VLA received 155 influenza viruses for confirmatory diagnosis. It also provided consultancy, diagnostic reagents and characterisation of the virus in a number of investigations across the world including Italy, Denmark, The Netherlands, North America and perhaps most significantly many countries in Asia. In the latter there was a considerable profile at international level, enhanced through direct transmission of the virus to humans. Several genes of the H5N1 virus, isolated from poultry in Thailand were found to be distinct from other H5 viruses circulating in the region during the last few years.

Chickens being sold in an Asian street market

VLA also carried out a national serologically based survey for H5 and H7 subtypes of AI on poultry populations as part of a wider EU initiative.

A small survey, looking for influenza A viruses from 50 different species of mainly shore birds was also carried out. One influenza A virus of H9N9 subtype was isolated from a Knot (Calidris canutus).

Research projects focused on improving our understanding of the molecular basis for pathogenicity, (a joint initiative by Defra and the EU) the correlation for interspecies transmission of influenza A viruses from avian to mammalian hosts and the development of improved rapid diagnostic assays specifically for H5 and H7 subtypes.

Genetic analysis was used to identify the changes that occur in AI viruses during host adaptation. After crossing the species barrier from wild aquatic birds to domestic poultry, AI strains can become highly pathogenic (HPAI). The analysis revealed a balance between changes in the haemagglutinin and the neuraminidase glycoproteins of the virus during adaptation which may lead to the emergence of viruses of high pathogenicity. The preliminary validation of a RT-PCR for the detection of influenza virus directly in clinical specimens from suspect disease outbreaks of HPAI was completed, which will complement existing techniques to aid the early diagnosis of such infections that are subject to statutory control.

Newcastle Disease

During 2003, VLA received 312 isolates for identification and confirmatory diagnosis of which 166 were avian paramyxoviruses.

Research studies focused on investigating the potential of avirulent Newcastle disease viruses, which are present in wild birds, to act as progenitor strains and to emerge as virulent viruses. An avirulent virus from a wild duck was adapted to growth in a number of cell culture types and clonal populations of the virus characterised. This should improve our knowledge of the risk factors for viruses in wild waterfowl and live vaccines following interspecies transmission.

Molecular epidemiology of pigeon paramyxovirus (PPMV-1) was carried out to investigate the virus population responsible for the ongoing panzootic in pigeons and other birds. The PPMV-1 virus responsible for ongoing disease in feral, show and racing pigeons over the last two decades has caused many outbreaks of disease in domestic poultry, pheasants, birds of prey, pet birds and some wild birds. This study confirms that this is an evolving strain of NDV that has been isolated in the UK as recently as 2003.

West Nile Virus

VLA has responded to new initiatives from both Defra and the Department of Health to develop uniform testing strategies for WNV surveillance and diagnosis in all host groups. A national reference method (RT-PCR) has been established which targets a conserved gene to detect active viral infection. The internationally recognised Plaque Reduction Neutralisation test has also been established and reference standards determined in conjunction with other UK laboratories including the Health Protection Agency. In collaboration with CSL, VLA has co-ordinated strategies for the detection and control of vector-borne diseases resulting in technology exchange specific to WNV.

Rabies

VLA is the UK National Reference Laboratory for rabies and a Communicable Disease Surveillance and Response World Health Organisation Collaborating Centre (WHO CC) for the characterisation of rabies and rabies-related viruses. VLA holds an extensive collection of rabies virus isolates from different countries including recent additions from Poland, Greenland, Czech Republic, Yugoslavia, Austria, South Africa, Sudan, Tanzania, Zimbabwe, Turkey, Russia and China. This extensive rabies database enables us to better understand rabies virus epidemiology in a global context, carry out phylogenetic analysis and determine the origin of human rabies cases and bat cases in the UK. The archive represents one of the largest global collections of lyssavirus strains (over 1300 isolates) and is considered to be a vital asset to the global rabies community.

Since the human fatality in GB in 2002, surveillance on European Bat Lyssavirus type 2 (EBLV-2) has been enhanced and preliminary data has confirmed a low level of antibodies in one species of British bat, the Daubenton’s bat (Myotis daubentonii). Oral swabs were subjected to reverse-transcriptase PCR (RT-PCR) testing for the presence of EBLV-2 specific RNA, indicative of the presence of virus. None of the Daubenton’s bats from the mFAVN positive pools were found to be RT-PCR positive and hence no evidence of active virus infection was detected.

Daubenton’s bat (Myotis daubentonii)

In Germany, the first case of an EBLV ‘spillover’ from an insectivorous bat into wildlife clearly indicated the role of bats as an important reservoir in the epidemiology of rabies. A rabies suspect stone marten (Martes foina) was sent for routine rabies diagnosis and in collaboration with the Federal Research Centre for Virus Diseases of Animals, Wusterhausen, Germany. VLA obtained sequence data from the first round RT-PCR products and the isolate was confirmed as European Bat Lyssavirus type-1.

Collaborations are vitally important and include:

AFSSA-Nancy, France

• pathogenesis and distribution of EBLVs in the red fox (Vulpes vulpes).

AFSSA-Nancy and Synbiotics Europe, France

• development of an ELISA, approved by OIE as a screen assay for rabies antibodies in dogs

Federal Research Centre for virus diseases of animals, Wusterhausen, Germany

• virulence of EBLVs in a related mustelid (Mustela putorius furo)

Pet Travel Scheme

Serological testing forms part of the UK Pet Travel Scheme and it is performed to strict quality assurance UKAS accreditation. Interestingly, the number of samples submitted to the VLA for testing has remained at similar levels to last year.

Classical Swine Fever

Joint research with the Institute of Animal Health, aimed at understanding the molecular basis of modulation of the host response, is utilising RT-PCR, ELISA, enzyme activity assays and western analysis to probe which of the individual components of these complex host response pathways are being targeted by the virus. Mechanisms by which this virus is able to prevent the host cell from undergoing apoptosis are also being investigated by studying alterations in the host cells transcriptional responses upon virus infection by microarray analysis.
In a complementary approach, the role of individual viral proteins in virus replication and evasion of the host’s immune response is also being investigated by identifying host proteins that interact with specific viral proteins. Identification of these viral and host protein interactions may provide insights into methods that could be exploited for control of this serious and highly contagious disease.

Equine Viral Arteritis (EVA)

A rapid one tube RT-PCR and a RT-PCR TaqMan® assay have been developed to test semen samples for EVA and to determine virus carrier status. Further validation with OIE reference laboratories for EVA, in Canada and Sweden has verified the tests and it is hoped that these methods will be adopted as the primary tests for diagnosis. Over 50 equine samples were received at VLA for EVA isolation and all were found to be negative.
Problems with the current virus neutralisation (VN) test have been experienced and in a joint project with the Animal Health Trust (AHT) results have indicated that there appears to be a significant correlation between vaccination and cytotoxic sera. We have carried out trials on two commercial ELISA kits as a possible alternative to the VN test and the results are being analysed.