Diagnosis of Equine Virus Diseases A report for the Rural Industries Research and Development Corporation By M.J Studdert, C.A.Hartley and E. Drummer Centre for Equine Virology School of Veterinary Science, University of Melbourne May 1999 RIRDC Publication No 99/27 RIRDC Project No UM-23A

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© 1999 Rural Industries Research and Development Corporation. All rights reserved. ISBN 0 642 57852 4 ISSN 1440-6845 Diagnosis of Equine Virus Diseases Publication No. 99/27 Project No. UM-23A The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186.

Researcher Contact Details Professor Michael J. Studdert Centre for Equine Virology School of Veterinary Science The University of Melbourne PARKVILLE VIC 3052 Phone: +61 3 9344 7373 Fax: +61 3 9344 7374 Email: m.studdert@vet.unimelb.edu.au Website:http://www.vet.unimelb.edu.au/research/CEV.html

RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6272 4539 Fax: 02 6272 5877 Email: rirdc@netinfo.com.au Website: http://www.rirdc.gov.au Published in May 1999 Printed on environmentally friendly paper by the AFFA Copy Centre

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Foreword After conducting surveys of the Australian horse industry to identify research needs, the RIRDC Equine Research and Development program has identified a number of key strategies of importance to the Australian horse industry. Of consistent and high priority need was found to be the need to improve the respiratory health of horses through improved diagnostic, treatment and preventative strategies. Viral diseases causing respiratory diseases and abortion are responsible for serious economic losses to the Australian horse industry. Viral respiratory diseases can require many expensive treatments, are responsible for poor performance and many lost training days. All of the viruses known to cause endemic viral diseases in Australia have been isolated and studied at the Centre for Equine Virology. The studies outlined in this report aim to improve the existing diagnostic technologies for these virus diseases by developing a comprehensive set of primers for use in polymerase chain reactions (PCR), which will be able to rapidly and sensitively detect the presence of these viruses in clinical samples. This report also describes work towards the development of a blood test and vaccine for equine rhinovirus type 1, a significant cause of respiratory and systemic illness of horses in Australia. The report, the latest addition to RIRDC’s diverse range of over 250 research publications, forms part of our Equine R&D Program which aims to assist in developing the Australian horse industry and enhance its export potential. Peter Core Managing Director Rural Industries Research and Development Corporation

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Contents Foreword .................................................................................... iii Abbreviations .................................................................................... vi Executive Summary .................................................................................... vii 1. INTRODUCTION 1.1 Diagnosis of equine virus diseases using polymerase chain reaction ..................................................................................... 1 1.2 Improved diagnosis and control of ERhV1 disease ........................ 1 2. OBJECTIVES .................................................................................... 3 3. METHODOLOGY 3.1 Polymerase chain reaction ........................................................ 4 3.2 ERhV1 - construction of ERhV1 cDNA expression cassettes and generation of recombinant baculoviruses .................................. 5 4. RESULTS 4.1 Diagnosis of equine virus diseases by PCR .................................... 7 4.2 Cloning and expression of the capsid proteins of ERhV1 in a baculovirus expression system...................................................... 13 5. DISCUSSION 5.1 Development of diagnostic PCRs for endemic equine virus diseases in Australia......................................................................... 18 5.2 Equine Rhinovirus............................................................................. 18 5.3 Implications....................................................................................... 19 5.4 Recommendations............................................................................ 20 5.5 Communications strategy ................................................................. 20 6. REFERENCES .................................................................................... 21

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Abbreviations bp base pairs cDNA complementary deoxyribonucleic acid CEV Centre for Equine Virology DNA deoxyribonucleic acid EAdV1 equine adenovirus 1 EAdV2 equine adenovirus 2 EAV equine arteritis virus EHV1 equine herpesvirus 1 (equine abortion virus) EHV2 equine herpesvirus 2 EHV3 equine herpesvirus 3 (equine coital exanthema virus) EHV4 equine herpesvirus 4 (equine rhinopneumonitis virus) EHV5 equine herpesvirus 5 EIV equine influenza virus ELISA enzyme linked immunosorbent assay ERhV1 equine rhinovirus 1 FMDV foot-and-mouth-disease virus gG glycoprotein G kbp kilobase pairs kDa kilodaltons MCS mulitple cloning site PCR polymerase chain reaction PCR-RFLP polymerase chains reaction linked restriction fragment length polymorphisms RNA ribonucleic acid RT-PCR reverse transcription linked polymerase chain reaction SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis SFM serum free medium TCID50 50% tissue culture infectious dose VLP virus-like particles VP1-4 virus proteins 1-4 (equine rhinovirus 1 structural proteins)

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Executive Summary The RIRDC Equine Research and Development program has identified a number of key strategies of importance to the development of the Australian horse industry. Of consistent and high priority need was found to be the need to improve the respiratory health of horses through improved diagnostic, treatment and preventative strategies. It is suggested that 50% of all calls veterinarians make to horses in racing are for viral respiratory disease. These diseases are responsible for many expensive treatments, poor performance and many lost training days. Diagnosis of endemic equine virus diseases is based on isolation of viruses from clinical samples linked to high powered electron microscopy, and a range of blood tests which measure the immune response of an infected animal to an invading virus. These techniques such as virus isolation are relatively slow, since the virus must be allowed time to replicate in cell culture in order to be detected. This can take as little as overnight for some viruses, but up to two weeks for other viruses. Indeed, some viruses cannot be grown in cell culture and therefore cannot be detected using this method. Since its discovery, polymerase chain reaction (PCR) technology has been introduced into many areas of medical and veterinary research. The enormous potential of PCR in diagnostic technology results from the ability of PCR to specifically detect minute amounts of target DNA. PCR involves repeated cycles of enzymatic synthesis of specific DNA sequences to result in an exponential accumulation of a specific DNA product, where the number of target DNA copies doubles every cycle. Thus 20 cycles of PCR yields about a million fold amplification of the original target DNA. The PCR offers several advantages over conventional techniques for the identification of unknown pathogens. It is highly sensitive and has been shown to be over 1000 times more sensitive than virus isolation in cell culture. Once established in the diagnostic laboratory, it is extremely rapid with testing completed within 8 hours. As the techniques are simple, many samples can be processed simultaneously. All of the viruses known to cause endemic viral respiratory diseases in Australia have been isolated and studied at the Centre for Equine Virology (CEV). Improved methods for the diagnosis of equine herpesvirus 1 (EHV1, equine abortion virus), EHV4 (equine rhinopneumonitis virus), EHV3 (equine coital exanthema virus) the equine gammaherpesviruses EHV2 and EHV5, equine adenovirus 1 (EAdV1), EAdV2, equine arteritis virus, equine rhinovirus (ERhV) are required. Equine rhinoviruses types 1-4 (ERhV1-4) are four of several viruses that cause respiratory and systemic disease in horses. Other virus diseases including particularly EHV1 and EHV4 and equine influenza (EIV), which is exotic to Australia, have taken priority in research efforts made around the world to reduce the costs involved in treatment and in the lost training days caused by these viruses. Our success in developing a vaccine for the control of EHV1 and EHV4 (Duvaxyn® EHV1,4; European Community License No. 159614170) and the absence of equine influenza in Australia means that ERhV should be of a high research priority for diagnosis and control.

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Respiratory disease caused by ERhV1 infection is of significant economic importance to the horse industry. In addition, ERhV1 can infect and cause disease in humans, seemingly in those in contact with horses. Currently, serodiagnosis of ERhV1 infection relies on conventional serum neutralisation assays which are time-consuming and costly. There is no vaccine available to prevent ERhV1 infection. It is now possible, using recombinant DNA technology based on the nucleotide sequence of ERhV1, to develop new and rapid serodiagnostic methods and vaccines. The objectives of this study were to improve existing diagnostic procedures by the development of a comprehensive set of PCR tests for all endemic equine virus diseases in Australia, and by the development recombinant DNA technology to engineer antigens for use in a blood test and vaccine for ERhV1. PCR tests have been developed for EHV1, EHV4, EHV3, EHV2, EHV5, ERhV1, EAdV1, EAdV2, equine arteritis virus and equine influenza virus. Of these tests the EHV1, EHV4, EHV2, EHV5 and ERhV1 tests have been implemented into our diagnostic laboratory at the CEV. The remaining tests require further rigorous validation on clinical material before they can be confidently used in this setting. Implementation of these PCR tests in the laboratory has reduced the amount of time required to obtain and report a result once a sample has been received. Furthermore, the use of PCR has enabled the retrospective diagnosis of ERhV1 as the cause of several outbreaks of febrile respiratory illness in horses in which no virus could be isolated. Substantial progress has also been made in the development of recombinant DNA technology to engineer antigens which could be used in a blood test and vaccine for ERhV1. ERhV1 grows only to low levels in cell culture, therefore obtaining sufficient quantities of native virus particles for the production of a blood test or vaccine would be difficult. Therefore DNA encoding the structural (capsid) proteins and some processing enzymes of ERhV1 were engineered into a vector to facilitate expression of unprocessed and fully processed ERhV1 capsid proteins in an insect cell system. This system has been shown for other proteins to enable extremely high levels of expression of recombinant proteins which are structurally very similar to their authentic counterparts. High levels of unprocessed ERhV1 capsid proteins have now been expressed in this system. Further work is currently under way to establish expression of the processed ERhV1 capsid proteins and also to establish their suitability for use as a blood test and vaccine.

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1. Introduction 1.1 Diagnosis of equine virus diseases using polymerase chain reaction. Virus diseases particularly those causing abortion and respiratory diseases are responsible for serious losses to the horse industry in Australia (and worldwide). The Centre for Equine Virology (CEV) plays an important role in the diagnosis of all endemic equine diseases that impact significantly on the horse industry in Australia. The CEV was designated an Australian Equine Reference Laboratory by the Animal Health Committee in 1989 and has since and before that time provided diagnostic support to the industry nationwide. The particular focus of the laboratory has been on equine abortion caused particularly by EHV1 and on equine respiratory disease viruses including EHV1, EHV4, EHV2 and EHV5, EAdV1 and 2 and ERhV1-4. Diagnosis of these virus diseases is based on virus isolation by cell culture linked to electron microscopy, and on a range of serological tests including serum neutralisation, haemagglutination inhibition and ELISA. A comprehensive set of standard viruses and serums is maintained at the CEV and these represent a major and invaluable/irreplaceable resource. Some of the viruses represent the first and only isolates recovered anywhere in the world e.g., EHV5 and EAdV2. Diagnostic techniques based on cell culture systems (virus isolation and serum neutralisation) are particularly time consuming and expensive, and generally require several days until a result can be confirmed and reported. Polymerase chain reaction (PCR) technology is a major technology which is being adopted for the diagnosis of many important medical and veterinary pathogens worldwide (for review see 17 ). PCR can offer several advantages over conventional cell culture based technologies including improved sensitivity of detection, reduced time required for confirmation of results and a potentially higher sample throughput capacity. The purpose of this project is to extend the capacity of the CEV to undertake rapid diagnosis based on PCR. A commitment to this technology by the CEV was underway prior to the commencement of this project, both for research and diagnostic purposes. Maintenance of a diagnostic capacity and its further development is judged a major priority need for the equine industry nationwide. It also provides very direct contact with the industry and a sharp focus and awareness of industry priority needs. 1.2 Improved diagnosis and control of ERhV1 disease. ERhV1 has been isolated from horses with quite severe illness at the CEV. Samples from which the virus was isolated originated from two outbreaks in South Australia, but we have published serological evidence that the virus infects horses nationwide. We believe that this virus and perhaps others of the four serotypes contribute significantly to the cause of respiratory disease and systemic illness in Australian horses. Based on biophysical properties, ERhV1 was classified as belonging to the Rhinovirus genus in the family Picornaviridae. However, subsequent studies indicated that several properties such as its nucleic acid density, base composition and apparent lack of antigenic diversity, differed

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from other members of the Rhinovirus genus. The entire coding region of the genome of our prototype virus ERhV1.393/75 (7500 bases) has been sequenced in our laboratory (12). Sequence analysis of this genome then showed that this virus is not related to members of the Rhinovirus genus as was previously thought, but is most closely related to foot-and-mouth-disease virus (FMDV). This finding is more consistent with the biophysical properties of the virus as well as some aspects of the pathogenesis of ERhV1. Both FMDV and ERhV1 infect via the upper respiratory tract, can cause viraemia, and can be shed in respiratory secretions, as well as in the faeces and urine in some cases for many years after primary infection. ERhV1 however, does not cause vesicular lesions like those associated with FMDV. Picornaviruses contain 60 copies of each of four capsid proteins (VP1 to VP4). These form an icosahedral particle encapsidating a positive-sense, single-stranded RNA genome. Examination of the capsid structure by monoclonal antibody analysis, nucleic acid sequencing of neutralisation-resistant mutants and X-ray crystallography reveal that all three major capsid proteins, VP1, VP2 and VP3, contribute to the antigenicity and immunogenicity of picornaviruses. Additionally, the major immunogenic sites on picornaviruses involve discontinuous epitopes (1, 8, 22). During the assembly of picornavirus structural proteins into mature virus particles, an intermediate capsid is formed which lacks nucleic acid. This is termed a virus-like particle (VLP). VLPs antigenically resemble native virus particles (7, 21, 23) thus, VLPs may induce protective responses similar to those elicited by the mature virions. Of course, as they contain no RNA genome, VLPs are not infectious and inactivation with chemical agents is not required. Recombinant baculoviruses have become increasingly popular vectors for the expression of recombinant proteins. Recombinant proteins have been reported to be abundantly expressed in this system and are usually functionally similar to their authentic counterparts. The Bac-to-Bac baculovirus expression system was used in attempts to produce VLPs and unprocessed capsid proteins of ERhV1. Recombinant proteins produced in this system can be secreted into the medium at high levels and therefore should be easily recovered from infected cells. In addition, insect cells grow well in suspension cultures and can be used in large scale reactors if required. This project seeks to clone the complementary DNA (cDNA) encoding the four structural (capsid) proteins of the virus into a baculovirus expression vector to produce ERhV1 VLPs to evaluate their potential as both an antigen for use in a diagnostic ELISA and for use in a vaccine.

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2. Objectives (i) Develop, expand and refine polymerase chain reactions for the diagnosis of equine virus diseases in Australia. (ii) To clone and express the capsid proteins of equine rhinovirus 1 (ERhV1) in a baculovirus system to provide a blood test (ELISA) and a vaccine.

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3. Methodology 3.1 Polymerase chain reaction PCR methodology is based on repeated cycles of oligonucleotide-directed DNA synthesis of target viral nucleic acid sequences. Specific oligonucleotide primers are designed to be complimentary to their annealing sites on the two different strands of a target double strand DNA sequence. The PCR reaction can be broken down into 3 steps. (i) The target DNA sequence (eg in this case the viral genome in our diagnostic clinical sample) is used as template and the two strands of DNA are separated by heat denaturation. (ii) In an annealing step the PCR primers bind to their complimentary sequences in the target DNA. (iii) A thermostable DNA polymerase then extends the primers and results in new primer binding sites. This cycle of denaturation, annealing and extension is performed 25-35 times and results in the exponential accumulation of a specific DNA fragment. The amplified product is then run in an agarose gel and stained with ethidium bromide to visualise the DNA. The primers are designed such that they are highly specific for the target sequence, a further advantage of PCR. The PCR can be made more sensitive by using nested PCR primers. In this method a first round of amplification is performed with a single primer pair, the amplification product is then transferred to a new tube for a second round of amplification using a second primer pair specific for the internal sequence amplified by the first pair. This technique offers several advantages. First, the sensitivity of the PCR is increased. Second, the internal primer pair verifies the specificity of the first round product. Thirdly, dilution of the first round product also dilutes out inhibitors that might be present in the sample initially. PCR products can also be used to examine restriction fragment length polymorphisms (PCR-RFLPs). The PCR product is cut with restriction endonucleases and the length of the restriction fragments are compared to that of the known amplified sequence. This technique is commonly used to differentiate serotypes of viruses and bacteria. PCR can also be used to expand and detect RNA targets. Table 1 lists the equine virus pathogens that are the focus of this report. Of the 8 pathogens, 3 are RNA viruses. RNA templates can be detected by PCR if the extracted RNA is first converted to DNA by using a retroviral reverse transcriptase. The resulting complimentary DNA is then used as the template in a conventional PCR reaction. PCR primers were designed for the viruses listed in Table 1. At the commencement of this project no sequence information was available for three of these viruses, EAdV1, EAdV2 and EHV3. A necessary adjunct to this project was therefore to undertake sequencing of these viruses in order to design virus-specific primers. The hexon genes of both EAdV1 and 2 were sequenced and PCR primers designed to these regions. In the case of EHV3, sequencing of the glycoprotein G gene was undertaken. This region has proven to be sufficiently divergent between EHV1 and EHV4 for specific diagnosis.

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Table 1. Respiratory pathogens of horses.

Virus Disease Genome Sequence (ref no.)

EHV4 Respiratory disease dsDNA AF030027* EHV1 Respiratory disease, abortion, encephalitis dsDNA (27) EHV3 Coital exanthema, respiratory disease dsDNA AF081188* EHV2 Poor performance dsDNA (26) EHV5 Unknown dsDNA (25) EAdV1 Respiratory disease, diarrhoea dsDNA (18) EAdV2 Respiratory disease, diarrhoea dsDNA (19) EIV Respiratory disease ssRNA (10) ERhV1 Respiratory disease ssRNA (12) EAV Congenital and respiratory disease, abortion ssRNA (6)

* Genbank Accession Numbers PCR primers were designed for each virus using the computer programs Amplify and GeneWorks 2.5 and tested on cell culture grown virus supernatants of reference virus strains (23). Optimal PCR conditions were determined on a log10 dilution series of template virus, known to be strongly positive at the lowest dilution and negative at the highest dilution. A series of reaction were performed to optimise PCR conditions in which variations to the following parameters were performed: annealing and extension time and temperature, concentration of primers, dinucleotide triphosphates (dNTPs), Mg2+ and Taq DNA polymerase. Reactions were performed in DNA polymerase enzyme buffer supplied by the manufacturer. Optimal conditions were chosen as those which produced the most sensitive PCR (ie. amplified specific product from tube containing the least template) based on repeatable production of a distinct band on agarose gel electrophoresis. Sensitivity of PCR reactions were determined on cloned PCR products or on virus supernatants of known concentration. PCRs were also tested on clinical material, where available. PCR conditions are shown in the results section for each of the viruses. 3.2 ERhV1 - construction of ERhV1 cDNA expression cassettes and generation of recombinant baculoviruses. Recombinant baculoviruses have become popular vectors for the expression of heterologous proteins, including those from viruses, fungi, plants and animals (11). Expression of foreign genes is usually driven by the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV), which is transcribed highly during the late stages of infection. The recombinant proteins are often expressed at high levels in cultured insect cells or infected larvae and are often functionally similar to their authentic counterparts. Recently, a rapid and efficient method to generate recombinant baculoviruses, designated as Bac-to-Bac baculovirus expression system (GIBCO-BRL), was developed (14). It is based on site-specific transposition of an expression cassette into a baculovirus shuttle vector (bacmid) propagated in E. coli. The recombinant bacmid replicates in E. coli as a large plasmid and remains infectious when introduced into insect cells. The procedure of site-specific transposition to insert foreign genes into a bacmid propagated in E. coli reduces the time it takes to identify and purify a recombinant virus from 4 to 6 weeks (typical for conventional methods, or slightly less if linearised parental virus DNA is used) to only 7 to 10

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days. Further, plaque purification of recombinant viruses is not required as bacmid DNA is isolated from a single colony of transformed E. coli and then transfected into insect cells (14). Construction of plasmids cDNAs from ERhV1.393/76 were used to construct the recombinant plasmids pP1.2A and pP1.2A.3C. In order to eliminate the L protease coding sequences of ERhV1 and to introduce an ATG codon preceding VP4, mutagenesis was carried out by PCR on the cDNAs, using the VP4F oligonucleotide (including a BamHI site and an ATG codon) which is complementary to the RNA sequence encoding the N-terminal part of VP4, and a RPN oligonucleotide (including a PstI site) which is complementary to the RNA sequence encoding the C-terminal part of 2A. The fragments generated (2451 bp) in the PCR was digested with PstI to make one end sticky with a PstI site and the other end blunt, and then ligated into pUC19 (Pharmacia) digested with SmaI/PstI to produce pP1.2A plasmid. Mutagenesis was also carried out by PCR on the cDNAs, using the 3CF oligonucleotide (including a PstI site and an ATG codon preceding the last two triplets of 3B) and 3CR oligonucleotide (including a HindIII site and a stop codon), to generate a 750 bp 3C fragment followed by the digestion with PstI/HindIII . PstI/HindIII digested 3C fragment was then ligated in to pUC19 cut with PstI/HindIII to obtain p3C plasmid. The pP1.2A.3C was constructed by subcloning the 3C fragment cut with PstI/HindIII into the PstI/HindIII digested pP1.2A in frame with P1.2A fragment downstream. All plasmids were analysed by restriction analysis to test the proper orientation and were sequenced across the junction between ERhV1 cDNA and pUC19 plasmid to ensure inserts were in correct reading frame. Generation of recombinant baculoviruses pBacP1.2A and pBacP1.2A.3C. To construct transfer plasmids pFastP1.2A and pFastP1.2A.3C, P1.2A and P1.2A.3C sequences were removed from pP1.2A and pP1.2A.3C plasmids by partial digestion with BamHI and complete digestion with HindIII , gel isolated and ligated into pFastBac1 digested with BamHI and HindIII . The recombinant pFastBac1 plasmids containing P1.2A or P1.2A.3C were transformed into DH10BAC competent cells. After 48 h incubation at 37˚C, large, white and kanamycin, gentamicin and tetracycline resistant colonies were selected. Using bacmid specific forward and reverse primers, and combinations of a bacmid primer and a viral specific primer PCRs were performed to screen the overnight culture derived from white colonies to confirm the size of inserts in the bacmid DNA. The recombinant baculoviruses containing P1.2A and P1.2A.3C were designated as pBacP1.2A and pBacP1.2A.3C, respectively. Transfections Recombinant bacmid plasmids (pBacP1.2A and pBacP1.2A.3C) were transfected into Sf9 cells using CellFECTIN Reagent (GibcoBRL). For each transfection, 5 x 105 cells were seeded in 35-mm wells of a 6-well plate (Nunc) and allowed to attach overnight. 5 µl (approx. 0.5 µg), 10 µl (approx. 1 µg) and 20 µl (approx. 2 µg) of recombinant bacmid plasmid were added separately to 95 µl, 90 µl and 80 µl of Sf-900 II SFM. 5 µl, 10 µl and 20 µl of CellFECTIN were also added into 95 µl, 90 µl and 80 µl of Sf-900 II SFM. The corresponding DNA and CellFECTIN were then combined to form lipid-DNA complexes followed by incubation for 45 min at room temperature with gentle shaking. The lipid-DNA complexes were diluted to 1 ml with Sf-900 II SFM and added to cells. Cells were incubated for 5 h at 27˚C. The transfection medium was removed and replaced with Sf-900 II SFM. The infected Sf9 cells were harvested 2 to 3 days post infection. Recombinant baculovirus particles was passaged a further two times before analysed for protein expression.

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4. Results 4.1 Diagnosis of equine virus diseases by PCR. PCR for the detection of EHV1 and EHV4 from clinical samples. Semi-nested PCR primers were designed to the glycoprotein B gene of EHV4 (20) and were used to amplify 509 bp and 323 bp products in first and second round PCR, respectively (Figure 1). Similarly, EHV1 PCR primers were designed to the glycoprotein H gene (27) and amplifies a 636 bp and 287 bp product in first and second round PCR, respectively (Figure 1). EHV1 and EHV4 reference strains 438/77 and 405/76 respectively were used as template in PCR reactions. Lane 1 2 3 4 5 6

Figure 1. Ethidium bromide stained agarose gel showing 1st (lanes 2 and 4) and 2nd round (lanes 3 and 5) products of EHV1 and EHV4 specific PCRs respectively. Size markers (pUC18/HaeIII) are shown in lanes 1 and 6. The following conditions were chosen as optimal based on repeated production of a distinct band on agarose gel electrophoresis after PCR amplification. DNA samples were amplified in 25µl reactions containing 1.5 mM MgCl2, 200µM each dNTP, 1.0µM each primer and 0.5U Taq DNA polymerase (Promega) and 5µl of sample. Amplification was performed in a thermal cycler (Hybaid) under the following conditions; 1 DNA denaturation cycle at 95°C for 5 min followed by 35 cycles of 30 s denaturation at 95°C, 30 s annealing at 60°C and 1 min extension at 72°C with an additional 5 min incubation at 72°C to complete all extensions. For the nested round of amplification, 0.5µl of the amplified PCR product from the first round was used as template under identical reaction conditions, except that the reverse nested primers replaced the reverse primers of the first round reaction. The EHV4 PCR amplified 5 diverse laboratory isolates of EHV4 but not EHV1. The PCR has been optimised to work on clinical samples. Nasal swabs collected from 43 horses showing signs of respiratory disease from which no virus could be isolated by conventional cell culture methods were tested using the PCR. Of these 43, 2 were found to be positive for EHV4 DNA by PCR. By comparison with conventional cell culture techniques the EHV4 PCR was found to be approximately 100 fold more sensitive than cell culture and is capable of detecting between 10 and 100 virus particles. The PCR is faster, more sensitive and comparable to the cost of cell culture isolation of EHV4. The EHV1 PCR is capable of amplifying EHV1 electropherotypes 1P and 1B as well neurological isolates of EHV1. The PCR does not amplify EHV4 DNA. The EHV1 PCR was found to be approximately 10 fold more sensitive than cell culture and is capable of detecting between 100 and 1000 virus particles. EHV1 PCR was found to effectively detect EHV1 DNA in foetal tissue homogenates without any further sample preparation than is required for virus isolation. The performance of the PCR was assessed using a collection of tissue samples from aborted foetuses that were confirmed EHV1 abortions by cell culture and the results are shown in Table 2.

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Table 2: Detection of EHV1 in diagnostic aborted foetal tissue samples by PCR and virus isolation.

Total Sample Number PCR/Virus Isolation +/+ +/- -/+ -/-

71 20*/20 1†/0 0/0 50/50 * 18 samples PCR positive at 10-2 dilution of homogenate and 2 samples PCR positive at 10-1 dilution of homogenate only. † PCR positive at 10-1 dilution only. Sample obtained from perinatal foal death. The size of the amplified products in first and second round PCR can be distinguished such that PCR for EHV1 and EHV4 can be performed simultaneously (multiplex PCR). There is no decrease in the sensitivity of the PCR in the multiplex format and it is more economical than performing PCR for both viruses separately. EHV2 and EHV5 PCR. A type specific PCR has been developed for the detection of EHV2 or EHV5 DNA in buffy coat cells or nasal washing’s from horses. The EHV2 and EHV5 PCR utilises primers specific to the glycoprotein B gene and the thymidine kinase gene, respectively. The PCR uses two rounds of amplification of target material to enhance the sensitivity and specificity. Following the first round of amplification, the EHV2 and EHV5 specific primers can detect approximately 10,000 genome copies. The sensitivity of the EHV2 PCR is increased 100-fold after a second round of amplification to detect 100 genome copies. There is no cross amplification of EHV5 DNA confirming the specificity of the PCR. Buffy coat cells containing leukocytes can be used as template for the reactions. To eliminate the effect of inhibition of the PCR reaction by red blood cells in the buffy coat, the cells are lysed overnight at 56°C in buffer containing detergent and proteinase. Alternatively, nasal washing’s from horses collected in minimal essential medium can be used directly in the PCR. A multiplex PCR can also be used to detect horses infected with either virus in the one PCR reaction without altering the sensitivity of detection of EHV5 but reducing the sensitivity for detection of EHV2 approximately 100-fold. The sensitivity can be enhanced by hybridisation of the amplified DNA to a radioactively labelled probe to the levels observed for single PCR reactions. Subsequent to the development and validation of these PCRs, a separate study in our laboratory undertook sequencing of the gB gene of three divergent EHV2 strains for comparison to the sequence of the prototype strain EHV2.86/67 (9). Alignment of these gB sequences revealed that the EHV2 gB primers designed for the diagnostic PCR contained several 3’ mismatches in the three divergent strains. These EHV2 diagnostic primers were subsequently shown to be unable to amplify a product from several of these strains. A new set of oligonucleotide primers were therefore designed to conserved regions of EHV2 gB (first and second round primers), and a separate set of primers were designed to EHV5 gB (first round primers only). The EHV2 gB primers were found to amplify 12 genomically divergent strains of EHV2 and did not amplify a product from EHV5 (Fig 2A). The first round PCR was able 10,000 template molecules of DNA, while the second round PCR improved the sensitivity 100-fold. Similarly the new EHV5 gB

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primers were found to amplify all three available Australian isolates of EHV5 and did not amplify EHV2 (Fig 2B). The EHV5 PCR could detect between 10 and 100 virus particles.

Figure 2. Ethidium bromide stained agarose gels showing amplification of (A) 1st (444 bp) and 2nd round (165 bp) PCR products of EHV2 specific PCR against 13 EHV2 strains (1-13) and a strain of EHV5 as a negative control, and (B) PCR products (293 bp) of the EHV5 specific PCR against three strains of EHV5. EHV2 is included as a negative control. M represents base pair size markers pUC18/HaeIII and ‘-’ represents the negative (H2O) control. Sequence determination of EHV3 glycoprotein G (gG) and development of a diagnostic PCR. Since no sequence data for EHV3 had previously been known, the complete nucleotide sequence of the EHV3 gG gene was determined in order to design EHV3 specific primers. The gG gene was chosen as the target for the diagnostic PCR since the gG homologue of EHV1 and EHV4 has proven to be divergent even between these two closely related viruses. Purified EHV3.334/74.SP viral DNA was cut with the restriction endonuclease HindIII and a 2.3 kbp fragment of DNA which mapped to the US region of the genome was cloned into the vector pUC18. Nucleotide sequence analysis of this cloned DNA revealed homology with the gG homologues of other alphaherpesviruses. The gG gene for EHV3 was then completely sequenced and revealed an open reading frame of 1344 base pairs encoding a predicted protein of 448 amino acids. PCR primers were designed which amplified a 337 base pair product from tissue culture supernatant of the prototype (sequenced) strain. These primers were found to be specific for EHV3, detecting a product of 337 base pairs product from all 12 available strains of EHV3 but not from EHV1 and EHV4 (Fig. 3). PCR reaction conditions were optimised in 25µl volumes containing 1.25 mM MgCl2, 200µM each dNTP, 1.0µM each primer, 0.5U Taq DNA polymerase (Promega) and 5µl of sample. The amplification cycles required 1 DNA denaturation cycle at 95°C for 5 min followed by 35 cycles of 30 s denaturation at 95°C, 30 s annealing at 62°C and 30 s extension at 72°C with an additional 5 min incubation at 72°C to complete all extensions. The EHV3 PCR could detect between 100 and 1000 molecules of target DNA.

1st round product

2nd round product

A 1 2 3 4 5 6 7 8 9 10 11 12 13 EHV5 - M

B M 1 2 3 EHV2 -

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Figure 3. Ethidium bromide stained agarose gel showing 337 bp products from EHV3 specific PCR against 8 different strains of EHV3 (1-8). EHV1 (E1), EHV4 (E4) and H2O (-) are included as negative controls. M indicates base pair size marker pUC18/HaeIII. EAdV1 and EAdV2 PCR At the commencement of this project, no sequence information was available for either EAdV1 or EAdV2. In order to design virus specific primers for a diagnostic PCR, the complete nucleotide sequence of the hexon genes of both EAdV1 and EAdV2 was determined. The location of hexon gene on the EAdV1 genome was first determined using Southern blot experiments by cross hybridisation with the hexon gene from human adenovirus type 1. Once located, the hexon gene was then sequenced to reveal a 2,742 base pair open reading frame encoding a predicated protein of 913 amino acids. Similarly, the EAdV2 hexon gene was identified by Southern blot but this time by cross hybridisation with the EAdV1 hexon gene. The EAdV2 hexon gene consists of a 2,712 nucleotide open reading frame encoding a predicted protein of 903 amino acids. The hexon genes of EAdV1 and 2 share 65% amino acid identity. Primers were designed to conserved regions of the hexon gene of each virus. Even though EAdV1 isolates are antigenically indistinguishable, genomic variability is known to occur. Therefore four sets of PCR primers were designed for diagnostic PCR of EAdV1. Only two of the 4 primer sets amplified all EAdV1 isolates collected between 1975 and 1982 at the CEV, and results from one primer set are shown in figure 4A. Primers designed to the EAdV2 hexon gene amplified a 201 base pair product from both Australian isolates of EAdV2, and did not amplify a product from EAdV1 strains (Fig. 4B).

Figure 4. Ethidium bromide stained agarose gel showing amplification products from (A) EAdV1 and (B) EAdV2 PCR. EAdV1 PCR was able to a amplify a 188 bp product from 14 different strains of EAdV1 (1-14), but not from EAdV2 (E2) or from the negative water control (-). Similarly, the EAdV2 PCR was able to amplify a 201 bp product from both isolates of EAdV2 (1 and 2) but not from EAdV1 (E1) or the water control (-). M indicates pUC18/HaeIII base pair size markers.

M 1 2 3 4 5 6 7 8 E1 E4 - M

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 E2 - A

M E1 1 2 -B

11

PCR conditions for both EAdV1 and EAdV2 primers have been optimised to the following conditions; 25µl reaction volumes containing 2.5 mM MgCl2, 200µM each dNTP, 2.0µM each primer and 0.5U Taq DNA polymerase and 5µl of sample. The amplification cycles required 1 DNA denaturation cycle at 95°C for 5 min followed by 35 cycles of 30 s denaturation at 95°C, 30 s annealing at 60°C (EAdV1) or 53°C (EAdV2) and 30 s extension at 72°C with an additional 5 min incubation at 72°C to complete all extensions. The primer sets have also been tested on nasopharyngeal swabs obtained from horses with signs of respiratory disease. EAdV1 was amplified in 5 of 6 horses from an outbreak of respiratory disease but not from two other unaffected horses. A four fold rise in serum neutralising antibody titre to EAdV1 was shown in 2 of the 5 PCR positive horses. Twenty three other nasal swabs collected from horses with signs of respiratory disease were all PCR negative. The EAdV1 and EAdV2 PCRs were used to examine 124 faecal samples for the presence of EAdV DNA. Using the EAdV1 primers set 28 of 124 faecal samples were positive for EAdV1. Forty of the 124 samples were also tested with the EAdV2 primers sets, and six of these samples were found to be positive. One sample positive for EAdV2 was also positive for EAdV1. EAV PCR. The primers for the EAV PCR were designed to the polymerase region of the Bucyrus strain of EAV (6). The RNA is first reverse transcribed to make a cDNA copy of the RNA template using murine molony virus reverse transcriptase. The reverse transcription conditions require 60 min incubation at 37°C of a 25µl reaction mix containing 4µM primers, 0.8mM dNTPs, 30U RNAGuard (RNase inhibitor) and 200U of reverse transcriptase. A single round of PCR is then performed to detect viral cDNA. The reaction mixture occurs in a 100µl volume containing 4µM primers, 190mM dNTPs, 4U Vent (exo-) polymerase and 3µl of the reverse transcription product. The reaction is then thermocycled for 5 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 60°C and 30 s at 72°C, with a final incubation of 5 min at 72°C to complete all extension reactions. Using these primers and conditions, tissue culture grown EAV gave a strongly positive 246 base pair product (Fig. 5) which was not seen when EHV1 or EHV4 were used as templates. When ERhV1 was used as template however, a product of approximately 270 base pairs was produced. This is larger product than that produced from an EAV template and does not hybridise to EAV in a Southern blot. The method is capable of detecting 10 tissue culture infective doses (TCID50)/ml of semen. The PCR was applied to semen collected from 7 Standardbred stallions from Victoria, none of which were EAV positive by PCR. This method requires further development in order to reduce the apparent cross amplification of ERhV1.

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Figure 5. Ethidium bromide stained gel showing products after RT-PCR amplification using EAV primers on RNA extracted from virus culture supernatants of EAV (EA), ERhV1 (ERh), EHV1 (E1) and EHV4 (E4). The negative water control (-) and ΦX/HaeIII size markers (M) are shown. Equine Influenza virus (EIV) PCR. PCR primers to detect the nucleoprotein gene of EIV have been designed. The PCR first utilises a reverse transcriptase reaction to make a cDNA copy of the nucleoprotein (NP) gene from which cDNA can be used as template for PCR. The primers were designed to highly conserved regions of the equine influenza NP gene, such that they also amplify human subtypes of type A viruses. The PCR was optimised in the laboratory using a human strain of influenza virus named X-31 (subtype H3N2). The PCR is highly sensitive and rapid and should be capable of amplifying equine subtypes of virus based on sequence analysis. However, access to equine influenza virus can only be gained via the Australian Animal Health Laboratories (AAHL) in Geelong, Victoria. Further work to refine the PCR on EIV needs to be performed to confirm its applicability to equine subtypes. No access to clinical material is possible in Australia outside AAHL. ERhV1 PCR The complete genome of equine rhinovirus serotype 1 (ERhV1) polyprotein was determined and published (12). The genome is 7.2kb and shows closest homology to Aphthoviruses of which FMDV is the sole member. Primers that span the VP1 and part of the 2A region of ERhV1 were designed and have been tested on nasopharyngeal swabs collected from 20 horses in two separate outbreaks of ERhV1 disease. The PCR results showed that all of the twenty horses suffering severe febrile respiratory disease had ERhV1 and this was confirmed by serum neutralisation tests on paired sera. Interestingly, the virus was not able to be cultivated from any of these swabs. This prompted us to perform a retrospective analysis of nasopharyngeal swabs collected from 9 other outbreaks of respiratory disease from horses displaying similar symptoms but from which no virus could be cultivated. Evidence for the involvement of ERhV1 was found by PCR in at least 2 of these outbreaks. We have concluded that the importance of ERhV1 as a pathogen has been underestimated due to the difficulties in cultivation on monolayer equine cell cultures. These findings form the basis of a scientific publication in a peer reviewed journal (13). Members of the Picornaviridae family are known to contain a large numbers of different serotypes and strains. In particular, the region of the genome encoding the structural capsid proteins of the virus are known to contain a high degree of genomic and antigenic variation. It is of critical importance therefore that any ERhV1 specific RT-PCR is able to amplify all available strains, otherwise such a PCR based diagnosis may overlook the presence of ERhV1. For this reason, the primers used above and 5 further sets of PCR primers were designed and tested against all available ERhV1 strains available in our laboratory. The primers were designed over a number

M - EA ERh E1 E4

13

of regions of the genome, including VP1, VP3, 2A, 2B and 3D. These primer sets were specific for ERhV1 and did not amplify ERhV2, but showed varying ability to amplify product from each of the ERhV1 strains. One ERhV1 strain (1331/5/96) was consistently negative in each RT-PCR reaction, despite the presence of high virus titres in the samples tested. This sample was again confirmed by immunofluorescence to be ERhV1. Primers designed by Dr R.W. Renshaw, College of Veterinary Medicine, Cornell University USA, complementary to the untranslated and leader region (UTR/L) of the genome were subsequently found to amplify a 261 bp product from all strains, including ERhV1.1331/5/96 (Fig. 6).

Figure 6. Ethidium bromide stained gel showing RT-PCR products amplified by ERhV1 specific primers on total RNA extracted from Vero cells infected with 7 different ERhV1 isolates (1-7) and ERhV2 (E2). An uninfected cell control (UI) and negative water controls from the reverse transcription (R-) and PCR (P-) step were also included. pUC18/HaeIII base pair size marker are shown (M). The RT-PCR was optimised with these primers to the following conditions; reverse transcription for production of cDNA occurs at 42°C for 50 minutes in 20µl volumes containing 1µM reverse primer, 2µl RNA template, 10µM dithiothreitol, 1 mM dNTPs and 100U Superscript II reverse transcriptase in Superscript II buffer. The PCR conditions required 25µl reaction volume containing 3 mM MgCl2, 1.2µM forward and reverse primers, 0.1µl cDNA (from RT reaction), 200µM dNTPs and 0.2U Taq DNA polymerase. The amplification cycles required 1 DNA denaturation cycle at 95°C for 5 min followed by 35 cycles of 30 s denaturation at 95°C, 30 s annealing at 53°C and 45 s extension at 72°C with an additional 5 min incubation at 72°C to complete all extensions. 4.2 Cloning and expression of the capsid proteins of ERhV1 in a baculovirus system. Construction of ERhV1 cDNA cassettes ERhV1 cDNA cassettes encoding the complete structural protein precursor P1.2A, and P1.2A.3C were derived from ERhV1.393/76 cDNA libraries and constructed as described in the methodology section. The sequencing of the first and last 300 bp of P1.2A and P1.2A.3C and restriction enzyme maps showed that the inserts were present in the correct orientation and did not detect any mutations. In these two plasmids, the last triplet of the L protein was included (adjacent to the N-terminus of VP4) and a new initiation codon was introduced preceding this triplet. The N-terminal 11 amino acids of 2B was included at the 3' end of P1.2A in both constructs. Also, the C-terminal 4 amino acids of 3B (a new initiation codon was introduced preceding this fragment) and the N-terminal 34 amino acids of 3D was included at the 5' end and 3' end of 3C, respectively. The ERhV1 cassettes in pP1.2A and pP1.2A.3C were subcloned into the donor plasmid pFastbac1 downstream of the polyhedrin promoter to give the vectors pFastP1.2A and pFastP1.2A.3C, respectively.

M 1 2 3 4 5 6 7 E2 UI R- P- M

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Isolation of recombinant bacmids pBacP1.2A and pBacP1.2A.3C Using site-specific transposition, expression cassettes containing P1.2A and P1.2A.3C were inserted into the bacmids and propagated in E. coli to obtain recombinant bacmids pBacP1.2A and pBacP1.2A.3C. White, gentamicin, tetracycline and kanamycin resistant colonies of pBacP1.2A and pBacP1.2A.3C were selected. PCR was used to screen white colonies to confirm the proper insertion of the ERhV1 cassettes into the bacmid. Using the bacmid reverse primer and the internal ERhV1 specific primer VP3F (located toward the 3' end of VP3), a 1135 bp fragment (835 bp sequence of VP1 and 2A of P1.2A insert plus 300 bp sequence of bacmid) and 1900 bp fragment (1600 bp sequence of VP1, 2A and 3C of P1.2A.3C insert plus 300 bp sequence of bacmid) were amplified from white, gentamicin, tetracycline and kanamycin resistant colonies individually containing pBacP1.2A and pBacP1.2A.3C. These results indicate that P1.2A and P1.2A.3C inserts were correctly transposed from pFastP1.2A and pFastP1.2A.3C into the bacmid under the control of the polyhedrin promoter. In addition, a bacmid forward primer and the internal viral primer R2A (located in the 3' end of 2A) also amplified the expected sizes for both pBacP1.2A and pBacP1.2A.3C. Characterisation of ERhV1 proteins expressed in Sf9 insect cells Transfection of Sf9 cells by pBacP1.2A and pBacP1.2A.3C is described in the methodology section. Recombinant pBacP1.2A and pBacP1.2A.3C were passaged twice in Sf9 cells. At each passage, following 48-96 h incubation, infected Sf9 cell culture monolayers were harvested. Following brief centrifugation, the cellular pellet was treated with phosphate buffered saline containing 0.5% Nonidet P40. Lysates were analysed under reducing conditions on SDS-PAGE and subjected to Western blotting. As shown in Figure 7, following transfection a protein of approximately 83 kDa was detected in Sf9 cells transfected with 0.5µg pBacP1.2A DNA. This protein was also detected after the second passage but not after the first passage (data not shown). Similar results were also found in Sf9 cells transfected with pBacP1.2A.3C in which a 83 kDa protein band was also identified after transfection (Fig. 7). The reason for the absences of the 83 kDa protein (which was presumably present but at levels below the detection limit) either in the interval passages or in subsequent passage, was probably due to differences in inoculum and/or incubation times between the passages. The 83 kDa band was not detected in Sf9 cells transfected with control bacmids containing wild type baculovirus. The 83 kDa protein corresponds to the predicted Mr for unprocessed P1 (capsid proteins) (85 kDa) and 2A (1.6 kDa). The predicted size of the pBacP1.2A.3C proteins in 112 kDa. Therefore, it was concluded that the 83 kDa band represented the unprocessed P1.2A precursor of ERhV1. A protein of 83 kDa, corresponding to P1.2A, is expected from either the pBacP1.2A or pBacP1.2A.3C construct. This was because the 2A protease cleavage site, located between 2A and 2B, was included in both constructs. However, it was also predicted that in the P1.2A.3C construct, P1.2A could be cleaved into individual capsid proteins by the 3C protease but no evidence of cleaved capsid protein was detected (Fig. 7).

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Figure 7. Western blot analysis of ERhV1 proteins expressed in Sf9 insect cells infected with pBac.P1.2A (1) or p.Bac.P1.2A.3C (2) 2 passages after transfection. Proteins contained in crude cell lysates were separated by 15% SDS-PAGE and transferred to polyvinylidine fluoride membrane. ERhV1 proteins were detected with polyclonal mouse antiserum raised to purified ERhV1.393/76 followed by horse radish peroxidase conjugated sheep anti-mouse immunoglobulins. Bound antibodies were then detected by reaction with 3’,3’-diaminobenzidine. A negative control recombinant bacmid (C) was included, along with a purified preparation of ERhV1.393/76 to illustrate the size of capsid proteins VP1 and VP2/3. Comparative analysis of ERhV1 proteins in Sf9 cells transfected with different amounts of pBacP1.2A and pBacP1.2A.3C indicated that either 0.5 µg or 1.0 µg of pBacP1.2A and pBacP1.2A.3C resulted in the expression of ERhV1 protein. Transfection using 2 µg of pBacP1.2A and pBacP1.2A.3C did not produce ERhV1 proteins in Sf9 insect cells probably due to toxicity of the transfection CellFECTIN reagent. When transfected with 1 µg of DNA the 83 kDa band is present in all three passages transfected and infected with pBacP1.2A, directly indicating that recombinant, infectious baculovirus expressing the P1.2A precursor of ERhV1 was produced and the different amount of expression within different passages was probably due to the explanations outlined above. Cleavage of the P1.2A protein by 3C in pBacP1.2A.3C To determine whether cleavage products of the P1.2A precursor processed by 3C were present in cells infected with recombinant pBacP1.2A.3C, a volume of cellular lysate equivalent to approximately 100 times the amount of protein loaded in figure 7 was used in SDS-PAGE and Western-blotting. The clarified supernatants of the infected insect cells (approximately 2 x107) were concentrated by ultra centrifugation at 200,000 x g for 2h at 4˚C and entire pellet was run on SDS-PAGE under reducing conditions and analysed in Western-blotting (data not shown). Apart from the presence of the 83 kDa protein, only in Sf9 cells infected with pBacP1.2A.3C was a protein of approximately around 26 kDa detected in both cellular lysates and pellets from supernatants. Given that these lanes were grossly overloaded and that clearly many proteins are non-specifically detected, this band may represent 3C flanked by small regions of 2B, 3B and 3D which has an expected size of 26 kDa. Bands corresponding to individual capsid proteins were not detected. The baculovirus produced pBacP1.2A and pBacP1.2A.3C capsid proteins do not form VLPs. The uncleaved capsid protein produced in insect cells was examined under electron microscopy. Despite the lack of cleavage, some folding of the precursor can occur and has been described in FMDV. However, we were unable to detect any capsid-like structures for equine rhinovirus. The formation of a capsid like structure for equine rhinovirus may require complete cleavage of the capsid protein.

C 1 2 V

83

27 24

kDa P1.2A

VP1 VP2/3

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Construction of a new series of baculovirus expression cassettes containing myristoylation consensus sequences. Efficiencies of processing of P1.2A by 3C from the products of the pBac.P1.2A.3C constructs, and assembly of VLPs were not as expected. There are four possible reasons as to why cleaved capsid proteins were not observed: (1) the 3C protease is active but does not cleave the P1.2A precursor (or does not efficiently cleave the P1.2A precursor), (2) the P1.2A precursor was cleaved but the individual viral proteins were unstable and were degraded, (3) the 3C protease is not acting as it requires more 3B or 3D flanking sequence and (4) the 3C protease contains a mutation which renders it non-functional. In order to address these possibilities, several new baculovirus expression cassettes were constructed with several modifications. A myristoylation consensus sequence was incorporated at the 5’ end of P1 in order to address (1) and (2) above. Myristoylation of picornavirus capsid proteins has been shown to be critical in the stabilisation of capsid structures and the correct processing and folding of the P1 precursor. It is possible that without a myristoylated P1 precursor 3C may be unable to efficiently process P1 into the structural proteins VP1-4, or that incorrect folding of a non-myristoylated P1 results in a substrate which cannot be recognised by the protease 3C. Secondly, to address (3) and (4) above, the 3C sequence was reamplified from RNA template to form a 967 bp product including 5’ flanking sequence incorporating half of 3A and all of 3B. Reamplification reactions were performed with Superscripts II RNase H- reverse transcriptase. This enzyme has improved proof reading capacity and thereby reduces the chance of RT-induced mutations in 3C. Three new bacmid constructs were prepared (1) pBac.mP1.2A - containing P1 (with myristoylation consensus sequence) and 2A regions, (2) pBac.mP1.2A.3C - containing P1 (with myristoylation consensus sequence), 2A, 3’ 120 bp region of 3A, all of 3B, 3C and the 5’ nucleotides of 3D, and (3) pBac.3C - the 3’ 120 bp region of 3A, all of 3B, 3C and the 5’ nucleotides of 3D. In addition, a fourth construct was prepared in the vector pFastBac Dual. This vector contains two separate polycloning sites under the control of two separate promoters. This construct, pBacDual.mP1.2A/3C contains P1 (with myristoylation consensus sequence) and 2A regions into BamH1/HindIII of multicloning site (MCS) I, and the 3’ 120 bp region of 3A, all of 3B, 3C and the 5’ nucleotides of 3D into Nco1/Nsi1 of MCS II. Transfection conditions were optimised for pBac.mP1.2A, pBac.3C and pBacDual.mP1.2A/3C requiring 20µg of bacmid DNA and 15µl of CellFECTIN reagent per transfection. These plasmids were transfected into insect cells and recombinant baculovirus passaged four times before cell lysates and cell culture supernatant were assayed for the production of ERhV1 viral proteins. 20µl of infected cell lysate, or 20µl of infected cell supernatant were separated by SDS-PAGE and transferred for Western blot. Using polyclonal mouse serum raised against purified ERhV1.393/76, 90 kDa bands were detected in both cell lysate and supernatant preparation of pBac.mP1.2A. These samples contained very high levels of P1 which peaked at 48 hours post infection (Fig. 8). Production of both secreted and cell associated P1 in this system appears to greatly exceed levels of native ERhV1.393/76 obtained by routine cell culture methods, but as yet no final quantitation has been performed. No bands were detected from pBac.3C and pBacDual.mP1.2A/3C infected cells (data not shown). Subsequent sequence analysis has shown the ATG start codon to be one base out of frame for the 3C containing constructs which would result in the lack of expression of 3C both of these clones.

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14799695743

2923

kDa P C P C P C P C PP24 48 72 24 48 72

supernatant cell lysate

Figure 8. Western blot analysis of ERhV1 proteins expressed in Sf9 cell supernatant or cell lysate infected with pBac.mP1.2A (P) or control plasmid (C) at 24, 48 and 72 hours post infection. 20µl of cell supernatant or crude cell lysate were separated on 15% SDS-PAGE and transferred to polyvinylidine difluoride membrane. ERhV1 proteins were detected with polyclonal mouse serum raised to purified ERhV1.393/76, followed by horse radish peroxidase labelled sheep anti-mouse immunoglobulins before reaction with enhanced chemiluminescent substrate and autoradiography.

P1.2A

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5. Discussion 5.1 Development of diagnostic PCRs for endemic equine virus diseases in Australia. The report describes the development and application of diagnostic PCRs for equine virus diseases that are endemic to Australia. Equine virus diseases are known to be the cause of significant economic loss to the industry in Australia and worldwide as a result of expensive treatments, poor performance and many lost training days. The development of these PCR technologies enables significant improvements to existing diagnostic technologies such as virus isolation. Once implemented in the laboratory, PCR has much greater throughput capacity and reduces time required to obtain a result from several days to 8 hours after receipt of sample. Of the PCRs described in this report those for EHV1, EHV4, EHV2, EHV5 and ERhV1 have been implemented into the diagnostic laboratory of the CEV. The PCR assays for EAdV1, EAdV2, EHV3, EAV and equine influenza virus require more rigorous validation by further optimisation and testing on clinical samples before they can be confidently used in this setting. The development of these PCRs is not intended to fully displace traditional virus diagnostic techniques, rather that these should be complementary to existing procedures. The advantages of incorporating PCR as a diagnostic technology was shown for ERhV1. Testing nasal swabs by PCR enabled the diagnosis of ERhV1 as the cause of febrile illness in three outbreaks of disease where no virus could be isolated. Diagnosis was subsequently confirmed by serum neutralisation studies on paired sera. This case illustrates the distinct advantages of PCR technologies over virus isolation for non-cultivable and slow growing viruses such as ERhV1, and also EAdV2, EHV2 and EHV5. It is intended to further refine the PCR technology whereby instead of each virus specific PCR being conducted separately, a single “multiplex” reaction could be used. In multiplex PCR, two or more primer pairs specific for different viruses are included in the same reaction. In this way different multiplex PCRs could be developed to screen for groups of viruses known to cause respiratory disease, abortion or diarrhoea. 5.2 Equine rhinovirus This report describes the construction and preliminary characterisation of the recombinant baculoviruses expressing ERhV1.393/76 proteins. Expression of pBacP1.2A and pBac.mP1.2A in Sf9 insect cells resulted in the synthesis of unprocessed P1.2A precursor of ERhV1. The unprocessed P1.2A of FMDV, either as a protomer or as a more complex structure, has been shown by other workers (24) to display discontinuous epitopes involved in virus neutralisation and which elicited a neutralising antibody response in guinea pigs, suggesting a new approach for the production of FMDV vaccines. Whether the unprocessed P1.2A precursor of ERhV1 elicits a protective immune response remains to be determined. It has been shown for all picornaviruses examined that the amino-terminal glycine of VP4 is myristoylated. Myristoylation is dependent on the consensus sequence (G-X-X-X-S/T) and modification of a single amino acid residue within this sequence at the N terminus will completely abolish the addition of myristate to the P1 precursor of picornaviruses. The blocking of myristoylation of poliovirus VP4 has been shown to inhibit the in vitro processing of the P1

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precursor whereas blocking of the myristoylation site in FMDV does not prevent efficient processing of the P1 precursor but results in less efficient assembly of VLPs (2-5, 15, 16). Furthermore, capsid precursor P1 without N-terminal myristoylation may prevent correct folding which is essential for correct recognition of 3C cleavage sites (24). Thus, the incorrect folding of P1 may render the precursor resistant to cleavage by 3C which results in the less efficient processing and assembly (24). We are yet to be able test our new 3C constructs against the myristoylated P1 protein to determine if myristoylated P1 is the correct substrate for 3C in this system. Further work will involve the reconstruction of the pBac.3C and pBacDual.mP1.2A/3C plasmids repairing the out of frame ATG codon, thereby allowing expression of the 3C in these constructs. Once expressed, 3C can then be tested against the myristoylated P1 precursor proteins. The cassettes constructed to date allow three options for 3C mediated processing of myristoylated P1 - (i) mP1 and 3C can either be transfected into insect cells in the same plasmid under the control of a single (pBac.mP1.2A.3C) or separate (pBacDual.mP1.2A/3C) promoters, (ii) insect cells can be transfected with two plasmids simultaneously (pBac.mP1.2A and pBac.3C), or (iii) insect cells can be separately transfected with pBac.mP1.2A and pBac.3C and resultant expressed proteins subsequently added together to enable 3C processing of mP1.2A. At least one, if not all of these methods should enable the correct processing of the mP1.2A precursor to produce high levels of ERhV1.393/76 antigen which could be tested for suitability as an antigen for diagnostic ELISA and vaccine. Expression of myristoylated P1 by the cassette pBac.mP1.2A appears to have improved the yield of the P1 precursor protein in this system. Transfection of insect cells with this construct results in high levels of expression of cell associated recombinant protein (as was seen in the unmyristoylated constructs) as well as mP1.2A which was secreted into the medium. The ability of mP1.2A to be secreted into the medium will ultimately enable more simple purification techniques than would be required if the protein remained cell associated. Furthermore, the finding that this system results in a higher level of expression of the capsid precursor than can be achieved by routine ERhV1.393/76 virus culture techniques, suggests that this method of obtaining antigen for diagnostic ELISAs or ERhV1 vaccines is worth pursuing further. 5.3 Implications (i) It has been agreed that viral respiratory diseases of horses are a major cause of economic loss to the industry not only in Australia but throughout the world. Viruses are also recognised causes of serious losses from abortion, perinatal foal mortalities and central nervous system disease. For many reasons, including need for complex methods, late collection of samples, difficulty in isolating a particular virus or the non-cultivability of some viruses, slowness in making clinically relevant diagnoses and the lack of suitable tests for detecting an immune response to a particular infection, improvements to existing diagnostic technologies are required. Whilst we have all of the standard methods for virus diagnosis in place, there is clear scope for improvement in diagnostic methods. We have invoked PCR as a common technology for the rapid diagnosis of all endemic equine virus diseases and are using recombinant DNA technologies to make appropriate antigens for rapid antibody (blood) tests for diagnosis. (ii) ERhV1 is a significant cause of acute respiratory disease. A vaccine to control this disease is expected to provide a major benefit. 5.4 Recommendations

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Some of the PCR diagnostic tests such as for EHV1, EHV4, EHV2, EHV5 and ERhV1 are fully developed and are being used for routine diagnosis. Others require further fine tuning and rigorous validation. Refinements in the technology whereby instead of each virus specific PCR being conducted separately, a single 'multiplex' reaction mixture could be used, for example, for all respiratory viruses, for viruses that cause abortion, or diarrhoea. These multiplex reactions are part of ongoing work. While we have achieved high level expression of ERhV1 proteins in the baculovirus system, the ERhV1 diagnostic ELISA and vaccine will require further work. 5.5 Communications Strategy It is expected that RIRDC will communicate salient features of the work to interested parties. The publication record of the project in refereed journals and presentations at national and international meetings provides a primary mode for all new scientific advances. The CEV Annual Report summarises RIRDC and other work. About 1000 copies are circulated each year to key industry people and all specialist equine veterinarians in Australia. A CEV web site is established and is updated as necessary.

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