Characterization of zoonotic flavi- and alphaviruses in sentinel animals in South Africa

Abstract

In South Africa (SA), the arboviruses West Nile virus (WNV), Wesselsbron virus (WSLV), Sindbis virus (SINV) and Middelburg virus (MIDV) are considered the most important flavi- and alphaviruses. Clinical presentation and importance of these viruses as animal pathogens in SA remains ambiguous. Although widely endemic in SA, lineage 2 (L2) WNV has rarely been associated with cases of neurological disease and was therefore assumed to be non-pathogenic. However, fatal encephalitis in a foal was diagnosed as L2 WNV in SA, 1996, leading to the thought that L2 cases were possibly being missed. As the above-mentioned arboviruses have the same transmission vectors, Culex mosquitoes for WNV and SINV and Aedes mosquitoes for WSLV and MIDV, co-screening for these viruses is important. We hypothesise that horses could be used as sentinels for virus activity in SA and cases of unexplained neurological disease or fever in animals overlooked, rather than being non-existent. To this end, the study aimed to screen horses displaying unexplained neurological disease or fever with Flavivirus family-specific RT-PCR. Additionally, samples were screened with an Alphavirus family-specific RT-PCR to determine whether co-circulating viruses could be responsible for neurological symptoms in horses. The results would aid in establishing the molecular epidemiology and disease description of each virus, virus distribution and disease seasonality in SA. In total 261 clinical specimens were collected from horses displaying these symptoms (2008 - 2010). Samples were screened with Flavi- and Alphavirus differential diagnostic RT-PCR and acute serum was screened for WNV-IgM and neutralizing antibodies. Serological screening (WNV haemagglutination inhibition, WNV IgG and/or WNV neutralization) identified 62 suspected WNV cases while 34 cases could be confirmed by RT-PCR (16/34), WNV IgM and neutralization assays (18/34) and virus isolation. Neurological disease made up 91% (31/34) of the cases, mortality was calculated at 44% (15/34). Phylogenetically 12/16 RT-PCR positives grouped with L2 SA strains. The first detection of L1 WNV and horse-associated abortion in SA was reported when a pregnant mare aborted her foetus in Ceres, Western Cape. The first cases of WSLV-associated disease in horses were identified by sequencing Flavivirus RT-PCR positive products from 2 horses displaying severe neurological disease; one being fatal. This suggests missed cases in the past. To elucidate virulence factors of WSLV, a human encephalitic strain AV259, was subjected to Roche FLX454 full-genome sequencing and compared to a previously sequenced febrile strain (H177). Several structural amino acid changes occurred in proteins NS2A, NS4B and NS5 of AV259; necessary for Flavivirus replication. Phylogenetically AV259, clinical horse strains and WSLV strains previously isolated from animals, humans and arthropods were similar. Additionally and in concurrence with other studies, WSLV clusters with Sepik virus (SEPV) within the YFV group of the Flaviviridae family. Alphavirus screening identified 17 cases; 6/17 SINV and 11/17 MIDV. SINV-WNV co-infections resulted in fatal neurological disease; remaining SINV cases recovered after displaying fever and/or mild neurological disease. MIDV symptoms varied from “three-day-stiffness” to severe neurological symptoms, with 2 fatalities. Co-infections with equine encephalosis and Shuni virus were identified. MIDV strains identified in this study were phylogenetically distinct from older strains. Results highlight the use of horses as sentinels for virus activity and suggest that these arboviruses may have been previously missed as horse pathogens in Africa. These viruses should be considered as the aetiological agents in animals displaying unexplained neurological or hepatic disease, fevers or abortions. Awareness of flavi- and alphaviruses and the disease manifestation they may have in horses was illustrated. These findings suggest that a WNV vaccine may be beneficial for horses in SA.