Direct evidence of H7N7 avian influenza virus mutation from low to high virulence on a single poultry premises during an outbreak in free range chickens in the UK, 2008.

H5 and H7 subtypes of low pathogenicity avian influenza viruses (LPAIVs) have the potential to evolve into highly pathogenic avian influenza viruses (HPAIVs), causing high mortality in galliforme poultry with substantial economic losses for the poultry industry. This study provides direct evidence of H7N7 LPAIV mutation to HPAIV on a single poultry premises during an outbreak that occurred in June 2008 in free range laying hens in Oxfordshire, UK. We report the first detection of a rare di-basic cleavage site (CS) motif (PEIPKKRGLF), unique to galliformes, that has previously been associated with a LPAIV phenotype. Three distinct HPAIV CS sequences (PEIPKRKKRGLF, PEIPKKKKRGLF and PEIPKKKKKKRGLF) were identified in the infected sheds suggesting molecular evolution at the outbreak premises. Further evidence for H7N7 LPAIV preceding mutation to HPAIV was derived by examining clinical signs, epidemiological descriptions and analysing laboratory results on the timing and proportions of seroconversion and virus shedding at each infected shed on the premises. In addition to describing how the outbreak was diagnosed and managed via statutory laboratory testing, phylogenetic analysis revealed reassortant events during 2006-2008 that suggested likely incursion of a wild bird origin LPAIV precursor to the H7N7 HPAIV outbreak. Identifying a precursor LPAIV is important for understanding the molecular changes and mechanisms involved in the emergence of HPAIV. This information can lead to understanding how and why only some H7 LPAIVs appear to readily mutate to HPAIV.

An Epizootiological Report of the Re-emergence and Spread of a Lineage of Virulent Newcastle Disease Virus into Eastern Europe.

A number of contemporary outbreaks of Newcastle disease (ND) in Israel, Turkey, Georgia and Bulgaria have all been caused by a very similar viruses related to lineage 5a (genotype VIIa). Comparison with published ND virus (NDV) sequences suggests that this virus strain originated in South-East Asia and on introduction has circulated widely in backyard poultry in the Middle East and into Eastern Europe. An intracerebral pathogenicity index of 1.9 was obtained for a representative isolate from Bulgaria. In addition, the International Reference Laboratory for ND has characterized a molecular epidemiologically linked virus that has been reported to have caused disease in well-vaccinated broiler chickens in Pakistan. In the 1990s, another strain from the 5a lineage NDV was introduced into Europe and spread across the continent causing numerous outbreaks up to 1999. Despite improved controls, including good diagnostic tests and widespread vaccination, in commercial poultry, the novel circulating NDV strains described here have been established widely in the region and represent an increased risk for similar disease outbreak events to reoccur within the EU.

Cytokine Expression at Different Stages of Influenza A(H1N1)pdm09 Virus Infection in the Porcine Lung, Using Laser Capture Microdissection.

Pandemic influenza A(H1N1)pdm09 virus has retained its ability to infect swine whilst developing the ability to transmit effectively between humans, thus making the pig a valuable model for studying disease pathogenesis in both species. Lung lesions in pigs caused by infection with influenza A viruses vary in both their severity and distribution with individual lung lobes exhibiting lesions at different stages of infection pathogenic development and disease resolution. Consequently, investigating interactions between the virus and host and their implications for disease pathogenesis can be complicated. Studies were undertaken to investigate the discrete expression of pro- and anti-inflammatory mediators during lung lesion formation in pigs during infection with influenza A(H1N1)pdm09 (A/Hamburg/05/09) virus. Laser capture microdissection was used to identify and select lung lobules containing lesions at different stages of development. Dissected samples were analysed using quantitative RT-PCR to assess pro- and anti-inflammatory cytokine mRNA transcripts. Differential expression of the immune mediators IL-8, IL-10 and IFN-γ was observed depending upon the lesion stage assessed. Upregulation of IFN-γ, IL-8 and IL-10 mRNA was observed in stage 2 lesions, whereas decreased mRNA expression was observed in stage 3 lesions, with IL-8 actively downregulated when compared with controls in both stage 3 and stage 4 lesions. This study highlighted the value of using laser capture microdissection to isolate specific tissue regions and investigate subtle differences in cytokine mRNA expression during lesion development in pigs infected with influenza A(H1N1)pdm09.

Identification of sensitive and specific avian influenza polymerase chain reaction methods through blind ring trials organized in the European Union.

Many different polymerase chain reaction (PCR) protocols have been used for detection and characterization of avian influenza (AI) virus isolates, mainly in research settings. Blind ring trials were conducted to determine the most sensitive and specific AI PCR protocols from a group of six European Union (EU) laboratories. In part 1 of the ring trial the laboratories used their own methods to test a panel of 10 reconstituted anonymized clinical specimens, and the best methods were selected as recommended protocols for part 2, in which 16 RNA specimens were tested. Both panels contained H5, H7, other AI subtypes, and non-AI avian pathogens. Outcomes included verification of 1) generic AI identification by highly sensitive and specific M-gene real-time PCR, and 2) conventional PCRs that were effective for detection and identification of H5 and H7 viruses. The latter included virus pathotyping by amplicon sequencing. The use of recommended protocols resulted in improved results among all six laboratories in part 2, reflecting increased sensitivity and specificity. This included improved H5/H7 identification and pathotyping observed among all laboratories in part 2. Details of these PCR methods are provided. In summary, this study has contributed to the harmonization of AI PCR protocols in EU laboratories and influenced AI laboratory contingency planning following the first European reports of H5N1 highly pathogenic AI during autumn 2005.

Induced increase in virulence of low virulence highly [corrected] pathogenic avian influenza by serial intracerebral passage in chickens.

Two highly pathogenic avian influenza (HPAI) virus clones that met the criteria for high-pathogenicity avian influenza viruses, by possessing a multibasic hemagglutinin (HA) cleavage site, were isolated from an H5N1 outbreak in Norfolk, England, in 1991-92. These two isolates, A/turkey/England/50-92/91 (50-92) and A/turkey/England/87-92/91 (87-92), displayed differences in virulence as determined by intravenous pathogenicity index-3 and -0, respectively. DNA sequencing of these two isolates identified 10 amino acid differences throughout the genome: three in HA and polymerase B2 (PB2) and two in polymerase B1 (PB1) and single mutations in nucleoprotein (NP) and polymerase A (PA). Serial intracerebral passages were performed in 1- or 2-day-old specific pathogen free (SPF) chicks with 87-92. Viruses reisolated from each bird passage displayed increases in intracerebral pathogenicity index values (from 0 to 1.9) and therefore virulence. Reverse transcriptase polymerase chain reaction and DNA sequencing on viruses isolated at each passage displayed nine out of the 10 mutations associated with the higher pathogenic genotype of 50-92, except for the mutation found in NP, which retained the amino acid residue associated with 87-92. Serial passage through 9-day-old SPF embryonated chicken eggs and serial intravenous passage in 6-wk-old birds could not reproduce these results. These results further highlight that nucleotide changes in the genome other than at the HA cleavage site can attenuate the virulence of HPAI viruses.

Recent epidemiology and ecology of influenza A viruses in avian species in Europe and the Middle East.

There have been at least ten distinct outbreaks of LPAI or HPAI in poultry caused by H5 or H7 viruses in the last eight years in Europe and the Middle East. There appears to be an increased occurrence of such episodes consistent with global trends. As a result, surveillance systems have been enhanced to facilitate early detection of infection in poultry, together with active surveillance of wild bird populations. These complementary activities have resulted in the detection of a number of viruses in wild bird populations, including some with high genetic similarity to newly detected viruses in poultry, for example, H7N3 in Italy and H7N7 in the Netherlands. Furthermore, there is evidence for continued circulation of H5 and H7 viruses in wild Anseriformes, thereby presenting a real and current threat for the introduction of viruses to domestic poultry, especially those reared in outdoor production systems. Viruses of H9N2 subtype continue to circulate widely in the Middle East and are associated with significant disease problems in poultry. The epidemiology has the potential to be complicated further by introduction of novel viruses through illegal importation of captive birds, such as was detected with H5N1 in Belgium in 2004. Continual genetic exchange in the avian virus gene pool and independent evolution of all gene segments either within an individual host species or among wild bird hosts suggests that these viruses are not in evolutionary stasis in the natural reservoir.