The molecular epidemiology of equine herpesvirus 1 (equine abortion virus) in Australasia 1975 to 1989 MJ STUDDERT,BS CRABB and N FICORILLI Equine Virus Laboratory, School of Veterinary Science, The University of Melbourne, Parhille, Victoria 3052 SUMMARY:The restriction endonuciease DNA fingerprints of 57 isolates of equine
herpesvirus1(EHV1; equine abortion virus) from abortion, perinatai foal mortalitles and encephalitis from 15 epidemics that occurred InAustralasia between 1975and 1989were examined using the enzymes Barn Hi, EcoRi and Bgl 11. There was a remarkable degree of uniformity Inthe restriction patterns; mobility differences were observed In only 14 of 52 (27%) of the fragments. Twelve of these 14 fragments were located wlthin the repeat structures that bracket the unique short region of the genome or were located at the left terminus of the 150 kilobase pair genome. Based on the Barn Hi fingerprints the commonest virus identifled In our study was EHVl .IF (PIs for prototype strain). There was a single notable exception In that the Barn Hi fingerprints of all 8 isolates from one of 3 Victorian farms that experienced abortion in 1989 resembled a variant EHV1.iB that was identifled as a cause of abortion in Central Kentucky in1970to 1974. We present evidence that EHVl .IB caused abortion in California in 1964and has remained unaltered In Its Barn HI restriction pattern. No antigenic differences were found among 4 distantly related EHVl isolates, including the variant IB, using a panel of 5 monoclonal antibodies to glycoprotein C (gC), a glycoprotein recognised to be highly variable. The uniformity of these unrelated EHVl isolates Is further evidence for a recent origin for EHVl and may help to explain the natural history of this virus in the horse in which It seems to be a cause of serious epidemics of abortlon and perinatai mortality, and less commonly of encephalitis. Aust Vet J: 69 104 - 111
Introduction
Materials and Methods
The horse is somewhat unusual in that it is host to 3 distinct alphaherpesviruses: equine herpesvirus 4 (EHv4; equine rhinopneumonitis virus), EHVl (equine abortion virus) and EHV3 (equine coital exanthema virus). Two of these viruses EHV4 and EHVl were considered until about 1980 the same virus (EHVl) or with considerable ambiguity subtypes of -1. Restriction endonuclease DNA fingerprinting unambiguously established that there were two distinct viruses (Sabine et ul1981; Studdert et ul1981) and led to a proposal to identify them as EHV4 and EHVl (Studdertetd 1981).ThenaturalhistoryofEHV4including latency (Browninget ul 1988) and transmissionfrom generation to generation usually associated with an annual Occurrence of respiratory disease (rhinopneumonitis) in foals conforms well to the natural history of other alphaherpesviruses.For EHV 1 the natural history seems to conform less well with that of other alphaherpesviruses in that it has been difficult to prove latency wington et ul 1985) and a regular, high incidence annual transmission pattern; it is assumed that EHVl is acquired as a respiratory infection leading to latency and that abortion occurs as an unpredictable sequel to reactivation in a carrier horse, although all abortions on a farm (storms), with the possible exception of the index case, appear to occur as an immediate consequence of horizontal transmission from the f i t case of abortion. A similar pattem, though at a much lower incidence, is followedin outbreaks of EHVl encephalitis,which appears to be caused by only certainEHV1strains. There is some evidence that the pattern of disease caused by EHVl may vary around the world. For example, EHVl has not been recovered from outbreaksof respiratory disease in Australia, where such isolates have been invariably EHVA Earlier studies (Allen et ul 1983; Studdert 1983) showed that for naturally occurring EHVl isolates there is considerable uniformity in therestriction endomicleaseDNA fingerprints.We report here an analysis of 57 EHVl isolates from abortions, perinatal foal deaths and encephalitis that occurred in Australia and New Zealand between 1975 and 1989.
The origin and designation of the 57 EHVl isolates are listed (2 to 58) in Table 1. EHV4.405 (virus 1 Table 1) was included for comparative purposes. V i e s were isolated and grown in fiith passage equine foetal kidney (EFK) monolayer cell cultures in which they were passaged 3 to 5 times. Also included in the study was DNA prepared from a virus EHVl.M13/65 that was isolated from an aborted foetus in California in 1964 (Studdert MJ 1964unpublished). A 175cm2plastic flask of EFK cells was inoculated with each virus and when cytopathology was complete the cell culture lysates were clarified by centrifugation at 4OC, 1600g for 10min. Virus was pelleted from the Supernatants by centrifugation at 50 OOO g in an SW28 head fur 1.5 h in a Beckman ultracentrifuge. The pellet was resuspended in a minimum volume and layered onto a 5 to 15% Fico114W gradient as described by Schrag et ul(1989). The viruscontaining band was collected in a fraction collector. DNA was extracted from purified virions using phenol-chloroform followed by ethanol precipitation. DNA was digested with Bum HI, Eco RI and Bgl II according to the manufacturer's recommendations and separated on 0.8% agarose els. Gels were stained with ethidium bromide and photographed . Southern transfers to nylon membranes were performed as described by Sambrwk et ul (1989). The membranes were probed with 32PdCTP-labelled whole viral DNA or with EHV1.25A cloned fragments (Whalley et ul1981; Whalley et id 1989) as indicated in Figure le and f. A panel of 5 monoclonal antibodies (MAbs) to gC was used in an ELISA to see if any antigenic (epitope) differences existed between 4 of the 57 EHVl isolates that were judged to be the most distantly related, that is, viruses 2,28,46 and 55 (Table 1). Two EHV4 isolates, virus 1, Table 1 and EHV4.3409 (Studdert 1983), were included in these assays. The method was based on
104
!
Phamaaa (Australia) Pty Ltd, North Ryde, New South Wales Polaroid(Australia) Pty Ltd, North Ryde, New South Wales
Australian Veterinary Journal, Vol69, No. 5, May 1992
TABLE 1 Australasian EHVl isolates 1975-1989 Group
Virus ID/year
Place of isolation
Reference
1
EHV4
405176
Flemington. Vic
Studdert and Blackney 1979
2 3 4
Australia
438177 440177 441177 441177 3105/11 714/18
Scone, NSW Scone, NSW Scone, NSW Scone, NSW Busselton, WA Diggers Rest, Vic Diggers Rest, Vic Oberon, NSW Scone, NSW Cobbiuy, NSW Cobbiuy, NSW Cobbitty, NSW Cobbitty, NSW Cobbitty, NSW Cobbitty, NSW Cobbitty, NSW Oberon, NSW Scone, NSW Scone, NSW NSW NSW Bacchus Marsh, Vic Bacchus Marsh, Vic
Studdert and Blackney 1979 Studdert and Blackney 1979 Studdert and Blackney 1979 Studdert and Blackney 1979 Peet ef a1 1978 Westbury et a1 1978 Westbury et QJ 1978 Studdert et a1 1983 Studdert et a1 1983 Studdert et a1 1983 Studdert et af 1983 Studdert et a1 1983 Studdert et a1 1983 Studdert et a1 1983 Studdert et a1 1983 Studdert et af 1983 Studdert et a1 1983 Studdert et a11983 Studdert et a1 1983 Studdert et a1 1983 Studdert et a1 1983 Studdert et a1 1984 Studdert et a1 1984
Nz Nz
-
Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic
Homer 1981 Homer, unpublished Homer, unpublished Homer, unpublished Homer,unpublished Homer, unpublished Homer, unpublished Homer, unpublished Homer, unpublished Homer, unpublished Homer, unpublished This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper
Farm 2
849 - 3/89 849 - 5/89 849 - 6/89 849 - 7/89 849 - 8/89 849 - 9/89 849 - 10/89 849 - 11/89
Metcalfe, Vic Metcalfe, Vic Metcalfe, Vic Metcalfe, Vic Metcalfe, Vic Metcalfe, Vic Metcalfe, Vic Metcalfe, Vic
This paper This paper This paper This paper This paper This paper This paper This paper
Farm 3
877189
Violet Town, Vic
This paper
number
Serial
1977-1982
5 6 7 8
118/18
9 10 11 12
7 140117 25/11 42/77 45/11 51/77 52/71 54/11 56/11 51/17 62/78 119178 123178 498181 526181 711N82 717C182 4592175 5521/11 4504/11 21548/88 21518188 12952188 20233A/88 20233B/88 20233Cl88 13261/88 25357188 848 - A189 848 - B/89 848 - c/89 848 - D/89 848 - W89 848 - F/89 848 GI89 848 - v89 848 1/89 848 - W89 848 - MI89 848 - Nl89 848 - 0189 848 P/89
13
14 15 16 17 18
19 20 21 22 23 24 25 26 27 28 29
New
Zealnnd 1975-1988
30 31
32 33 34 35 36 37
Farm 1
38
39 40 41 . 42 43 44 45 46 47
-
-
48
49 50 51 52 53 54 55 56 51
58
Australian VeterinaryJournal, Vol69, No. 5, May 1992
Nz. Nz.
Nz Nz
m Nz Nz Nz. Nz.
105
thatpreviouslydescribed(Crabbef ul1991). In this assay antigen was prepared from infected cell culture supernatantsby diluting them 1:2 in PBS containing 1% "-40, mixing by whirling for 30 s and centrifuging at 12 OOO g for 1 min. The supernatant was then diluted a further 1:2 in PBS yontaining 5 clghnl BSA and 0.05% Tween-20 to a final dilution of 1:4. Antigen was added toplates (100 pl/well) that had beencoated overnightwithEHVl horse antiserum diluted 1:400 in 0.1M NaC03/NaHC03 buffer (pH 9.6). Monoclonal antibodies and horse-radish peroxidase conjugated rabbit anti-mouse IgG were then added as described (Crabb ef ul1991).
TABLE 2 EUSA showlng reactivity of giycoprotdn C speclfk monoclonal antlbodles with dlfferent Isolates of EHVl and M V 4
MAb' 26A5 14H7 41D3 43H12 20F3
EHV1' 438ff7 2154W88 848189 849189 5.2* 4.8 5.2 4.1
herpesvirus1(EHV1; equine abortion virus) from abortion, perinatai foal mortalitles and encephalitis from 15 epidemics that occurred InAustralasia between 1975and 1989were examined using the enzymes Barn Hi, EcoRi and Bgl 11. There was a remarkable degree of uniformity Inthe restriction patterns; mobility differences were observed In only 14 of 52 (27%) of the fragments. Twelve of these 14 fragments were located wlthin the repeat structures that bracket the unique short region of the genome or were located at the left terminus of the 150 kilobase pair genome. Based on the Barn Hi fingerprints the commonest virus identifled In our study was EHVl .IF (PIs for prototype strain). There was a single notable exception In that the Barn Hi fingerprints of all 8 isolates from one of 3 Victorian farms that experienced abortion in 1989 resembled a variant EHV1.iB that was identifled as a cause of abortion in Central Kentucky in1970to 1974. We present evidence that EHVl .IB caused abortion in California in 1964and has remained unaltered In Its Barn HI restriction pattern. No antigenic differences were found among 4 distantly related EHVl isolates, including the variant IB, using a panel of 5 monoclonal antibodies to glycoprotein C (gC), a glycoprotein recognised to be highly variable. The uniformity of these unrelated EHVl isolates Is further evidence for a recent origin for EHVl and may help to explain the natural history of this virus in the horse in which It seems to be a cause of serious epidemics of abortlon and perinatai mortality, and less commonly of encephalitis. Aust Vet J: 69 104 - 111
Introduction
Materials and Methods
The horse is somewhat unusual in that it is host to 3 distinct alphaherpesviruses: equine herpesvirus 4 (EHv4; equine rhinopneumonitis virus), EHVl (equine abortion virus) and EHV3 (equine coital exanthema virus). Two of these viruses EHV4 and EHVl were considered until about 1980 the same virus (EHVl) or with considerable ambiguity subtypes of -1. Restriction endonuclease DNA fingerprinting unambiguously established that there were two distinct viruses (Sabine et ul1981; Studdert et ul1981) and led to a proposal to identify them as EHV4 and EHVl (Studdertetd 1981).ThenaturalhistoryofEHV4including latency (Browninget ul 1988) and transmissionfrom generation to generation usually associated with an annual Occurrence of respiratory disease (rhinopneumonitis) in foals conforms well to the natural history of other alphaherpesviruses.For EHV 1 the natural history seems to conform less well with that of other alphaherpesviruses in that it has been difficult to prove latency wington et ul 1985) and a regular, high incidence annual transmission pattern; it is assumed that EHVl is acquired as a respiratory infection leading to latency and that abortion occurs as an unpredictable sequel to reactivation in a carrier horse, although all abortions on a farm (storms), with the possible exception of the index case, appear to occur as an immediate consequence of horizontal transmission from the f i t case of abortion. A similar pattem, though at a much lower incidence, is followedin outbreaks of EHVl encephalitis,which appears to be caused by only certainEHV1strains. There is some evidence that the pattern of disease caused by EHVl may vary around the world. For example, EHVl has not been recovered from outbreaksof respiratory disease in Australia, where such isolates have been invariably EHVA Earlier studies (Allen et ul 1983; Studdert 1983) showed that for naturally occurring EHVl isolates there is considerable uniformity in therestriction endomicleaseDNA fingerprints.We report here an analysis of 57 EHVl isolates from abortions, perinatal foal deaths and encephalitis that occurred in Australia and New Zealand between 1975 and 1989.
The origin and designation of the 57 EHVl isolates are listed (2 to 58) in Table 1. EHV4.405 (virus 1 Table 1) was included for comparative purposes. V i e s were isolated and grown in fiith passage equine foetal kidney (EFK) monolayer cell cultures in which they were passaged 3 to 5 times. Also included in the study was DNA prepared from a virus EHVl.M13/65 that was isolated from an aborted foetus in California in 1964 (Studdert MJ 1964unpublished). A 175cm2plastic flask of EFK cells was inoculated with each virus and when cytopathology was complete the cell culture lysates were clarified by centrifugation at 4OC, 1600g for 10min. Virus was pelleted from the Supernatants by centrifugation at 50 OOO g in an SW28 head fur 1.5 h in a Beckman ultracentrifuge. The pellet was resuspended in a minimum volume and layered onto a 5 to 15% Fico114W gradient as described by Schrag et ul(1989). The viruscontaining band was collected in a fraction collector. DNA was extracted from purified virions using phenol-chloroform followed by ethanol precipitation. DNA was digested with Bum HI, Eco RI and Bgl II according to the manufacturer's recommendations and separated on 0.8% agarose els. Gels were stained with ethidium bromide and photographed . Southern transfers to nylon membranes were performed as described by Sambrwk et ul (1989). The membranes were probed with 32PdCTP-labelled whole viral DNA or with EHV1.25A cloned fragments (Whalley et ul1981; Whalley et id 1989) as indicated in Figure le and f. A panel of 5 monoclonal antibodies (MAbs) to gC was used in an ELISA to see if any antigenic (epitope) differences existed between 4 of the 57 EHVl isolates that were judged to be the most distantly related, that is, viruses 2,28,46 and 55 (Table 1). Two EHV4 isolates, virus 1, Table 1 and EHV4.3409 (Studdert 1983), were included in these assays. The method was based on
104
!
Phamaaa (Australia) Pty Ltd, North Ryde, New South Wales Polaroid(Australia) Pty Ltd, North Ryde, New South Wales
Australian Veterinary Journal, Vol69, No. 5, May 1992
TABLE 1 Australasian EHVl isolates 1975-1989 Group
Virus ID/year
Place of isolation
Reference
1
EHV4
405176
Flemington. Vic
Studdert and Blackney 1979
2 3 4
Australia
438177 440177 441177 441177 3105/11 714/18
Scone, NSW Scone, NSW Scone, NSW Scone, NSW Busselton, WA Diggers Rest, Vic Diggers Rest, Vic Oberon, NSW Scone, NSW Cobbiuy, NSW Cobbiuy, NSW Cobbitty, NSW Cobbitty, NSW Cobbitty, NSW Cobbitty, NSW Cobbitty, NSW Oberon, NSW Scone, NSW Scone, NSW NSW NSW Bacchus Marsh, Vic Bacchus Marsh, Vic
Studdert and Blackney 1979 Studdert and Blackney 1979 Studdert and Blackney 1979 Studdert and Blackney 1979 Peet ef a1 1978 Westbury et a1 1978 Westbury et QJ 1978 Studdert et a1 1983 Studdert et a1 1983 Studdert et a1 1983 Studdert et af 1983 Studdert et a1 1983 Studdert et a1 1983 Studdert et a1 1983 Studdert et a1 1983 Studdert et af 1983 Studdert et a1 1983 Studdert et a11983 Studdert et a1 1983 Studdert et a1 1983 Studdert et a1 1983 Studdert et a1 1984 Studdert et a1 1984
Nz Nz
-
Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic Diggers Rest, Vic
Homer 1981 Homer, unpublished Homer, unpublished Homer, unpublished Homer,unpublished Homer, unpublished Homer, unpublished Homer, unpublished Homer, unpublished Homer, unpublished Homer, unpublished This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper
Farm 2
849 - 3/89 849 - 5/89 849 - 6/89 849 - 7/89 849 - 8/89 849 - 9/89 849 - 10/89 849 - 11/89
Metcalfe, Vic Metcalfe, Vic Metcalfe, Vic Metcalfe, Vic Metcalfe, Vic Metcalfe, Vic Metcalfe, Vic Metcalfe, Vic
This paper This paper This paper This paper This paper This paper This paper This paper
Farm 3
877189
Violet Town, Vic
This paper
number
Serial
1977-1982
5 6 7 8
118/18
9 10 11 12
7 140117 25/11 42/77 45/11 51/77 52/71 54/11 56/11 51/17 62/78 119178 123178 498181 526181 711N82 717C182 4592175 5521/11 4504/11 21548/88 21518188 12952188 20233A/88 20233B/88 20233Cl88 13261/88 25357188 848 - A189 848 - B/89 848 - c/89 848 - D/89 848 - W89 848 - F/89 848 GI89 848 - v89 848 1/89 848 - W89 848 - MI89 848 - Nl89 848 - 0189 848 P/89
13
14 15 16 17 18
19 20 21 22 23 24 25 26 27 28 29
New
Zealnnd 1975-1988
30 31
32 33 34 35 36 37
Farm 1
38
39 40 41 . 42 43 44 45 46 47
-
-
48
49 50 51 52 53 54 55 56 51
58
Australian VeterinaryJournal, Vol69, No. 5, May 1992
Nz. Nz.
Nz Nz
m Nz Nz Nz. Nz.
105
thatpreviouslydescribed(Crabbef ul1991). In this assay antigen was prepared from infected cell culture supernatantsby diluting them 1:2 in PBS containing 1% "-40, mixing by whirling for 30 s and centrifuging at 12 OOO g for 1 min. The supernatant was then diluted a further 1:2 in PBS yontaining 5 clghnl BSA and 0.05% Tween-20 to a final dilution of 1:4. Antigen was added toplates (100 pl/well) that had beencoated overnightwithEHVl horse antiserum diluted 1:400 in 0.1M NaC03/NaHC03 buffer (pH 9.6). Monoclonal antibodies and horse-radish peroxidase conjugated rabbit anti-mouse IgG were then added as described (Crabb ef ul1991).
TABLE 2 EUSA showlng reactivity of giycoprotdn C speclfk monoclonal antlbodles with dlfferent Isolates of EHVl and M V 4
MAb' 26A5 14H7 41D3 43H12 20F3
EHV1' 438ff7 2154W88 848189 849189 5.2* 4.8 5.2 4.1
