Dokaz i kvantificiranje ovčjeg herpesvirusa 2 u Hrvatskoj

Ovine herpesvirus-2 (OvHV-2), a gammaherpesvirus (genus Rhadinovirus) causes a severe disease known as sheep-associated malignant catarrhal fever (SA-MCF) in certain ruminants, such as cow, deer, bison and water buffalo. Suspected cases of SA-MCF in cows without identififi cation of the agent have been reported in Croatia in the past. In June 2005, on a farm in northwest Croatia, where 17 Simmental diary cows and 2 heifers shared stables and meadows with sheep, a 13 month-old heifer showed symptoms reminiscent of SA-MCF, including anorexia, high fever, nasal discharge, and neurological symptoms, such as ataxia, tremor, convulsions and hyperesthesia. The animal died within 14 days. Gross necropsy fifi ndings were sharply demarcated erosions on mucosal surfaces, including the tongue, oral mucosa, esophagus, abomasum, jejunum, colon, caecum and urinary bladder. Histopathology revealed extremely severe perivascular and intramural arterial infifi ltrations with mononuclear cells, mostly lymphocytes. These lesions were seen in almost every organ, especially the brain and lungs. Formaldehyde fixed samples from the brain, cerebellum, spleen and lymph nodes were obtained and subjected to DNA extraction procedures. Fluorogenic real-time PCR (Polymerase chain reaction) amplififi cation specific to OvHV-2 DNA was performed and OvHV-2 DNA was detected in the brain, cerebellum and spleen, as well as in the lymph nodes. These data indicate that the animal had been infected with OvHV-2, the agent of SA-MCF. For the first time, OvHV-2 was identified and quantified in a Croatian heifer as the causative agent of SA-MCF.Ovčji herpesvirus 2 (OvHV-2) iz roda Rhadinovirus, potporodice Gammaherpesvirinae uzročnik je zarazne korice goveda u nekih vrsta domaćih i divljih preživača. Dosad su u Republici Hrvatskoj zabilježeni mnogi slučajevi sumnje na pojavu zarazne korice u goveda temeljeni na kliničkoj metodi dijagnosticiranja, ali bez dokaza uzročnika bolesti. U lipnju 2005. godine na jednoj farmi u sjeverozapadnoj Hrvatskoj zabilježena je pojava zarazne korice goveda u jedne junice, simentalske pasmine, u dobi od 13 mjeseci, koja je bila smještena u istoj staji s još 17 mliječnih krava i jednom junicom simentalske pasmine te manjim stadom ovaca. Životinja je naglo oboljela uz znakove gubitka apetita, visoke vrućice, seroznog iscjetka iz nosa te pojavom znakova središnjega živčanoga sustava, ataksije, tremora, grčeva i hiperestezije. Životinja je uginula 14. dan od početka prvih znakova bolesti. Razudbom je utvrđena prisutnost oštro ograničenih erozija na sluznici jezika, usne šupljine, jednjaka, sirišta, tankoga, debeloga i slijepoga crijeva te mokraćnoga mjehura. Histopatološki je gotovo u svakom organu, a posebno u mozgu i plućima, utvrđena jaka perivaskularna i intramuralna infiltracija arterija mononuklearnim stanicama, većinom limfocitima. Iz formalinom fiksiranih uzoraka mozga, maloga mozga, slezene i limfnih čvorova izdvojena je DNK i podvrgnuta fluorogenoj real-time PCR amplifikaciji specifičnoj za OvHV-2. OvHV-2 dokazan je i kvantificiran u svim pretraživanim organima. Dobiveni rezultat upućuje na to da je uginula životinja bila zaražena s OvHV-2 što je ujedno i prvi dokaz uzročnika zarazne korice goveda u Hrvatskoj

Ovine hepresvirus 2 infection and the immune resonse in American bison

Ovine herpesvirus 2 (OvHV-2) infects sheep subclinically and causes malignant catarrhal fever (MCF) in poorly adapted species such as bison and cattle. Although MCF is generally fatal once clinical signs develop, subclinical infection can occur in poorly adapted species. This project was initially aimed at developing an experimental, subclinical OvHV-2 infection model in bison for studying adaptive immunity. OvHV-2 has never been propagated in vitro which limited experimental studies until a method of intranasal nebulization with virus from sheep nasal secretions was developed. A dose response study in bison using this method was designed to induce subclinical OvHV-2 infection and determine if this conferred resistance to lethal dose challenge. Results indicated that minimal infectious and minimal lethal doses overlapped. Therefore, experimental production of subclinically infected bison is unlikely to be practical. However, five bison seropositive for antibody against the MCF virus group before inoculation died of MCF after low dose nebulization, indicating that previous exposure to an MCF group virus did not provide resistance to OvHV-2-induced MCF. Lacking a subclinical challenge model, we next compared OvHV-2 infection and immunity in bison to that reported in sheep in order to identify differences applicable to vaccine development. A previous study demonstrated transient OvHV-2 replication in sheep lung after nebulization concurrent with an increase in local transcription of immune response genes. Subsequent dissemination was followed by maintenance of latent virus. For comparison, 40 bison nebulized with OvHV-2 were euthanized between 1 and 35 days post-inoculation. Transient viral replication, indicated by increased OvHV-2 DNA level and transcription of two lytic cycle genes, occurred in bison lung 7 to 12 days post-inoculation but was not associated with increased immune response gene transcription. Subsequent systemic dissemination was followed by lytic replication in tissues concurrent with lesion development. Based on these results, we propose that OvHV-2 replication in bison lung is self-limiting rather than controlled by the host immune response. We also propose that differential immunomodulation by the virus may be the reason for differences in the pulmonary immune response and could explain why widespread lytic replication and lesion development occurs in bison but not in sheep

Malignant Catarrhal Fever in Indonesia and Its Control Strategy

Malignant catarrhal fever (MCF) is an immunoproliferative and lethal disease of many species of the order Artiodactyla (such as families Bovidae, Cervidae and Suidae) caused by a member of the MCF virus (MCFV) group belongs to the genus Macavirus in the subfamily Gammaherpesvirinae. There are two types of MCF i.e. Wildebeest-Associated MCF (WA-MCF) which is caused by Alcelaphine Herpesvirus-1 (AlHV-1) with wildebeest as reservoir animal; and Sheep-Associated MCF (SA-MCF) which is caused by Ovine herpesvirus 2 (OvHV-2) with sheep and goats as reservoir animals. AlHV-1 virus has already been isolated whereas OvHV-2 has not been isolated so that vaccines are not yet available. Both types cannot be differentiated by clinical and pathological findings. This disease was previously diagnosed based on the epidemiological information and clinicopathological findings, but now it can be diagnosed by using molecular biological tests. This paper describes the epidemiology of MCF virus, MCF cases in Indonesia and efforts to control this disease. In Indonesia, SA-MCF cases have been reported almost in all provinces as endemic as well as epidemic nature. Separation of reservoir animal with susceptible species, "producing" a SA-MCF virus free sheep and attempt to develop a recombinant vaccine against SA-MCF is the main control strategy that can be suggested

Gammaherpesviren bei kleinen Wiederkäuern, Zoo- und Wildtieren

Bösartiges Katarrhalfieber (BKF) ist eine meist tödlich verlaufende, meldepflichtige Erkrankung von Paarhufern, die durch verschiedene Macaviren der Unterfamilie Gammaherpesvirinae aus der Familie Herpesviridae ausgelöst wird. In der Regel handelt es sich dabei um eine Einzeltiererkrankung. Seit den ersten Fällen von Blauzungenkrankheit in Deutschland hat der Nachweis von Macaviren im Rahmen der Differenzialdiagnostik an Bedeutung gewonnen. Ungezielte Methoden zur Virusdiagnostik wie die Virusanzucht in Zellkultur und die Elektronenmikroskopie sind im Hinblick auf Macaviren nicht erfolgversprechend. Für eine Labordiagnose ist die PCR Mittel der Wahl. Bisher wird hierfür von den meisten Labors eine OvHV-2-spezifische PCR bei Rinderproben eingesetzt. Ein Ziel dieser Arbeit war es, die diagnostischen Möglichkeiten so zu erweitern, dass ein möglichst breites Spektrum von potentiellen BKF-Erregern detektiert werden kann. Weiterhin sollte ein System zur Vermehrung von CpHV-2 gefunden werden. Bezüglich der BKF-Diagnostik wurden zwei neue konventionelle PCR-Verfahren mit breitem Nachweisspektrum für Macaviren und eine OvHV-2-CpHV-2-diskriminierende Echtzeit PCR etabliert. Weiterhin wurden zwei serologische Verfahren, ELISA und Serumneutralisationstest, miteinander verglichen. Dafür wurden Proben von 276 Wildwiederkäuern, 230 Ziegen und 58 Schafen untersucht. Bei der Antikörperbestimmung ist der kompetitive ELISA dem SNT im Hinblick auf Arbeitszeit, Aufwand und Qualität der Testergebnisse deutlich überlegen. Für den Einsatz in der Routinediagnostik wird der ELISA vor allem für Hauptwirte empfohlen. Der Bedarf breit angelegter Detektionsmethoden für die BKF-Diagnostik konnte im PCR-Bereich anhand von 18 positiven Resultaten bei Wildtierproben demonstriert werden. Diese 18 Proben führten zu negativen Ergebnissen beim Einsatz der OvHV-2-spezifischen PCR, aber zu positiven Ergebnissen mit den PAN-Macavirus-PCR-Verfahren. Klonierung, Sequenzierung und eine phylogenetische Analyse der Amplifikate führten zu den Nachweisen von CpHV-2, CpHV-2-ähnlichem und BoHV-6-ähnlichem Macavirus. Erstmals konnte CpHV-2-induziertes BKF bei zwei Banteng-Rindern (Bos javanicus) festgestellt werden. Bisher wurde CpHV-2 nur als Auslöser der Erkrankung bei Hirschen und Elchen angesehen. Die Daten zeigen, dass die bisher ausschließlich durchgeführte OvHV-2-Diagnostik im Rinderbereich zu falsch negativen Befunden führen kann. Die beschriebene Etablierung einer OvHV-2-CpHV-2-diskriminierenden Echtzeit PCR ermöglicht eine schnelle Unterscheidung zwischen den beiden in Europa vorkommenden Hauptverursachern von BKF. Diese Methode ist für den Einsatz in der Routinediagnostik für kleine Wiederkäuer und Rinder gut geeignet. Für den Einsatz bei Wildtieren sind die breiter angelegten konventionellen PCR-Methoden vorzuziehen. Anzuchtversuche ausgehend von CpHV-2-positiven ZNS-Proben von Hirschen wurden auf unterschiedlichen Zelllinien durchgeführt. Weiterhin konnten Primär- und Sekundärkulturen aus Ziegen-Lymphozyten etabliert werden. Abschließend wurde ein vergleichbarer Virusanzuchtversuch auf diesen Sekundärkulturen durchgeführt. Bei allen Ansätzen kam es jedoch zum Verlust der virusspezifischen DNS. Zusätzlich wurde ein Transfektionsversuch mit Nukleinsäure aus dem oben angeführten ZNS von erkrankten Hirschen auf PT-Zellen durchgeführt und Überstände sowie Zellen mittels Echtzeit PCR untersucht. Auch dieser Ansatz erlaubte nicht die Kultivierung von CpHV-2.Malignant catarrhal fever (MCF) is a fatal, notifiable disease of Artiodactyla, which is caused by different Macaviruses of the subfamily Gammaherpesvirinae of the family Herpesviridae. MCF usually occurs only sporadically. Within the framework of differential diagnostics the detection of Macaviruses has become more important since the first cases of bluetongue disease were noticed in Germany. Undirected virological methods like virus propagation in cell culture and electron microscopy are not promising with regard to Macaviruses. PCR is the method of choice for laboratory diagnosis. Until now most laboratories use the OvHV-2 specific PCR for samples of cattle. One objective of this work was to improve the diagnostic tools for the detection of a broad spectrum of potential MCF-viruses. Furthermore experiments towards the establishment of a system for the cultivation of CpHV-2 were performed. With regard to MCF-diagnostics two new conventional PCR-methods and an OvHV-2-CpHV-2-discriminating realtime PCR were established, which allowed to detect a broad spectrum of macaviruses. Furthermore two antibody-test-systems, ELISA and serum neutralisation assay (SNA), were compared to each other. Samples of 276 wild ruminants, 230 goats and 58 sheep were examined by these methods. Concerning time, effort and quality of result the competitive ELISA was clearly superior to SNA. ELISA is recommended for routine diagnostic use, especially for reservoir hosts. The importance of broadly reactive PCR detection methods could be demonstrated by 18 positive results of wild animal samples. All 18 cases led to negative results with OvHV-2-specific PCR, but reacted positive in the PAN-Macavirus-PCR. Cloning, sequencing and phylogenetic analyses of the amplificates led to the detection of CpHV-2, CpHV-2-resembling and BoHV-6-resembling Macaviruses. For the first time CpHV-2-induced MCF was detected in two Banteng-cattle (Bos javanicus). Until now, CpHV-2 has been considered as a cause of MCF only in deer and moose. This implies that routine OvHV-2-specific-diagnostic in cattle is insufficient. The establishment of an OvHV-2-CpHV-2-discriminating realtime PCR allowed rapid differentiation between the two most common causative agents of MCF in Europe. This method is suitable for application in routine diagnostics of small ruminants and cattle. For the application in wild ruminants the broadly reactive conventional PCR is more suited. Using CpHV-2-positive CNS-samples of MCF-diseased deer as starting material, it was attempted to cultivate the virus in different cell lines. Furthermore, primary and secondary cultures of goat lymphocytes were established and used for propagation trials. However, after prolonged incubation Macavirus specific DNA could not be detected. Finally PT-cells were transfected with nucleic acid prepared from the above mentioned CpHV-2 positive deer-CNS and supernatant as well as cells were tested by realtime PCR. This approach did also not lead to detectable replication of CpHV-2

Identification and characterization of Ovine Herpesvirus 2 microRNAs

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

Virus life cycle and the parthenogenesis of malignant catarrhal fever

Malignant catarrhal fever (MCF) is caused by two closely associated gamma herpes viruses namely alcelaphine herpesvirus 1 (AlHV-1) and ovine herpesvirus 2 (OvHV-2) and characterised with lymphocyte infiltration in non-lymphoid tissues, vasculitis and epithelial damage. The mechanism by which the viruses cause the disease is not fully understood. The hypothesis of this project was that MCF is initiated by aberrant gene expression in endothelium, epithelium and infected T cells of susceptible animals, because they are not the natural hosts for the viruses and the viruses will not have evolved in them. The first goal was to examine whether rabbit epithelium and bovine endothelium can be infected in vitro and in vivo with AlHV-1 using q PCR and, if infected whether viral transcripts could be identified in these tissue cells using q PCR and in situ hybridisation (ISH). The results revealed that endothelium and epithelium can be infected and latent infection can be established in them. This suggests the likelihood of establishing a similar type of infection in vivo. Secondly, the trial to identify latency-associated transcripts using 5-azacitidine treatment on bovine turbinate fibroblast (BT) cells and rabbit large granular lymphocytes (LGLs) was only partially successful. However, pan T antigen was expressed in 5-azacitidine treated but not untreated LGLs cells. This may indicate a function of the drug either directly or through the latency state. Transcriptome analysis in the infected and treated LGLs and BT cells showed that several pathways were affected by 5-aza although a possible latency (low transcript levels) was only seen in the BTs. Transcriptome analysis revealed similar pathways to those described for MCF in the tissues in vivo, and an effect of 5-aza on these. Viral transcripts analysis showed that genes related to productive/lytic cycles were higher than latent ones on day 17 of the in vivo experiment demonstrating that the virus may replicate at this stage of the disease. The attempt to localize the viral transcripts on the rabbit infected tissues using ISH was unsuccessful due to a lack of time

Virus life cycle and the parthenogenesis of malignant catarrhal fever

Malignant catarrhal fever (MCF) is caused by two closely associated gamma herpes viruses namely alcelaphine herpesvirus 1 (AlHV-1) and ovine herpesvirus 2 (OvHV-2) and characterised with lymphocyte infiltration in non-lymphoid tissues, vasculitis and epithelial damage. The mechanism by which the viruses cause the disease is not fully understood. The hypothesis of this project was that MCF is initiated by aberrant gene expression in endothelium, epithelium and infected T cells of susceptible animals, because they are not the natural hosts for the viruses and the viruses will not have evolved in them. The first goal was to examine whether rabbit epithelium and bovine endothelium can be infected in vitro and in vivo with AlHV-1 using q PCR and, if infected whether viral transcripts could be identified in these tissue cells using q PCR and in situ hybridisation (ISH). The results revealed that endothelium and epithelium can be infected and latent infection can be established in them. This suggests the likelihood of establishing a similar type of infection in vivo. Secondly, the trial to identify latency-associated transcripts using 5-azacitidine treatment on bovine turbinate fibroblast (BT) cells and rabbit large granular lymphocytes (LGLs) was only partially successful. However, pan T antigen was expressed in 5-azacitidine treated but not untreated LGLs cells. This may indicate a function of the drug either directly or through the latency state. Transcriptome analysis in the infected and treated LGLs and BT cells showed that several pathways were affected by 5-aza although a possible latency (low transcript levels) was only seen in the BTs. Transcriptome analysis revealed similar pathways to those described for MCF in the tissues in vivo, and an effect of 5-aza on these. Viral transcripts analysis showed that genes related to productive/lytic cycles were higher than latent ones on day 17 of the in vivo experiment demonstrating that the virus may replicate at this stage of the disease. The attempt to localize the viral transcripts on the rabbit infected tissues using ISH was unsuccessful due to a lack of time