Proc Natl Acad Sci U S A 2006 May 22; (Read article online)

Park MS, Steel J, García-Sastre A, Swayne D, Palese P

Avian influenza viruses of the H5 and H7 hemagglutinin subtypes, and Newcastle disease virus (NDV), are important pathogens in poultry worldwide. Specifically, the highly pathogenic H5N1 avian influenza virus is a particular threat because it has now occurred in more than 40 countries on several continents. Inasmuch as most chickens worldwide are vaccinated with a live NDV vaccine, we embarked on the development of vaccine prototypes that would have dual specificity and would allow a single immunization against both avian influenza and Newcastle disease. Using reverse genetics, we constructed a chimeric avian influenza virus that expressed the ectodomain of the hemagglutinin-neuraminidase gene of NDV instead of the neuraminidase protein of the H5N1 avian influenza virus. Our second approach to creating a bivalent vaccine was based on expressing the ectodomain of an H7 avian influenza virus hemagglutinin in a fusogenic and attenuated NDV background. The insertion into the NDV genome of the foreign gene (containing only its ectodomain, with the transmembrane and cytoplasmic domains derived from the F protein of NDV) resulted in a chimeric virus with enhanced incorporation of the foreign protein into virus particles. A single immunization of chickens with this improved vaccine prototype virus induced not only a 90% protection against an H7N7 highly pathogenic avian influenza virus, but also complete immunity against a highly virulent NDV. We propose that chimeric constructs should be developed for convenient, affordable, and effective vaccination against avian influenza and Newcastle disease in chickens and other poultry.

Nat Struct Mol Biol 2006 May 21; (Read article online)

Bornholdt ZA, Prasad BV

The nonstructural protein NS1 of influenza virus is an antagonist of host immune responses and is implicated in virulence. It has two domains, an N-terminal double-stranded RNA-binding domain (RBD) and an effector domain crucial for RBD function, for nuclear export and for sequestering messenger RNA-processing proteins. Here we present the crystallographic structure of the effector domain, which has a novel fold and suggests mechanisms for increased virulence in H5N1 strains.

Vaccine 2006 May 2; (Read article online)

Ernst WA, Kim HJ, Tumpey TM, Jansen AD, Tai W, Cramer DV, Adler-Moore JP, Fujii G

The recent emergence of multiple avian influenza A subtypes that cause human disease (i.e., H5N1, H9N2 and H7N7), coupled with the fear that one of these strains might precipitate a new pandemic, underscores the need to develop new technological approaches to immunization which elicit protective immune responses against multiple subtypes of influenza A. In response to this demand, several matrix 2 protein ectodomain segments (M2eA) corresponding to the H1N1, H5N1 and H9N2 influenza strains were formulated using a novel liposome-based vaccine technology and evaluated as potential immunogens for developing a "universal" influenza vaccine. Mice immunized with liposomal M2eA survived homologous challenges with H1N1 (100% survival) or H9N2 (80% survival) influenza strains. There were significant reductions in their lung viral load as well as in immunized mice challenged with the H5N1 subtype. The mice vaccinated with an M2eA segment corresponding to the H1N1 and H6N2 (a reassortant influenza A virus carrying the M2eA from PR8/34) strains elicited elevated IgG ELISA antibody titers to this M2eA epitope segment and antiserum from these immunized mice provided passive protection (100% survival) to naïve mice receiving a lethal dose of H6N2 influenza virus. These results provide the first evidence that recombinant M2eA epitopes to multiple subtypes elicited immune protection against a homologous challenge and provides further evidence in favor of the development of a "universal" influenza vaccine based on M2eA.

Virology 2006 May 17; (Read article online)

Smith GJ, Naipospos TS, Nguyen TD, de Jong MD, Vijaykrishna D, Usman TB, Hassan SS, Nguyen TV, Dao TV, Bui NA, Leung YH, Cheung CL, Rayner JM, Zhang JX, Zhang LJ, Poon LL, Li KS, Nguyen VC, Hien TT, Farrar J, Webster RG, Chen H, Peiris JS, Guan Y

Highly pathogenic avian influenza virus H5N1 is endemic in poultry in East and Southeast Asia with disease outbreaks recently spreading to parts of central Asia, Europe and Africa. Continued interspecies transmission to humans has been reported in Vietnam, Thailand, Cambodia, Indonesia and China, causing pandemic concern. Here, we genetically characterize 82 H5N1 viruses isolated from poultry throughout Indonesia and Vietnam and 11 human isolates from southern Vietnam together with sequence data available in public databases to address questions relevant to virus introduction, endemicity and evolution. Phylogenetic analysis shows that all viruses from Indonesia form a distinct sublineage of H5N1 genotype Z viruses suggesting this outbreak likely originated from a single introduction that spread throughout the country during the past two years. Continued virus activities in Indonesia were attributed to transmission via poultry movement within the country rather than through repeated introductions by bird migration. Within Indonesia and Vietnam, H5N1 viruses have evolved over time into geographically distinct groups within each country. Molecular analysis of the H5N1 genotype Z genome shows that only the M2 and PB1-F2 genes were under positive selection, suggesting that these genes might be involved in adaptation of this virus to new hosts following interspecies transmission. At the amino acid level 12 residues were under positive selection in those genotype Z viruses, in the HA and PB1-F2 proteins. Some of these residues were more frequently observed in human isolates than in avian isolates and are related to viral antigenicity and receptor binding. Our study provides insight into the ongoing evolution of H5N1 influenza viruses that are transmitting in diverse avian species and at the interface between avian and human hosts.

Emerg Infect Dis 2006 Apr; 12(4): 575-81 (Read article online)

Songserm T

In addition to causing 12 human deaths and 17 cases of human infection, the 2004 outbreak of H5N1 influenza virus in Thailand resulted in the death or slaughter of 60 million domestic fowl and the disruption of poultry production and trade. After domestic ducks were recognized as silent carriers of H5N1 influenza virus, government teams went into every village to cull flocks in which virus was detected; these team efforts markedly reduced H5N1 infection. Here we examine the pathobiology and epidemiology of H5N1 influenza virus in the 4 systems of duck raising used in Thailand in 2004. No influenza viruses were detected in ducks raised in "closed" houses with high biosecurity. However, H5N1 influenza virus was prevalent among ducks raised in "open" houses, free-ranging (grazing) ducks, and backyard ducks.

J Infect Dis 2006 Jun 15; 193(12): 1626-9 (Read article online)

Cheung CL, Rayner JM, Smith GJ, Wang P, Naipospos TS, Zhang J, Yuen KY, Webster RG, Peiris JS, Guan Y, Chen H

We examined the distribution of genetic mutations associated with resistance to the M2 ion channel-blocking adamantane derivatives, amantadine and rimantadine, among H5N1 viruses isolated in Vietnam, Thailand, Cambodia, Indonesia, Hong Kong, and China. More than 95% of the viruses isolated in Vietnam and Thailand contained resistance mutations, but resistant mutants were less commonly isolated in Indonesia (6.3% of isolates) and China (8.9% of isolates), where human infection was recently reported. The dual mutation motif Leu26Ile-Ser31Asn (leucine-->isoleucine at aa 26 and serine-->asparagine at aa 31) was found almost exclusively in all resistant isolates from Vietnam, Thailand, and Cambodia, suggesting the biological selection of these mutations.

Emerg Infect Dis 2006 Apr; 12(4): 681-3 (Read article online)

Songsermn T

We report H5N1 virus infection in a domestic cat infected by eating a pigeon carcass. The virus isolated from the pigeon and the cat showed the same cluster as the viruses obtained during the outbreak in Thailand. Since cats are common house pets, concern regarding disease transmission to humans exists.

Virology 2006 May 9; (Read article online)

Webster RG, Webby RJ, Hoffmann E, Rodenberg J, Kumar M, Seiler P, Krauss S, Songserm T

H5N1 avian influenza viruses are continuing to spread in waterfowl in Eurasia and to threaten the health of avian and mammalian species. The possibility that highly pathogenic (HP) H5N1 avian influenza is now endemic in both domestic and migratory birds in Eurasia makes it unlikely that culling alone will control H5N1 influenza. Because ducks are not uniformly killed by HP H5N1 viruses, they are considered a major contributor to virus spread. Here, we describe a reverse genetics-derived high-growth H5N3 strain containing the modified H5 of A/chicken/Vietnam/C58/04, the N3 of A/duck/Germany/1215/73, and the internal genes of A/PR/8/34. One or two doses of inactivated oil emulsion vaccine containing 0.015 to 1.2 mug of HA protein provide highly efficacious protection against lethal H5N1 challenge in ducks; only the two dose regimen has so far been tested in chickens with high protective efficacy.

Tex Nurs 2006 Jan; 80(1): 6-10 (Read article online)

Pascoe N

If an influenza pandemic struck today, borders might close, the global economy would be severely impacted, international vaccine supplies and health are systems would be overwhelmed, and some people might panic. To limit the fallout, the industrialized world must create a detailed response strategy involving the public and private sectors. Some experts feel we are overdue for a flu pandemic and the SARS pandemic of 2003 could have been the wake up call to begin preparations. Fortunately there is some assistance coming from the federal government. On January 12, U.S. Department of Health & Human Services Secretary Mike Leavitt announced funding to assist in the preparation for a pandemic flu response. $100 million is being provided initially with another $250 million due later this year to assist states in pandemic flu preparedness. Texas' initial allocation is $5,875,044. While some believe that the AI (H5N1) causing illness and deaths in Asia and Turkey will be the pandemic flu strain, there is no guarantee that will occur. Thus, without knowing which strain may lead to a pandemic, development and manufacturing of a vaccine is delayed.

Br Med Bull 2006; 75-76: 63-80 (Read article online)

Stephenson I, Democratis J

Influenza is an infectious respiratory pathogen causing annual outbreaks and infrequent pandemics, resulting in significant morbidity, mortality and burdens on the delivery of health care. The geographical spread of highly pathogenic avian influenza (HPAI) H5N1 among poultry and wild bird populations is unprecedented. Growing numbers of sporadic avian influenza infections are occurring in humans, increasing the threat of the next influenza pandemic. Vaccines are the principle means of combating influenza, and a number of studies with H5N1 vaccine candidates are underway. Antiviral agents can be used to treat influenza infection and can be taken as chemoprophylaxis during influenza outbreaks. Oseltamivir has been stockpiled as part of influenza pandemic preparedness planning; however, the emergence of drug resistance may limit its clinical use.

J Infect Chemother 2006 Apr; 12(2): 73-9 (Read article online)

Nagayama A

Recently we have heard warnings of an outbreak of a highly pathogenic avian influenza virus (H5N1). Although, to prevent such infections we must prepare anti-viral drugs and type-specific vaccines against influenza, we need various simple and effective protection methods, such as the use of face masks for public health. Also, in any consideration of bacterial infections, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and multidrug-resistant Pseudomonas aeruginosa (MDRP) also pose serious concerns which must be addressed. I examined the antiviral activity of gentian violet (GV) and GV-dyed cloth against the influenza A (H1N1) virus. Time-kill studies were carried out, and the virus titer was determined based on the 50% tissue culture infective dose (TCID(50)). The minimum inhibitory concentrations (MICs) of GV against bacteria were also determined, and the killing activities of the GV-dyed cloth were judged from viable cell counts. GV immediately killed the influenza A virus and this was confirmed by electron microscopy. Moreover, cloth dyed with a combination of GV and copper showed not only excellent antiviral activity but also prominent bactericidal activities.

Zhonghua Jie He He Hu Xi Za Zhi 2006 Jan; 29(1): 9-13 (Read article online)

Zhou C, Fang P, Liu YN, Hu B, Ding HM, Xu XL, Wu H, Wang J, Lin L, Pan H, Wu TS, Song YL

OBJECTIVE: To describe the clinical features of the infection caused by the highly pathogenic avian influenza A (H(5)N(1)). METHODS: A previously healthy 24 year old woman presented to our hospital on November 7, 2005. She was confirmed to be an H(5)N(1) infected case after death. The clinical, radiological and epidemiological data were analyzed. RESULTS: The patient had a history of direct contact with diseased and dead poultry (chicken and duck). The disease course was 10 days from onset of illness to death, and fever preceded dyspnea by 5 days. On admission, the striking characteristics were acute community-acquired pneumonia (CAP) and acute respiratory distress syndrome (ARDS), and the major radiographic abnormalities included extensive infiltration bilaterally, focal consolidation and air bronchograms. The radiographic and clinical deterioration was rapid, and the patient died in less than 3 days after hospitalization. The diagnosis of influenza A (H(5)N(1)) was confirmed by means of reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR on specimens of the lower respiratory tract, performed by Chinese Center for Disease Control. The postmortem examination showed bronchial hyperemia, extensive consolidation, serous cavity effusions, disseminated intravascular coagulation (DIC) and multiple organ failure (MOF). CONCLUSIONS: Human infection by the highly pathogenic avian influenza A (H(5)N(1)) is a fatal communicable disease. Information of avian influenza A (H(5)N(1)) virus, more attention to the epidemiologic data, and early intervention are critical in reducing the mortality.

Biochem Biophys Res Commun 2006 Jun 9; 344(3): 1048-55 (Read article online)

Wei DQ, Du QS, Sun H, Chou KC

The highly pathogenic H5N1 influenza virus, which is rapidly mutating and becoming increasingly drug-resistant, was investigated by means of structure-activity relationship between NA (neuraminidase) and three inhibitors, i.e., DANA (2,3-didehydro-2-deoxy-N-acetylneuraminic acid), zanamivir, and oseltamivir. A homology model of the H5N1-NA from the highly pathogenic chicken H5N1 A viruses isolated during the 2003-2004 influenza outbreaks in Japan was built based on the crystal structure of N9-NA complexed with DANA (PDB code: 1F8B). It was found that the traditional constituent residues around the active site of NA family are highly conserved in the H5N1-NA. However, a partially lipophilic pocket composed by Ala248 and Thr249 in N9-NA becomes a hydrophilic pocket because the two residues in the H5N1-NA are replaced by hydrophilic residues Ser227 and Asn228, respectively. On the other hand, two hydrophilic residues Asn347 and Asn348 in the N9-NA are replaced by two lipophilic residues Ala323 and Tyr324 in the H5N1-NA, respectively, leading to the formation of a new lipophilic pocket. This kind of subtle variation not only destroys the original lipophilic environment but also changes the complement interaction between the H5N1-NA and DANA. Such a finding might provide insights into the secret why some of H5N1 strains bear high resistance for existing NA inhibitors, and stimulate new strategies for designing new drugs against these viruses.

Indian Pediatr 2006 Apr; 43(4): 317-25 (Read article online)

Lahariya C, Sharma AK, Pradhan SK

Avian flu is affecting the poultry animals world over since first outbreak in 1997 in Hong Kong and has resulted in 92 human deaths and culling of more than 150 million poultry animals in Asia and Europe. The loss to the economy has also been enormous. 13 new countries, including India, reported occurrence of the disease in poultry animals in February 2006 only, to the World Health Organisation. This rapid rate of spread of virus along with notoriety of the virus for frequent genetic re-assortment, which might enable H5N1 to infect human beings, threatens of possible influenza pandemic since the last pandemic in 1968. The human influenza caused by this subtype of the virus (H5N1) has high case fatality of 54% and majority of affected humans are between the age of 5 to 23 years. Lack of effective vaccine, poor knowledge about treatment, and with scarcity of public health measures in developing countries are major causes of concern. The real threat of impending pandemic can be avoided only if we act immediately on the basis of currently available source of information and apply scientific knowledge rationally for containment and prevention of bird flu and treat human cases promptly.

Virus Res 2006 Apr 18; (Read article online)

Kash JC, Goodman AG, Korth MJ, Katze MG

Influenza virus is a major public health problem with annual deaths in the US of 36,000 with pandemic outbreaks, such as in 1918, resulting in deaths exceeding 20 million worldwide. Recently, there is much concern over the introduction of highly pathogenic avian influenza H5N1 viruses into the human population. Influenza virus has evolved complex translational control strategies that utilize cap-dependent translation initiation mechanisms and involve the recruitment of both viral and host-cell proteins to preferentially synthesize viral proteins and prevent activation of antiviral responses. Influenza virus is a member of the Orthomyxoviridae family of negative-stranded, segmented RNA viruses and represents a particularly attractive model system as viral replication strategies are closely intertwined with normal cellular processes including the host defense and stress pathways. In this chapter, we review the parallels between translational control in influenza virus infected cells and in stressed cells with a focus on selective translation of viral mRNAs and the antagonism of the dsRNA and host antiviral responses. Moreover, we will discuss how the use of genomic technologies such as DNA microarrays and high through-put proteomics can be used to gain new insights into the control of protein synthesis during viral infection and provide a near comprehensive view of virus-host interactions.

Rev Sci Tech 2005 Dec; 24(3): 933-44 (Read article online)

Nishiguchi A, Yamamoto T, Tsutsui T, Sugizaki T, Mase M, Tsukamoto K, Ito T, Terakado N

An outbreak of highly pathogenic avian influenza (HPAI), caused by the virus sub-type H5N1, occurred at four premises in three prefectures in Japan during January and March 2004. As a result, 274,654 poultry died or were slaughtered. This was the first outbreak of HPAI in Japan since 1925. (The earlier outbreak was caused by H7N7.) The disease was successfully eradicated within three-and-a-half months, following an eradication campaign that included depopulating the affected premises, implementing movement controls and intensive surveillance. Control measures were conducted in accordance with the National Manual of HPAI Control. However, during the eradication campaign, some key issues arose, such as delays in notification by the affected farmers. As a result of these experiences, the relevant laws and HPAI Control Manual have been appropriately revised.

Nature 2006 Apr 27; 440(7088): E9-E10 (Read article online)

Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG

We have proposed that the virus responsible for the 1918 human influenza pandemic was avian-like, which to us is a reasonable interpretation of all the available data from phylogenetic, sequence, serological and historical analysis, combined with what is known of influenza A virus biology. However, Gibbs and Gibbs and Antonovics et al. question our inferred origin of the 1918 influenza virus.

Vaccine 2006 Mar 29; (Read article online)

Mingxiao M, Ningyi J, Zhenguo W, Ruilin W, Dongliang F, Min Z, Gefen Y, Chang L, Leili J, Kuoshi J, Yingjiu Z

cDNAs of the HA genes of subtype H5N1 AIV were fused to form a single open reading frame, designated H5HA-H7HA. The H5HA-H7HA cDNA and chicken Interleukin-18 (IL18) were inserted into the fowlpox virus (FPV) expression vector pUTA-16-LacZ to produce pUTAL-H5-H7-IL18. cDNA of H5N1 AIV HA was inserted into the FPV expression vector pUTA2 to create the recombinant expression plasmid pUTA2-H5. Plasmids were then co-transected into CEF cells. The two recombinant fowlpox viruses (rFPV) were produced by three cycles with the BrdU and verified by RT-PCR, IFA and Western blotting. One-day-old specific pathogen free (SPF) chickens and 7-day-old commercial Leghorn egg-laying chickens were inoculated with 10(6)PFU recombinant or parental fowlpox vaccine viruses by wing-web puncture. Hemagglutination inhibition (HI) antibody titer and nonspecific cellular immunity level were assessed after 1-3 weeks post-immunization. We found that all rFPV-vaccinated groups produced HI-specific antibodies, and the level of cellular immunity induced by the rFPV-H5-H7-IL18 strain was significantly higher than that induced by rFPV-H5HA. At 3 weeks post-inoculation, immunized SPF and Leghorn chickens were challenged with H5N1 HP AIV. The rFPV-H5-H7-IL18 vaccine strains were able to induce complete (10/10) protection, while the rFPV-H5HA vaccine strain induced (9/10) protection. Cloacal swabbing samples were collected from immunized leghorn chickens during the first week post-challenge; no shedding was found in the rFPV-H5-H7-IL18 vaccinated group. The rFPV-H5-H7-IL18 vaccinated group displayed significantly increased weight gain relative to the rFPV-H5HA group. This study reports a significant step in the further development of new AIV vaccines.

Med Hypotheses 2006 Apr 16; (Read article online)

Friel H, Lederman H

By early February 2006, the World Health Organization had reported 165 human cases of H5N1 influenza since December 2003, with 88 fatalities. However, the avian H5N1 influenza virus apparently is not yet efficiently transmitted between humans. Though a near-term possibility of a global H5N1 influenza pandemic remains, currently there is no vaccine or anti-viral drug that is proven to be safe and effective in preventing or treating H5N1 influenza in humans. There is thus a compelling public interest in developing alternative prophylaxis and treatment strategies for H5N1 influenza, which would need to address the complex pathogenesis of H5N1 influenza that is responsible for its apparently unusually high virulence. The authors present here a significant body of medical and scientific evidence to support the prophylactic use of a carefully designed nutritional supplement formulation that may antagonize the major pathogenic processes of H5N1 influenza in humans. Through several independently-mediated mechanisms, the formulations may: (a) degrade H5N1 virulence by directly affecting the virus itself, (b) inhibit H5N1 viral replication by maintaining cellular redox equilibrium in host cells, (c) inhibit H5N1 replication by a blockade of the nuclear-cytoplasmic translocation of the viral ribonucleoproteins and reduced expression of late viral proteins related to the inhibition of protein kinase C activity and its dependent pathways, (d) down-regulate activation and proliferation of proinflammatory cytokines in respiratory epithelial cells and macrophages that are implicated in the pathogenesis of H5N1 influenza, and (e) protect the lungs and other vital organs from virus- and cytokine-induced oxidative stress by supplying and maintaining sufficient levels of exogenous and endogenous antioxidants. Key mediators in these processes include selenium, vitamin E, NAC/glutathione, resveratrol, and quercetin. Taken prophylactically, and throughout the duration and recovery of an H5N1 infection, the nutritional supplement formula may aid humans infected with H5N1 influenza to survive with a reduced likelihood of major complications, and may provide a relatively low-cost strategy for individuals as well as government, public-health, medical, health-insurance, and corporate organizations to prepare more prudently for an H5N1 pandemic. Some evidence also indicates that the supplement formulation may be effective as an adjunctive to H5N1 vaccine and anti-viral treatments, and should be tested as such.

J Mol Biol 2006 Mar 22; (Read article online)

Tarnovitski N, Matthews LJ, Sui J, Gershoni JM, Marasco WA

Rapid elucidation of neutralizing antibody epitopes on emerging viral pathogens like severe acute respiratory syndrome (SARS) coronavirus (CoV) or highly pathogenic avian influenza H5N1 virus is of great importance for rational design of vaccines against these viruses. Here we combined screening of phage display random peptide libraries with a unique computer algorithm "Mapitope" to identify the discontinuous epitope of 80R, a potent neutralizing human anti-SARS monoclonal antibody against the spike protein. Using two different types of random peptide libraries which display cysteine-constrained loops or linear 13-15-mer peptides, independent panels containing 42 and 18 peptides were isolated, respectively. These peptides, which had no apparent homologous motif within or between the peptide pools and spike protein, were deconvoluted into amino acid pairs (AAPs) by Mapitope and the statistically significant pairs (SSPs) were defined. Mapitope analysis of the peptides was first performed on a theoretical model of the spike and later on the genuine crystal structure. Three clusters (A, B and C) were predicted on both structures with remarkable overlap. Cluster A ranked the highest in the algorithm in both models and coincided well with the sites of spike protein that are in contact with the receptor, consistent with the observation that 80R functions as a potent entry inhibitor. This study demonstrates that by using this novel strategy one can rapidly predict and identify a neutralizing antibody epitope, even in the absence of the crystal structure of its target protein.