Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever (MCF) in susceptible ruminants. Through an unknown mechanism, presence of the virus leads to proliferation of NK-like T cells that are not targetrestricted by the MHC class molecules. These host cells cause the symptoms found in MCF; fever, swollen lymph nodes, and necrotic lesions of the nasal, conjunctival, and oral mucosa, which usually leads to death of the host. MicroRNAs (miRNAs) are ~22 nt RNA molecules expressed by eukaryotes and viruses that regulate genes post-transcriptionally. Viral miRNAs have been found to regulate cellular genes to control the cell cycle and have a role in pathogenesis. It was hypothesised that OvHV-2 expresses miRNAs and these play a role in MCF pathogenesis. The aim of this project was to determine if OvHV-2 encodes miRNAs. Bioinformatic analysis was conducted on deep sequencing data from RNA of OvHV-2- immortalised T cells. Candidate miRNAs were selected if they adhered to miRNA secondary structure. 46 candidate miRNAs were found, with three clusters on the minus strand; one at the 5’ end and the other two in a 9.3 kb region that contains no predicted open reading frames. The 8 most abundant candidates were successfully validated by northern hybridisation for small RNAs. The majority of the predicted targets for the 8 validated OvHV-2 miRNAs were from the OvHV-2 genome. This study adds OvHV-2 to the list of herpesviruses that encode miRNAs and provides another tool for studying the pathogenesis of MCF.

Malignant catarrhal fever (MCF), a disease that is usually fatal in cattle, is caused by two distinct but related bovine herpesviruses which are members of the genus Macavirus. The wildebeest-associated alcelaphine herpesvirus-1 (AlHV-1) occurs mainly in East and southern Africa, whereas the sheep-associated ovine herpesvirus-1 (OvHV-2) has an almost worldwide distribution. The natural hosts or carriers of these two viruses are subclinically infected. The 130 kilobase pair (kbp) AlHV-1 double stranded DNA genome consists of 18 open reading frames (ORFs) coding for structural proteins and approximately 50 ORFs coding for non-structural proteins. The 18 structural ORFs encode for 4 capsid proteins, 5 tegument proteins, 8 glycoproteins and a minor capsid scaffold protein. ORF8 encoding for glycoprotein B, is the most conserved of the proteins amongst gammaherpesviruses, whereas the minor capsid protein encoded by ORF65, is amongst the most variable. Thus, the minor capsid protein is one of the antigens of choice for the development of an ELISA for detection of AlHV-1 reactive antibodies and glycoprotein B could be of importance in developing a cross-protective vaccine for gammaherpesviruses. The naming and annotation of most of the AlHV-1 ORFs is based on comparison with related gammaherpesviruses and bioinformatics. Most of these ORFs are putative as there is no direct experimental evidence confirming that they code for any particular protein. In order to investigate whether the ORFs code for any proteins, two ORFs were targeted for in vitro heterologous expression. AlHV-1, isolate C500, was grown in fetal bovine turbinate (BT) cell culture and viral genomic DNA extracted. ORF8, the putative glycoprotein B, was amplified with a PCR assay and inserted into a mammalian expression vector, pCI. VERO cells were transfected with the recombinant vector. Expression of ORF8 was confirmed by an indirect immunofluorescence assay (IFA) with AlHV-1 polyclonal sera and rabbit anti-bovine IgG (whole molecule) FITC conjugate. Truncated forms of ORF8 were further expressed as baculovirus recombinants using the Bac-to-Bac baculovirus expression system. Expression of the truncated ORF8 was confirmed by SDS-PAGE and Western blot. AlHV-1 ORF65, the minor capsid protein gene, was amplified with a PCR assay from the viral genomic DNA and cloned in frame with a histidine tag in a bacterial expression vector, pCOLD I. Expression of the minor capsid protein was confirmed by SDS-PAGE and Western blot with the histidine tag monoclonal as well as AlHV-1 polyclonal sera. Orf65 was expressed in large quantities and column purified using the histidine tag. Orf65 was also expressed as a baculovirus recombinant using the Bac-to-Bac baculovirus expression system. Expression of the protein was confirmed by SDS-PAGE and Western blot with the histidine tag and AlHV-1 polyclonal sera. ORF65 expression in the baculovirus Bac-to-Bac expression system was up-scaled and the expressed protein column purified. Antibodies raised in chicken against the purified antigen were used successfully in an indirect immunoassay to detect AlHV-1 infected cells. An indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against AlHV-1 was developed. It is based on the use of the AlHV-1 minor capsid protein as the capture antigen for antibodies. The primary antibodies are detected by the addition of enzymelabelled (horseradish peroxidase) protein G which detects bovid, ovid and wildebeest antibodies. Addition of a substrate of the enzyme, in this case, 3,3’,5,5’- tetramethylbenzidine (TMB), results in a colour reaction which is measured using spectrophotometric procedures. At a selected cut-off point of 18, the ELISA test has a sensitivity of 100% and a specificity of 100% and has been shown to detect AlHV-1 antibodies in cattle and wildebeest. The ELISA showed no cross-reactivity with sera raised in cattle against related viruses such as ovine herpesvirus 2, bovine herpesvirus 1, 2 and 4. The two expressed proteins used in this study were found to be amongst the antigens expressed in cattle suffering from malignant catarrhal fever. The experimental AlHV-1 indirect ELISA needs further validation and this research may be extended to determine the performance of these antigens as candidate subunit vaccines. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Veterinary Tropical Diseases / unrestricted

Malignant catarrhal fever (MCF) is a lymphoproliferative and inflammatory syndrome affecting primarily ruminant species. The disease, which is often fatal, is most often described as affecting bovids and cervids. No vaccines are available for prevention of MCFV infection. The primary method to control spread of disease is to prevent contact between carriers and clinically susceptible species. There is no known method to control infection of malignant catarrhal fever virus-white-tailed deer variant (MCFV-WTD), as the carrier animal of this virus is unknown. To determine the prevalence of malignant catarrhal fever viruses in Tennessee ruminant populations, blood and/or lymph node samples were collected from farms, animal processing and disposal facilities, and hunter check-in stations from 2006-2008 from several species of animals including cervids, cattle, and goats. Strain-specific real time PCR was developed to detect ovine herpesvirus-2 (OvHV-2), caprine herpesvirus-2 (CpHV-2), and MCFV-WTD DNA. MCFV DNA was detected in all species of ruminants sampled. Although disease related to infection with MCFV-WTD and CpHV-2 has not been reported in Tennessee cattle or cervid populations, MCFV-WTD DNA was detected in 3 percent of cervid samples, and MCFV-WTD and CpHV-2 DNA was detected in 27 and 3 percent respectively of cattle samples from animal disposal facilities that process dead or debilitated animals. One hunter harvested deer (n=781) and 25 cattle (n=165) tested from animal disposal facilities were positive for OvHV-2 DNA. This study demonstrated that healthy cattle and cervids can be infected with OvHV-2 and MCFV-WTD without apparent disease, and dead or debilitated cattle were infected with OvHV-2, MCFV-WTD and CpHV-2 at a higher percentage than healthy herd animals. Prevalence of CpHV-2 in Tennessee goat populations (7%) was significantly lower than reported in other goat populations (73%). Low prevalence of CpHV-2 in Tennessee goat populations likely explains why no evidence of infection was found in cervids tested, and the low prevalence of CpHV-2 infection in dead or debilitated cattle compared to prevalence of infection with OvHV-2 and MCFV-WTD. The discovery of infection in cattle with CpHV-2 and MCFV-WTD opens a new avenue of investigation into the pathology and virulence of MCFV’s in domestic cattle.

Bösartiges Katarrhalfieber ist eine unheilbare Virusinfektion bei Paarhufern, die wiederholt in zoologischen Gärten auftrat, ohne dass die erkrankten Fehlwirte Kontakt zu Reservoirwirten hatten. Die BKF-auslösenden Gammaherpesviren sind eng miteinander verwandt und werden von verschiedenen klinisch gesunden Reservoirwirten latent beherbergt und ausgeschieden. Einige dieser Reservoirwirte sind seit längerem bekannt, andere wurden erst kürzlich identifiziert und es wird vermutet, dass es noch weitere unerkannte Reservoirwirte für BKF-Viren gibt. Hervorzuheben ist, dass die Viren normalerweise eng an ihre Reservoirwirte gebunden sind. Es traten in letzter Zeit jedoch immer wieder Fälle auf, in denen auch Fehlwirte zwar infiziert waren, aber nicht erkrankten oder das Virus sogar ausschieden. Der Zusammenhang zwischen dem Verhalten der BKF-Viren bei Fehl- und Reservoirwirten und den ungeklärten BKF-Fällen in zoologischen Gärten wurde in der hier vorliegenden Studie näher untersucht. Es sollte herausgefunden werden, ob Wildwiederkäuer, die bisher nicht als Reservoirwirte für BKF-Viren galten, diese Viren ausscheiden und so möglicherweise für die oben erwähnten BKF-Fälle verantwortlich waren. / Malignant catarrhal fever (MCF) is an incurable infectious disease in even-toed ungulates, which occurred repeatedly in zoological gardens in Europe without any contact between known hosts and animals with clinical MCF. The causative agents are closely related viruses of the family gamma-herpesviridae, which are latently carried and shed by different clinically healthy ruminant species. Some of the hosts for MCF viruses have been known for many years, while others have been identified only recently. Yet, there are probably still more host species to be discovered. It has to be pointed out that generally MCF viruses are strictly associated with their hosts. However, it has been reported that known susceptible species were infected with MCF viruses without showing any signs of MCF, some of which even excreted the virus. This present study investigates the relationship between the behaviour of MCF viruses in hosts and susceptible species and the nebulous cases of MCF in zoological gardens. The goal was to determine whether wild ruminants, which are normally not known as hosts for MCF, shed these viruses and are possibly responsible for MCF cases mentioned above.

<p>Вирус катаралне грознице оваца наноси велике штете сточарској производњи због чега су правовремено препознавање болести и брза дијагноза есенцијални предуслови за адекватну реакцију ветеринарске службе. Из тих разлога, предмет ове докторске дисертације су анализе патолошких и хистолошких промена код животиња, серолошких и молекуларних метода потребних за идентификацију и ближу карактеризацију узрочника што је од примарног значаја за ефективност предузетих мера у борби против катаралне грознице оваца.<br />Серолошки, током активног надзора, пргледано је 944 узорка крвног серума оваца и 953 узорка крвног серума говеда, а потом, током пасивног надзора, 114 узорака крвног серума говеда, 302 узорка крвног серума оваца и 22 узорка крвног серума коза. Коришћена је компетитивна имуноензимска ELISA метода, којом је доказано присуство специфичних антитела код говеда, оваца и коза. Из пуне крви серопозитивних јединки, ланчаном реакцијом полимеразе уз коришћење Pan-BTV rRT-PCR протокола заснованог на публикацији Toussiant и сар., (2007) је доказано присуство секвенце вирусне РНК. У случају позитивних резултата спроведена је и конвенционална PCR метода, употребом One step PCR кита, уз коришћење два пара прајмера за серотип BTV-4 чиме је доказано да је овај серотип узрочник обољења у Црној Гори. Секвенцирањем и филогенетском анализом изолата утврђено је да припада западном топотипу BTV-4 и да је сродан, готово идентичан са изолатима земаља из окружења.<br />Од 302 овце, којима је узета крв за серолошку анализу, уочене су каратеристичне макроскопске и микроскопске промене код свих угинулих од катаралне грознице оваца. Испитивањем је обухваћено 20 угинулих оваца које су претходно, током испитивања за живота, поред манифестне клиничке слике, имале позитивне резултате серолошких и молекуларних испитивања. Након угинућа, овце су обдуковане при чему су констатоване и сликане макроскопске промене те издвојена ткива за хистолошке анализе. Ткива су фиксирана у 10% пуферизованом неутралном формалину, а потом уклапљена у парафин. Парафински исечци дебљине 5 микрометара су бојени хематоксилин-еозин методом. На промењеним органима доминирају едем, хиперемија и крварења. Хистолошки се запажају интензивне хеморагичне инфилтрације свих промењених органа, а поред тога и местимични некротични процеси у срцу и језику као и периваскуларни едеми лимфних органа са израженом<br />лимфоцитном деплецијом у лимфним чворовима, слезини и тимусу.</p> / <p>Virus kataralne groznice ovaca nanosi velike štete stočarskoj proizvodnji zbog čega su pravovremeno prepoznavanje bolesti i brza dijagnoza esencijalni preduslovi za adekvatnu reakciju veterinarske službe. Iz tih razloga, predmet ove doktorske disertacije su analize patoloških i histoloških promena kod životinja, seroloških i molekularnih metoda potrebnih za identifikaciju i bližu karakterizaciju uzročnika što je od primarnog značaja za efektivnost preduzetih mera u borbi protiv kataralne groznice ovaca.<br />Serološki, tokom aktivnog nadzora, prgledano je 944 uzorka krvnog seruma ovaca i 953 uzorka krvnog seruma goveda, a potom, tokom pasivnog nadzora, 114 uzoraka krvnog seruma goveda, 302 uzorka krvnog seruma ovaca i 22 uzorka krvnog seruma koza. Korišćena je kompetitivna imunoenzimska ELISA metoda, kojom je dokazano prisustvo specifičnih antitela kod goveda, ovaca i koza. Iz pune krvi seropozitivnih jedinki, lančanom reakcijom polimeraze uz korišćenje Pan-BTV rRT-PCR protokola zasnovanog na publikaciji Toussiant i sar., (2007) je dokazano prisustvo sekvence virusne RNK. U slučaju pozitivnih rezultata sprovedena je i konvencionalna PCR metoda, upotrebom One step PCR kita, uz korišćenje dva para prajmera za serotip BTV-4 čime je dokazano da je ovaj serotip uzročnik oboljenja u Crnoj Gori. Sekvenciranjem i filogenetskom analizom izolata utvrđeno je da pripada zapadnom topotipu BTV-4 i da je srodan, gotovo identičan sa izolatima zemalja iz okruženja.<br />Od 302 ovce, kojima je uzeta krv za serološku analizu, uočene su karateristične makroskopske i mikroskopske promene kod svih uginulih od kataralne groznice ovaca. Ispitivanjem je obuhvaćeno 20 uginulih ovaca koje su prethodno, tokom ispitivanja za života, pored manifestne kliničke slike, imale pozitivne rezultate seroloških i molekularnih ispitivanja. Nakon uginuća, ovce su obdukovane pri čemu su konstatovane i slikane makroskopske promene te izdvojena tkiva za histološke analize. Tkiva su fiksirana u 10% puferizovanom neutralnom formalinu, a potom uklapljena u parafin. Parafinski isečci debljine 5 mikrometara su bojeni hematoksilin-eozin metodom. Na promenjenim organima dominiraju edem, hiperemija i krvarenja. Histološki se zapažaju intenzivne hemoragične infiltracije svih promenjenih organa, a pored toga i mestimični nekrotični procesi u srcu i jeziku kao i perivaskularni edemi limfnih organa sa izraženom<br />limfocitnom deplecijom u limfnim čvorovima, slezini i timusu.</p> / <p>The Bluetongue virus (BTV) incurs great damage to the production of livestock, due to which timely recognition of the resultant disease and rapid diagnosis are essential prerequisites for an adequate reaction by the veterinary service. For these reasons, the subject of this doctoral dissertation are the analyses of pathological and histological alterations in animals, the serological and molecular methods necessary for identifying and closely characterizing the causative agent which is of primary concern for the efficacy of the measures taken in the fight against bluetongue disease.<br />Serologically, during active surveillance, 944 samples of sheep blood serum as well as 953 samples of cattle blood serum were examined, followed by 114 samples of cattle, 302 samples of sheep and 22 samples of goat blood serum during passive surveillance. Competitive ELISA was utilized to detect the presence of specific antibodies in cattle, sheep and goats. The presence of viral RNA sequences was confirmed in whole blood samples of seropositive individuals using the Pan-BTV rRT-PCR protocol as described in Toussiant et al. (2007). In the case of positive results conventional PCR analysis was also performed, using the One Step PCR kit with two primer pairs specific to the BTV- serotype, which showed that this serotype is the cause of the disease in Montenegro. Sequencing and phylogenetic analysis of the isolate determined that it belongs to the western topotype of BTV-4 and that it is related and practically identical to the isolates from neighbouring countries.<br />This work describes the macroscopic and microscopic changes found in sheep which died due to affliction with bluetongue disease. The study investigated a sample of 20 sheep for which premortem analysis, apart from the manifested clinical features, had confirmed positive serologic and molecular test results. The animals were dissected postmortem during which macroscopic alterations were identified and imaged, followed by tissue isolation for histological analysis. Tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections of 5&mu;m thickness were cut and stained with haematoxylin/eosin. The predominant organ changes identified included edema, hyperaemia and bleeding. Histologically intensive hemorrhagic infiltrations of all affected organs were observed as well as sporadic necrotic processes in the heart and tongue accompanied by perivascular edemas of the lymphoid organs with pronounced lymphocyte depletion within the lymph nodes,<br />spleen and thymus.</p>

Bösartiges Katarrhalfieber ist eine unheilbare Virusinfektion bei Paarhufern, die wiederholt in zoologischen Gärten auftrat, ohne dass die erkrankten Fehlwirte Kontakt zu Reservoirwirten hatten. Die BKF-auslösenden Gammaherpesviren sind eng miteinander verwandt und werden von verschiedenen klinisch gesunden Reservoirwirten latent beherbergt und ausgeschieden. Einige dieser Reservoirwirte sind seit längerem bekannt, andere wurden erst kürzlich identifiziert und es wird vermutet, dass es noch weitere unerkannte Reservoirwirte für BKF-Viren gibt. Hervorzuheben ist, dass die Viren normalerweise eng an ihre Reservoirwirte gebunden sind. Es traten in letzter Zeit jedoch immer wieder Fälle auf, in denen auch Fehlwirte zwar infiziert waren, aber nicht erkrankten oder das Virus sogar ausschieden. Der Zusammenhang zwischen dem Verhalten der BKF-Viren bei Fehl- und Reservoirwirten und den ungeklärten BKF-Fällen in zoologischen Gärten wurde in der hier vorliegenden Studie näher untersucht. Es sollte herausgefunden werden, ob Wildwiederkäuer, die bisher nicht als Reservoirwirte für BKF-Viren galten, diese Viren ausscheiden und so möglicherweise für die oben erwähnten BKF-Fälle verantwortlich waren. / Malignant catarrhal fever (MCF) is an incurable infectious disease in even-toed ungulates, which occurred repeatedly in zoological gardens in Europe without any contact between known hosts and animals with clinical MCF. The causative agents are closely related viruses of the family gamma-herpesviridae, which are latently carried and shed by different clinically healthy ruminant species. Some of the hosts for MCF viruses have been known for many years, while others have been identified only recently. Yet, there are probably still more host species to be discovered. It has to be pointed out that generally MCF viruses are strictly associated with their hosts. However, it has been reported that known susceptible species were infected with MCF viruses without showing any signs of MCF, some of which even excreted the virus. This present study investigates the relationship between the behaviour of MCF viruses in hosts and susceptible species and the nebulous cases of MCF in zoological gardens. The goal was to determine whether wild ruminants, which are normally not known as hosts for MCF, shed these viruses and are possibly responsible for MCF cases mentioned above.