ETIOLOGICAL AND MOLECULAR BIOLOGICAL INVESTIGATION OF CAPRINE HERPESVIRUS 1 ISOLATED IN BULGARIA

From goat and bucks with gen i tal dis or ders and abor tions the at tempts for iso la tion of caprine herpesvirus 1 (CHV 1) were car ried out. For vi rus ex al ta tion Dexametazone was used. For vi ruses iso la tion vag i nal, na sal, rec tal, preputial swabs and or gan sam ples were used. Pri mary and con tin u ous cell cul tures rab bit kid ney (RK), Madin Darby bo vine kid ney (MDBK), and embrional bo vine tra chea (EBTR) were used for cul ti va tion. For de ter mi na tion of DNA type and lipid en velop 60 μg/ml iod desoxiuridine (JDUR) and the ether treat ment was used. Neu tral iza tion by spe cific hyperimmune se rum ob tained from Swit zer land was per formed. Five CHV 1 strains were iso lated by cell cul tures and iden ti fied as goat herpesviruses from dif fer ent Bul gar ian re gions. Af ter elec tron mi cros copy the vi ral agents with typ i cal size and mor phol ogy for herpesviruses were es tab lished. For dem on stra tion gC gene of CHV 1 the poly mer ase chain re ac tion (PCR) with prim ers de signed from se quences de pos ited in gene bank were de vel oped. Iso lated on cell cul tures herpesviruses were proved as caprine herpesvirus 1 by us ing ap plied PCR vari ant. The prod ucts af ter gC gene am pli fi ca tion from Bul gar ian iso lates were sep a rated on the same place as the amplicons of ref er ence CHV 1 strains.


INTRODUCTION
Caprine herpesvirus 1 (CHV 1) is a DNA con tain ing vi rus with a di am e ter 120-150 nm and lipid en ve lope. The an ti genic pe cu liar ity of vi rus is not well stud ied. Ac cord ing to Engels et al. (1987) vi ral DNA has a high homology with bo vine herpesvirus 1 DNA. Af ter in ves ti ga tion of agent by neu tral iza tion test, ELISA and restrictase frag ment pat tern anal y sis is de ter mined that CHV 1 is im mu no log i cally dif fer ent from herpesviruses in big ru mi nants and elks (5,10,18). Glycoproteins with mo lec u lar weight 74 and 91kD are re spon si ble for cross neu tral iza tion be tween bovine and caprine herpesvirus. De spite the dif fer ences in mo lec u lar weight it was de -ter mined by PAGE that the polypeptides of goat and bo vine herpesviruses have sim i lar mo bil ity (2).
The first re port for CHV1 co mes in early 1974 from Saito et al. (13) in Cal i for nia who de scribed the dis ease with high per cent mor tal ity among an gora goats. In young an i mals CHV 1 caused gen er al ized in fec tion dam ag ing gas tro in tes ti nal and re spi ratory tracts (1). Mac ro scop i cally are vis i ble ul cer and necroses in all gas tro in tes ti nal chan nels, changes in lungs, uri nary blad der and liver. Pathohystologically are observed heavy necrotizing en ter i tis, as well as thick en ing of al ve o lar septa and ne crotic bronchoalveolitis (12). Clear mi cro scopic dam ages are ob served also in liver, uri nary blad der, spleen, thy mus, mesenterial lymph nodes and kid ney. The dis ease is ac compa nied with di ar rhea, rhi ni tis, tracheitis, breathing disturbance and later supurative nasal discharges.
In bucks the vi rus caused balanopostitis and penopostitis and in goats vulvovaginitis (8,14,15). From dam aged gen i tal tract the in fec tion is trans mit ted by breed ing. Abor tions are ob served, too (17,19).
The num ber of iso lated CHV 1 in the world is lim ited. Some parts of ep i de mi ology, pathogenesis and spread ing of the dis ease are not well elu ci dat ing. This is the reason to iso late eti o log i cal agent and study the cul tural and ge nome characteristics.
In this pa per is de scribed the iso la tion and iden ti fi ca tion of CHV 1 caus ing goat in fec tion and by us ing the physicochemical and mo lec u lar bi o log i cal meth ods to study some bi o log i cal pe cu liar ity of the virus.

MATERIAL AND METHODS
Na sal, vag i nal and preputial swabs, lung lavages, 10% sus pen sion in phos phate buf fered sa line (PBS) from in ter nal or gans, probang test, buffy coats, fe cal and milk sam ples were used for vi rus iso la tion. To tally 163 sam ples were checked. The sam ples orig i nated from sev eral coun try re gions: Troian, Suhindol, Kustendil, P.Bania, Smolian, Haskovo, Yambol.
For herpesvirus iso la tion the method for ex al ta tion by dexa meth a sone (DMSO) was used (3).

Cell cul tures, me dia and so lu tions
For vi rus iso la tion and iden ti fi ca tion of vi ruses pri mary and per ma nent cell cultures were used. The pri mary cell cul tures were ob tained new born rab bit kid ney and as per ma nent cell lines Madin Darby bo vine kid ney (MDBK), (AUBEK), Geor gia bovine kid ney (GBK), em bry onic bo vine tra chea (EBTR) and calf tra chea (TTr). For wash ing cell cul ture PBS with rN 7.4 or nor mal sa line were used. As grow ing me dia Ea gle's Min i mum Es sen tial Me dium (MEM) in Hanks bal anced salts so lu tion supple mented with 10% fe tal calf sera (FCS) and as a sup port ive me dia the same me dia with 2% FCS were used. As ad di tives pen i cil lin 100 UI/mL, strep to my cin 100 g/mL, 0.2 M/L L -glutamine and NaHCO 3 were added.

Iso la tion and iden ti fi ca tion of CHV 1 on cell cul tures
For in fec tion cell cul tures with com plete monolayer were used. Af ter elim i na tion of me dia and wash ing with PBS or nor mal sa line 0.2 mL vi rus in ocu lums were added. De pend ing on the type of sam ples ad sorp tion var ied be tween 60 and 120 min at temper a ture of 37°S. Af ter wash ing of cell monolayers and add ing the main tain ing me dia the tubes were placed in a roller at 37°S. Si mul ta neously, with the in ves ti gated samples non in fected con trol cells were used in the same con di tion.
Mi cro scop i cally, the iso lates growth was con trolled daily by de ter mi na tion the pres ence of cytopatic ef fect (CPE) on monolayers. In at tempts for pri mary iso la tion the cell cul tures were ob served mi cro scop i cally 7 days af ter the in fec tion. Three consec u tive pas sages were per formed when CPE was miss ing and more pas sages -when CPE was vis i ble. Be cause of the dif fi cul ties con nected to the pri mary iso la tion of CHV 1 other meth ods for cul ti va tion were used -in fec tion on fresh monolayer, young 24 h cell cultures or in cell suspension.
Iden ti fi ca tion of vi ral agents in the in fected cell monolayers with CPE were accom plished by the meth ods de scribed by Pay ment and Trudel (1993). To de ter mine the nu cleic acid type the vi ral iso lates were treated with 60 g/mL 5-jodo-2-deoxyuridine (JDUR), and for lipid pres ence treated with 20% ether.
In neu tral iza tion re ac tion b-vari ant the ref er ence hyperimmune sera from Switzer land had ti ter of 8 log 2. The obeseved CPE was sim i lar to CHV. The se rum par tic ipated in main tain me dia at con stant di lu tion 1:4, and in ves ti gated iso lates in 10 times in creas ing di lu tion. As con trol the ref er ence CHV 1 strain E/CH with a ti ter 10 7.33 TCID 50 /mL on MDBK was ap plied. As con trol heterologous RNA strain paramyxo parainfluencae 3 vi rus "Svetovrachene" strain was used.

Elec tron mi cros copy
Elec tron mi cros copy in ves ti ga tion was per formed with di rect elec tron mi croscopy (DEM) af ter slow speed centrifugation of vi ral iso lates 2000 rpm/20 min. Supernatants were dif fer en tially cen tri fuged for 1h at 30000xg and the ob tained pel let were dis solved in min i mal vol ume ster ile dis tilled wa ter -0.5 ml, af ter which new slow speed centrifugation was car ried out at 2000 rpm/20 min. For per for mance of DEM the clear supernatants were added onto butvar and car bon coated coo per grids with 400 mesh and neg a tive stain ing were per formed with 2% so dium phosphovolframate rN 5.8 or 2% uranil acetate rN 4.2-4.5.
The in ves ti ga tions were car ried out by elec tron mi cro scope JEM 1200 EX with accel er ated ten sion 80 kV and in stru men tal en large ment 40000 to 75000X.

Mo lec u lar bi o log i cal in ves ti ga tion
The vi ruses were cul ti vated in cell cul ture and in 80% vis i ble CPE they were harvested. Com mer cial kit GIAamp DNA mini kit Giagen, GmbH, Hilden Ger many was used for iso la tion of DNA fol low ing the kit in struc tion. The DNA amount was con -trolled spec tro pho to met ri cally with Jenway ap pa ra tus (Genova) and by gel elec tropho re sis and was am pli fied by poly mer ase chain re ac tion (PCR) us ing the fol low ing pairs of prim ers cor re spond ing to se quences 759-779 and 1172-1154 from the gene bank: For ward R 1 5'-AGGGCGCCGGTGGATGCTCTG -3' Re verse R 2 5' -GGCGGGCGGTGCGTCGTGA -3' Re ac tion was con ducted in a vol ume of 25 µL, us ing Fidely tag PCR Mas ter mix (2X), for ward primer P 1 , re verse primer P 2 and DNA tem plate. Thermocycler QB -96 (LKB) and the pro gram de scribed from Hecht et al. (1995) were used for PCR reac tions.
Serological in ves ti ga tion by mi cro vi rus neu tral iza tion test (MVNT) Sera from goats with clin i cal symp toms typ i cal for CHV 1 in fec tion and re cov ered were in ves ti gated for an ti bod ies against CHV 1 by mi cro vi rus neu tral iza tion test (MVNT)b vari ant. The sera were tem per a ture treated at 56°S for 30 min, af ter that se rial two fold di lu tion in main te nance me dia MEM Ea gle, pen i cil lin 100 UI/mL, strep to my cin 100 µg/mL, 2 mM/L L-glutamin, 1.5 g/L so dium bi car bon ate were performed. Hun dred tis sue cul ture in fec tious dose 50/mL (TCID 50 /mL) from ref er ence strain E/CH with a ti ter 10 7.3 was added to di lute sera. The mix tures were in cu bated at 37°S for 2 h. Af ter that the in di ca tion sys tem -cell line MDBK at quan tity 4h10 -4 cells/mL was added.
The ac count of re sults was per formed till 72 h. The high est di lu tions of sera giv ing com plete growth sup pres sion of in di ca tor vi rus were ac cepted as serum titer.

RESULTS
Vi ral agents were iso lated only af ter dexametazone treat ment from an i mals with an ti bod ies against CHV 1. The vi ruses were iso lated only from vag i nal and preputial swabs. The vi rus iso lates have cul tural and bio chem i cal char ac ter is tics typ i cal for caprine herpesvirus 1. From goats and bucks with prob lems in breed ing 5 CHV 1 strains were iso lated: Troian, Suhindol, Kustendil, P. Bania and Biser.
The citopatic ef fect on pri mary and per ma nent cell cul tures de pended on used cell lines and started with round ing of cells. Be tween 6 and 12 h af ter in fec tion of monolayers only sin gle cells were rounded. Cytopatic ef fect on 24 h be came more diffuse and great num ber of cells was dam aged (Fig. 1A) af ter which the speed changes on monolayers were vis i ble. At 48 h on cell monolayers were ob served ex tended empty places as a re sult of the vi ral growth (Fig. 1B) and af ter 48 h the part of cell monolayers was de tached from the tube walls and af ter 72 h a de struc tion of monolayers was vis i ble (Fig.1 C). In con trol non in fected MDBK cell cul tures the cytopatic ef fect was not observed (Fig. 1 D The ob tained re sults dur ing in ves ti ga tion for iden ti fi ca tion of agents in in fected cell cul tures are shown in Table 1. Ta ble 1. Data from bio chem i cal and mo lec u lar bi o log i cal stud ies for de ter mi na tion of nu cleic acid type, ef fect of HIS against CHV 1, pres ence of lipid coat, im pact of low and high pH buff ers with five CHV 1 iso lates, ref er ence E/CH strain and heterologous Pi-3 strain "Svetovrachene". Af ter treat ment with 60 g/mL 5-jodo-2-deoxyuridine (JDUR) for the de ter mi nation of nu cleic acid type in 5 iso lates a sup pres sion of growth with more than 3-4log 10 was es tab lished. In treat ment of ref er ent strain E/SN a sup pres sion of growth -5 log 10 in com par i son with non treated vi rus. Sup pres sion of vi ral ti ter was not found in RNA strain Paramyxovirus PI-3 "Svetovrachene". Af ter neu tral iza tion test with pos i tive hyperimmune sera from Swit zerland in all new iso lated strains a sup pression be tween 4-6 log 10 was ob tained. In ref er ence strain E/CH also was de ter mined re duc tion in vi ral ti ter with 6 log 10 . In heterologous strain "Svetovrachene" such de crease in vi ral ti ter was not ob served.
The five iso lates, ref er ent E/CH and con trol RNA vi ral strain were not grow ing on cell cul ture af ter treat ment with 20% ether.
Af ter elec tron mi cros copy the vi ral parti cles with di am e ter ap prox i mately 150-180 nm and typ i cal for herpesviruses mor phology were ob served (Fig. 2). In mo lec u lar bi o log i cal in ves ti ga tion the quan tity of DNA ob tained by Giagen columns var ied be tween 163.5 ng/µl and 198.2 ng/µl. Af ter us ing PCR mas ter mix with the de scribed pro gram mul ti pli ca tion of gC gene was pos si ble. Prod ucts with size 414 bp for all 5 caprine herpesviruses iso lated in Bul garia and for all ref er ence strain from Eu rope and USA were ob tained af ter per form ing PCR re ac tion (Fig. 3). There was not mul ti pli ca tion of ge nome of in ves ti gated IBR "Ozet" strain in us ing the same prim ers and pro ce dure for PCR spec i fic ity. Fig. 3. Af ter serological in ves ti ga tion in five coun try re gions from which the vi ruses were iso lated an in crease of an ti body titers two and more log 2 in paired sera was de termined against ref er ent E/CH strain (Fig. 4). The high est per cent pos i tive sam ple in Kustendil (100%) fol lowed by Biser (79.9%), Suhindol (75%), P. Bania (50%), and Troian (22.75%) were found (Fig. 4). The num ber of pos i tive bucks in creased af ter breed ing cam paign, and also the ti ter of an ti bod ies was in creased in com par i son with an ti body titers be fore the cam paign. In some goats were de ter mined re peat ing twice and three time heat es pe cially in Troyan, Suhindol and P. Bania farms. Ad di tion ally titers against chlamidophilla pur sue in fection were de ter mined for Troyan (3.44%), Suhindol (12.5%), P. Bania (3.57%), Biser (21%) and against coxiella burnety in Suhindol (12.5%) and P. Bania (21.42%) only.

DISCUSSION
Us ing the scheme (Buonavoglia et al., 1996) for ex al ta tion of caprine herpesvirus 1 we suc ceed to iso late five CHV 1 strains from vag i nal and preputial swabs. The vi rus iso lated only from vag i nal swabs, but not from na sal, rec tal swabs and buffy coat. The iso la tion of goat and bucks vi ruses was pos si ble with high DMSO doses be tween 3 and 9 days af ter fin ish ing the treat ment of pos i tive for an ti bod ies an i mals. Smaller DMSO doses were not enough for vi rus iso la tion.
The cytopatogenic ef fect in the ad ap ta tion and cul ti va tion of strains was with mild changes with round ing of the cells af ter that the monolayer was de structed as described by Engels et al. (1983) changes. The grow rate of new iso lated CHV 1 strains by us was more rapid in com par i son with that of bo vine herpesvirus 1 as Engels et al. (1983) es tab lished for CHV 1 strain. Af ter la tent pe riod of 5 h ex po nen tial phases of vi rus grow ing 6 to 12 h af ter in fec tion was ob served. The cytopatic ef fect of iso lated vi ral agents on cell cul tures with rab bit and bo vine or i gin was more dif fuse than the Af ter treat ment with JDUR and the ap ply ing of MVNT with hyperimmune sera from Swit zer land against ref er ence CHV 1 strain the vi ral titers were de creased with more than 2-4log 2 . This is the ev i dence that the iso lated vi ral agents were CHV 1. The com plete sup pres sion of vi ral growth af ter treat ment with ether con firmed that the new iso lated sam ples and ref er ence con trols are with lipids bilayers of mem brane. This is sup ported by the fact that the heterologous Pi 3 vi rus is sup pressed from a treat ment with 20% ether. At lower and higher pH so lu tions the growth of vi ral isolates is also sup pressed. The prob a ble rea son for this is the re pres sion of con tacts between vi ral an ti gens and cell re cep tors and the im pos si bil ity for en trance and mul ti pli ca tion of causative agents in cell cultures.
At elec tron mi cros copy stud ies vi ral agents with typ i cal size and mor phol ogy for herpesviruses were de ter mined.
The ob served bi o log i cal pe cu liar i ties of all 5 Bul gar ian iso lates by per formed PCR re ac tion sim i lar to ref er ence CHV 1 strains E/CH and McKercher are the ev i dence that all five iso lates are CHV 1 strain. This per mit us to con clude that PCR re ac tion with the de scribed prim ers and pro ce dures can be used for rapid and exact diagnosis.
Af ter in ves ti ga tion by MVNT of sera orig i nat ing from an i mals in acute and con vales cent pe riod of the dis ease from the five farms we de tected an in crease in an ti body titers with two and more log a rithms against ref er ence CHV 1 strain which is an in dica tion for CHV 1 cir cu la tion in flocks. On some farms the es tab lished an ti body titers were a re sult of sin gle in fec tion, while on other farms it was ac com pa nied with chlamydophilla and q-fe ver in fec tions. The dis ease elapsed more heavily in the presence of these ac com pa ny ing in fec tions and re cov er ing pe riod of an i mals was long last ing and of ten in next breeding season the breedings were hampered. CONCLUSIONS 1. Iso la tion and ad ap ta tion of five goat herpesviruses were pos si ble only af ter treatment with DMSO. 2. CHV 1 vi ral strains can be suc cess fully adapted on primar and per ma nent cell cul ture of bo vine or i gin. 3. Iso lated vi ral agents have cul tural, bio chem i cal and mor pho log i cal char ac ter istics typ i cal for CHV 1.