Isolation and identification of malignant catarrhal fever virus in cell cultures

Abstract

Vet. Med., 19, No 4,[263][264][265][266][267][268][269][270][271][272][273] Malignant catarrhal fever (MCF) is a fatal disease syndrome responsible for mortality in domestic and wild ruminant species. MCF is caused by gammaherpesviruses -the ovine herpesvirus type 2 causing sheep-associated malignant catarrhal fever (SA-MCF) and alcelaphine herpesvirus type 1 known as wildebeest-associated MCF (WA-MCF). The present study described the cultural peculiarities of MCF virus isolated from different samples originated from dead and alive wild animals from the Sofia Zoo. MDBK, EBTR, RK, MA-104 and VERO cell cultures were used for the isolation of MCF virus. The best growth of viruses was observed on MDBK cell cultures. The CPE was characterised with forming of the syncytium and destruction of the monolayers 2-3 days after the virus adaptation. The CPE was different for obtained isolates. The isolates from gaur formed a bigger cell syncytium than that in the cell cultures infected with buffy coat from bisons where a cell syncytium of smaller size and shape was observed. The virus identification was performed by biochemical methods and PCR.

Isolation and characterization ofOvine gammaherpesvirus type 2from an outbreak of Malignant Catarrhal Fever inArtiodactylaand horses in Mexico

“…Finally, OvHV-2 was identified in the primary cell cultures through endpoint nested PCR targeting a 238 bp fragment of the ORF75 gene, with a greater intensity starting in the third and fourth passages in all isolates from the different affected species. These results indicate that to generate many free viruses and achieve adaptability of the virus to the cell, several blind passes are needed, coinciding with the report by Hristov et al (2016)(18) The definitive diagnosis confirmation of OvHV-2 infection was performed using partial sequencing of the 422-bp fragment of the ORF75 gene. With this approach, it was possible to determine that all obtained isolates had 98 to 99% nucleotide identity among them and with the OvHV-2 sequences reported in different regions of the world.…”

“…We identified perinuclear and cytoplasmic acidophilic inclusion bodies by H&E and immunohistochemistry, in the liver of deer that died during the SA-MCF outbreak and in primary cell cultures from rabbit testes infected with OvHV-2 isolates. (18). The presence of perinuclear and cytoplasmic acidophilic inclusion bodies observed in the present study could be due to sites of accumulation and retention of overproduced viral and cellular proteins; these macromolecular structures called "dense bodies" are very common in human cells infected by cytomegalovirus.…”

“…In our study, we used buffy coats from affected animals to isolate OvHV-2, which is similar to the study by Hristov et al (2016), who used bison buffy coats and whole blood, lung, and spleen samples from different affected animals during an outbreak of SA-MCF (18). From the third passage in the primary cell cultures of rabbit testis, a marked cytopathic effect was observed at 72 hours post-infection consisting of small foci of refractile cytomegalic cells.…”

“…From the third passage in the primary cell cultures of rabbit testis, a marked cytopathic effect was observed at 72 hours post-infection consisting of small foci of refractile cytomegalic cells. This CPE was also during the isolation of OvHV-2 from camels affected by SA-MCF (18,32).…”

“…PCR is a sensitive and specific test that can support the diagnosis of SA-MCF (17). Buffy coats and lymphoid tissues are ideal sources to isolate the OvHV-2 virus and to perform PCR tests (18). SA-MCF cases have been documented in North America, and the losses due to this disease forced bison producers to leave the market (19).…”