Project
title / Equine viral arteritis: virulence and immunity
/ MAFF
project code / SE0750

ministry of agriculture, fisheries and food CSG 15

Research and Development

Final Project Report

(Not to be used for LINK projects)

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Project title / Equine viral arteritis: virulence and immunity
MAFF project code / SE0750
Contractor organisation and location / Veterinary Laboratories Agency (Weybridge), Woodham Lane, New Haw, ADDLESTONE, Surrey. KT15 3NB
Total MAFF project costs / £ 259,835
Project start date / 01/04/97 / Project end date / 31/03/02
Executive summary (maximum 2 sides A4)
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CSG 15 (1/00) 3

Project
title / Equine viral arteritis: virulence and immunity
/ MAFF
project code / SE0750

There is no information on why some strains of EAV are more virulent than others. Genetic comparisons of field strains of differing virulence is unrewarding, because virulence markers, which may be very subtle, are masked by other differences that are of no relevance. In order to circumvent this difficulty, it is necessary either to obtain or to generate virus pairs that are very closely related, but which have differences in virulence. Genetic comparison of such pairs can identify the determinants of virulence. Once pinpointed in this way, the markers can be readily looked for in other viruses to assess the generality of their impact. Recent work on a related virus of pigs (PRRS) has demonstrated that virulent and non-virulent variants can be identified within a strain on the basis of plaque size in vitro.

A better understanding of the immune response to infection could lead to improved methods for distinguishing between vaccinated and non-vaccinated seropositive stallions and between carrier and non-carrier seropositive stallions. If such discrimination could be made on the basis of a blood test this would be a tremendous advantage. Very little is known concerning cell-mediated immune responses, but it is likely that killed vaccines of the type used in Britain would not induce the same type of immunity as wild-type infection and, similarly, virus persistence may be linked to a deficit in the immune response. A first step in investigating cell-mediated immunity is to establish in vitro methods for its quantification and to test the methodology on horses experimentally infected with the equine arteritis virus.

The advantages of RT-PCR as a means of detecting viral agents in semen are established. Various assay formats have been developed for EVA and it has been shown that RT-PCR can be more rapid and sensitive than the alternative of virus isolation in cell culture. Our own experience confirms this, in that parallel testing has revealed two stallions from which virus was only detected in semen by RT-PCR. Nevertheless, statutory acceptance of the test has been slow, because of concern that not all genomic variants of EAV may be detected; and because it is difficult to ensure that false positive and false negative results do not occasionally occur. A large and diverse collection of EAV isolates has now been characterised at the genetic level and this offers an opportunity to reassess the specificity of RT-PCR primers used for diagnostic purposes. Additional tools for improving the reliability of RT-PCR have also become available, including novel techniques and an infectious clone of EAV. It is intended to use these to establish a highly reproducible method with a high probability of gaining international acceptance.

This project was supported by matched funding from the European Union (FAIR6-CT98-4123; Key action 5). Specific collaboration was identified between VLA Weybridge and AHT Newmarket, who were also a su-contractor within this ROAME.

The virulent horse passaged “pleural fluid” form of the Bucyrus strain of equine arteritis virus (EAV) was obtained from America and was sub-cloned in vitro to derive a number of homogeneous progeny viruses. Two of these clones which have different plaque sizes (small and large) when grown in cell culture, were inoculated into horses. Both produced clear evidence of disease and of extensive in vivo replication. The most serious illness and profound temperature elevation was seen with the small plaque variant. It is considered that this clone can be used as a representative “virulent” virus. A third clone was derived from the “pleural fluid” virus by extensive passage in a heterologous cell culture system. This “vero adapted” variant is a candidate “avirulent” virus. In the second year, we completed two further experimental horse infection studies. In the first of these, the “vero adapted” virus was inoculated into ponies and shown to be avirulent, causing no illness and replicating to only a very limited extent. In a second experiment, the original “pleural fluid” isolate was inoculated into ponies and its virulence in our challenge model was confirmed. These studies demonstrate that we have successfully generated closely related viruses with striking phenotypic differences. The next stage is to try to relate the phenotypic differences to genotypic markers. To this end the viruses were supplied to partner 1, where the complete genetic sequences of each have been determined.

Determination of the full-length sequence of a virulent derivative of the large plaque (LP) variant, LP3A.1 was initiated and performed by various partners to extend this comparative analysis. Sequencing of products of independent RT-PCR assays were used instead of cloned RT-PCR products (as was done before). P1 prepared and sent the RT-PCR fragments to P2, P3, and P5 for sequencing.

In year one, we compared the plaque sizes of viruses of differing virulence to see if this attribute could correlate to virulence. Similar determinations were carried out for some additional viruses in year two and three, but overall no clear correlation was found. Another possible marker was thought to be rate of in vitro replication, and in year one, one-step growth curves were established for each of our in vitro generated viral variants. In year three again in vitro replication studies were repeated on Bucyrus cloned variants in an attempt to see if there was any difference between low and high virulence cloned viruses. Again, no decisive differences were noted between virulent and avirulent isolates. In year two, we completed these studies by looking at growth efficiency at different temperatures. It was found that all of the viruses analysed grew better at 37ºC than at either 34ºC or 40ºC. There was a suggestion that low virulent viruses grew relatively better at 34ºC than high virulence ones, but more work is needed to confirm a definite association. In conclusion, thus far, a clear in vitro marker for in vivo virulence has not been found.

The different RT-PCR methods described for EAV have been tabulated, and the different methods have been compared in our laboratory. A single, suitable method has been selected for optimisation and the incorporation of a TaqMan fluorogenic probe has given consistent results. The development of a control mimic, followed by a final validation of the TaqMan on different strains of EAV, semen and clinical samples to confirm the chosen method’s reliability has also been completed.

This project has provided DEFRA with excellent added value, since the development of a cytotoxic T cell assay was an opportunistic activity, dependent on horse experiments carried out as part of an EU project. If paid for by DEFRA just for this work, it would have been prohibitively expensive. Additionally, the collaboration of AHT in this work was invaluable and reaped great dividends for both institutes.

CSG 15 (1/00) 3

Project
title / Equine viral arteritis: virulence and immunity
/ MAFF
project code / SE0750
Scientific report (maximum 20 sides A4)
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CSG 15 (1/00) 3

Project
title / Equine viral arteritis: virulence and immunity
/ MAFF
project code / SE0750

The three objectives within the project were as follows:

01 / Identification of virulence determinants in the genetic sequence of EAV by selection of closely related viruses with differing virulence, followed by complete genomic comparison.
02 / Establish an in vitro assay for measurement of the cell-mediated immunity (CMI) to EAV and use it to examine CMI in experimentally infected horses.
03 / Establish and validate a second generation RT-PCR test for detection of EAV in semen, using fluorescent probes to improve specificity, and a mimic system to improve quality control.

Objective 1. Identification of virulence determinants in the genetic sequence of EAV by selection of closely related viruses with differing virulence, followed by complete genomic comparison.

Our strategy towards identification of virulence markers relied on the production of pairs of similar viruses with differing virulence, derived from a virulent precursor virus. We employed two approaches to this end. The first approach utilises a method successfully employed with a related virus, porcine reproductive and respiratory syndrome virus (PRRS), by isolation of large and small plaque variants, so-called because one produces large foci of infected cells in cell culture, whereas the other produces only small foci. The PRRS study showed that large plaque variants are more virulent than small plaque variants. Our second approach to produce related high and low virulence pairs has employed the strategy of passaging a virulent virus several times in a cell type from an abnormal host - African Green Monkey cells, in an attempt to attenuate it.

Methodology

a) Development of method for examination and selection of viral plaques

Plaque purification of virus stocks was required in order to provide a homogeneous viral population for meaningful genotypic and phenotypic comparisons to be made. Secondly it was planned to evaluate differences in plaque morphology and to see if this correlated to viral virulence.

A method of observing EAV plaques has been reported by Hyllseth (1969). Our method was an adaptation of this and of the methods of Park (1996). It involved inoculating 6 well plates of equine embryonic lung cells (EELs) with EAV. After allowing for adsorption, the cells were washed and then over- laid with maintenance medium in a low melting point agarose to facilitate individual plaque growth. After 3 days incubation the plaques were visualised either by neutral red staining (if the plaques were to be picked and a stock grown for further plaquing) or by staining with Crystal violet for permanence prior to measurement and photography.

b) Selection of viral stock from which to derive clones

Most isolates of EAV are not very virulent when inoculated into horses. The most well characterised virulent EAV is the velogenic Bucyrus strain in the form of pleural fluid derived from experimentally infected horses. This is said to cause fatal illness in a proportion of experimentally inoculated animals. A stock of this virus was obtained from Dr McCollum in Kentucky. Our hypothesis is that this will be a mixed population of closely related virus variants from which we will attempt to derive high and low virulence virus pairs. Because such viruses are expected to have few genetic differences, there is a more realistic possibility of determining which of the differences are significant with respect to virulence.

c) Selection of large and small plaque variants

Because it is expected that cell culture passage may attenuate EAV virulence, a minimum number of passages should be used to derive clonal virus sub-populations, and an equine cell culture system should be used.

Description of cloning and selection procedure:

A stock of the velogenic Bucyrus strain of EAV was obtained by inoculating an 800ml flask of EEL cells with the pleural fluid isolate, and after 3 days when a cytopathic effect was observed the virus supernatant

was harvested. The virus stock was divided into two.One half of the stock was aliquoted into 1ml tubes and frozen at -800C and the other half aliquoted into 1ml glass vials and freeze dried.

The stock Bucyrus virus was titrated out and inoculated into a 6 well plate of EEL cells. The virus was adsorbed for 1 hour in 5% CO2, washed and then over- laid with MEM 2% FCS in 1% low melting point agarose. After 3 days the cultures were examined for discrete plaques. The small and large plaques were picked separately and each inoculated separately into 25cm flasks of just confluent EEL cells, to allow for growth of separate stocks of increasingly purer small and large plaque viruses. These stocks were in turn titrated and plaques picked twice more as shown in the diagram below, before final stocks of small and large plaques were cultured for horse challenge experiments. The final stocks were centrifuged, filtered to remove cellular debris and then aliquoted in 1ml amounts for storage at -800C.

d) Selection of culture attenuated variant

Description of selection and subsequent cloning procedure:

The first passage stock of Bucyrus virus above was passaged 20x in Vero cells, 3x in Baby hamster kidney cells (BHK-21) and then a further 6 passages in Vero cells. A final stock was cloned and plaque picked as above, for use in the equine challenge experiments.

e) Sequence analysis of selected clones

Analysis of the large and small plaque variants and the Vero-adapted variant was performed by sequencing the GL gene. The method used was the Perkin Elmer ABI PRISM Dye Terminator Sequencing Ready reaction Kit, with AmpliTaq DNA Polymerase.

f) Growth curves for supernatant and cell-associated viruses

In vitro growth curves were determined in cell cultures for supernatant and cell-associated virus. Viruses tested were the three clones derived from the velogenic Bucyrus virus as described above, the ARVAC vaccine strain, and the Vienna isolate.

For each virus, flasks of EEL cells (25cm2) were inoculated with 1ml of virus (100 TCID50), allowed to adsorb for 1hour, then washed once in MEM without serum and then over-laid with MEM containing 2% FCS. Starting at time 0 and then at mainly 2 hour intervals the flasks were harvested for supernatant and cell associated virus. The cell associated virus was harvested by first collecting the supernatant virus, then the cells were washed twice with MEM and then 2ml of MEM added to the flask before freezing at -80oC.