Ilaria Capua

Avian influenza: recent developments

This paper reviews the worldwide situation regarding avian influenza infections in poultry from 1997 to March 2004. The increase in the number of primary introductions and the scientific data available on the molecular basis of pathogenicity have generated concerns particularly for legislative purposes and for international trade. This has led to a new proposed definition of ‘avian influenza’ to extend all infections caused by H5 and H7 viruses regardless of their virulence as notifiable diseases, although this has encountered some difficulties in being approved. The paper also reviews the major outbreaks caused by viruses of the H5 or H7 subtype and the control measures applied. The zoonotic aspects of avian influenza, which until 1997 were considered to be of limited relevance in human medicine, are also discussed. The human health implications have now gained importance, both for illness and fatalities that have occurred following natural infection with avian viruses, and for the potential of generating a reassortant virus that could give rise to the next human influenza pandemic.

Replication and transmission of H9N2 influenza viruses in ferrets: evaluation of pandemic potential.

H9N2 avian influenza A viruses are endemic in poultry of many Eurasian countries and have caused repeated human infections in Asia since 1998. To evaluate the potential threat of H9N2 viruses to humans, we investigated the replication and transmission efficiency of H9N2 viruses in the ferret model. Five wild-type (WT) H9N2 viruses, isolated from different avian species from 1988 through 2003, were tested in vivo and found to replicate in ferrets. However these viruses achieved mild peak viral titers in nasal washes when compared to those observed with a human H3N2 virus. Two of these H9N2 viruses transmitted to direct contact ferrets, however no aerosol transmission was detected in the virus displaying the most efficient direct contact transmission. A leucine (Leu) residue at amino acid position 226 in the hemagglutinin (HA) receptor-binding site (RBS), responsible for human virus-like receptor specificity, was found to be important for the transmission of the H9N2 viruses in ferrets. In addition, an H9N2 avian-human reassortant virus, which contains the surface glycoprotein genes from an H9N2 virus and the six internal genes of a human H3N2 virus, showed enhanced replication and efficient transmission to direct contacts. Although no aerosol transmission was observed, the virus replicated in multiple respiratory tissues and induced clinical signs similar to those observed with the parental human H3N2 virus. Our results suggest that the establishment and prevalence of H9N2 viruses in poultry pose a significant threat for humans.

Genome Analysis Linking Recent European and African Influenza (H5N1) Viruses

Although linked, these viruses are distinct from earlier outbreak strains.

Newcastle disease outbreaks in recent years in Western Europe were caused by an old (VI) and a novel genotype (VII)

SummaryNewcastle disease virus (NDV) strains, isolated from outbreaks during epizootics between 1992 and 1996 in Western European countries, were compared by restriction enzyme cleavage site mapping of the fusion (F) protein gene between nucleotides 334 and 1682 and by sequence analysis between nucleotides 47 and 435. Both methods revealed that NDV strains responsible for these epizootics belong to two distinct genotypes. Strains derived from sporadic cases in Denmark, Sweden, Switzerland and Austria were classified into genotype VI [6], the same group which caused outbreaks in the Middle East and Greece in the late 1960’s and in Hungary in the early 1980’s. In contrast, viruses that caused epizootics in Germany, Belgium, The Netherlands, Spain and Italy could be classified into a novel genotype (provisionally termed VII), hitherto undetected in Europe. It is possible that the genotype VII viruses originated in the Far East because they showed a high genetic similarity (97%) to NDV strains isolated from Indonesia in the late 1980’s.

The avian influenza epidemic in Italy, 1999—2000: A review

During 1999, northern Italy has been affected by an epidemic of low pathogenicity avian influenza (LPAI) caused by a virus of the H7N1 subtype. Due to the characteristics of the poultry industry in the area and to the absence of specific legislative tools to eradicate infection, the virus continued to circulate for several months until a highly pathogenic virus of the same subtype emerged. The highly pathogenic virus had caused death, at the time of writing, of over 13 million birds in 3 months. The consequences of the highly pathogenic avian influenza (HPAI) epidemic appear to be devastating for the poultry industry and the social community. Several conditions generated the current situation, including the high density of susceptible animals and the structure of the poultry industry in the infected area. In addition, the circulation of LPAI virus for a number of months inevitably delayed the prompt identification of HPAI and complicated the interpretation of diagnostic results. A reconsideration of current European legislation and a reorganization of the poultry industry are suggested to prevent the occurrence of similar situations in countries of the European Union.

Comparison of three rapid detection systems for type A influenza virus on tracheal swabs of experimentally and naturally infected birds.

The present paper reports of the comparison between three rapid virus detection systems and virus isolation (VI) from pooled tracheal swabs collected from naturally and experimentally infected birds with a low pathogenicity avian influenza virus of the H7N3 subtype. The relative sensitivity, specificity and agreement (K value) were calculated for a commercial antigen capture enzyme immunoassay (AC-EIA) and for two nucleic acid detection tests, a one-step reverse transcriptase-polymerase chain reaction (RT-PCR) and a real-time RT-PCR (RRT-PCR), both targeting the M gene. The results indicate that in experimentally infected turkeys VI was positive from the pooled tracheal swabs collected from day 3 to day 10. One-step RT-PCR was able to detect influenza RNA from samples collected from day 3 to day 12, while RRT-PCR amplified influenza RNA in swabs collected from day 3 to day 15. The AC-EIA test yielded positive results between day 5 and day 10 post-infection. On field samples, the K value between the AC-EIA and VI tests was 0.82. Compared with VI, the relative sensitivity of this test was 88.9% (CI95=85.2–92.6) and the relative specificity was 95.7% (CI95=93.7–97.7). The K value between the RT-PCR and VI tests was 0.88. Compared with virus isolation, the relative sensitivity of the one-step RT-PCR was 95.6% (CI95=93.1–98.0) and the relative specificity was 96.3% (CI95=94.4–98.1). The K value between the RRT-PCR and VI tests was 0.92. Compared with virus isolation, the relative sensitivity and specificity of RRT-PCR was 93.3% (CI95=90.4–96.3) and 98.4% (CI95=97.2–99.6), respectively. Generally speaking, comparison between virus isolation, the AC-EIA test and the two nucleic acid detection methods indicated excellent agreement. Data obtained from both experimental and field study suggest a higher sensitivity of the PCR-based methods compared with the AC-EIA. The economical and practical implications of using one of the rapid tests as an alternative to VI during an avian influenza epidemic are discussed.

Control of Avian Influenza in Poultry

Vaccines that enable detection of field exposure to any AI would be ideal.

H7N1 avian influenza in Italy (1999 to 2000) in intensively reared chickens and turkeys

From the end of March to the beginning of December 1999, an epidemic of low pathogenicity avian influenza (LPAI) affected the industrial poultry population of northern Italy. The virus responsible for the epidemic was subtyped as H7N1 with an intravenous pathogenicity index (IVPI) of 0.0, and a deduced amino acid sequence of the region coding for the cleavage site of the haemagglutinin molecule typical of low pathogenicity viruses. The circulation of the virus in a susceptible population for several months caused the emergence of a highly pathogenic virus with an IVPI of 3.0 and the presence of multiple basic amino acids in the deduced amino acid sequence for the cleavage site of the haemagglutinin molecule. Over 13 million birds were affected by the epidemic and, in the present paper, we report the results of the clinical, virological and histopathological investigations performed on affected chickens and turkeys. Clinical, gross and microscopic lesions caused by LPAI were more severe in turkeys than in chickens, while highly pathogenicity avian influenza (HPAI) caused similar mortality rates in both species. Current European legislation considers LPAI and HPAI as two completely distinct diseases, not requiring any compulsory eradication policy for LPAI but enforcing eradication for HPAI. In the Italian 1999 to 2000 epidemic, LPAI mutated to HPAI in a densely populated area, causing great economic losses. A reconsideration of the current European Union legislation on avian influenza, including LPAI of the H5 and H7 subtypes, could possibly be an aid to avoiding devastating epidemics for the poultry industry.

Evidence of infection by H5N2 highly pathogenic avian influenza viruses in healthy wild waterfowl

The potential existence of a wild bird reservoir for highly pathogenic avian influenza (HPAI) has been recently questioned by the spread and the persisting circulation of H5N1 HPAI viruses, responsible for concurrent outbreaks in migratory and domestic birds over Asia, Europe, and Africa. During a large-scale surveillance programme over Eastern Europe, the Middle East, and Africa, we detected avian influenza viruses of H5N2 subtype with a highly pathogenic (HP) viral genotype in healthy birds of two wild waterfowl species sampled in Nigeria. We monitored the survival and regional movements of one of the infected birds through satellite telemetry, providing a rare evidence of a non-lethal natural infection by an HP viral genotype in wild birds. Phylogenetic analysis of the H5N2 viruses revealed close genetic relationships with H5 viruses of low pathogenicity circulating in Eurasian wild and domestic ducks. In addition, genetic analysis did not reveal known gallinaceous poultry adaptive mutations, suggesting that the emergence of HP strains could have taken place in either wild or domestic ducks or in non-gallinaceous species. The presence of coexisting but genetically distinguishable avian influenza viruses with an HP viral genotype in two cohabiting species of wild waterfowl, with evidence of non-lethal infection at least in one species and without evidence of prior extensive circulation of the virus in domestic poultry, suggest that some strains with a potential high pathogenicity for poultry could be maintained in a community of wild waterfowl.

Evidence for differing evolutionary dynamics of A/H5N1 viruses among countries applying or not applying avian influenza vaccination in poultry

Highly pathogenic avian influenza (HPAI) H5N1 (clade 2.2) was introduced into Egypt in early 2006. Despite the control measures taken, including mass vaccination of poultry, the virus rapidly spread among commercial and backyard flocks. Since the initial outbreaks, the virus in Egypt has evolved into a third order clade (clade 2.2.1) and diverged into antigenically and genetically distinct subclades. To better understand the dynamics of HPAI H5N1 evolution in countries that differ in vaccination policy, we undertook an in-depth analysis of those virus strains circulating in Egypt between 2006 and 2010, and compared countries where vaccination was adopted (Egypt and Indonesia) to those where it was not (Nigeria, Turkey and Thailand). This study incorporated 751 sequences (Egypt n=309, Indonesia n=149, Nigeria n=106, Turkey n=87, Thailand n=100) of the complete haemagglutinin (HA) open reading frame, the major antigenic determinant of influenza A virus. Our analysis revealed that two main Egyptian subclades (termed A and B) have co-circulated in domestic poultry since late 2007 and exhibit different profiles of positively selected codons and rates of nucleotide substitution. The mean evolutionary rate of subclade A H5N1 viruses was 4.07×10(-3) nucleotide substitutions per site, per year (HPD 95%, 3.23-4.91), whereas subclade B possessed a markedly higher substitution rate (8.87×10(-3); 95% HPD 7.0-10.72×10(-3)) and a stronger signature of positive selection. Although the direct association between H5N1 vaccination and virus evolution is difficult to establish, we found evidence for a difference in the evolutionary dynamics of H5N1 viruses among countries where vaccination was or was not adopted. In particular, both evolutionary rates and the number of positively selected sites were higher in virus populations circulating in countries applying avian influenza vaccination for H5N1, compared to viruses circulating in countries which had never used vaccination. We therefore urge a greater consideration of the potential consequences of inadequate vaccination on viral evolution.