋白为H5N1禽流感病毒HA蛋白的假病毒,对其生物学特性进行研究,并将其初步应用于H5N1禽流感病毒的血清检测.方法 将我国分离的高致病性H5N1禽流感病毒的HA基因插入真核表达质粒,得到pLP-HA,与假病毒构建体系的三种质粒pLP1,pLF2和pEmGFP,瞬时共转染人胚肾细胞293T,48 h收集假病毒上清,对其感染性,血凝活性进行测定,并应用于微量中和实验.同时,构建了优化HA基因的假病毒以及一株含有越南禽流感病毒HA基因的假病毒,进行比较.结果 电镜下观察到假病毒颗粒的存在;Western-Blot表明HA蛋白存在于假病毒颗粒中;HA假病毒与野生型活病毒的微量中和实验相比,两者结果具有很好的相关性.结论 成功构建了不同高致病性H5N1禽流感病毒HA蛋白的假病毒,所构建的假病毒可以应用于微量中和实验.研究发现不同禽流感病毒株HA蛋白假病毒的包装效率不同,并且真核表达优化基因并不能显著提高假病毒颗粒包装效率.     

Characterization of H5N1 influenza A viruses isolated from domestic green-winged teal

Two avian influenza virus strains, A/domestic green-winged teal/Hunan/67/2005 (H5N1) (D-GWT/67) and A/domestic green-winged teal/Hunan/79/2005 (H5N1) (D-GWT/79), were isolated from healthy domestic green-winged teals (Anas crecca) in Hunan Province, South China. Genomic analysis showed that both isolates were reassortants. The hemagglutinin (HA) genes of the two isolates were closely related to that of an H5N1 strain isolated from tree sparrow (A/tree sparrow/Henan/1/04). The neuraminidase (NA) genes and the internal protein genes of both isolates were closely related to those from A/chicken/Shantou/4231/2003-like (H5N1) viruses, with exception of the matrix (M) gene of D-GWT/79, which was closely related to that of the H7N3 strain A/mallard/Netherlands/12/2000 isolated from wild mallard duck. The virulence of the two isolates was examined in chickens, ducks, and mice. Both strains were found to be highly pathogenic in chickens and ducks, but showed low pathogenicity in mice. These findings contribute to the realization that domestic green-winged teals carrying the H5N1 virus may play an important role in transmitting the virus among birds.     

Inactivated North American and European H5N2 avian influenza virus vaccines protect chickens from Asian H5N1 high pathogenicity avian influenza virus.

High-pathogenicity (HP) avian influenza (AI) virus of the H5N1 subtype has caused an unprecedented epizootic in birds within nine Asian countries/regions since it was first reported in 1996. Vaccination has emerged as a tool for use in managing the infection in view of future eradication. This study was undertaken to determine whether two divergent H5N2 commercial vaccine strains, one based on a European and the other a North American low-pathogenicity AI virus, could protect chickens against a recent Asian H5N1 HPAI virus. The North American and European vaccine viruses had 84 and 91% deduced amino acid sequence similarity to the HA1 segment of haemagglutinin protein of Indonesia H5N1 HPAI challenge virus, respectively. Both vaccine strains provided complete protection from clinical signs and death. The vaccines reduced the number of chickens infected and shedding virus from the respiratory and intestinal tracts at the peak of virus replication. In addition, the quantity of virus shed was reduced by 10(4) to 10(5) median embryo infectious doses. The use of specific neuraminidase inhibition tests allowed identification of infected chickens within the vaccinated groups. These data indicate that the currently available H5 vaccines of European and North American lineages will protect chickens against the Asian H5N1 HPAI virus and reduce environmental contamination by the H5N1 HPAI virus. They will be an adjunct to biosecurity measures to reduce virus transmission.     

Dogs are highly susceptible to H5N1 avian influenza virus

Replication of avian influenza viruses (AIVs) in dogs may facilitate their adaptation in humans; however, the data to date on H5N1 influenza virus infection in dogs are conflicting. To elucidate the susceptibility of dogs to this pathogen, we infected two groups of 6 beagles with 10⁶ 50% egg-infectious dose of H5N1 AIV A/bar-headed goose/Qinghai/3/05 (BHG/QH/3/05) intranasally (i.n.) and intratracheally (i.t.), respectively. The dogs showed disease symptoms, including anorexia, fever, conjunctivitis, labored breathing and cough, and one i.t. inoculated animal died on day 4 post-infection. Virus shedding was detected from all 6 animals inoculated i.n. and one inoculated i.t. Virus replication was detected in all animals that were euthanized on day 3 or day 5 post-infection and in the animal that died on day 4 post-infection. Our results demonstrate that dogs are highly susceptible to H5N1 AIV and may serve as an intermediate host to transfer this virus to humans.     

禽流感H5N1型病毒对NIH小鼠的致病敏感性研究

目的观察禽流感H5N1型病毒对NIH小鼠的致病性。方法将NIH小鼠随机分为接毒组（20只）和对照组（10只）,在负压感染实验室,接毒组于乙醚麻醉后给予AIVH5N1型病毒滴鼻,对照组予正常阴性尿囊液滴鼻,观察14d,记录小鼠的体温、体重、临床症状、死亡情况、病理变化、肺指数及抗体变化。结果NIH小鼠感染禽流感H5N1型病毒后第1天就出现精神不振,病程主要集中在第3—7天,临床症状主要表现为弓背、蜷缩、竖毛、颤抖、反应迟钝、活动减少、爱扎堆,体重（下降）出现减轻,体温降低,死亡率为40％;接毒组死亡小鼠肺指数为（2．21±0．40）,较对照组的（0．44±0．23）明显增高,差异有统计学意义（P〈0．01）;肺组织病理严重改变,第8天才能检出抗体（OD值等于0．231）。结论禽流感H5N1型病毒对NIH小鼠有一定的致病性。     

Emergence of amantadine-resistant avian influenza H5N1 virus in India

This study reports the genetic characterization of highly pathogenic avian influenza (HPAI) virus (subtype H5N1) isolated from poultry in West Bengal, India. We analyzed all the eight genome segments of two viruses isolated from chickens in January 2010 to understand their genetic relationship with other Indian H5N1 isolates and possible connection between different outbreaks. The hemagglutinin (HA) gene of the viruses showed multiple basic amino acids at the cleavage site, a marker for high virulence in chickens. Of greatest concern was that the viruses displayed amino acid substitution from serine-to-asparagine at position 31 of M2 ion channel protein suggesting emergence of amantadine-resistant mutants not previously reported in HPAI H5N1 outbreaks in India. Amino acid lysine at position 627 of the PB2 protein highlights the risk the viruses possess to mammals. In the phylogenetic trees, the viruses clustered within the lineage of avian isolates from India (2008-2009) and avian and human isolates from Bangladesh (2007-2009) in all the genes. Both these viruses were most closely related to the viruses from 2008 in West Bengal within the subclade 2.2.3 of H5N1 viruses.