Rift Valley Fever virus M and L genome segment detection: a comparison of field-deployable reverse transcription insulated isothermal PCR (RT-iiPCR) and laboratory-based multiplex reverse transcription real-time PCR.

Rift Valley Fever phlebovirus (RVFV) is a mosquito-borne zoonotic pathogen that causes major agricultural and public health problems in Africa and the Arabian Peninsula. It is considered a potential agro-bioterrorism agent for which limited countermeasures are available. To address diagnostic needs, here we describe a rapid and sensitive molecular method immediately employable at sites of suspected outbreaks in animals that commonly precede outbreaks in humans. The strategy involves the concurrent detection of two of the three RVFV genome segments (large and medium) using reverse transcription insulated isothermal PCR (RT-iiPCR) performed on a portable, touch screen nucleic acid analyzer, POCKIT. The analytical sensitivity for both the RT-iiPCR and a laboratory-based L and M multiplex reverse transcription real-time PCR assay was estimated at approximately 0.1-3 copies/reaction using synthetic RNA or viral RNA. The diagnostic sensitivity and specificity of detection of RVFV on the POCKIT, determined using sera from sheep and cattle (n = 181) experimentally infected with two strains of RVFV (SA01 and Ken06), were 93.8% and 100% (kappa = 0.93), respectively. Testing of ruminant field sera (n = 193) in two locations in Africa demonstrated 100% diagnostic sensitivity and specificity. We conclude that the POCKIT dual-gene RVFV detection strategy can provide reliable, sensitive, and specific point-of-need viral RNA detection. Moreover, the field detection of RVFV in vectors or susceptible animal species can aid in the surveillance and epidemiological studies to better understand and control RVFV outbreaks. IMPORTANCE: The content of this manuscript is of interest to the diverse readership of the Journal of Clinical Microbiology, including research scientists, diagnosticians, healthcare professionals, and policymakers. Rift Valley Fever virus (RVFV) is a zoonotic mosquito-borne pathogen that causes major agricultural and public health problems. Current and most sensitive diagnostic approaches that are molecular-based are performed in highly specialized molecular diagnostic laboratories. To address diagnostic needs, we developed a novel, rapid, and sensitive molecular method using a portable PCR machine, POCKIT, capable of immediate deployment at sites of suspected outbreaks. Here, we demonstrate that field-deployable RVFV detection can provide reliable, sensitive, and specific point-of-need viral RNA detection that could be used for diagnostic investigations and epidemiological studies, and can be performed in the field.

Detection of Rift Valley Fever Virus in Aedes (Aedimorphus) durbanensis, South Africa.

Rift Valley fever virus (RVFV) is a mosquito-borne, zoonotic phlebovirus-causing disease in domestic ruminants and humans in Africa, the Arabian Peninsula and some Indian Ocean islands. Outbreaks, characterized by abortion storms and a high morbidity rate in newborn animals, occur after heavy and prolonged rainfalls favouring the breeding of mosquitoes. However, the identity of the important mosquito vectors of RVFV is poorly known in most areas. Mosquitoes collected in the Ndumo area of tropical north-eastern KwaZulu-Natal (KZN), South Africa, were tested for RVFV nucleic acid using RT-PCR. The virus was detected in a single pool of unfed Aedes (Aedimorphus) durbanensis, indicating that this seasonally abundant mosquito species could serve as a vector in this area of endemic RVFV circulation. Phylogenetic analysis indicated the identified virus is closely related to two isolates from the earliest outbreaks, which occurred in central South Africa more than 60 years ago, indicating long-term endemicity in the region. Further research is required to understand the eco-epidemiology of RVFV and the vectors responsible for its circulation in the eastern tropical coastal region of southern Africa.

Neutralizing antibodies against Rift Valley fever virus in wild antelope in far northern KwaZulu-Natal, South Africa, indicate recent virus circulation.

Rift Valley fever (RVF) is a zoonotic viral disease of domestic ruminants in Africa and the Arabian Peninsula caused by a mosquito-borne Phlebovirus. Outbreaks in livestock and humans occur after heavy rains favour breeding of vectors, and the virus is thought to survive dry seasons in the eggs of floodwater-breeding aedine mosquitoes. We recently found high seroconversion rates to RVF virus (RVFV) in cattle and goats, in the absence of outbreaks, in far northern KwaZulu-Natal (KZN), South Africa. Here, we report the prevalence of, and factors associated with, neutralizing antibodies to RVFV in 326 sera collected opportunistically from nyala (Tragelaphus angasii) and impala (Aepyceros melampus) culled during 2016-2018 in two nature reserves in the same area. The overall seroprevalence of RVFV, determined using the serum neutralization test, was 35.0% (114/326; 95%CI: 29.8%-40.4%) and tended to be higher in Ndumo Game Reserve (11/20; 55.0%; 95%CI: 31.5%-76.9%) than in Tembe Elephant Park (103/306; 33.6%; 95%CI: 28.4%-39.3%) (p = .087). The presence of antibodies in juveniles (6/21; 28.6%; 95%CI: 11.3%-52.2%) and sub-adults (13/65; 20.0%; 95%CI: 11.1%-37.8%) confirmed that infections had occurred at least until 2016, well after the 2008-2011 RVF outbreaks in South Africa. Odds of seropositivity was higher in adults than in sub-adults (OR = 3.98; 95%CI: 1.83-8.67; p = .001), in males than in females (OR = 2.66; 95%CI: 1.51-4.68; p = .001) and in animals collected ≤2 km from a swamp or floodplain compared with those collected further away (OR = 3.30; 95%CI: 1.70-6.38; p < .001). Under similar ecological conditions, domestic and wild ruminants may play a similar role in maintenance of RVFV circulation and either or both may serve as the mammalian host in a vector-host reservoir system. The study confirms the recent circulation of RVFV in the tropical coastal plain of northern KZN, providing the basis for investigation of factors affecting virus circulation and the role of wildlife in RVF epidemiology.

High seroconversion rate to Rift Valley fever virus in cattle and goats in far northern KwaZulu-Natal, South Africa, in the absence of reported outbreaks.

BACKGROUND: Rift Valley fever (RVF) is a mosquito-borne zoonotic disease characterized in South Africa by large epidemics amongst ruminant livestock at very long, irregular intervals, mainly in the central interior. However, the presence and patterns of occurrence of the virus in the eastern parts of the country are poorly known. This study aimed to detect the presence of RVF virus (RVFV) in cattle and goats in far northern KwaZulu-Natal province and to estimate the prevalence of antibodies to the virus and the incidence rate of seroconversion. METHODOLOGY: Cross-sectional studies were performed in communally farmed cattle (n = 423) and goats (n = 104), followed by longitudinal follow-up of seronegative livestock (n = 253) 14 times over 24 months, representing 160.3 animal-years at risk. Exposure to RVFV was assessed using an IgG sandwich ELISA and a serum neutralization test (SNT) and seroconversion was assessed using SNT. Incidence density was estimated and compared using multivariable Poisson models and hazard of seroconversion was estimated over time. PRINCIPAL FINDINGS: Initial overall seroprevalence was 34.0% (95%CI: 29.5-38.8%) in cattle and 31.7% (95%CI: 22.9-41.6%) in goats, varying by locality from 18-54%. Seroconversions to RVFV based on SNT were detected throughout the year, with the incidence rate peaking during the high rainfall months of January to March, and differed considerably between years. Overall seroconversion rate in cattle was 0.59 per animal-year (95% CI: 0.46-0.75) and in goats it was 0.41 per animal-year (95% CI: 0.25-0.64), varying significantly over short distances. CONCLUSIONS/SIGNIFICANCE: The high seroprevalence in all age groups and evidence of year-round viral circulation provide evidence for a hyperendemic situation in the study area. This is the first study to directly estimate infection rate of RVFV in livestock in an endemic area in the absence of reported outbreaks and provides the basis for further investigation of factors affecting viral circulation and mechanisms for virus survival during interepidemic periods.

Reassortment of bluetongue virus vaccine serotypes in cattle.

Bluetongue is primarily a disease of sheep in South Africa, while cattle and goats are mostly subclinically infected. The viraemia of bluetongue virus in cattle lasts much longer than in sheep and the role of cattle in the epidemiology of bluetongue in South Africa is poorly understood. Bluetongue virus has a segmented double-stranded ribonucleic acid genome and reassortment of genomes is a common feature. The aim of the study was to investigate whether reassortment occurs between vaccine and field strains when simultaneously administered to cattle. Six cattle between the ages of 6 and 12 months were infected with five strains of modified live vaccine bluetongue virus and a virulent field isolate of bluetongue virus 4. Blood samples were subsequently collected daily from these animals from day 1 to day 39 post-inoculation. Viruses were directly isolated during viraemia from the buffy coat on Vero cells using the plaque forming unit method. Analysis of plaques indicated that no reassortants between virulent field and vaccine strains occurred and the virulent bluetongue virus 4 was identified as the predominant virus strain. However, a reassortant virus between two bluetongue virus vaccine strains was isolated from the buffy coat. Whole genome sequences from the vaccine viruses were compared to the suspected reassortant and it was found that segment 8 exchanged between the bluetongue virus 8 and bluetongue virus 9 vaccine strains. The use of the live-attenuated bluetongue virus multivalent vaccine in South Africa causes circulation of different vaccine serotypes in Culicoides spp. and susceptible hosts and cattle might provide the ideal host for reassortment to occur.

Complete Genome Sequences of Five Bluetongue Virus (BTV) Vaccine Strains from a Commercial Live Attenuated Vaccine, a BTV-4 Field Strain from South Africa, and a Reassortant Strain Isolated from Experimentally Vaccinated Cattle.

This is a report of the complete genome sequences of plaque-selected isolates of each of the five virus strains included in a South African commercial trivalent bluetongue virus (BTV) attenuated live virus vaccine, a BTV-4 field strain isolated from Rustenburg, South Africa, in 2011, and a bluetongue reassortant (bluetongue virus 4 strain 4/O. aries-tc/ZAF/11/OBP-115) isolated from experimentally vaccinated cattle. Full-genome sequencing and phylogenetic analyses show that the bluetongue virus 9 strain 9/B. taurus-tc/ZAF/15/Onderstepoort_B02b is a reassortant virus containing segments from both BTV-9 and BTV-8.