Molecular characterization of a porcine picobirnavirus RNA-dependent RNA polymerase

Picobirnavirus is an unclassified dsRNA virus, which is associated with viral gastroenteritis in humans and animals. Picobirnavirus dsRNA has been detected in many cases when diagnostic PAGE screening for rotavirus dsRNA is performed. During this routine diagnosis, picobirnavirus dsRNA has been detected in the faeces of patients with and without viral gastroenteritis. Despite the common occurrence of picobirnavirus infection in humans and animals, its direct involvement in causing gastroenteritis has not been established. No molecular studies have been done on picobirnavirus except sequencing and epidemiology studies. Like all RNA viruses, picobirnavirus encodes a RNA-dependent RNA polymerase. The picobirnavirus RNA-dependent RNA polymerase has only been identified on the basis of its amino acid sequence. The catalytic activity of the polymerase has not been studied to date. In this study, a porcine picobirnavirus was studied at a molecular level to establish the activity of the protein encoded by segment 2 of its genome. To determine the identity of this putative picobirnavirus RNA-dependent RNA polymerase, its open reading frame (ORF) was successfully amplified by PCR, cloned and sequenced. Subsequently the ORF was successfully sub-cloned into baculovirus and bacterial expression vectors. The protein encoded by picobirnavirus segment 2 was successfully expressed as a recombinant protein in a soluble form in both baculovirus and bacterial expression systems. In the baculovirus system, two recombinant baculoviruses were constructed. One recombinant baculovirus expressing a histidine tagged protein and another one expressing an untagged protein. In bacterial expression systems, a recombinant protein fused to a Glutathione-S-Transferase (GST) tag at the N terminal end was expressed. The GST tag allowed easy purification of the expressed GST fusion protein by affinity chromatography on immobilized glutathione. Subsequently the GST tag could be removed from the purified recombinant protein by proteolysis with thrombin. Both tagged and untagged putative picobirnavirus RNA-dependent RNA polymerase from the bacterial expression system were shown to have an affinity for heparin. This implies that the protein might have an affinity for nucleic acids. Picobirnavirus genome segment 2 ssRNA was generated from full length picobirnavirus genome segment 2 cDNA by in vitro transcription. The recombinant E.coli expressed proteins (tagged and untagged) was tested for ssRNA binding and RNA replicase activity. No RNA binding or replicase activity was observed with either tagged or untagged recombinant protein. This study reports the first evidence other than conserved polymerase motifs, that the protein encoded by segment 2 of picobirnavirus is most likely a RNA-dependent RNA polymerase. CopyrightDissertation (MSc)--University of Pretoria, 2008.Veterinary Tropical Diseasesunrestricte

Lumpy skin disease of cattle : an emerging problem in the Sultanate of Oman

Lumpy skin disease (LSD) is a highly infectious disease of cattle caused by a virus belonging to the Capripoxvirus genus of the family Poxviridae. The purpose of this study is to place on record the first confirmation of LSD in the Sultanate. The disease was diagnosed and confirmed using polymerase chain reaction, histopathology, transmission electron microscopy and serum neutralization testing. The epizootic occurred in 2009 involving a large number of animals and covering a wide area including Nezwa, Alqabel, Sohar, Saham and Burimi. Morbidity and mortality rates of 29.7 and 26.3 %, and 13.6 and 15.4 % were observed at Nezwa and Sohar, respectively. The clinical signs were much more severe in Holstein–Friesian cattle compared to indigenous breeds and were characterized by multiple skin nodules covering the neck, back, perineum, tail, limbs and genital organs. Affected animals also exhibited lameness, emaciation and cessation of milk production. Oedema of limbs and brisket, and superficial lymph node enlargement were highly prominent. It is not known from where the virus originated, or how it spread to the Sultanate. The disease has become endemic in the country and is liable to extend to other Gulf Cooperation Council Countries and cause a pandemic. It is of major concern to the Omani dairy industry. Due to the widespread presence of screw worm, serious economic losses can follow outbreaks.South African ARChttp://link.springer.com/journal/11250hb201

A comparative genome analysis of Rift Valley Fever virus isolates from foci of the disease outbreak in South Africa in 2008-2010

Rift Valley fever (RVF) is a re-emerging zoonotic disease responsible for major losses in livestock production, with negative impact on the livelihoods of both commercial and resource-poor farmers in sub-Sahara African countries. The disease remains a threat in countries where its mosquito vector thrives. Outbreaks of RVF usually follow weather conditions which favour increase in mosquito populations. Such outbreaks are usually cyclical, occurring every 10–15 years. Recent outbreaks of the disease in South Africa have occurred unpredictably and with increased frequency. In 2008, outbreaks were reported in Mpumalanga, Limpopo and Gauteng provinces, followed by 2009 outbreaks in KwaZulu-Natal, Mpumalanga and Northern Cape provinces and in 2010 in the Eastern Cape, Northern Cape, Western Cape, North West, Free State and Mpumalanga provinces. By August 2010, 232 confirmed infections had been reported in humans, with 26 confirmed deaths.To investigate the evolutionary dynamics of RVF viruses (RVFVs) circulating in South Africa, we undertook complete genome sequence analysis of isolates from animals at discrete foci of the 2008–2010 outbreaks. The genome sequences of these viruses were compared with those of the viruses from earlier outbreaks in South Africa and in other countries. The data indicate that one 2009 and all the 2008 isolates from South Africa and Madagascar (M49/08) cluster in Lineage C or Kenya-1. The remaining of the 2009 and 2010 isolates cluster within Lineage H, except isolate M259_RSA_09, which is a probable segment M reassortant. This information will be useful to agencies involved in the control and management of Rift Valley fever in South Africa and the neighbouring countries.S1 Table. Previously published RVF virus genome sequences used in the study.http://www.plosNTDS.org/hj2020Veterinary Tropical Disease

A comparative genome analysis of Rift Valley Fever virus isolates from foci of the disease outbreak in South Africa in 2008-2010.

Rift Valley fever (RVF) is a re-emerging zoonotic disease responsible for major losses in livestock production, with negative impact on the livelihoods of both commercial and resource-poor farmers in sub-Sahara African countries. The disease remains a threat in countries where its mosquito vector thrives. Outbreaks of RVF usually follow weather conditions which favour increase in mosquito populations. Such outbreaks are usually cyclical, occurring every 10-15 years. Recent outbreaks of the disease in South Africa have occurred unpredictably and with increased frequency. In 2008, outbreaks were reported in Mpumalanga, Limpopo and Gauteng provinces, followed by 2009 outbreaks in KwaZulu-Natal, Mpumalanga and Northern Cape provinces and in 2010 in the Eastern Cape, Northern Cape, Western Cape, North West, Free State and Mpumalanga provinces. By August 2010, 232 confirmed infections had been reported in humans, with 26 confirmed deaths.To investigate the evolutionary dynamics of RVF viruses (RVFVs) circulating in South Africa, we undertook complete genome sequence analysis of isolates from animals at discrete foci of the 2008-2010 outbreaks. The genome sequences of these viruses were compared with those of the viruses from earlier outbreaks in South Africa and in other countries. The data indicate that one 2009 and all the 2008 isolates from South Africa and Madagascar (M49/08) cluster in Lineage C or Kenya-1. The remaining of the 2009 and 2010 isolates cluster within Lineage H, except isolate M259_RSA_09, which is a probable segment M reassortant. This information will be useful to agencies involved in the control and management of Rift Valley fever in South Africa and the neighbouring countries