**5. Conclusions**

Increased equine WNV-positive case numbers in RSA in 2017 were largely attributed to environmental factors favouring the breeding habits of the vector. The largest proportion of cases during 2016–2017 was reported in the temperate to warm, eastern inland

RSA plateau, at intermediate elevation above sea level, during March–April. The WNVassociated case fatality rate and neuroinvasive disease proportions from 2016 to 2017 were consistent with those reported in previous local and international studies. Most of the cases displayed neurological signs, which were significantly associated with WNV infection, and approximately half of the cases had fever. Fever was marginally associated with recovery from WNV and may potentially be used as a prognostic indicator. Vaccination against WNV was significantly protective, and the risk of developing clinical WNV significantly decreased with increasing age, which was likely due to increased immunity from repeated long-term, low grade field exposure.

Therefore, it is advisable that owners with competitive horses or those younger than two to five years old, especially the highly purebred breeds (such as Thoroughbreds, Warmbloods, and Arabians) residing in the eastern temperate to warm parts of RSA with high summer rainfall, or travelling between provinces, should practice routine, complete vaccination against WNV. These vaccines should be given annually during spring, in order to decrease morbidity and mortality by timeously increasing immunological resistance against WNV.

**Author Contributions:** All three authors contributed to the conceptualisation, methodology, and resources for the study. Investigation and data curation were performed by F.-M.B. and M.V., while F.-M.B. and P.N.T. were responsible for the analysis and mapping software, formal analysis and visualisation. F.-M.B. prepared and wrote the original draft, and M.V. and P.N.T. supervised the project, validated the results, reviewed and edited the manuscript and contributed to funding acquisition. M.V. is the owner of the database and all specimens that had been tested for arboviruses under the Zoonotic Arbo and Respiratory Viruses Programme which had been approved under section 20 approval by the Department Agriculture Forestry and Fisheries and the University of Pretoria Animal Ethics Committee. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research including WNV testing and laboratory investigations were funded through M.V.'s National Research Foundation incentive fund and University of Pretoria Development fund during 2016-2017 and were done at no extra expense to either the owner or veterinarian.

**Institutional Review Board Statement:** The study was conducted according to the ARRIVE guidelines and approved by the Animal Ethics Committee of the University of Pretoria (V080-18, 9 October 2018; H012-16, 25 July 2016). The study was also conducted according to the guidelines of the Declaration of Helsinki, and approved by the University of Pretoria, Faculty of Health ethics committee (protocol code 155/2019, 12 April 2019). Title: "One Health approach to detect zoonotic arboviruses through surveillance in animals and vectors, and development of molecular and serological assays to define their epidemiology in South Africa".

**Informed Consent Statement:** All animal specimens were submitted to the surveillance program under informed consent that the data will be used as part of arbovirus surveillance and research. The study did not involve human subjects thus human informed consent was not applicable.

**Data Availability Statement:** The data were collected as part of the research and surveillance program by the ZARV programme in the Centre for Viral Zoonoses, University of Pretoria. The data is not publicly available to protect the identity of the owners. The minimal dataset that supports the central findings of a published study may be obtained from the corresponding author, M.V. under agreemen<sup>t</sup> with the University of Pretoria.

**Acknowledgments:** The authors would like to express sincere appreciation to the owners, stud and stable managers, and veterinarians who provided information, and condolences to those who lost a beloved horse. We would also like to thank and acknowledge all staff in the Zoonotic Arbo and Respiratory Viruses Programme in the CVZ who participated in laboratory testing, as well as June Williams of the Department of Veterinary Pathology, University of Pretoria, who was personally involved in post-mortem examination and test requisition of many of the WNV-positive cases.

**Conflicts of Interest:** The authors declare no conflict of interest.
