Genomic signatures of human versus avian influenza A viruses.
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Position-specific entropy profiles created from scanning 306 human and 95 avian influenza A viral genomes showed that 228 of 4591 amino acid residues yielded significant differences between these 2 viruses. We subsequently used 15,785 protein sequences from the National Center for Biotechnology Information (NCBI) to assess the robustness of these signatures and obtained 52 "species-associated" positions. Specific mutations on those points may enable an avian influenza virus to become a human virus. Many of these signatures are found in NP, PA, and PB2 genes (viral ribonucleoproteins [RNPs]) and are mostly located in the functional domains related to RNP-RNP interactions that are important for viral replication. Upon inspecting 21 human-isolated avian influenza viral genomes from NCBI, we found 19 that exhibited > or =1 species-associated residue changes; 7 of them contained > or =2 substitutions. Histograms based on pairwise sequence comparison showed that NP disjointed most between human and avian influenza viruses, followed by PA and PB2. (+info)
Characterization of avian H9N2 influenza viruses from United Arab Emirates 2000 to 2003.
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Our aim was to establish the phylogenetic relation of H9N2 avian viruses in the Middle East to other Asian H9N2 lineages by characterization of 7 viruses isolated from United Arab Emirates (2000-2003). All these viruses had an additional basic amino acid at the hemagglutinin-connecting peptide; 6 contained a mutation associated with increased affinity toward human-like sialic acid substrates. The viruses' surface glycoproteins and most internal genes were >90% similar to those of A/Quail/Hong Kong/G1/97 (H9N2) lineage. The hemadsorbing site of neuraminidase had up to 4 amino acid substitutions, as do human pandemic viruses. M2 sequence analysis revealed amino acid changes at 2 positions, with increasing resistance to amantadine in cell culture. They replicated efficiently in inoculated chickens and were successfully transmitted to contacts. They continue to maintain H5N1-like genes and may augment the spread of H5N1 viruses through regional co-circulation and inapparent infection. These viruses may present as potential pandemic candidates themselves. (+info)
Evolution and molecular epidemiology of H9N2 influenza A viruses from quail in southern China, 2000 to 2005.
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H9N2 influenza viruses have become established and maintain long-term endemicity in terrestrial poultry in Asian countries. Occasionally these viruses transmit to other mammals, including humans. Increasing epidemiological and laboratory findings suggest that quail may be an important host, as they are susceptible to different subtypes of influenza viruses. To better understand the role of quail in influenza virus ecology and evolution, H9N2 viruses isolated from quail during 2000 to 2005 were antigenically and genetically characterized. Our results showed that H9N2 viruses are prevalent year-round in southern China and replicate mainly asymptomatically in the respiratory tract of quail. Genetic analysis revealed that both the G1-like and Ck/Bei-like H9N2 lineages were cocirculating in quail since 2000. Phylogenetic analyses demonstrated that most of the isolates tested were double- or multiple-reassortant variants, with four G1-like and 16 Ck/Bei-like genotypes recognized. A novel genotype of G1-like virus became predominant in quail since 2003, while multiple Ck/Bei-like genotypes were introduced into quail, wherein they incorporated G1-like gene segments, but none of them became established in this host. Those Ck/Bei-like reassortants generated in quail have then been introduced into other poultry. These complex interactions form a two-way transmission system between quail and other types of poultry. The present study provides evidence that H9N2 and H5N1 subtype viruses have also exchanged gene segments to generate currently circulating reassortants of both subtypes that have pandemic potential. Continuing influenza virus surveillance in poultry is critical to understanding the genesis and emergence of potentially pandemic strains in this region. (+info)
Detection of H5, H7 and H9 subtypes of avian influenza viruses by multiplex reverse transcription-polymerase chain reaction.
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Subtype-specific multiplex reverse transcription-polymerase chain reaction (RT-PCR) was developed to simultaneously detect three subtypes (H5, H7 and H9) of avian influenza virus (AIV) type A. The sensitivity of the multiplex RT-PCR was evaluated and compared to that of RT-PCR-enzyme-linked immunosorbent assay (ELISA) and conventional RT-PCR. While the sensitivity of the multiplex RT-PCR is as sensitive as the conventional RT-PCR, it is 10 times less sensitive than RT-PCR-ELISA. The multiplex RT-PCR is also as sensitive as the virus isolation method in detecting H9N2 from tracheal samples collected at day 3 and 5 post inoculation. Hence, the developed multiplex RT-PCR assay is a rapid, sensitive and specific assay for detecting of AIV subtypes. (+info)
Amino acid 226 in the hemagglutinin of H9N2 influenza viruses determines cell tropism and replication in human airway epithelial cells.
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Influenza A viruses of the H9N2 subtype are endemic in poultry in many Eurasian countries and have occasionally caused clinical respiratory diseases in humans. While some avian H9N2 viruses have glutamine (Q) at amino acid position 226 of the hemagglutinin (HA) receptor-binding site, an increasing number of isolates have leucine (L) at this position, which has been associated with the establishment of stable lineages of the H2 and H3 subtypes of viruses in humans. Little is known about the importance of this molecular trait in the infection of H9N2 viruses in humans. We show here that during the course of a single cycle of infection in human airway epithelial (HAE) cells cultured in vitro, the L-226-containing H9N2 viruses displayed human virus-like cell tropisms (preferentially infecting nonciliated cells) different from the tropisms showed by Q-226-containing H9N2 isolates (which infect both ciliated and nonciliated cells at ratios of 1:1 to 3:2) or other waterfowl viruses (which preferentially infect ciliated cells). During multiple cycles of replication in HAE cultures, L-226-containing H9N2 isolates grew consistently more efficiently and reached approximately 100-fold-higher peak titers than those containing Q-226, although peak titers were significantly lower than those induced by human H3N2 viruses. Our results suggest that the variation in residue 226 in the HA affects both cell tropism and replication of H9N2 viruses in HAE cells and may have implications for the abilities of these viruses to infect humans. (+info)
The quail and chicken intestine have sialyl-galactose sugar chains responsible for the binding of influenza A viruses to human type receptors.
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The receptor specificity of influenza viruses is one factor that allows avian influenza viruses to cross the species barrier. The recent transmissions of avian H5N1 and H9N2 influenza viruses from chickens and/or quails to humans indicate that avian influenza viruses can directly infect humans without an intermediate host, such as pigs. In this study, we used two strains of influenza A virus (A/PR/8/34, which preferentially binds to an avian-type receptor, and A/Memphis/1/71, which preferentially binds to a human-type receptor) to probe the receptor specificities in host cells. Epithelial cells of both quail and chicken intestines (colons) could bind both avian- and human-type viruses. Infected cultured quail colon cells expressed viral protein and allowed replication of the virus strain A/PR/8/34 or A/Memphis/1/71. To understand the molecular basis of these phenomena, we further investigated the abundance of sialic acid (Sia) linked to galactose (Gal) by the alpha2-3 linkage (Siaalpha2-3Gal) and Siaalpha2-6Gal in host cells. In glycoprotein and glycolipid fractions from quail and chicken colon epithelial cells, there were some bound components of Sia-Gal linkage-specific lectins, Maackia amurensis agglutinin (specific for Siaalpha2-3 Gal) and Sambucus nigra agglutinin (specific for Siaalpha2-6Gal), indicating that both Siaalpha2-3Gal and Siaalpha2-6Gal exist in quail and chicken colon cells. Furthermore, we demonstrated by fluorescence high-performance liquid chromatography (HPLC) analysis that 5-N-acetylneuraminic acid was the main molecular species of Sia, and we demonstrated by multi-dimensional HPLC mapping and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis that bi-antennary complex-type glycans alpha2-6 sialylated at the terminal Gal residue(s) are major (more than 79%) sialyl N-glycans expressed by intestinal epithelial tissues in both the chicken and quail. Taken together, these results indicate that quails and chickens have molecular characterization as potential intermediate hosts for avian influenza virus transmission to humans and could generate new influenza viruses with pandemic potential. (+info)
Antigenic and genetic characterization of H9N2 swine influenza viruses in China.
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As pigs are susceptible to infection with both avian and human influenza A viruses, they have been proposed to be an intermediate host for the generation of pandemic virus through reassortment. Antigenic and genetic characterization was performed for five swine H9N2 influenza viruses isolated from diseased pigs from different farms. The haemagglutinin (HA) antigenicity of swine H9N2 viruses was different from that of chicken H9N2 viruses prevalent in northern China. Genetic analysis revealed that all five isolates had an RLSR motif at the cleavage site of HA, which was different from those of A/duck/Hong Kong/Y280/97 (Dk/HK/Y280/97)-like viruses established in chickens in China. Phylogenetic analyses indicated that the five swine H9N2 viruses formed novel HA and neuraminidase sublineages that were related closely to those of earlier chicken H9 viruses and were also consistent with the extent of the observed antigenic variation. The six internal genes of the isolates possessed H5N1-like sequences, indicating that they were reassortants of H9 and H5 viruses. The present results indicate that avian to porcine interspecies transmission of H9N2 viruses might have resulted in the generation of viruses with novel antigenic and genetic characteristics; therefore, surveillance of swine influenza should be given a high priority. (+info)
A new generation of modified live-attenuated avian influenza viruses using a two-strategy combination as potential vaccine candidates.
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In light of the recurrent outbreaks of low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI), there is a pressing need for the development of vaccines that allow rapid mass vaccination. In this study, we introduced by reverse genetics temperature-sensitive mutations in the PB1 and PB2 genes of an avian influenza virus, A/Guinea Fowl/Hong Kong/WF10/99 (H9N2) (WF10). Further genetic modifications were introduced into the PB1 gene to enhance the attenuated (att) phenotype of the virus in vivo. Using the att WF10 as a backbone, we substituted neuraminidase (NA) for hemagglutinin (HA) for vaccine purposes. In chickens, a vaccination scheme consisting of a single dose of an att H7N2 vaccine virus at 2 weeks of age and subsequent challenge with the wild-type H7N2 LPAI virus resulted in complete protection. We further extended our vaccination strategy against the HPAI H5N1. In this case, we reconstituted an att H5N1 vaccine virus, whose HA and NA genes were derived from an Asian H5N1 virus. A single-dose immunization in ovo with the att H5N1 vaccine virus in 18-day-old chicken embryos resulted in more than 60% protection for 4-week-old chickens and 100% protection for 9- to 12-week-old chickens. Boosting at 2 weeks posthatching provided 100% protection against challenge with the HPAI H5N1 virus for chickens as young as 4 weeks old, with undetectable virus shedding postchallenge. Our results highlight the potential of live att avian influenza vaccines for mass vaccination in poultry. (+info)