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Equine Influenza Virus

Equine influenza virus is an example from a large group of viruses that naturally infect wild aquatic birds, which can act as a reservoir of virus for transmission into other species. Cross-species transmission is a rare event and is restricted by virus subtype – so that only certain types of influenza viruses are generally found in hosts such as horses, humans and pigs.

The diagram below shows the structure of the influenza A virus. It has a segmented RNA genome, which is coated in protein. On the surface are the two glycoproteins haemagglutinin (HA) and neuraminidase (NA) that are recognized by the host immune system. These proteins are involved in virus entry and exit of host cells and are used to subtype influenza viruses.

Antibodies that we raise to the haemaglutinin glycoprotein protect from future infection by the same virus. They also protect against closely related viruses. However, influenza viruses gradually change with time through a process called antigenic drift. By changing the amino acids in haemagglutin, the virus can escape our immune system. This is the reason why the virus strains included in vaccines, both equine and human, are updated. The diagram below shows the structure of the haemagglutinin molecule, which is made of three HA units. Marked in red are the amino acid changes between the virus that caused an outbreak of equine influenza in 2003, compared with the vaccine strain Newmarket/1/93.

Haemagglutinin molecule showing amino acid changes (marked in red) between the Newmarket/1/93 vaccine strain and the Newmarket 2003 outbreak strain

Equine influenza viruses belong to the H7N7 and H3N8 subtypes, those currently circulating are H3N8. By sequencing the HA gene of isolates that are sent in to the Animal Health Trust, we can follow the evolution of the virus with time. The diagram below shows a phylogenetic tree for equine influenza H3N8 virus from 1963 to the present day, showing the different branches, or sublineages, of the virus. In the late 1980s the virus haemagglutinin sequences diverged into two separate branches, the American and the European sublineages. The American branch has further subdivided to form the ‘variant American’ sublineage and most of the viruses circulating in the UK belong to this branch. These are slightly different to those found in North America, which also belong to this branch.

The test that we use to antigenically characterize equine influenza viruses is routinely used all over the world. It is the haemagglutination–inhibition test and is based on recognition of virus by a panel of reference antibodies. Influenza viruses bind to red blood cells using the haemagglutinin molecule and agglutinate them, a process which is easily seen if virus and red blood cells are mixed together in the correct proportions and plated out in a 96-well plate. By serially diluting specific antibodies and adding these to the virus it is possible to block this interaction and measure how closely the virus is related to the antisera and to previous strains. In the diagram below, the top row of wells shows a strong interaction between virus and antiserum (titre = 256), the bottom row shows a weak interaction (titre = 4)

Antigenic analysis: Haemagglutination-inhibition test.

In addition to the surveillance work on equine influenza virus carried out at the Animal Health Trust, our research interests include development of antigenic maps, cross species transmission to dogs, and pathogenicity. We use reverse genetics to carry out some of our research and are in the process of developing a new system for equine influenza virus.

Equine Influenza Surveillance

The Animal Health Trust has long been renowned for its work on the prevention, diagnosis and treatment of equine infectious diseases. In particular, we have focused on monitoring and surveillance and, with funding from the Horserace Betting Levy Board, we have established a programme for detailed surveillance of equine influenza in the United Kingdom. To enhance the achievements of this programme we have developed a sentinel practice scheme through which equine veterinary practitioners throughout the UK participate and contribute to our overall surveillance endeavours. This scheme is kindly supported by Schering-Plough Animal Health.

Practices which become members of the scheme are supplied with nasopharyngeal swabs and virus transport medium, and are asked to submit swabs taken from horses suspected of having influenza to our diagnostic unit where the samples will be tested free of charge for the presence of equine influenza using our NP ELISA. The results of this test are then reported back to the submitting veterinary practice.

If the sample proves positive, this sets in motion a chain of events which starts with our disease surveillance veterinarians contacting other equine veterinary practices in the area that the virus was isolated, alerting them to the presence of influenza in their region, and offering advice and guidance in an outbreak situation.

The positive sample is then passed on to members of our research team, who attempt to isolate and grow the virus for characterisation. This characterisation is carried out both antigenically, to look at how antibodies raised against particular strains behave towards the new isolate, and genetically to enable the comparison of the virus’s genome against previous strains and the current vaccine strains.

This characterisation allows us to monitor the evolution of the virus and to provide the OIE (World Organisation for Animal Health) with the relevant data to decide whether an update of the vaccine strains is necessary.

The findings from our investigations into the new isolate are reported back to the submitting practice, and members of the scheme are kept up to date by the publication of our frequent newsletters.

Each new isolate is also reported on our surveillance website www.equiflunet.org.uk, which forms a network with other equine influenza diagnostic and research laboratories worldwide, aiding the global surveillance of equine influenza.

The veterinarians in the field, who submit samples to us, play a crucial role in our surveillance of equine influenza and, ultimately, in ensuring that available vaccines contain the most appropriate strains. Veterinary practitioners who are interested in learning more about our surveillance programme, or would like to join the sentinel practice scheme, should e-mail equiflunet@aht.org.uk

Reverse genetics and equine influenza virus

Reverse genetics is an important tool used to study the interaction of the virus with the host cell and the host immune response. Currently, reverse genetics is used to generate vaccines against the influenza virus.

The genetic machinery which allows the multiplication of the influenza virus is split into segments encoding different genes which work together to create the new virus particle. Reverse genetics allows the infection of host cells with different strains of certain segments of the virus and helps investigate the effects of these different segments on the host. One example of reverse genetics in action is in the development of vaccines against the H5N1 avian influenza viruses infecting humans. At the AHT we are developing our own reverse genetics system for equine influenza to help us understand how and why certain strains of the virus are more pathogenic to horses than other strains.

Useful references on reverse genetics:

Enami, M., W. Luytjes, et al. (1990). "Introduction of site-specific mutations into the genome of influenza virus." Proc Natl Acad Sci U S A 87(10): 3802-5.

Fodor, E., L. Devenish, et al. (1999). "Rescue of influenza A virus from recombinant DNA." J Virol 73(11): 9679-82.
Garcia-Sastre, A. and P. Palese (1993). "Genetic manipulation of negative-strand RNA virus genomes." Annu Rev Microbiol 47: 765-90.

Luytjes, W., M. Krystal, et al. (1989). "Amplification, expression, and packaging of foreign gene by influenza virus." Cell 59(6): 1107-13.

Neumann, G., T. Watanabe, et al. (1999). "Generation of influenza A viruses entirely from cloned cDNAs." Proc Natl Acad Sci U S A 96(16): 9345-50.

Neumann, G., T. Watanabe, et al. (2000). "Plasmid-driven formation of influenza virus-like particles." J Virol 74(1): 547-51.

Pleschka, S., R. Jaskunas, et al. (1996). "A plasmid-based reverse genetics system for influenza A virus." J Virol 70(6): 4188-92.