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PubMedDate not supplied

Isolation of avian influenza virus (subtype H5N2) from chicken eggs during a natural outbreak.

Avian influenza virus (A/Chicken/Pennsylvania/83; H5N2) was recovered from the yolk, albumen, and shell surface of eggs obtained from naturally infected chicken flocks in Pennsylvania and Virginia. These findings represent the first reported isolation of avian influenza virus from the internal contents of eggs from naturally infected flocks. The need for adequate safeguards to prevent spread of the virus during commercial movement of table and hatching eggs, cracked and "checked" eggs, and egg flats and other materials is emphasized.

Animals↗

PubMedDate not supplied

Presence of avian influenza virus (AIV) subtypes H5N2 and H7N1 in emus (Dromaius novaehollandiae) and rheas (Rhea americana): virus isolation and serologic findings.

Avian influenza virus (AIV) subtypes H5N2 and H7N1 were isolated from emus (Dromaius novaehollandiae) and rheas (Rhea americana) in Texas and North Carolina. All the rheas and emus had a history of respiratory disease except one emu, which was clinically normal. The isolates were not pathogenic for chickens and turkeys under the conditions of the experiment. Humoral antibodies to all known hemagglutinin (H) subtypes except H10, H13, and H14 and to all nine neuraminidase (N) subtypes were found in emus and rheas in 11 states. Therefore, emus and rheas are susceptible to infection with several AIV subtypes.

Animals↗

PubMed2006

Avian influenza A virus subtype H5N2 in a red-lored Amazon parrot.

CASE DESCRIPTION: A 3-month-old red-lored Amazon parrot (Amazona autumnalis autumnalis) was evaluated for severe lethargy. CLINICAL FINDINGS: Avian influenza virus hemagglutinin subtype H5N2 with low pathogenicity was characterized by virus isolation, real-time reverse transcriptase PCR assay, chicken intravenous pathogenicity index, and reference sera. The virus was also determined to be closely related to a virus lineage that had been reported only in Mexico and Central America. TREATMENT AND OUTCOME: The chick was admitted to the hospital and placed in quarantine. Supportive care treatment was administered. Although detection of H5 avian influenza virus in birds in the United States typically results in euthanasia of infected birds, an alternative strategy with strict quarantine measures and repeated diagnostic testing was used. The chick recovered from the initial clinical signs after 4 days and was released from quarantine 9 weeks after initial evaluation after 2 consecutive negative virus isolation and real-time reverse transcriptase PCR assay results. CLINICAL RELEVANCE: To the authors' knowledge, this is the first report of H5N2 avian influenza A virus isolated from a psittacine bird and represents the first introduction of this virus into the United States, most likely by illegal importation of psittacine birds. Avian influenza A virus should be considered as a differential diagnosis for clinical signs of gastrointestinal tract disease in psittacine birds, especially in birds with an unknown history of origin. Although infection with avian influenza virus subtype H5 is reportable, destruction of birds is not always required.

Amazona↗

PubMed2005

[Generation of attenuated H5N1 and H5N2 subtypes of influenza virus recombinants by reverse genetics system].

The HA connecting peptide at cleavage site, PQRERRKKR / GL, of an H5N1 subtype avian influenza virus (AIV) was replaced with PQRESR / GL, and then the modified HA gene was cloned into the transcription/expression vector, pHW2000, constructing a plasmid named pHW524-HA. The NA (N1) gene from the H5N1 virus and the NA (N2) gene from an H9N2 AIV were also cloned into pHW2000 separately, resulting in plasmids pHW506-NA and pHW206-NA. With the organization of pHW524-HA, pHW506-NA or pHW206-NA, and six plasmids containing internal genes from A/WSN/33 backbone virus, two transfectants, H5N1/WSN and H5N2/WSN, were subsequently generated by eight-plasmid system. After 15 consecutive passages in embryonated eggs, the two recombinants grew up to high titers of 1:2(9) in hemagglutination test with no changes in nucleotide sequences of the surface genes detected. Both the recombinant viruses belonged to mildly pathogen when evaluated by the pathogenicity test in six-week-old SPF chickens. H5N2/WSN recombinant virus was obviously less pathogenic than H5N1/WSN virus for embryonated chicken eggs. This presentation showed that the reverse genetics system is a very useful tool for studying the construction and function of individual genes and for the generation of virus as vaccine candidate.

Animals↗

PubMed2006

The panorama of the diversity of H5 subtype influenza viruses.

To elucidate the global diversity of H5 influenza viruses from a dynamic view, haemagglutinin (HA) sequences of 170 isolates were selected and analyzed in this study. Our results showed that H5 influenza isolates could be divided into two distinct lineages that circulated in the Eastern Hemisphere and the Western Hemisphere, respectively. This may be due to the separate migration routes and habitats of birds in the two hemispheres. The two distinct lineages, having existed at least for decades, possibly began divergence in 1850s. Each of the two distinct HA lineages could be further divided into some sublineages, but there was little correlation between the minor lineages and their isolation places, isolation time, neuraminidase subtypes, host species or virulence. The panorama of the diversity of H5 influenza viruses presented here integrated all known H5 epidemics including the current severe H5N1 avian epidemics in the Eastern Hemisphere and suggested that H5 virulent viruses could originate from multiple sublineages and associate with multiple NA subtypes. Our study provided a framework for the studies on the evolution and epidemiology of H5 influenza viruses.

Genetic Variation↗

PubMed2006

Large-scale sequence analysis of avian influenza isolates.

The spread of H5N1 avian influenza viruses (AIVs) from China to Europe has raised global concern about their potential to infect humans and cause a pandemic. In spite of their substantial threat to human health, remarkably little AIV whole-genome information is available. We report here a preliminary analysis of the first large-scale sequencing of AIVs, including 2196 AIV genes and 169 complete genomes. We combine this new information with public AIV data to identify new gene alleles, persistent genotypes, compensatory mutations, and a potential virulence determinant.

Animals↗

PubMed2006

Development and use of fowlpox vectored vaccines for avian influenza.

The avian influenza (AI) vaccine designated TROVAC-AIV H5 (TROVAC-H5) contains a live recombinant fowlpox rec. (FP) recombinant (recFP), expressing the hemagglutinin (HA) gene of an AI H5 subtype isolate. This recombinant vaccine was granted a license in the United States for emergency use in 1998 and full registration in Mexico, Guatemala, and El Salvador where over 2 billion doses have been administered. One injection of TROVAC-H5 protects chickens against AI-induced mortality and morbidity for at least 20 weeks, and significantly decreases shedding after challenge with a wide panel of H5-subtype AI strains, regardless of neuraminidase subtype. Recently, excellent protection was demonstrated against 2003 and 2004 Asian highly pathogenic H5N1 isolates. Whereas TROVAC-H5 AI H5 efficacy was not inhibited by anti-AI or anti-fowlpox maternal antibodies (passive immunity), protection to AI was significantly decreased in chickens previously vaccinated or infected with FP (active immunity). Advantages of the TROVAC-H5 vaccine over inactivated AI vaccines are: (a) single administration at 1 day of age and early onset (1 week) of protection, (b) easy monitoring of AI infection in vaccinated flocks with agar gel precipitation (AGP) and enzyme-linked immunosorbent assay (ELISA) used as tests to differentiate infected from vaccinated animals (DIVA tests), and (c) no residue problem due to adjuvant. These features make TROVAC-H5 an ideal AI vaccine for routine administration of day-of-age chicks in hatcheries. RecFP expressing HA from three lineages of H7 subtype (Eurasian, American, and Australian) were also tested for efficacy against a highly pathogenic avian influenza (HPAI) Eurasian HPAI H7N1. Only the recFP expressing the Eurasian H7 gene provided sufficient protection indicating that the breadth of protection induced by recFP is apparently restricted for H7 isolates. The fowlpox vector technology can also be used for the production of an emergency vaccine: once the HA sequence of an emerging AI virus is known, recFP can be rapidly generated. TROVAC-H5 has recently been shown to be immunogenic in cats and could therefore also be considered for use in mammals.

Animals↗

PubMed2005

No detection of avian influenza A viruses of the subtypes H5 and H7 and isolation of lentogenic avian paramyxovirus serotype 1 in passerine birds during stopover in the year 2001 on the island Helgoland (North Sea).

A total of 543 migrating passerines were captured during their stopover on the island of Helgoland (North Sea) in spring and autumn 2001. They were sampled for the detection of avian influenza A viruses (AIV) subtypes H5 and H7, and for avian paramyxoviruses serotype 1 (APMV-1). The goal of the study was to examine the role of migrating birds as potential vectors for these zoonotic viral diseases. For virus detection samples were taken from a) short-distance migrants such as chaffinches (Fringilla coelebs, n = 131) and song trushes (Turdus philomelos, n = 169), and b) long-distance migrants such as garden warbler (Sylvia borin, n = 142) and common redstarts (Phoenicurus phoenicurus, n = 101). Virus detection was done on conjunctival, choanal cleft and cloacal swabs. Embryonated SPF chicken eggs were used to isolate and propagate virus followed by virus identification in a hemagglutination test, hemagglutination inhibition test and in an agar gel diffusion test. In none of the tested samples AIV was detected. Therefore, we conclude that the tested four species of passerines were infected by these pathogens. Six out of 543 birds (1.1 %) were found to carry non-pathogenic and lentogenic strains of APMV-1. This indicates that the passerine species examined in this study may play only a minor role as potential vectors of APMV-1.

Animal Migration↗

PubMed2006

Reassortment and modification of hemagglutinin cleavage motif of avian/WSN influenza viruses generated by reverse genetics that correlate with attenuation.

Avian influenza associated with H9N2 and H5N1 subtypes of avian influenza viruses (AIVs) has raised great concerns in China. To study this problem, reverse genetics has been employed. Three reassortants, rgH9N2, rgH5N1 and rgH5N2, were prepared and compared. Their hemagglutinin (HA) and neuraminidase (NA) genes originated from Chinese AIV isolates of H9N2 or H5N1 subtype, while the rest of their genes were derived from A/WSN/33(H1N1) virus (WSN). In the H5 HA reassortants, the multibasic cleavage site was converted to a monobasic one. The results demonstrated that the reassortants did not produce CPE on MDCK cells in the absence of trypsin, showed egg-adaptation phenotype and stability of HA and NA during consecutive egg passages, and were not lethal to chickens and mice. However, the rgH5N1 reassortant exhibited a residual virulence in terms of lethality to chick embryos and pathogenesis in chickens. It can be concluded that (i) the genetic modification of H5 HA attenuated the H5 reassortants, (ii) the presence of internal WSN proteins contributed to the attenuated properties of the reassortants independently on H5 HA, and (iii) also the overall genome composition contributed to virulence differences. This report provides further contribution of reverse genetics to the knowledge of virulence of influenza viruses.

Animals↗

PubMed2006

Resistance of immune-suppressed pigeons to subtypes H5N2 and H6N1 low pathogenic avian influenza virus.

Resistance to infection with avian influenza virus (AIV) was studied in healthy and immune-suppressed pigeons, which were treated with the immunosuppressant cyclophosphamide (Cy) before infection. Two subtypes of low pathogenic AIV (LPAIV; CK/TW/H5 and CK/TW/H6) were inoculated via the oculonasal route. Nested reverse transcriptase-polymerase chain reaction (nested RT-PCR) and virus isolation were used as detection methods. The Cy-treated and -untreated pigeons in both experiments did not shed viruses or become antibody positive throughout the 21-day observation period. All pigeons were negative for AIV RNA when trachea, lung, pancreas, spleen, kidney, and rectum tissues were examined. Negative results were also obtained in uninoculated contact chickens, which were housed together with H6N1 AIV-inoculated pigeons. Therefore, it was concluded that the pigeons are resistant to infection with these two LPAIVs and do not serve as transmission hosts, even in the presence of immune dysfunction.

Animals↗

PubMed1996

An Arg-Lys insertion at the hemagglutinin cleavage site of an H5N2 avian influenza isolate.

Recent isolations of H5N2 subtype avian influenza (AI) viruses in North America have raised questions concerning their origin, transmission to commercial poultry, and potential for virulence. One ratite-origin isolate of low pathogenicity, A/emu/TX/39924/93 (H5N2), was subjected to a procedure that rapidly selects and/or amplifies highly pathogenic (HP) strains. The resulting highly virulent derivative had an altered hemagglutinin (HA) gene containing an additional six nucleotides at position 970-975 in the HA1 coding region. This resulted in an arg-lys insertion near the proteolytic cleavage site of the HA protein. The remainder of the HA sequence differed by an additional seven amino acids from the parent. The HA precursor of the derivative, but not the parent, was readily cleaved during replication in cell culture without addition of trypsin. In experimentally infected chickens, the derivative produced lesions typical of highly pathogenic avian influenza. A reverse transcriptase-polymerase chain reaction (RT-PCR) primer set was designed to amplify exclusively from molecules with the inserted six nucleotides. The set yielded product only from the selected derivative samples and not the parent. Thus, the levels of the HP variants in the parent stock were undetectable, or the insertion occurred rapidly during the selection process.

Animals↗

PubMed2000

Prevention and treatment of bronchopneumonia in mice caused by mouse-adapted variant of avian H5N2 influenza A virus using monoclonal antibody against conserved epitope in the HA stem region.

The effects of monoclonal antibody (MAb) C179 recognizing a conformational epitope in the middle of the hemagglutinine (HA) stem region were examined in a mouse model in the experiments of prevention and treatment of lethal bronchopneumonia caused by influenza A virus of H5 subtype. To model the lethal infection, avian nonpathogenic strain A/mallard duck/Pennsylvania/10218/84 (H5N2) was adapted to mice. This resulted in highly pathogenic pneumovirulent mouse-adapted (MA) variant, which was characterized. Three amino acid changes were found in the HA1 subunit of HA of MA virus. One of these was located inside the region of the conformational epitope recognized by MAb C179. However, this substitution was not significant for the recognition of HA and virus neutralization by MAb C179 in vitro and in vivo. Intraperitoneal administration of two different concentrations of MAb C179 one day before or two days after the virus challenge significantly decreased mortality rate. These results suggest that MAb C179 is efficient not only in the prevention and treatment of H1 and H2 influenza virus bronchopneumonia, as was reported previously, but also of H5-induced bronchopneumonia as well, and demonstrate in vivo the existence of a common neutralizing epitope in the HAs of these three subtypes.

Animals↗

PubMed2006

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.

Animals↗

PubMedDate not supplied

Influenza viruses and paramyxoviruses in ducks in the Atlantic flyway, 1977-1983, including an H5N2 isolate related to the virulent chicken virus.

From 1977 to 1983, waterfowl migrating along the Atlantic flyway were annually monitored for orthomyxoviruses and paramyxoviruses in an area in central New York State. A total of 168 influenza isolates were obtained from 1,430 waterfowl. Twenty-four combinations of hemagglutinin and neuraminidase subtypes were detected, with as many as 12 found in a single year. One combination, an H5N2 isolate in 1982, was closely related to the virulent chicken virus that appeared in Pennsylvania in 1983. The prevalence of influenza varied greatly among the common waterfowl species: mallards 42%, black ducks 30%, blue-winged teal 11%, wood ducks 2%, and Canada geese 0%. A total of 89 paramyxoviruses were also from these waterfowl. In contrast to findings with influenza virus, the prevalence of paramyxoviruses did not differ significantly among the duck species. Serotype 1 (Newcastle disease virus) was predominant; three other serotypes were also identified. These findings indicated that ducks in the Atlantic flyway continually harbor influenza viruses and paramyxoviruses. The viruses may be a source of infection for other species.

Animals↗

PubMed2006

Protective avian influenza in ovo vaccination with non-replicating human adenovirus vector.

Protective immunity against avian influenza virus was elicited in chickens by single-dose in ovo vaccination with a non-replicating human adenovirus vector encoding an H5N9 avian influenza virus hemagglutinin. Vaccinated chickens were protected against both H5N1 (89% hemagglutinin homology; 68% protection) and H5N2 (94% hemagglutinin homology; 100% protection) highly pathogenic avian influenza virus challenges. This vaccine can be mass-administered using available robotic in ovo injectors which provide a major advantage over current vaccination regimens. In addition, this class of adenovirus-vectored vaccines can be produced rapidly with improved safety since they do not contain any replication-competent adenoviruses. Furthermore, this mode of vaccination is compatible with epidemiological surveys of natural avian influenza virus infections.

Adenoviruses, Human↗

PubMed2006

Vaccines developed for H5 highly pathogenic avian influenza in China.

Since the first detection of highly pathogenic H5N1 avian influenza virus from sick goose in Guangdong province in China in 1996, scientists in China started to develop vaccines for avian influenza pandemic preparedness. An H5N2 inactivated vaccine was produced from a low pathogenic virus, A/turkey/England/N-28/73, and was used for the buffer zone vaccination in the H5N1 outbreaks in 2004 in China. We also generated a low pathogenic H5N1 reassortant virus A/Harbin/Re-1/2003 (Re-1) that derives its HA and NA genes from GSGD/96 virus and six internal genes from the high-growth A/Puerto Rico/8/34 (PR8) virus by using plasmid-based reverse genetics. The inactivated vaccine derived from Re-1 strain could induce more than 10 months protective immune response in chickens after one dose inoculation, and most importantly, this vaccine is immunogenic for geese and ducks. An H5N1 fowlpox vectored live vaccine was also generated by inserting the HA and NA genes of GSGD/96 virus in the genome of a fowlpox vaccine strain. Laboratory tests indicated that after one dose of immunization of this vaccine, chickens could develop an over than 40 weeks protective immune response against H5N1 virus challenge.

Animals↗

PubMed2006

Characterization of an influenza A H5N2 reassortant as a candidate for live-attenuated and inactivated vaccines against highly pathogenic H5N1 viruses with pandemic potential.

We generated a high-growth 7:1 reassortant (Len17/H5) that contained the hemagglutinin (HA) gene from non-pathogenic A/Duck/Potsdam/1402-6/86 (H5N2) virus and other genes from the cold-adapted (ca) attenuated A/Leningrad/134/17/57 (H2H2) strain. Len17/H5 demonstrated an attenuated phenotype in mice and did not infect chickens. Mice administered Len17/H5 either as a live-attenuated intranasal vaccine or as an inactivated intramuscular vaccine were substantially protected from lethal challenge with highly pathogenic A/Hong Kong/483/97 (H5N1) virus and were protected from pulmonary infection with antigenically distinct A/Hong Kong/213/2003 (H5N1) virus. The cross-protective effect correlated with the levels of virus-specific mucosal IgA and/or serum IgG antibodies. Our results suggest a new strategy of using classical genetic reassortment between a high-growth ca H2N2 strain and antigenically related non-pathogenic avian viruses to prepare live-attenuated and inactivated vaccines for influenza pandemic.

Animals↗

PubMed1996

Heterogeneity in the haemagglutinin gene and emergence of the highly pathogenic phenotype among recent H5N2 avian influenza viruses from Mexico.

Molecular changes in the haemagglutinin (HA)-coding regions and proteolytic cleavage sites from multiple H5N2 subtype viruses isolated during a recent outbreak of avian influenza (AI) in central Mexico have been characterized. Eighteen isolates, collected during a 15 month period (October 1993 to January 1995) from six central states, were sequenced. None of the 18 predicted HA1 amino acid sequences were identical and changes were not restricted to a specific region of the sequence. Phylogenetic analyses of the HA1 sequences demonstrated two virus lineages, designated Puebla and Jalisco, with sequence variation as high as 10.5 percent for amino acid and 6.2 percent for nucleotide sequences. During the latter months of the surveillance period, highly pathogenic (HP) strains of AI emerged causing lethal disease in commercial poultry flocks. In each of the HP strains isolated, the HA protein was cleaved in chicken embryo fibroblast cells in the absence of trypsin, and two alterations not found in earlier non-HP isolates were detected. In the HA protein, HP strains all had a glutamic acid --> lysine substitution at amino acid position 324 and an insertion of arginine and lysine as new residues 325 and 326. The insertion appears to be due to a duplication of the nucleotide sequence AAAGAA at nucleotide positions 965-970 of the HA1-coding region. Computer-assisted secondary structure analyses place the target for the insertion in a predicted RNA stem-loop structure. A mechanism is suggested by which the polymerase duplicates the sequence.

Amino Acid Sequence↗