Jill Banks

Recombination Resulting in Virulence Shift in Avian Influenza Outbreak, Chile

Influenza A viruses occur worldwide in wild birds and are occasionally associated with outbreaks in commercial chickens and turkeys. However, avian influenza viruses have not been isolated from wild birds or poultry in South America. A recent outbreak in chickens of H7N3 low pathogenic avian influenza (LPAI) occurred in Chile. One month later, after a sudden increase in deaths, H7N3 highly pathogenic avian influenza (HPAI) virus was isolated. Sequence analysis of all eight genes of the LPAI virus and the HPAI viruses showed minor differences between the viruses except at the hemagglutinin (HA) cleavage site. The LPAI virus had a cleavage site similar to other low pathogenic H7 viruses, but the HPAI isolates had a 30 nucleotide insert. The insertion likely occurred by recombination between the HA and nucleoprotein genes of the LPAI virus, resulting in a virulence shift. Sequence comparison of all eight gene segments showed the Chilean viruses were also distinct from all other avian influenza viruses and represent a distinct South American clade.

A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene

A sequence 375 nucleotides in length, which included the region encoding the cleavage activation site and signal peptide of the fusion protein gene, was determined for 174 isolates of Newcastle disease virus (avianparamyxovirus type 1). These were compared with the sequences of 164 isolates published on GenBank, and the resulting alignment was analysed phylogenetically using maximum likelihood. The results are presented asunrooted phylogenetic trees. Briefly, the isolates divided into six broadly distinct groups (lineages 1 to 6).Lineages 3 and 4 were further subdivided into four sublineages (a to d) and lineage 5 into five lineages (a to e).Considerable genetic heterogeneity was detected within avian paramyxoviruses type 1, which appears to beinfluenced by host, time and geographical origin. It is concluded that by using this dataset it will be possible totype future virus isolates rapidly on the basis of their nucleotide sequence and make inferences about theirorigins.

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].

A review of RT-PCR technologies used in veterinary virology and disease control: sensitive and specific diagnosis of five livestock diseases notifiable to the World Organisation for Animal Health

Real-time, reverse transcription polymerase chain reaction (rRT-PCR) has become one of the most widely used methods in the field of molecular diagnostics and research. The potential of this format to provide sensitive, specific and swift detection and quantification of viral RNAs has made it an indispensable tool for state-of-the-art diagnostics of important human and animal viral pathogens. Integration of these assays into automated liquid handling platforms for nucleic acid extraction increases the rate and standardisation of sample throughput and decreases the potential for cross-contamination. The reliability of these assays can be further enhanced by using internal controls to validate test results. Based on these advantageous characteristics, numerous robust rRT-PCRs systems have been developed and validated for important epizootic diseases of livestock. Here, we review the rRT-PCR assays that have been developed for the detection of five RNA viruses that cause diseases that are notifiable to the World Organisation for Animal Health (OIE), namely: foot-and-mouth disease, classical swine fever, bluetongue disease, avian influenza and Newcastle disease. The performance of these tests for viral diagnostics and disease control and prospects for improved strategies in the future are discussed.

Phylogenetic analysis of H7 haemagglutinin subtype influenza A viruses

Summary. A 945 nucleotide region (bases 76–1020) of the HA1 part of the HA gene was obtained for 31 influenza viruses of H7 subtype isolated primarily from Europe, Asia and Australia over the last 20 years. These were analysed phylogenetically and compared with sequences of the same region from 23 H7 subtype viruses available in Genbank. The overall results showed two geographically distinct lineages of North American and Eurasian viruses with major sublineages of Australian, historical European and equine viruses. Genetically related sublineages and clades within these major groups appeared to reflect geographical and temporal parameters rather than being defined by host avian species. Viruses of high and low virulence shared the same phylogenetic branches, supporting the theory that virulent viruses are not maintained as a separate entity in waterfowl.

Characterisation of an avian influenza A virus isolated from a human – is an intermediate host necessary for the emergence of pandemic influenza viruses?

SummaryThe partial sequencing of the internal and the neuraminidase genes of isolate 268/96 obtained from a woman with conjunctivitis showed all seven to have closest homology with avian influenza viruses. The entire nucleotide sequence of the haemagglutinin gene of 268/96 had close, 98.2%, homology with an H7N7 virus isolated from turkeys in Ireland in 1995. This appears to be the first reported case of isolation of an influenza A virus from a human being infected as a result of direct natural transmission of an avian influenza virus from birds.

Nucleic acid sequence-based amplification methods to detect avian influenza virus

Abstract Infection of poultry with highly pathogenic avian influenza virus (AIV) can be devastating in terms of flock morbidity and mortality, economic loss, and social disruption. The causative agent is confined to certain isolates of influenza A virus subtypes H5 and H7. Due to the potential of direct transfer of avian influenza to humans, continued research into rapid diagnostic tests for influenza is therefore necessary. A nucleic acid sequence-based amplification (NASBA) method was developed to detect a portion of the haemagglutinin gene of avian influenza A virus subtypes H5 and H7 irrespective of lineage. A further NASBA assay, based on the matrix gene, was able to detect examples of all known subtypes (H1–H15) of avian influenza virus. The entire nucleic acid isolation, amplification, and detection procedure was completed within 6h. The dynamic range of the three AIV assays was five to seven orders of magnitude. The assays were sensitive and highly specific, with no cross-reactivity to phylogenetically or clinically relevant viruses. The results of the three AIV NASBA assays correlated with those obtained by viral culture in embryonated fowl’s eggs.

Phylogenetic analysis of influenza A viruses of H9 haemagglutinin subtype.

A 380 nucleotide region (bases 613 to 992) of the HA1 part of the haemagglutinin (H) gene was obtained for 35 influenza viruses of H9 subtype isolated from around the world over the past 33 years. These were analyzed phylogenetically and compared with sequences from 19 H9 subtype viruses available in the Genbank database. These viruses do not show such clear geographical lineages as other subtypes (i.e. H5 or H7) and there is a high degree of variation at the cleavage site of the haemagglutinin. Genetically distinct lineages of H9 viruses have circulated contemporaneously in different locations. Thus, it is likely that the numerous infections of poultry and other birds with H9 subtype influenza viruses during the 1990s originate from separate introductions from feral birds. The observed heterogeneity of these viruses may reflect the gene pool for H9 viruses, which is maintained in shorebirds and gulls (Charadriiformes).

Pathogenesis of highly pathogenic avian influenza A/turkey/Turkey/1/2005 H5N1 in Pekin ducks (Anas platyrhynchos) infected experimentally

Asian H5N1 (hereafter referred to as panzootic H5N1) highly pathogenic avian influenza (HPAI) virus has caused large numbers of deaths in both poultry and wild-bird populations. Recent isolates of this virus have been reported to cause disease and death in commercial ducks, which has not been seen with other HPAI viruses. However, little is known about either the dissemination of this H5N1 within the organs or the cause of death in infected ducks. Nineteen 4-week-old Pekin ducks were infected with 106.7 median egg infectious doses of HPAI A/turkey/Turkey/1/05 (H5N1, clade 2.2) in 0.1ml via the intranasal and intraocular routes. Cloacal and oropharyngeal swabs were taken daily before three animals were selected randomly and killed humanely for postmortem examination, when samples of tissues were taken for real-time reverse transcriptase-polymerase chain reaction, histopathological examination and immunohistochemistry. Clinical signs were first observed 4 days post infection (d.p.i.) and included depression, reluctance to feed, in-coordination and torticollis resulting in the death of all the birds remaining on 5d.p.i. Higher levels of virus shedding were detected from oropharyngeal swabs than from cloacal swabs. Real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry identified peak levels of virus at 2d.p.i. in several organs. In the spleen, lung, kidney, caecal tonsils, breast muscle and thigh muscle the levels were greatly reduced at 3d.p.i. However, the highest viral loads were detected in the heart and brain from 3d.p.i. and coincided with the appearance of clinical signs and death. Our experimental results demonstrate the systemic spread of this HPAI H5N1 virus in Pekin ducks, and the localization of virus in the brain and heart tissue preceding death.

Role for migratory wild birds in the global spread of avian influenza H5N8

Migration of influenza in wild birds Virus surveillance in wild birds could offer an early warning system that, combined with adequate farm hygiene, would lead to effective influenza control in poultry units. The Global Consortium for H5N8 and Related Influenza Viruses found that the H5 segment common to the highly pathogenic avian influenza viruses readily reassorts with other influenza viruses (see the Perspective by Russell). H5 is thus a continual source of new pathogenic variants. These data also show that the H5N8 virus that recently caused serious outbreaks in European and North American poultry farms came from migrant ducks, swans, and geese that meet at their Arctic breeding grounds. Because the virus is so infectious, culling wild birds is not an effective control measure. Science, this issue p. 213; see also p. 174 High pathogenicity avian H5 influenza disperses around the Northern Hemisphere in long-distant migrant geese and ducks. Avian influenza viruses affect both poultry production and public health. A subtype H5N8 (clade 2.3.4.4) virus, following an outbreak in poultry in South Korea in January 2014, rapidly spread worldwide in 2014–2015. Our analysis of H5N8 viral sequences, epidemiological investigations, waterfowl migration, and poultry trade showed that long-distance migratory birds can play a major role in the global spread of avian influenza viruses. Further, we found that the hemagglutinin of clade 2.3.4.4 virus was remarkably promiscuous, creating reassortants with multiple neuraminidase subtypes. Improving our understanding of the circumpolar circulation of avian influenza viruses in migratory waterfowl will help to provide early warning of threats from avian influenza to poultry, and potentially human, health.