Genome Sequencing of Historical Encephalomyocarditis Viruses from South Africa Links the Historical 1993/4 Savanna Elephant (Loxodonta africana) Outbreak to Cryptic Mastomys Rodents.

From 1993 to 1994, 64 free-ranging elephants (Loxodonta africana) succumbed to encephalomyocarditis in the Kruger National Park, South Africa, of which 83% were adult bulls. Mastomys rodents were implicated as the reservoir host of the Encephalomyocarditis virus (EMCV) based on serology and RT-PCR. However, in the absence of sequence-confirmation of both the virus and the rodent host, definitive links between the elephant outbreak strains and rodent reservoir could not be established. In this study, we generate the first reference genome sequences for three historical EMCVs isolated from two Mastomys rodents and one Mastomys-associated mite, Laelaps muricola, in Gauteng Province, South Africa, in 1961. In addition, near-complete genome sequences were generated for two elephant outbreak virus strains, for which data were previously limited to the P1 and 3D genome regions. The consensus sequence of each virus was determined using a PCR-Sanger sequencing approach. Phylogenetic analysis confirmed the three near-identical (99.95-99.97%) Mastomys-associated viruses to be sister to the two near-identical (99.85%) elephant outbreak strains, differing from each other at 6.4% of sites across the ~7400-nucleotide region characterised. This study demonstrates a link between Mastomys-associated viruses and the historical elephant outbreak strains and implicates Mastomys as reservoirs of EMCV in South Africa.

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 African Swine Fever Virus in Ornithodoros Tick Species Associated with Indigenous and Extralimital Warthog Populations in South Africa.

We investigated the possibility that sylvatic circulation of African swine fever virus (ASFV) in warthogs and Ornithodoros ticks had extended beyond the historically affected northern part of South Africa that was declared a controlled area in 1935 to prevent the spread of infection to the rest of the country. We recently reported finding antibody to the virus in extralimital warthogs in the south of the country, and now describe the detection of infected ticks outside the controlled area. A total of 5078 ticks was collected at 45 locations in 7/9 provinces during 2019-2021 and assayed as 711 pools for virus content by qPCR, while 221 pools were also analysed for tick phylogenetics. Viral nucleic acid was detected in 50 tick pools representing all four members of the Ornithodoros (Ornithodoros) moubata complex known to occur in South Africa: O. (O.) waterbergensis and O. (O.) phacochoerus species yielded ASFV genotypes XX, XXI, XXII at 4 locations and O. (O.) moubata yielded ASFV genotype I at two locations inside the controlled area. Outside the controlled area, O. (O.) moubata and O. (O.) compactus ticks yielded ASFV genotype I at 7 locations, while genotype III ASFV was identified in O. (O.) compactus ticks at a single location. Two of the three species of the O. (O.) savignyi complex ticks known to be present in the country, O. (O.) kalahariensis and O. (O.) noorsveldensis, were collected at single locations and found negative for virus. The only member of the Pavlovskyella subgenus of Ornithodoros ticks known to occur in South Africa, O. (P.) zumpti, was collected from warthog burrows for the first time, in Addo National Park in the Eastern Cape Province where ASFV had never been recorded, and it tested negative for the viral nucleic acid. While it is confirmed that there is sylvatic circulation of ASFV outside the controlled area in South Africa, there is a need for more extensive surveillance and for vector competence studies with various species of Ornithodoros ticks.

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.

Extension of Sylvatic Circulation of African Swine Fever Virus in Extralimital Warthogs in South Africa.

Sylvatic circulation of African swine fever virus (ASFV) in warthogs and Ornithodoros ticks that live in warthog burrows historically occurred in northern South Africa. Outbreaks of the disease in domestic pigs originated in this region. A controlled area was declared in the north in 1935 and regulations were implemented to prevent transfer of potentially infected suids or products to the rest of the country. However, over the past six decades, warthogs have been widely translocated to the south where the extralimital animals have flourished to become an invasive species. Since 2016, there have been outbreaks of ASF in pigs outside the controlled area that cannot be linked to transfer of infected animals or products from the north. An investigation in 2008-2012 revealed that the presence of Ornithodoros ticks and ASFV in warthog burrows extended marginally across the boundary of the controlled area. We found serological evidence of ASFV circulation in extralimital warthogs further south in the central part of the country.

Updated distribution and host records for the argasid tick Ornithodoros (Pavlovskyella) zumpti: A potential vector of African swine fever virus in South Africa.

African swine fever virus (ASFV) causes a lethal and contagious disease of domestic pigs. In South Africa, the virus historically circulated in warthogs and ornithodorid ticks that were only found in warthog burrows in the north of the country. Regulations implemented in 1935 to prevent transfer of infected animals or products to the south initially proved effective but from 2016 there have been outbreaks of disease in the south that cannot be traced to transfer of infection from the north. From 1963 there were widespread translocations of warthogs to the south, initially from a source considered to be free of ornithodorid ticks. We undertook to determine whether sylvatic circulation of ASFV occurs in the south, including identification of potential new vectors, through testing extralimital warthogs for antibody and ticks for virus. Results of testing warthogs for antibody and other species of ticks for virus will be presented separately. Here we report finding Ornithodoros (Pavlovskyella) zumpti ticks in warthog burrows for the first time. This occurred in the Eastern Cape Province (ECP) in 2019. Since African swine fever was recognised in the ECP for the first time in 2020 and outbreaks of the disease in domestic pigs continue to occur there, priority should be given to determining the distribution range and vector potential of O. (P.) zumpti for ASFV.

Mammarenaviruses of Rodents, South Africa and Zimbabwe.

We conducted a survey for group-specific indirect immunofluorescence antibody to mammarenaviruses by using Lassa fever and Mopeia virus antigens on serum specimens of 5,363 rodents of 33 species collected in South Africa and Zimbabwe during 1964-1994. Rodents were collected for unrelated purposes or for this study and stored at -70°C. We found antibody to be widely distributed in the 2 countries; antibody was detected in serum specimens of 1.2%-31.8% of 14 species of myomorph rodents, whereas 19 mammarenavirus isolates were obtained from serum specimens and viscera of 4 seropositive species. Phylogenetic analysis on the basis of partial nucleoprotein sequences indicates that 14 isolates from Mastomys natalensis, the Natal multimammate mouse, were Mopeia virus, whereas Merino Walk virus was characterized as a novel virus in a separate study. The remaining 4 isolates from 3 rodent species potentially constitute novel viruses pending full characterization.

Human-Pathogenic Kasokero Virus in Field-Collected Ticks.

Kasokero virus (KASV; genus Orthonairovirus) was first isolated in 1977 at Uganda Virus Research Institute from serum collected from Rousettus aegyptiacus bats captured at Kasokero Cave, Uganda. During virus characterization studies at the institute, 4 laboratory-associated infections resulted in mild to severe disease. Although orthonairoviruses are typically associated with vertebrate and tick hosts, a tick vector of KASV never has been reported. We tested 786 Ornithodoros (Reticulinasus) faini tick pools (3,930 ticks) for KASV. The ticks were collected from a large R. aegyptiacus bat roosting site in western Uganda. We detected KASV RNA in 43 tick pools and recovered 2 infectious isolates, 1 of which was derived from host blood-depleted ticks. Our findings suggest that KASV is maintained in an enzootic transmission cycle involving O. (R.) faini ticks and R. aegyptiacus bats and has the potential for incidental virus spillover to humans.

Theoretical risk of genetic reassortment should not impede development of live, attenuated Rift Valley fever (RVF) vaccines commentary on the draft WHO RVF Target Product Profile.

In November 2019, The World Health Organization (WHO) issued a draft set of Target Product Profiles (TPPs) describing optimal and minimally acceptable targets for vaccines against Rift Valley fever (RVF), a Phlebovirus with a three segmented genome, in both humans and ruminants. The TPPs contained rigid requirements to protect against genomic reassortment of live, attenuated vaccines (LAVs) with wild-type RVF virus (RVFV), which place undue constraints on development and regulatory approval of LAVs. We review the current LAVs in use and in development, and conclude that there is no evidence that reassortment between LAVs and wild-type RVFV has occurred during field use, that such a reassortment event if it occurred would have no untoward consequence, and that the TPPs should be revised to provide a more balanced assessment of the benefits versus the theoretical risks of reassortment.

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.

Phylogenetic Characterization of the Palyam Serogroup Orbiviruses.

The Palyam serogroup orbiviruses are associated with abortion and teratogenesis in cattle and other ruminants. Of the 13 different serotypes that have been identified, the full genome sequence of only one, Kasba, has been published. We undertook to perform Next Generation Sequencing (NGS) and phylogenetic analysis on 12 Palyam serotypes plus field isolates of the African serotypes in our possession. The Palyam serogroup was found to be most closely related to the African horse sickness virus group and showed the most distant evolutionary relationship to the equine encephalosis viruses (EEV). Amino acid sequence analysis revealed that the gene encoding VP7 was the most conserved within serotypes and VP2 and VP5 showed the highest degree of variation. A high degree of sequence identity was found for isolates from the same geographical region. The phylogenetic analysis revealed two clades where the African serotypes were all very closely related in one clade and the other clade contained the Australian and Asian serotypes and one African serotype, Petevo. It was evident from the sequence data that the geographical origin of Palyam serogroup viruses played an important role in the development of the different serotypes.

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.

Mosquito-borne arboviruses of African origin: review of key viruses and vectors.

Key aspects of 36 mosquito-borne arboviruses indigenous to Africa are summarized, including lesser or poorly-known viruses which, like Zika, may have the potential to escape current sylvatic cycling to achieve greater geographical distribution and medical importance. Major vectors are indicated as well as reservoir hosts, where known. A series of current and future risk factors is addressed. It is apparent that Africa has been the source of most of the major mosquito-borne viruses of medical importance that currently constitute serious global public health threats, but that there are several other viruses with potential for international challenge. The conclusion reached is that increased human population growth in decades ahead coupled with increased international travel and trade is likely to sustain and increase the threat of further geographical spread of current and new arboviral disease.

Genome Sequences of Crimean-Congo Hemorrhagic Fever Virus Strains Isolated in South Africa, Namibia, and Turkey.

We report here the full-length sequences of 16 historical isolates of Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) obtained in Turkey, Namibia, and South Africa. The strains may be useful for future work to develop molecular diagnostics or viral evolution studies.

Ecology of Filoviruses.

Filoviruses can cause severe and often fatal disease in humans. To date, there have been 47 outbreaks resulting in more than 31,500 cases of human illness and over 13,200 reported deaths. Since their discovery, researchers from many scientific disciplines have worked to better understand the natural history of these deadly viruses. Citing original research wherever possible, this chapter reviews laboratory and field-based studies on filovirus ecology and summarizes efforts to identify where filoviruses persist in nature, how virus is transmitted to other animals and ultimately, what drivers cause spillover to human beings. Furthermore, this chapter discusses concepts on what constitutes a reservoir host and highlights challenges encountered while conducting research on filovirus ecology, particularly field-based investigations.

Biosafety standards for working with Crimean-Congo hemorrhagic fever virus.

In countries from which Crimean-Congo haemorrhagic fever (CCHF) is absent, the causative virus, CCHF virus (CCHFV), is classified as a hazard group 4 agent and handled in containment level (CL)-4. In contrast, most endemic countries out of necessity have had to perform diagnostic tests under biosafety level (BSL)-2 or -3 conditions. In particular, Turkey and several of the Balkan countries have safely processed more than 100 000 samples over many years in BSL-2 laboratories. It is therefore advocated that biosafety requirements for CCHF diagnostic procedures should be revised, to allow the tests required to be performed under enhanced BSL-2 conditions with appropriate biosafety laboratory equipment and personal protective equipment used according to standardized protocols in the countries affected. Downgrading of CCHFV research work from CL-4, BSL-4 to CL-3, BSL-3 should also be considered.

Anomalous High Rainfall and Soil Saturation as Combined Risk Indicator of Rift Valley Fever Outbreaks, South Africa, 2008-2011.

Rift Valley fever (RVF), a zoonotic vectorborne viral disease, causes loss of life among humans and livestock and an adverse effect on the economy of affected countries. Vaccination is the most effective way to protect livestock; however, during protracted interepidemic periods, farmers discontinue vaccination, which leads to loss of herd immunity and heavy losses of livestock when subsequent outbreaks occur. Retrospective analysis of the 2008-2011 RVF epidemics in South Africa revealed a pattern of continuous and widespread seasonal rainfall causing substantial soil saturation followed by explicit rainfall events that flooded dambos (seasonally flooded depressions), triggering outbreaks of disease. Incorporation of rainfall and soil saturation data into a prediction model for major outbreaks of RVF resulted in the correctly identified risk in nearly 90% of instances at least 1 month before outbreaks occurred; all indications are that irrigation is of major importance in the remaining 10% of outbreaks.

High prevalence of IgG antibodies to Ebola virus in the Efé pygmy population in the Watsa region, Democratic Republic of the Congo.

BACKGROUND: Factors related to the natural transmission of Ebola virus (EBOV) to humans are still not well defined. Results of previous sero-prevalence studies suggest that circulation of EBOV in human population is common in sub-Saharan Africa. The Efé pygmies living in Democratic Republic of the Congo are known to be exposed to potential risk factors of EBOV infection such as bush meat hunting, entry into caves, and contact with bats. We studied the pygmy population of Watsa region to determine seroprevalence to EBOV infection and possible risks factors. METHOD: Volunteer participants (N = 300) aged 10 years or above were interviewed about behavior that may constitute risk factors for transmission of EBOV, including exposures to rats, bats, monkeys and entry into caves. Samples of venous blood were collected and tested for IgG antibody against EBOV by enzyme-linked immunosorbent assay (ELISA). The χ2-test and Fisher's exact test were used for the comparison of proportions and the Student's t-test to compare means. The association between age group and anti-EBOV IgG prevalence was analysed by a nonparametric test for trend. RESULTS: The prevalence of anti-EBOV IgG was 18.7 % overall and increased significantly with age (p = 0.023). No association was observed with exposure to risk factors (contacts with rats, bats, monkeys, or entry into caves). CONCLUSIONS: The seroprevalence of IgG antibody to EBOV in pygmies in Watsa region is among the highest ever reported, but it remains unclear which exposures might lead to this high infection rate calling for further ecological and behavioural studies.

Sindbis and Middelburg Old World Alphaviruses Associated with Neurologic Disease in Horses, South Africa.

Old World alphaviruses were identified in 52 of 623 horses with febrile or neurologic disease in South Africa. Five of 8 Sindbis virus infections were mild; 2 of 3 fatal cases involved co-infections. Of 44 Middelburg virus infections, 28 caused neurologic disease; 12 were fatal. Middelburg virus likely has zoonotic potential.

Orthobunyavirus antibodies among humans in selected parts of the Rift Valley and northeastern Kenya.

Ngari, Bunyamwera, Ilesha, and Germiston viruses are among the mosquito-borne human pathogens in the Orthobunyavirus genus, family Bunyaviridae, associated with febrile illness. Although the four orthobunyaviruses have been isolated from mosquito and/or tick vectors sampled from different geographic regions in Kenya, little is known of human exposure in such areas. We conducted a serologic investigation to determine whether orthobunyaviruses commonly infect humans in Kenya. Orthobunyavirus-specific antibodies were detected by plaque reduction neutralization tests in 89 (25.8%) of 345 persons tested. Multivariable analysis revealed age and residence in northeastern Kenya as risk factors. Implementation of acute febrile illness surveillance in northeastern Kenya will help to detect such infections.