Direct typing and molecular evolutionary analysis of field samples of foot-and-mouth disease virus collected in Viet Nam between 2006 and 2007.

In this study, we used universal or duplex serotype-specific (O and Asia 1) RT-PCR to analyze clinical field samples of foot-and-mouth disease virus (FMDV) or virus isolates collected in Viet Nam between 2006 and 2007. We found viral serotypes O and Asia 1 circulating concurrently during this period. Direct sequencing of type-specific RT-PCR products revealed the existence of three different topotypes of serotype O: Southeast Asia (SEA), Middle East-South Asia (ME-SA), and Cathay. Of these, SEA was most prevalent during the period. All samples of serotype Asia 1 belonged to genetic group V. Based on the rooted maximum likelihood phylogenetic trees inferred from the VP1 region, new lineages in topotype SEA were originating from Viet Nam, and group V strains of Asia 1 have undergone fewer passages from the common ancestor, compared with other genetic groups. The co-circulation of different types of FMDV may complicate the individual or population genomic structures of FMDV and make conventional multiplex diagnostic methods and phylogenetic analyses with relevant evolutionary models essential in Viet Nam.

Detection and characterization of clade 7 high pathogenicity avian influenza H5N1 viruses in chickens seized at ports of entry and live poultry markets in Vietnam.

High pathogenicity avian influenza H5N1 has become an endemic poultry disease in several Asian countries, including Vietnam. Recently, dade 7 H5N1 viruses of the Eurasian lineage were isolated from chickens seized at ports of entry in Lang Son Province, Vietnam. Extensive nucleotide and amino acid divergence across the hemagglutinin (HA) protein gene of these isolates in comparison to previously described clade 7 viruses was identified. Clade 7 viruses are antigenically distinct from contemporary strains of H5N1 known to circulate in Vietnamese poultry (clade 1 and clade 2.3.4). Subsequent surveillance of sick poultry in live poultry markets in Hai Duong Province identified additional clade 7 isolates with HA genes very similar to the group B virus cluster detected previously at the Lang Son Province border. Antigenic analysis of the isolates from the live bird markets revealed significant cross-reactivity only between those clade 7 viruses belonging to the same subgroups. To meet pandemic response preparedness objectives, we have developed a reassortant virus from A/chicken/Vietnam/NCVD-016/2008, which could be used as a new prepandemic vaccine candidate for veterinary or human vaccination, should the need arise. Findings from these studies indicate that viruses with clade 7 HA have continued to evolve in Southeast Asian poultry, leading to significant antigenic drift relative to other H5N1 viruses currently circulating in Vietnam.

Prophylactic and therapeutic efficacy of avian antibodies against influenza virus H5N1 and H1N1 in mice.

BACKGROUND: Pandemic influenza poses a serious threat to global health and the world economy. While vaccines are currently under development, passive immunization could offer an alternative strategy to prevent and treat influenza virus infection. Attempts to develop monoclonal antibodies (mAbs) have been made. However, passive immunization based on mAbs may require a cocktail of mAbs with broader specificity in order to provide full protection since mAbs are generally specific for single epitopes. Chicken immunoglobulins (IgY) found in egg yolk have been used mainly for treatment of infectious diseases of the gastrointestinal tract. Because the recent epidemic of highly pathogenic avian influenza virus (HPAIV) strain H5N1 has resulted in serious economic losses to the poultry industry, many countries including Vietnam have introduced mass vaccination of poultry with H5N1 virus vaccines. We reasoned that IgY from consumable eggs available in supermarkets in Vietnam could provide protection against infections with HPAIV H5N1. METHODS AND FINDINGS: We found that H5N1-specific IgY that are prepared from eggs available in supermarkets in Vietnam by a rapid and simple water dilution method cross-protect against infections with HPAIV H5N1 and related H5N2 strains in mice. When administered intranasally before or after lethal infection, the IgY prevent the infection or significantly reduce viral replication resulting in complete recovery from the disease, respectively. We further generated H1N1 virus-specific IgY by immunization of hens with inactivated H1N1 A/PR/8/34 as a model virus for the current pandemic H1N1/09 and found that such H1N1-specific IgY protect mice from lethal influenza virus infection. CONCLUSIONS: The findings suggest that readily available H5N1-specific IgY offer an enormous source of valuable biological material to combat a potential H5N1 pandemic. In addition, our study provides a proof-of-concept for the approach using virus-specific IgY as affordable, safe, and effective alternative for the control of influenza outbreaks, including the current H1N1 pandemic.

Characterization of a highly pathogenic avian influenza H5N1 virus sublineage in poultry seized at ports of entry into Vietnam.

Highly pathogenic avian influenza H5N1 virus was detected in poultry seized at two ports of entry located in Lang Son Province, Vietnam. Sequence analysis of the hemagglutinin (HA) genes from five H5N1 virus isolates and ten PCR amplicons from chicken cloacal samples revealed their close phylogenetic relationship to clade 7 H5N1 HA genes. However, these HA genes exhibited extensive genetic divergence at both the nucleotide and amino acid levels in comparison to previously described clade 7 viruses; e.g., A/chicken/Shanxi/2/2006. In addition, hemagglutination inhibition tests revealed antigenic differences between these and previously isolated H5N1 viruses from Vietnam. These results indicate that viruses with clade 7 HA are evolving rapidly in poultry in Southeast Asia.

Evolution of highly pathogenic H5N1 avian influenza viruses in Vietnam between 2001 and 2007.

Highly pathogenic avian influenza (HPAI) H5N1 viruses have caused dramatic economic losses to the poultry industry of Vietnam and continue to pose a serious threat to public health. As of June 2008, Vietnam had reported nearly one third of worldwide laboratory confirmed human H5N1 infections. To better understand the emergence, spread and evolution of H5N1 in Vietnam we studied over 300 H5N1 avian influenza viruses isolated from Vietnam since their first detection in 2001. Our phylogenetic analyses indicated that six genetically distinct H5N1 viruses were introduced into Vietnam during the past seven years. The H5N1 lineage that evolved following the introduction in 2003 of the A/duck/Hong Kong/821/2002-like viruses, with clade 1 hemagglutinin (HA), continued to predominate in southern Vietnam as of May 2007. A virus with a clade 2.3.4 HA newly introduced into northern Vietnam in 2007, reassorted with pre-existing clade 1 viruses, resulting in the emergence of novel genotypes with neuraminidase (NA) and/or internal gene segments from clade 1 viruses. A total of nine distinct genotypes have been present in Vietnam since 2001, including five that were circulating in 2007. At least four of these genotypes appear to have originated in Vietnam and represent novel H5N1 viruses not reported elsewhere. Geographic and temporal analyses of H5N1 infection dynamics in poultry suggest that the majority of viruses containing new genes were first detected in northern Vietnam and subsequently spread to southern Vietnam after reassorting with pre-existing local viruses in northern Vietnam. Although the routes of entry and spread of H5N1 in Vietnam remain speculative, enhanced poultry import controls and virologic surveillance efforts may help curb the entry and spread of new HPAI viral genes.

Influenza A H5N1 clade 2.3.4 virus with a different antiviral susceptibility profile replaced clade 1 virus in humans in northern Vietnam.

BACKGROUND: Prior to 2007, highly pathogenic avian influenza (HPAI) H5N1 viruses isolated from poultry and humans in Vietnam were consistently reported to be clade 1 viruses, susceptible to oseltamivir but resistant to amantadine. Here we describe the re-emergence of human HPAI H5N1 virus infections in Vietnam in 2007 and the characteristics of the isolated viruses. METHODS AND FINDINGS: Respiratory specimens from patients suspected to be infected with avian influenza in 2007 were screened by influenza and H5 subtype specific polymerase chain reaction. Isolated H5N1 strains were further characterized by genome sequencing and drug susceptibility testing. Eleven poultry outbreak isolates from 2007 were included in the sequence analysis. Eight patients, all of them from northern Vietnam, were diagnosed with H5N1 in 2007 and five of them died. Phylogenetic analysis of H5N1 viruses isolated from humans and poultry in 2007 showed that clade 2.3.4 H5N1 viruses replaced clade 1 viruses in northern Vietnam. Four human H5N1 strains had eight-fold reduced in-vitro susceptibility to oseltamivir as compared to clade 1 viruses. In two poultry isolates the I117V mutation was found in the neuraminidase gene, which is associated with reduced susceptibility to oseltamivir. No mutations in the M2 gene conferring amantadine resistance were found. CONCLUSION: In 2007, H5N1 clade 2.3.4 viruses replaced clade 1 viruses in northern Vietnam and were susceptible to amantadine but showed reduced susceptibility to oseltamivir. Combination antiviral therapy with oseltamivir and amantadine for human cases in Vietnam is recommended.