Molecular Epidemiology and Characterisation of Wesselsbron and Co-circulating in Sentinel Animals in South Africa

3 4 Human S.1, Gerdes T , Stroebel J. C. , and Venter M1, 2*

1 Department of Medical Virology, Faculty of Health Sciences, University of Pretoria, South Africa; 2 National Health Laboratory Services, Tshwane Academic Division; 3 Onderstepoort Veterinary Institute, South Africa; 4 Western Cape Provincial Veterinary Laboratory * Contact person: Dr M Venter. Senior lecturer, Department of Medical Virology, Faculty of Health Sciences, University of Pretoria/NHLS Tshwane Academic Division, P.O Box 2034, Pretoria, 0001, South Africa. Tel: +27 12 319 2660, Fax: +27 12 319 5550, e-mail: [email protected], [email protected]

ABSTRACT

Flavi and alphaviruses are a significant cause of morbidity and mortality in both humans and animals worldwide. In order to determine the contribution of flavi and alphaviruses to unexplained neurological hepatic or fever cases in horses and other animals in South Africa, cases resembling these symptoms were screened for Flaviviruses (other than West Nile virus) and Alphaviruses. The results show that both Wesselsbron and alphaviruses (Sindbis-like and Middelburg virus) can contribute to neurological disease and fevers in horses.

KEYWORDS

Wesselsbron virus disease, Sindbis-like virus, Middelburg virus

INTRODUCTION

In South Africa (SA) the most important mosquito borne are West Nile Virus (WNV) and Wesselsbron virus (Flaviviridae) and the Sindbis and Middelburg viruses (Togaviridae). These viruses are transmitted by Culex spp. (WNV and ) and Aedes spp. (Wesselsbron and Middelburg virus) mosquitoes.

Wesselsbron (WSLB) virus was first discovered in 1955 in the Wesselsbron district of the Free State province in SA where it was isolated from an eight-day-old lamb (Weiss et al., 1956). WSLB virus disease causes an acute, biphasic, febrile illness in livestock and has a broad host range occasionally causing illness in ostriches (Verwoed, 2000; Allwright et al, 1995) and man. In adult animals it is sub-clinical, except in the case of pregnant ewes where it may cause hepatitis, haemorrhages and abortions associated with hydrops amnii (Smithburn et al, 1957). In newborn lambs WSLB virus primarily causes a fatal disease. Liver pathology in lambs is constant and typical with features including fatty infiltration, bile pigmentation, death of hepatocytes as well as the infiltration of neutrophils and lymphocytes (Coetzer et al, 1978). Outbreaks of abortions amongst sheep and goats as well as substantial mortality among newborn lambs and kids attract considerable attention, especially as these losses lead to a heavy economic burden amongst farmers.

Alphaviruses are important pathogens of livestock and humans worldwide (Attoui et al, 2007). Until the 1974 outbreaks in the Karoo and Northern Cape Province in SA (Jupp et al, 1986), Sindbis virus (SINV) was considered to be of little medical importance (Sammels et al, 1999) as it causes a self-limiting febrile disease. Another epidemic occurred in 1983/84 in the Witwatersrand/Pretoria regions of SA, where hundreds of human cases were reported (Jupp et al, 1986). Middelburg virus was thought to be non-pathogenic until it was isolated from a spleen of a horse in Zimbabwe that died of symptoms clinically similar to African Horse Sickness (Attoui et al, 2007).

We aimed to screen horses with unexplained neurological and/or hepatic disease for flaviviruses other than WNV. As alphaviruses are transmitted by the same vectors as flaviviruses, these viruses were screened for as well.

MATERIALS AND METHODS

Clinical Specimen Collection Serum, plasma, cerebrospinal fluid (CSF), formalin fixed and fresh organ specimens were collected from horses and other animals displaying unexplained hepatic and/or neurological disease or fever in 2008/2009. Samples were received from the Onderstepoort Veterinary Institute (OVI), University of Pretoria Faculty of Veterinary Science and by private equine veterinarians from the Gauteng, Kwa-Zulu Natal and the Northern and Western Cape provinces.

Extraction of Viral RNA, Flavivirus and Screening and Cloning Viral RNA was extracted from serum/plasma/CSF/culture using the QIA-amp viral RNA mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instruction. A 30mg piece of tissue was used for extraction from fresh and formalin fixed tissues using the RNeasy Plus mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. A nested Flavivirus family RT-PCR was used to screen specimens for Flaviviruses. Similarly, an alphavirus family-specific nested RT-PCR was used to screen clinical specimens (Sanchez-Seco et al, 2001). PCR products were visualised following agarose gel electrophoresis and UV transillumination. Positive alphavirus PCR products were purified and cloned.

Sequencing and Phylogenetic Analysis Positive PCR products and recombinant clones were gel purified and sequenced. Sequences were compiled using Sequencher v4.6 and aligned with MAFFT v6 (http://align.bmr.kyushu-u.ac.jp/mafft/software/). Aligned sequences were edited with BioEdit v7.0.9.0. Neighbor-joining trees were generated with the Kimura 2- parameter model for 1000 bootstrap replicates using Mega v4.

RESULTS

RT-PCR and Nucleotide Sequencing

Out of a total of 80 specimens screened in 2008, of which 48 had fever and 32 were neurological, 2 horses (SAE118/08 and SAE12/08) with neurological symptoms were shown to be Wesselsbron virus by DNA sequence analysis. Phylogenetic analysis clustered them within the Unassigned and Yellow Fever virus group within the Flaviviridae. Both cases had severe neurological disease with symptoms similar to WNV, one which was fatal. Symptoms included ataxia, weak hind and/or forelimb and paresis; complete paralysis, jaundice and/or hepatitis. SAE118/08 was grouped with H117 a Wesselsbron strain that caused a febrile illness in a human (Smithburn et al, 1957) and SAE122/08 clustered most closely to AR2209, a Wesselsbron strain isolated from mosquitoes.

Alphavirus Screening Of the 43 cases screened this year, 5 cases were PCR positive (SAE30/09, SAE31/09, SAE40/09, SAE55/09 and SAE68/09). Sequencing confirmed four specimens to be Sindbis-like virus and one as Middelburg virus. SAE40/09 and SAE55/09 had a co-infection with WNV and had to be euthanized due to severe neurological symptoms including weak hindquarters, multiple skin abrasions on the head and limbs, increased respiratory and heart rate, severe ataxia, weak facial reflexes and recumbency. SAE30/09 had symptoms such as fever and jaundice and survived, whilst SAE31/09 was mildly choleric, mildly ataxic, had pale mucous membranes, petechia and mild neurological symptoms but recovered. The horse diagnosed with Middelburg virus (SAE68/09) recovered after displaying severe neurological symptoms and ataxia.

Phylogenetically, the Sindbis-like specimens grouped with the SA strains of Sindbis-like virus as. The average number of nucleotide differences within the Sindbis-like specimens was 0.7% and differed with the known Sindbis-like and prototype Sindbis viruses by 1.7% and 8% respectively. Meanwhile, the positive Middelburg specimen differed by 3.4% to the known strain of Middelburg virus; and to the known Sindbis-like viruses by 29% and the prototype Sindbis virus by 32%.

DISCUSSION

Mosquito borne viruses such as WSLB, Middelburg and Sindbis viruses have the potential to infect a large number of farm animals as well as horses during conditions that favour the breeding of the mosquito vectors, and have the ability to infect farm workers and owners during zoonotic transmission (McIntosh B.M., 1986) or through mosquito bites. This may create a burden on not only the farmer or animal owner, but also the country’s economy if large outbreaks occur. Using sentinel animals such as horses to screen for WSLB virus is an easy method of outbreak prevention by prediction of an increase in cases and initiating vector control. Since sheep and cattle farming contribute most to the agricultural sector in SA and makes up approximately four percent of SA’s gross domestic product (GDP), it is important to control outbreaks in herds of these animals (www.southafrica.info/business/economy/sectors/542547.html). The positive WSLB specimens were horses that presented clinically with severe neurological symptoms fever, jaundice, profuse urination and recumbency. This is the first study to describe Wesselsbron in horses in South Africa. Alphavirus screening revealed 5 (SAE30/09, SAE31/09, SAE40/09, SAE55/09 and SAE68/09) positive cases. Two horses (SAE40/09 and SAE55/09) had a co-infection with WNV and had to be euthanised due to severe neurological infection. These positive PCR results came from brain specimens, while the rest were diagnosed from blood samples. SAE30/09 survived and had symptoms such as fever and jaundice, whilst SAE31/09 was mildly choleric and displayed mild neurological symptoms but recovered. This suggests that a neurotropic virus such as WNV may allow the alphaviruses to cross the blood-brain-barrier (BBB) and that infection with only Sindbis did not develop severe neurological disease. The horse diagnosed with Middelburg virus (SAE68/09) had more severe neurological symptoms and ataxia but recovered. This suggests that Middelburg virus may be more pathogenic and neurotropic than Sindbis-like viruses in horses and is able to cross the BBB, resulting in severe neurological disease. Further investigations in mice are necessary to confirm this.

CONCLUSION Flaviviruses and alphaviruses in SA may be significantly underdiagnosed as these viruses are not thought of when neurological disease is presented in horses. Wesselsbron virus is able to cause severe neurological disease in horses as shown by our study, which is the first to document this disease in horses. Middelburg virus identified in the current study resulted in more severe neurological disease than the Sindbis-like viruses; this suggests that this virus is more pathogenic and neurotropic. Using horses as sentinel animals to detect an increase in zoonotic virus infections could potentially help to prevent outbreaks of these diseases in humans and other animals.

ACKNOWLEDGEMENTS We would like to thank the following veterinarians that supplied us with the specimens described in this study: Dr H Setzkorn of Chartwell Equine Clinic, Midrand; Dr G Rous of the Karoo Veterinary Clinic, Colesburg; Dr D.C. Triegaardt of Bergview Animal Consulting Rooms, Tulbagh, Western Cape and Dr R Katzwinkel of Summerveld Equine Clinic, Natal.

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