Richard M. Irvine

Replication, Pathogenesis and Transmission of Pandemic (H1N1) 2009 Virus in Non-Immune Pigs

The declaration of the human influenza A pandemic (H1N1) 2009 (H1N1/09) raised important questions, including origin and host range [1], [2]. Two of the three pandemics in the last century resulted in the spread of virus to pigs (H1N1, 1918; H3N2, 1968) with subsequent independent establishment and evolution within swine worldwide [3]. A key public and veterinary health consideration in the context of the evolving pandemic is whether the H1N1/09 virus could become established in pig populations [4]. We performed an infection and transmission study in pigs with A/California/07/09. In combination, clinical, pathological, modified influenza A matrix gene real time RT-PCR and viral genomic analyses have shown that infection results in the induction of clinical signs, viral pathogenesis restricted to the respiratory tract, infection dynamics consistent with endemic strains of influenza A in pigs, virus transmissibility between pigs and virus-host adaptation events. Our results demonstrate that extant H1N1/09 is fully capable of becoming established in global pig populations. We also show the roles of viral receptor specificity in both transmission and tissue tropism. Remarkably, following direct inoculation of pigs with virus quasispecies differing by amino acid substitutions in the haemagglutinin receptor-binding site, only virus with aspartic acid at position 225 (225D) was detected in nasal secretions of contact infected pigs. In contrast, in lower respiratory tract samples from directly inoculated pigs, with clearly demonstrable pulmonary pathology, there was apparent selection of a virus variant with glycine (225G). These findings provide potential clues to the existence and biological significance of viral receptor-binding variants with 225D and 225G during the 1918 pandemic [5].

Suppression of Avian Influenza Transmission in Genetically Modified Chickens

Transgenic birds expressing a short hairpin RNA that blocks viral polymerase hinder influenza transmission. Infection of chickens with avian influenza virus poses a global threat to both poultry production and human health that is not adequately controlled by vaccination or by biosecurity measures. A novel alternative strategy is to develop chickens that are genetically resistant to infection. We generated transgenic chickens expressing a short-hairpin RNA designed to function as a decoy that inhibits and blocks influenza virus polymerase and hence interferes with virus propagation. Susceptibility to primary challenge with highly pathogenic avian influenza virus and onward transmission dynamics were determined. Although the transgenic birds succumbed to the initial experimental challenge, onward transmission to both transgenic and nontransgenic birds was prevented.

Real time reverse transcription (RRT)-polymerase chain reaction (PCR) methods for detection of pandemic (H1N1) 2009 influenza virus and European swine influenza A virus infections in pigs.

Please cite this paper as: Slomka et al. (2010) Real time reverse transcription (RRT)‐polymerase chain reaction (PCR) methods for detection of pandemic (H1N1) 2009 influenza virus and European swine influenza A virus infections in pigs. Influenza and Other Respiratory Viruses 4(5), 277–293.

First reported incursion of highly pathogenic notifiable avian influenza A H5N1 viruses from clade 2.3.2 into European poultry.

This study reports the first incursion into European poultry of H5N1 highly pathogenic notifiable avian influenza A (HPNAI) viruses from clade 2.3.2 that affected domestic poultry and wild birds in Romania and Bulgaria, respectively. Previous occurrences in Europe of HPNAI H5N1 in these avian populations have involved exclusively viruses from clade 2.2. This represents the most westerly spread of clade 2.3.2 viruses, which have shown an apparently expanding range of geographical dispersal since mid-2009 following confirmation of infections in wild waterfowl species in Mongolia and Eastern Russia. During March 2010, AI infection was suspected at post-mortem examination of two hens from two backyard flocks in Tulcea Country, Romania. HPNAI of H5N1 subtype was confirmed by reverse transcription polymerase chain reaction (RT-PCR). A second outbreak was confirmed 2 weeks later by RT-PCR, affecting all hens from another flock located 55 km east of the first cluster. On the same day, an H5N1 HPNAI virus was detected from a pooled tissue sample collected from a dead Common Buzzard found on the Black Sea coast in Bulgaria. Detailed genetic characterization of the haemagglutinin gene revealed the cleavage site of the isolates to be consistent with viruses of high pathogenicity belonging to clade 2.3.2 of the contemporary Eurasian H5N1 lineage. Viruses from a clade other than 2.2 have apparently spread to wild birds, with potential maintenance and spread through such populations. Whilst the scale of threat posed by the apparent westward spread of the clade 2.3.2 viruses remains uncertain, ongoing vigilance for clinical signs of disease as part of existing passive surveillance frameworks for AI, and the prompt reporting of suspect cases in poultry is advised.

Influenza A (H1N1) infection in pigs

We wish to report the preliminary findings of an experimental study in pigs infected with a strain of the recently emerged influenza A (H1N1) virus associated with the current global epidemic in humans ([Irvine and Brown 2009][1]). The study is funded by the European Commission (DG SANCO) and Defra

Infection dynamics of highly pathogenic avian influenza and virulent avian paramyxovirus type 1 viruses in chickens, turkeys and ducks

A range of virus doses were used to infect 3-week-old chickens, turkeys and ducks intranasally/intraocularly, and infection was confirmed by the detection of virus shedding from the buccal or cloacal route by analysis of swabs collected using real-time reverse transcriptase-polymerase chain reaction assays. The median infectious dose (ID50) and the median lethal dose (LD50) values for two highly pathogenic avian influenza (HPAI) viruses of H5N1 and H7N1 subtypes and one virulent Newcastle disease virus (NDV) were determined for each virus and host combination. For both HPAI viruses, turkeys were >100-fold more susceptible to infection than chickens, while both these hosts were >10-fold more susceptible to H5N1 virus than the H7N1 virus. All infected chickens and turkeys died. Ducks were also much more readily infected with the H5N1 virus (ID50≤101 median embryo infective dose [EID50]) than the H7N1 virus (ID50=104.2 EID50). However, the most notable difference between the two viruses was their virulence for ducks, with a LD50 of 103 EID50 for the H5N1 virus, but no deaths in ducks being attributed to infection with H7N1 virus even at the highest dose (106 EID50). For both HPAI virus infections of ducks, the ID50 was lower than the LD50, indicating that infected birds were able to survive and thus excrete virus over a longer period than chickens and turkeys. The NDV strain used did not appear to establish infection in ducks even at the highest dose used (106 EID50). Some turkeys challenged with 106 EID50, but not other doses, of NDV excreted virus for a number of days (ID50=104.6 EID50), but none died. In marked contrast, chickens were shown to be extremely susceptible to infection and all infected chickens died (ID50/LD50=101.9 EID50).

Outbreak of Newcastle disease due to pigeon paramyxovirus type I in grey partridges (Perdix perdix) in Scotland in October 2006.

In October 2006, following an initially non-statutory disease investigation affecting 12-week-old grey partridges (Perdix perdix), an outbreak of Newcastle disease due to infection with the avian paramyxovirus type 1 virus responsible for the current panzootic in pigeons (PPMV-1) was confirmed in Scotland. Two pens of partridges were affected by signs including loss of condition, diarrhoea, progressive neurological signs and mortality totalling approximately 24 per cent, and laboratory evidence of the infection was obtained only in these groups. The premises had approximately 17,000 poultry including a collection of 375 birds of rare breeds, containing endangered breeds of significant conservation value, which were not culled but subjected to a health monitoring and testing programme. Investigations suggested that a population of feral pigeons living above the affected pens of partridges was the likely source of the outbreak. Laboratory and genetic analyses confirmed that the isolate recovered from the clinically affected partridges was PPMV-1, belonging to genetic lineage 4b. However, the virus could not be isolated from or detected in dead pigeons collected from the affected buildings.

Development and Validation of RT-PCR Tests for the Detection and S1 Genotyping of Infectious Bronchitis Virus and Other Closely Related Gammacoronaviruses Within Clinical Samples

Summary Two tests were developed that allow the detection and genotyping of infectious bronchitis virus (IBV) and other closely related gammacoronaviruses. The first test employs a one‐step, reverse transcription‐polymerase chain reaction (RT‐PCR) assay in which the amplification is monitored in real time using a TaqMan® probe. This real‐time RT‐PCR test was used to examine a panel of field samples and its performance compared to virus isolation in embryonated fowls’ eggs. A total of 323 field samples were tested; 176 samples were positive using the real‐time RT‐PCR method, but only three were positive by virus isolation. Sequencing was used to confirm the positive real‐time RT‐PCR results for a subset of samples. The test is suitable for swabs and post‐mortem samples and has been shown to be highly sensitive and specific. The second test, a genotyping method, was developed for identification of the strain of IBV present in field samples based on nucleotide variations within the gene encoding the S1 subunit of the surface spike (S) glycoprotein. This method was developed to provide a tool to inform vaccination decisions and for ongoing surveillance to detect new and emerging strains of IBV within the UK. The performance of the test was evaluated using laboratory isolates of IBV and field samples. Both tests are suitable for use in a high‐throughput diagnostic laboratory.

Role of Real-Time RT-PCR Platform Technology in the Diagnosis and Management of Notifiable Avian Influenza Outbreaks: Experiences in Great Britain

Abstract Diagnosis and management of avian influenza outbreaks now include the use of validated real-time reverse transcription PCR (RRT-PCR) methods in many countries, including all member states of the European Union. Two outbreaks in poultry of notifiable avian influenza (H5 and H7 subtypes) that occurred in Great Britain during 2007 will serve as examples in which RRT-PCR demonstrated its value in 1) rapid diagnosis and confirmation of disease by sensitive and specific laboratory testing of samples derived from the index cases and 2) high-volume, rapid testing of surveillance samples. The two poultry outbreaks followed the incursion of a H7N2 low-pathogenicity notifiable avian influenza (LPNAI) virus (May–June 2007) and a Eurasian lineage H5N1 highly pathogenic notifiable avian influenza (HPNAI) virus (November 2007). Coupled with the use of high-throughput, robotic RNA extraction methods, a total of approximately 9300 and 20,300 field samples were tested by appropriate, validated RRT-PCR assays during the 4- and 5-wk duration of the H7N2 LPNAI and H5N1 HPNAI outbreaks, respectively. Fundamental features of the validated RRT-PCR assays used included their high degree of sensitivity, specificity, and rapidity, attributes that were invaluable in providing timely and accurate information for notifiable AI outbreak management.

Outbreak of highly pathogenic avian influenza caused by Asian lineage H5N1 virus in turkeys in Great Britain in January 2007.

UNTIL recently, it appeared that the epidemiology of avian influenza consisted of the perpetuation of low pathogenicity avian influenza (LPAI) viruses of all 16 haemagglutinin (H) subtypes in wild birds, where they caused little or no disease and spread on occasion to poultry. Very occasionally, introductions of LPAI viruses of the H5 or H7 subtype into poultry resulted in the mutation of these viruses to virulent viruses that caused highly pathogenic avian influenza (HPAI). This is how the four HPAI outbreaks in poultry in Great Britain between 1959 and 2006 were thought to have occurred. These HPAI outbreaks were one in chickens in Scotland in 1959, caused by virus of H5N1 subtype, and three in turkeys in Norfolk, one in 1963 caused by virus of H7N3 subtype, one in 1979 caused by H7N7 virus, and the most recent in 1991 caused by a virus of H5N1 subtype (Alexander 1992, Alexander and others 1993). Since the mid-1990s, the epidemiology of HPAI has become more complicated. In 1996, the progenitor virus of the Asian lineage H5N1 HPAI virus was reported in geese in Guandong Province, China (Xu and others 1999). This virus and its descendents proceeded to spread to poultry in most countries in south-east Asia, becoming endemic in several of them. Following reports of the spread of the Asian lineage H5N1 HPAI virus to wild birds, the virus was reported in wild birds and poultry throughout Asia, Europe and into Africa (Alexander 2007), probably as a result of movements of infected poultry or poultry products and infected wild birds. There had been two isolated incursions of the Asian lineage H5N1 HPAI virus into Great Britain before 2007. The first occurred in October 2005 in a quarantine facility in England. Statutory postimportation investigations of deaths in captive caged birds, supposedly from Taiwan, showed them to be the result of Asian lineage H5N1 HPAI virus infection (DEFRA 2005). This virus was genetically closest to contemporaneous viruses isolated in China. The second case, in April 2006, involved a dead whooper swan (Cygnus cygnus) found in the sea at Cellardyke, Fife, which proved to be infected (Blissit 2007). This H5N1 virus was genetically closest to viruses isolated from swans and other wild birds in Germany, including those infecting birds on islands in the Baltic Sea. This short communication describes the outbreak of HPAI caused by an Asian lineage H5N1 virus in turkeys in Holton, Suffolk, that began at the end of January 2007, and the characterisation of the causative virus. The outbreak occurred on a large commercial meat turkey site consisting of 24 houses, two of which were empty, stocked with 159,000 birds. There was also a slaughter and meat processing plant adjacent to the site. Suspicion of notifiable disease was reported to DEFRA following a very sudden deterioration in health, with rapidly increasing mortality, of sevento eight-week-old turkeys in only one house (house 10), with an original placement of 7119 day-old poults. Mortality and cull figures for the five days beginning January 29 were eight, 71, 186, 860 and 1580, that is, in this period 38 per cent of the birds originally placed had died or had been culled. The clinical picture was of exceptionally high morbidity, with over 90 per cent of birds sitting down, some with the legs out to one side, some birds showing a fine head tremor, and others were moribund. The flock was very quiet. There was virtually no feeding or drinking, and the birds were uninterested in people. At postmortem examination of typically affected turkeys, there were no specific changes suggestive of avian influenza virus infection. Carcases were generally dehydrated, although there was no macroscopic evidence of striking kidney pathology. Gizzards were small and flabby, and contained more litter than feed; crops were empty. There was no upper respiratory tract involvement, and, grossly, the lungs appeared unremarkable. A few birds showed a mild airsacculitis, mainly of the lower abdominal air sacs. Livers appeared congested. Spleens were enlarged, pale and mottled. There were no haemorrhages in the internal organs or any mucosal surfaces of the respiratory or alimentary tracts. Suspicion of notifiable disease was reached only due to the unexplained high and escalating mortality, and exceptionally high morbidity. At the time of reporting to DEFRA, and up to the time of culling of all the birds on the site, there was no evidence of clinical disease in any other house on the premises. Ten carcases, 17 cloacal swabs and 20 blood samples were submitted from the affected turkey house to the Veterinary Laboratories Agency (VLA) – Weybridge, and examined using the diagnostic methods prescribed in Directive 2005/94/EC and the EU diagnostic manual for avian influenza (CEC 2006a, b). The swabs, tissues and samples from the carcases were tested for the presence of infectious avian influenza virus by inoculation of nineto 10-day-old embryonated specific pathogenfree fowls’ eggs. The samples were also tested by real-time reverse transcriptase-PCR (rRT-PCR) to detect the presence of the influenza A matrix gene (Spackman and others 2002), and any positives were further tested by rRT-PCR to detect H5 or H7 genes (Slomka and others 2007). The rRT-PCR results were positive for H5 and, subsequently, virus was isolated. This was shown to have the HA0 cleavage site amino acid motif PQGERRRKKR*GLF, which is typical of the Asian lineage H5N1 HPAI viruses seen in Europe in 2006. The isolated virus was identified as H5N1 subtype using conventional techniques (Alexander and Spackman 1981) and subjected to an intravenous pathogenicity index test (CEC 2006a, b), in which a maximum value of 3·00 was recorded. Following the decision to cull all the turkeys on the site, 20 oropharyngeal swabs, 20 cloacal swabs and 20 blood samples were submitted to VLA – Weybridge from turkeys in each of the other 21 populated houses. Despite the lack of clinical signs, HPAI H5N1 virus was detected in samples in three further houses by rRT-PCR, and H5N1 HPAI virus isolates were obtained from birds in these three houses. None of the blood samples from the 21 houses was positive for H5 antibodies. Phylogenetic analysis of the nucleotide sequences of the HA1 gene of the viruses isolated showed them to be of the Asian H5N1 HPAI lineage and the Lake Qinghai sublineage, that is, closely related to the viruses isolated from poultry and wild birds in Europe in 2006. The nucleotide sequence of the HA1 gene of a HPAI H5N1 virus isolated from farmed domestic geese in Hungary in January 2007 was identical to the viruses isolated from the Suffolk outbreak. Sequences of the whole genome (13,558 nucleotides) of representative viruses A/turkey/England/250/07 (virus isolated from house 10), A/goose/Hungary/2823/07 (derived from the first reported Hungarian outbreak in January 2007) and Veterinary Record (2007) 161, 100-101