Influenza surveillance in Hong Kong: results of a trial Physician Sentinel Programme

The H5N1 influenza outbreak in Hong Kong at the end of 1997 emphasised the need for viral surveillance so that new influenza epidemics can be foreseen. Although South China is regarded as the regional epicentre of influenza epidemics, there has been little epidemiological documentation of the disease there. A sentinel physician network was established in Hong Kong in 1993 to estimate the incidence, severity, and seasonality of influenza-like illnesses and to provide data on the demand for health care that is related to this illness. Influenza-like illness occurred throughout the year of the survey, peaking from March through May and accounting for 15% of doctor visits. The incidence was approximately 117 in 1000 patients and was greatest among children aged 1 to 4 years. Ongoing physician surveillance with appropriate coverage of the general population supported by a laboratory virus isolation capability may help control future influenza outbreaks.published_or_final_versio

Genetic analysis of porcine H3N2 viruses originating in southern China

From immunological and phylogenetic analyses of H3 influenza viruses isolated from pigs and ducks in the People's Republic of China (China), Hong Kong, Taiwan and Japan, between 1968 and 1982, we arrived at the following conclusions. The H3 haemagglutinin and N2 neuraminidase genes from swine isolates can be segregated into four mammalian lineages, including: (i) the earliest human strains; (ii) early swine strains including Hong Kong isolates from 1976-1977; (iii) an intermediate strain between the early swine and recent human strains; and (iv) recent human strains. In this study we found an unusual swine strain (sw/Hong Kong/127/82) belonging to the third lineage which behaved like those of the early swine-like lineage in the haemagglutination inhibition test; but neuraminidase inhibition profiles with monoclonal antibodies indicated that this virus is related to late human strains. On the basis of pairwise comparisons of complete or partial nucleotide sequences the genes encoding the three polymerase proteins (PB2, PB1, PA), the nucleoprotein, the membrane protein and possibly the nonstructural proteins of sw/Hong Kong/127/82 are of the swine H1N1 lineage, whereas genes encoding the two surface glycoproteins belong to the human H3N2 lineage. In contrast, all RNA segments of one swine isolate (sw/Hong Kong/81/78) are similar to those of recent human H3N2 viruses. This study indicated that frequent interspecies infections between human and swine hosts appeared to occur during 1976-82. Although the evolutionary rates of human (0.0122/site/year), swine (0.0127/site/year) and avian (0.0193/site/year) virus genes are similar when based upon synonymous substitutions, nonsynonymous substitutions indicated that viral genes derived from human and swine viruses evolved about three times faster (0.0026-0.0027/site/year) than those of avian viruses (0.0008/site/year). Furthermore, the evolutionary mechanism by which human and swine H3 haemagglutinin genes evolve at a similar rate, based on nonsynonymous substitutions, appeared to be quite different from previous evidence which showed that human H1 haemagglutinin genes evolved three times faster than those of swine viruses. However, comparison of the number of nonsynonymous substitutions in the antigenic sites (A-E) of haemagglutinin molecules demonstrated that swine viruses evolve at a rate that is about one fifth to one tenth that of human viruses, reflecting the conservative nature of the antigenic structure in the former.published_or_final_versio

Characterization of H3N2 influenza viruses isolated from pigs in southern China

Poster Presentations: Animal Influenza EcologyHuman-like H3N2 influenza viruses have repeatedly transmitted to domestic pigs in different regions of the world, but it is still not certain whether any of those variants have become established in pig populations. The detection of different subtypes of avian influenza viruses from pigs makes it an ideal candidate for the genesis of a possible reassortant virus with both human and avian gene segments. However, whether pigs could act as a “mixing vessel” for a possible pandemic virus remains unanswered. Long-term influenza surveillance in pigs in southern China revealed that H3N2 influenza viruses were regularly detected from domestic pigs from 1998 to 2003. Antigenic analysis of representative strains revealed that two distinguishable groups of H3N2 influenza viruses were present in pigs during this period: a contemporary human-like viruses (represented by Sydney/5/97), and Port Chalmers/1/73-like (PC-like) viruses. Phylogenetic analysis of the representative strains confirmed those two groups. In general, the PC-like viruses were most closely related to those H3N2 reassortants recognized from European pigs since the mid-1980s, while the remaining isolates were most closely related to those contemporary human H3N2 viruses. It is interesting to note that one PC-like isolate contained a classical swine H1N1-like NP gene, Sw/HK/1197/02, suggesting that after introduction to pigs in southern China the European swine H3N2 virus further reassorted with local swine virus. The contemporary humanlike H3N2 viruses isolated from pig appeared to have resulted from repeated introduction from humans to pigs. Interestingly, one isolate (Sw/HK/NS1128/03) clustered with those human isolates detected in the early 1990s. These findings suggesting that some recent human H3N2 variants may be maintained long-term in pig populations in southern China. The present study provides updated information on the role of pigs in the interspecies transmission and genetic reassortment of influenza viruses in this region.postprin

Workshop 1: Surveillance issues of pandemic influenza

published_or_final_versio

Characterization of low pathogenic H5 subtype influenza viruses from Eurasia: Implications for the origin of highly pathogenic H5N1 viruses

Oral Presentations - Genetic and Antigenic EvolutionHighly pathogenic avian influenza (HPAI) H5N1 viruses are now endemic in many Asian countries. The immediate precursor of these HPAI viruses was recognized as A/Goose/Guangdong/1/96 (Gs/Gd)-like H5N1 HPAI viruses first detected in Guangdong in 1996. However, precursors of the Gs/GD-like viruses and their subsequent reassortants have not been fully determined. Here we characterize low pathogenic avian influenza (LPAI) H5 subtype viruses isolated from poultry and migratory birds in southern China and Europe from the 1970s to the 2000s. Phylogenetic analyses revealed that Gs/GD-like virus was likely derived from an LPAI H5 virus in migratory birds. However, its variants arose from multiple reassortments between Gs/GD-like virus and viruses from migratory birds, or with those Eurasian viruses isolated in the 1970s. It is of note that unlike HPAI H5N1 viruses, those recent LPAI H5 viruses have not become established in aquatic or terrestrial poultry. Phylogenetic analyses revealed the dynamic nature of the influenza gene pool in Eurasia with repeated transmissions between the eastern and western extremities of the continent. The data also shows reassortment between influenza viruses from domestic and migratory birds in this region that has contributed to the expanded diversity of the influenza gene pool among poultry in Eurasia ...postprin

Spatial Distribution and Risk Factors of Highly Pathogenic Avian Influenza (HPAI) H5N1 in China

Highly pathogenic avian influenza (HPAI) H5N1 was first encountered in 1996 in Guangdong province (China) and started spreading throughout Asia and the western Palearctic in 2004–2006. Compared to several other countries where the HPAI H5N1 distribution has been studied in some detail, little is known about the environmental correlates of the HPAI H5N1 distribution in China. HPAI H5N1 clinical disease outbreaks, and HPAI virus (HPAIV) H5N1 isolated from active risk-based surveillance sampling of domestic poultry (referred to as HPAIV H5N1 surveillance positives in this manuscript) were modeled separately using seven risk variables: chicken, domestic waterfowl population density, proportion of land covered by rice or surface water, cropping intensity, elevation, and human population density. We used bootstrapped logistic regression and boosted regression trees (BRT) with cross-validation to identify the weight of each variable, to assess the predictive power of the models, and to map the distribution of HPAI H5N1 risk. HPAI H5N1 clinical disease outbreak occurrence in domestic poultry was mainly associated with chicken density, human population density, and elevation. In contrast, HPAIV H5N1 infection identified by risk-based surveillance was associated with domestic waterfowl density, human population density, and the proportion of land covered by surface water. Both models had a high explanatory power (mean AUC ranging from 0.864 to 0.967). The map of HPAIV H5N1 risk distribution based on active surveillance data emphasized areas south of the Yangtze River, while the distribution of reported outbreak risk extended further North, where the density of poultry and humans is higher. We quantified the statistical association between HPAI H5N1 outbreak, HPAIV distribution and post-vaccination levels of seropositivity (percentage of effective post-vaccination seroconversion in vaccinated birds) and found that provinces with either outbreaks or HPAIV H5N1 surveillance positives in 2007–2009 appeared to have had lower antibody response to vaccination. The distribution of HPAI H5N1 risk in China appears more limited geographically than previously assessed, offering prospects for better targeted surveillance and control interventions

Investigating poultry trade patterns to guide avian influenza surveillance and control: a case study in Vietnam

Live bird markets are often the focus of surveillance activities monitoring avian influenza viruses (AIV) circulating in poultry. However, in order to ensure a high sensitivity of virus detection and effectiveness of management actions, poultry management practices features influencing AIV dynamics need to be accounted for in the design of surveillance programmes. In order to address this knowledge gap, a cross-sectional survey was conducted through interviews with 791 traders in 18 Vietnamese live bird markets. Markets greatly differed according to the sources from which poultry was obtained, and their connections to other markets through the movements of their traders. These features, which could be informed based on indicators that are easy to measure, suggest that markets could be used as sentinels for monitoring virus strains circulating in specific segments of the poultry production sector. AIV spread within markets was modelled. Due to the high turn-over of poultry, viral amplification was likely to be minimal in most of the largest markets. However, due to the large number of birds being introduced each day, and challenges related to cleaning and disinfection, environmental accumulation of viruses at markets may take place, posing a threat to the poultry production sector and to public health

Transmission of Avian Influenza A Viruses among Species in an Artificial Barnyard

Waterfowl and shorebirds harbor and shed all hemagglutinin and neuraminidase subtypes of influenza A viruses and interact in nature with a broad range of other avian and mammalian species to which they might transmit such viruses. Estimating the efficiency and importance of such cross-species transmission using epidemiological approaches is difficult. We therefore addressed this question by studying transmission of low pathogenic H5 and H7 viruses from infected ducks to other common animals in a quasi-natural laboratory environment designed to mimic a common barnyard. Mallards (Anas platyrhynchos) recently infected with H5N2 or H7N3 viruses were introduced into a room housing other mallards plus chickens, blackbirds, rats and pigeons, and transmission was assessed by monitoring virus shedding (ducks) or seroconversion (other species) over the following 4 weeks. Additional animals of each species were directly inoculated with virus to characterize the effect of a known exposure. In both barnyard experiments, virus accumulated to high titers in the shared water pool. The H5N2 virus was transmitted from infected ducks to other ducks and chickens in the room either directly or through environmental contamination, but not to rats or blackbirds. Ducks infected with the H7N2 virus transmitted directly or indirectly to all other species present. Chickens and blackbirds directly inoculated with these viruses shed significant amounts of virus and seroconverted; rats and pigeons developed antiviral antibodies, but, except for one pigeon, failed to shed virus

Early apoptosis of porcine alveolar macrophages limits avian influenza virus replication and proinflammatory dysregulation

Pigs are evidently more resistant to avian than swine influenza A viruses, mediated in part through frontline epithelial cells and alveolar macrophages (AM). Although porcine AM (PAM) are crucial in influenza virus control, their mode of control is unclear. To gain insight into the possible role of PAM in the mediation of avian influenza virus resistance, we compared the host effects and replication of two avian (H2N3 and H6N1) and three mammalian (swine H1N1, human H1N1 and pandemic H1N1) influenza viruses in PAM. We found that PAM were readily susceptible to initial infection with all five avian and mammalian influenza viruses but only avian viruses caused early and extensive apoptosis (by 6 h of infection) resulting in reduced virus progeny and moderated pro- inflammation. Full length viral PB1-F2 present only in avian influenza viruses is a virulence factor that targets AM for mitochondrial associated apoptotic cell death. With the use of reverse genetics on an avian H5N1 virus, we found that full length PB1-F2 contributed to increased apoptosis and pro-inflammation but not to reduced virus replication. Taken together, we propose that early apoptosis of PAM limits the spread of avian influenza viruses and that PB1-F2 could play a contributory role in the process

Broad Clade 2 Cross-Reactive Immunity Induced by an Adjuvanted Clade 1 rH5N1 Pandemic Influenza Vaccine

The availability of H5N1 vaccines that can elicit a broad cross-protective immunity against different currently circulating clade 2 H5N1 viruses is a pre-requisite for the development of a successful pre-pandemic vaccination strategy. In this regard, it has recently been shown that adjuvantation of a recombinant clade 1 H5N1 inactivated split-virion vaccine with an oil-in-water emulsion-based adjuvant system also promoted cross-immunity against a recent clade 2 H5N1 isolate (A/Indonesia/5/2005, subclade 2.1). Here we further analyse the cross-protective potential of the vaccine against two other recent clade 2 isolates (A/turkey/Turkey/1/2005 and A/Anhui/1/2005 which are, as defined by WHO, representatives of subclades 2.2 and 2.3 respectively).Two doses of the recombinant A/Vietnam/1194/2004 (H5N1, clade 1) vaccine were administered 21 days apart to volunteers aged 18-60 years. We studied the cross-clade immunogenicity of the lowest antigen dose (3.8 microg haemagglutinin) given with (N = 20) or without adjuvant (N = 20). Immune responses were assessed at 21 days following the first and second vaccine doses and at 6 months following first vaccination. Vaccination with two doses of 3.8 microg of the adjuvanted vaccine induced four-fold neutralising seroconversion rates in 85% of subjects against A/turkey/Turkey/1/2005 (subclade 2.2) and 75% of subjects against A/Anhui/1/2005 (subclade 2.3) recombinant strains. There was no response induced against these strains in the non-adjuvanted group. At 6 months following vaccination, 70% and 60% of subjects retained neutralising antibodies against the recombinant subclade 2.2 and 2.3 strains, respectively and 40% of subjects retained antibodies against the recombinant subclade 2.1 A/Indonesia/5/2005 strain.In addition to antigen dose-sparing, adjuvantation of inactivated split H5N1 vaccine promotes broad and persistent cross-clade immunity which is a pre-requisite for a pre-pandemic vaccine.ClinicalTrials.gov NCT00309634