Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia.

A highly pathogenic avian influenza virus, H5N1, caused disease outbreaks in poultry in China and seven other east Asian countries between late 2003 and early 2004; the same virus was fatal to humans in Thailand and Vietnam. Here we demonstrate a series of genetic reassortment events traceable to the precursor of the H5N1 viruses that caused the initial human outbreak in Hong Kong in 1997 (refs 2-4) and subsequent avian outbreaks in 2001 and 2002 (refs 5, 6). These events gave rise to a dominant H5N1 genotype (Z) in chickens and ducks that was responsible for the regional outbreak in 2003-04. Our findings indicate that domestic ducks in southern China had a central role in the generation and maintenance of this virus, and that wild birds may have contributed to the increasingly wide spread of the virus in Asia. Our results suggest that H5N1 viruses with pandemic potential have become endemic in the region and are not easily eradicable. These developments pose a threat to public and veterinary health in the region and potentially the world, and suggest that long-term control measures are required.

Persistence of Hong Kong influenza virus variants in pigs.

The A/Hong Kong/1/68 (H3N2) influenza virus which has not been isolated from man for several years, was recently isolated from pigs in Hong Kong. Influenza viruses similar to A/Victoria/3/75, which are currently circulating in man, were also isolated from pigs. Both above-mentioned viruses could be transmitted readily from pig to pig in experimental studies. The isolation of influenza viruses similar to A/Hong Kong/68 from pigs in 1976 suggests that pigs may serve as a potential reservoir for future human pandemics as well as a possible source of genetic information for recombination between human and porcine strains of influenza virus.

Diffusion coefficient and molecular weight of type 5 adenovirus by photon-correlation spectroscopy.

The technique of photon-correlation spectroscopy (intensity fluctuation spectroscopy) is applied to light scatter from type 5 adenovirus undergoing Brownian motion in solution and the translation diffusion coefficient (D20,w) measured to be 0,367 +/- 0.003 Fick units. Using Svedberg's equation with previously determined parameters, a molecular weight of 165 - 10(6) +/- 5 - 10(6) is obtained.

A unique phosphatidylinositol bearing a novel branched-chain fatty acid from Rhodococcus equi binds to influenza virus hemagglutinin and inhibits the infection of cells.

From the aquatic bacterium Rhodococcus equi strain S(420), we isolated a substance that strongly binds to influenza viruses. Structural analyses revealed that it is a unique type of phosphatidylinositol (PtdIns) bearing a branched-chain fatty acid (14-methyloctadecanoic acid). In a TLC/virus-binding immunostaining assay, this PtdIns bound to all subtypes of hemagglutinin (HA) of influenza A viruses tested, isolated from humans, ducks and swine, and also to human influenza B viruses. Furthermore, the PtdIns significantly prevented the infection of MDCK cells by influenza viruses, and also inhibited the virus-mediated hemagglutination and low pH-induced hemolysis of human erythrocytes, which represents the fusogenic activities of the viral HA. We also used purified hemagglutinin instead of virions to examine the interaction between viral HA and PtdIns, showing that the PtdIns binds to hemagglutinin. These findings indicate that the inhibitory mechanism of PtdIns on the influenza virus infection may be through its binding to viral HA spikes and host cell endosomal/lysosomal membranes, which are mediated by the function of viral HA.

Duck and human pandemic influenza A viruses retain sialidase activity under low pH conditions.

The majority of influenza A viruses isolated from wild birds, but not humans, can replicate in the duck intestinal tract. Here we demonstrate that all duck isolates tested universally retain sialidase activities under low pH conditions independent of their neuraminidase (NA) subtypes. In contrast, the sialidase activities of most isolates from humans and pigs practically disappear below pH 4.5, with the exception of four human pandemic viruses isolated in 1957 and 1968. Sequence comparisons among duck, human, and swine N2 NA subtypes indicate that amino acids at positions 153, 253, 307, 329, 344, 347, 356, 368, 390, and 431 may be associated with the low pH stability of duck and human pandemic N2 NAs. This finding suggests that the low pH stability of duck influenza A virus NA may be a critical factor for replication in the intestinal tract through the digestive tract of ducks, and that the properties of NAs are important for understanding the epidemiology of the influenza virus.

Rubella virus serology:detection of residual lipoprotein inhibitors of haemagglutination using sensitive indicator arboviruses.

Immune and non-immune rubella sera were examined in the haemagglutination inhibition (HAI) test to evaluate the removal of lipoprotein non-specific inhibitors (NSI) of haemagglutination after kaolin and manganous chloride/heparin treatments. The sera were titrated fresh or after lipoprotein deterioration brought about by ageing the samples at 4 degrees C for various periods of time and by freezing at -20 degrees C with subsequent thawing. Deterioration was seen as altered electrophoretic mobility while the lipoproteins in treated sera were detected by indicator arboviruses whose haemagglutination is known to be strongly inhibited by the native macromolecules. After both treatments, notably manganous chloride/heparin, residual NSI activity was detected in deteriorated samples, particularly with group B arboviruses such as Japanese encephalitis and west Nile viruses but generally less so or not at all with the group A arboviruses employed. Absolutely fresh sera are considered highly desirable for rubella virus HAI assay, and it is suggested that the efficiency of lipoprotein NSI removal regardless of treatment protocol could be monitored in parallel HAI tests using carefully chosen indicator arboviruses. This could be done in conjunction with density gradient centrifugation of doubtful sera should ultracentrifuging facilities be available. The suitability of the monitoring procedure would be dependent to some extent on whether certain arboviruses are known to be endemic in a particular area.

Removal of non-specific serum inhibitors of haemagglutination of rubella virus by treatment with dodecylamine-gel.

The suitability of using dodecylamine-gel for removing the serum non-antibody-like inhibitors of haemagglutination by rubella was studied. Compared with kaolin and MnCl2/heparin treatment this new procedure appears to have a higher specificity since it removes the non-antibody-like inhibitors from serum without affecting the immunoglobulin level significantly. The potential application of this procedure in routine serological analysis for rubella virus infection is discussed.

A note on the use of trypsinized human group O erythrocytes in arbovirus titrations.

Twenty-two different arbovirus antigens derived from five antigenic groups were examined for their ability to agglutinate normal and trypsinized human group O erythrocytes. Trypsinization rendered the cells susceptible to agglutination by 18 viruses,chiefly in the pH 5.85 to 6.2 range, yielding hemagglutinin titers comparable with those obtained with gander cells at their optimal pH.

Serological evidence of Hantaan-related viruses in Hong Kong.

Sera from rats (Rattus norvegicus), domestic animals and man in Hong Kong were studied by immunofluorescent antibody assay and plaque reduction neutralization tests for evidence of infection by Hantaan-related viruses. Antibody probably arising from infection by Seoul or a Seoul-like virus was detected in rat and human sera (and one pig serum), highlighting the potential public health importance of this group of viruses in Hong Kong, and in the region.

Interspecies transmission of influenza viruses: H5N1 virus and a Hong Kong SAR perspective.

This account takes stock of events and involvements, particularly on the avian side of the influenza H5N1 'bird flu' incident in Hong Kong SAR in 1997. It highlights the role of the chicken in the many live poultry markets as the source of the virus for humans. The slaughter of chicken and other poultry across the SAR seemingly averted an influenza pandemic. This perspective from Hong Kong SAR marks the coming-of-age of acceptance of the role of avian hosts as a source of pandemic human influenza viruses and offers the prospect of providing a good baseline for influenza pandemic preparedness in the future. Improved surveillance is the key. This is illustrated through the H9N2 virus which appears to have provided the 'replicating' genes for the H5N1 virus and which has since been isolated in the SAR from poultry, pigs and humans highlighting its propensity for interspecies transmission.

Avian influenza in Hong Kong 1997-2002.

In 1997, a high-pathogenicity H5N1 avian influenza virus caused serious disease in both man and poultry in Hong Kong, China. Eighteen human cases of disease were recorded, six of which were fatal. This unique virus was eliminated through total depopulation of all poultry markets and chicken farms in December 1997. Other outbreaks of high-pathogenicity avian influenza (HPAI) caused by H5N1 viruses occurred in poultry in 2001 and 2002. These H5N1 viruses isolated had different internal gene constellations to those isolated in 1997. No new cases of infection or disease in man due to these or other H5N1 viruses have been reported. This paper provides an overview and chronology of the events in Hong Kong relating to avian influenza, covering the period from March 1997 to March 2002.

Multiple genotypes of nonpathogenic H6N2 influenza viruses isolated from chickens in California.

From February 2000 through September 2001, a limited number of H6N2 influenza viruses were isolated from chickens in California. This report describes the genetic characterization of nine of these H6N2 viruses. All of the viruses analyzed had phylogenetically similar hemagglutinin (HA) and neuraminidase molecules that suggested the viruses shared a recent common ancestor. The analysis of the HA sequence of these viruses with all available H6 viruses from different hosts and locations showed that these genes do not separate into well-defined North American and Eurasian lineages. The neuraminidase genes of the California viruses contain an 18 amino acid deletion, a possible adaptation to growth in chickens. Analysis of the remaining gene segments of the California viruses revealed that three distinct genotypes of H6N2 viruses were present.

Reassortants of H5N1 influenza viruses recently isolated from aquatic poultry in Hong Kong SAR.

The H5N1 virus (H5N1/97) that caused the bird flu incident in Hong Kong in 1997 has not been isolated since the poultry slaughter in late 1997. But the donor of its H5 hemagglutinin gene, Goose/Guangdong/1/96-like (Gs/Gd/96-like) virus, established a distinct lineage and continued to circulate in geese in the area. In 2000, a virus from the Goose/Guangdong/1/96 lineage was isolated for the first time from domestic ducks. Subsequently, it has undergone reassortment, and these novel reassortants now appear to have replaced Gs/Gd/96-like viruses from its reservoir in geese and from ducks. The internal gene constellation is also different from H5N1/97, but these variants have the potential for further reassortment events that may allow the interspecies transmission of the virus.

The impact of a monthly rest day on avian influenza virus isolation rates in retail live poultry markets in Hong Kong.

Retail live poultry markets (LPMs) may act as a reservoir of avian influenza viruses (AIV). In this study we test the hypothesis that a rest day in the LPMs where the stalls are completely emptied of poultry, cleansed, and restocked will reduce the isolation rates of avian influenza viruses. The isolation rate of H9N2 subtype viruses from chicken was significantly lower after the rest day than prior to it, indicating its impact in reducing transmission. In contrast, Newcastle disease virus (NDV) isolation rates appear unaffected by this intervention, possibly reflecting differences in herd immunity or virus transmission dynamics.

An update on avian influenza in Hong Kong 2002.

An outbreak of highly pathogenic avian influenza caused by multiple genotypes of H5N1 virus occurred in Hong Kong, commencing in January 2002. Infection in local chicken farms was preceded by the detection of virus in multiple retail markets and the main poultry wholesale market. The first case of this disease on a local farm was detected on February 1, 2002. By February 9, 2002, 15 farms were infected, and by late March a total of 22 infected farms had been identified. Three main clusters of infected farms were seen, suggesting multiple incursions of virus, and subsequent limited lateral spread to neighboring firms. Control of this disease has been effected through a combination of quarantine, tightening of biosecurity measures, and depopulation of infected and contact farms. About 950,000 birds have been destroyed. Vaccination using a killed H5 vaccine was introduced in April 2002 to farms in one zone where infection has persisted. None of the viruses isolated contained the internal genes found in the 1997 H5N1 virus.

Cost-effectiveness study on influenza prevention in Hong Kong.

INTRODUCTION: Recent studies confirm that influenza vaccination confers health benefits and reduces direct and indirect costs associated with the illness. However, these studies did not examine the situation in southern China, a hypothetical influenza epicentre for the emergence of pandemic influenza viruses. METHODS: Surveillance data were collected in Hong Kong in 1993/94 and used economic model was used to estimate the medical and social costs associated with influenza-like illness (ILI) and to predict the cost-effectiveness of implementing an influenza prevention programme. FINDINGS: The estimated ILI incidence was 110/1000. It was highest in those between 1 and 25 years of age while the rate of hospitalization was highest in the elderly. Influenza occurred throughout the study period, which was a mild influenza year. The model predicted more than 660000 ILI cases in a non-epidemic year, in which influenza B virus predominated, with an average ILI-associated cost of HK$283/case (US$36) and vaccination-associated costs of HK$74 (US$9.50) per vaccinated individual. CONCLUSION: The medical, social and monetary costs of ILI in Hong Kong were not observed to be large when compared with those in more developed countries where there is a clearly defined influenza season and recognized disease burden. From the perspective of a susceptible individual, the vaccine was cost-effective but from the perspective of society it was not, even with the most cost-effective strategy of targeting the elderly. However, if the vaccine were effective in controlling newly emerging and highly virulent strains, targeted vaccination programmes might be highly cost-effective.

Isolation of ortho- and paramyxoviruses from domestic poultry in Hong Kong between November 1977 and October 1978 and comparison with isolations made in the preceding two years.

Regular surveillance of domestic poultry from Hong Kong and southern China for influenza A viruses was conducted in Hong Kong from November 1977 to October 1978. Tracheal or cloacal swabs taken from 2844 ducks, geese and fowls yielded 141 influenza viruses of which 135 were derived from ducks. The isolation rate from ducks was twice as high from the cloaca (12.1 per cent) as from the trachea (5.8 per cent). The overall frequency of isolation was 10.3 per cent and 2.3 per cent for poultry originating from southern China and Hong Kong, respectively. Fourteen of the influenza antigenic combinations recorded in this period of surveillance had been obtained in the previous two years while five were new--H2N2, H2Nav6, Hav5N2, Hav6Nav1 and Hav7Nav4. The number of combinations recorded over three years was 39, the most frequent being Hav4Nav1 which comprised one fifth of the total. All the combinations found in geese and fowl isolates, apart from Hav7Nav6 found in a fowl, have been recorded in duck isolates. Faecal/oral transmission of virus from infected pond water was thought to be an important factor in the high incidence of influenza in ducks. The existence of a large number of combinations of haemagglutinin and neuraminidase antigens and the isolation of two viruses from the cloaca suggest that recombination is occurring in these birds. Reasons for the limited isolation of some antigenic combinations are considered. The paramyxoviruses, Newcastle disease virus and the Hong Kong avian paramyxovirus, were also isolated. Over the three year period the isolation rate of influenza and paramyxoviruses from ducks appeared to be cyclical and seasonal. Influenza was found mainly in the summer and paramyxoviruses in the winter.

Multiple avian influenza infection: selection of a non-avid virus by a heterologous avian host.

Experimental infection of domestic fowl, ducks and geese with an influenza A virus (H7N2) isolated from a domestic duck showed that this virus was apathogenic for these poultry. A second virus (H6N2), also apathogenic and more 'non-avid' than any such isolates previously recognised in surveillance of domestic poultry in Hong Kong, was isolated from one goose after H7N2 shedding had ceased. This goose, in effect, acted as a selective isolation system for the H6N2 virus whose presence in the field isolate could not be detected in spite of multiple passage in embryonated eggs.

Newcastle disease virus surveillance in Hong Kong on local and imported poultry.

Surveillance of apparently healthy ducks, geese and fowl originating in Hong Kong and the People's Republic of China at a poultry dressing plant in Hong Kong yielded 67 isolates of Newcastle disease virus. More than twice as many viruses were isolated from the cloaca than from the trachea. Twelve representative isolates were examined in virulence tests--all six of the fowl isolates and two of five duck isolates behaved as velogenic strains, the other four were lentogenic.