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Shuni virus is associated with neurologic and febrile illness in animals and humans. To determine potential vectors, we collected mosquitoes in South Africa and detected the virus in species of the genera Mansonia, Culex, Aedes, and Anopheles. These mosquitoes may be associated with Shuni virus outbreaks in Africa and emergence in other regions.

We describe Shuni virus (SHUV) detection in human neurologic disease cases in South Africa. SHUV RNA was identified in 5% of cerebrospinal fluid specimens collected during the arbovirus season from public sector hospitals. This finding suggests that SHUV may be a previously unrecognized cause of human neurologic infections in Africa.

Shuni virus (SHUV) (Peribunyaviridae: Orthobunyavirus) was isolated in the 1960s from livestock, Culicoides midges, and a febrile child in Nigeria. In South Africa, SHUV was identified as the causative agent of neurologic disease in horses; seropositivity was also demonstrated in 3.0% of veterinarians, suggesting human exposures. SHUV was subsequently identified in aborted livestock and cattle with neurologic disease in Israel, suggesting an extended range beyond Africa. We investigated other potential susceptible species in South Africa.

We detected Shuni virus in horses and ovine fetuses and Shamonda virus in a caprine fetus in South Africa. We identified a Shuni/Shamonda virus reassortant in a horse and Shuni/Caimito, Shamonda/Caimito, and Shamonda/Sango virus reassortants in Culicoides midges. Continued genomic surveillance will be needed to detect orthobunyavirus infections in Africa.

Culicoides-borne viruses such as bluetongue, African horse sickness, and Schmallenberg virus cause major economic burdens due to animal outbreaks in Africa and their emergence in Europe and Asia. However, little is known about the role of Culicoides as vectors for zoonotic arboviruses. In this study, we identify both veterinary and zoonotic arboviruses in pools of Culicoides biting midges in South Africa, during 2012–2017. Midges were collected at six surveillance sites in three provinces and screened for Alphavirs, Flavivirus, Orthobunyavirus, and Phlebovirus genera; equine encephalosis virus (EEV); and Rhaboviridae, by reverse transcription polymerase chain reaction. In total, 66/331 (minimum infection rate (MIR) = 0.4) pools tested positive for one or more arbovirus. Orthobunyaviruses, including Shuni virus (MIR = 0.1) and EEV (MIR = 0.2) were more readily detected, while only 2/66 (MIR = 0.1) Middelburg virus and 4/66 unknown Rhabdoviridae viruses (MIR = 0.0) were detected. This study suggests Culicoides as potential vectors of both veterinary and zoonotic arboviruses detected in disease outbreaks in Africa, which may contribute to the emergence of these viruses to new regions.

The Orthobunyavirus genus, family Peribunyaviridae, contains several important emerging and re-emerging arboviruses of veterinary and medical importance. These viruses may cause mild febrile illness, to severe encephalitis, fetal deformity, abortion, hemorrhagic fever and death in humans and/or animals. Shuni virus (SHUV) is a zoonotic arbovirus thought to be transmitted by hematophagous arthropods. It was previously reported in a child in Nigeria in 1966 and horses in Southern Africa in the 1970s and again in 2009, and in humans with neurological signs in 2017. Here we investigated the epidemiology and phylogenetic relationship of SHUV strains detected in horses presenting with febrile and neurological signs in South Africa. In total, 24/1820 (1.3%) horses submitted to the zoonotic arbovirus surveillance program tested positive by real-time reverse transcription (RTPCR) between 2009 and 2019. Cases were detected in all provinces with most occurring in Gauteng (9/24, 37.5%). Neurological signs occurred in 21/24 (87.5%) with a fatality rate of 45.8%. Partial sequencing of the nucleocapsid gene clustered the identified strains with SHUV strains previously identified in South Africa (SA). Full genome sequencing of a neurological case detected in 2016 showed 97.8% similarity to the SHUV SA strain (SAE18/09) and 97.5% with the Nigerian strain and 97.1% to the 2014 Israeli strain. Our findings suggest that SHUV is circulating annually in SA and despite it being relatively rare, it causes severe neurological disease and death in horses.

Viruses in the Orthobunyavirus genus, Peribunyaviridae family, are associated with encephalitis, birth defects and fatalities in animals, and some are zoonotic. Molecular diagnostic investigations of animals with neurological signs previously identified Shuni virus (SHUV) as the most significant orthobunyavirus in South Africa (SA). To determine if other orthobunyaviruses occur in SA, we screened clinical specimens from animals with neurological signs, abortions, and acute deaths from across SA in 2021 using a small (S) segment Simbu serogroup specific TaqMan real-time reverse transcription polymerase chain reaction (RT-PCR). Positive cases were subjected to Sanger sequencing and phylogenetic analysis to identify specific viruses involved, followed by next-generation sequencing (NGS) and additional PCR assays targeting the medium (M) segment and the large (L) segment. In total, 3/172 (1.7%) animals were PCR positive for Simbu serogroup viruses, including two horses with neurological signs and one aborted goat fetus in 2021. Phylogenetic analyses confirmed that the two horses were infected with SHUV strains with nucleotide pairwise (p-) distances of 98.1% and 97.6% to previously identified strains, while the aborted goat fetus was infected with a virus closely related to Shamonda virus (SHAV) with nucleotide p-distances between 94.7% and 91.8%. Virus isolation was unsuccessful, likely due to low levels of infectious particles. However, phylogenetic analyses of a larger fragment of the S segment obtained through NGS and partial sequences of the M and L segments obtained through RT-PCR and Sanger sequencing confirmed that the virus is likely SHAV with nucleotide p-distances between 96.6% and 97.8%. This is the first detection of SHAV in an aborted animal in SA and suggests that SHAV should be considered in differential diagnosis for abortion in animals in Southern Africa.

The genus Orthobunyavirus (family Peribunyaviridae, order Bunyavirales) comprises over 170 named mosquito- and midge-borne viruses, several of which cause severe disease in animals or humans. Their three-segmented genomes enable reassortment with related viruses, which may result in novel viruses with altered host or tissue tropism and virulence. One such reassortant, Schmallenberg virus (SBV), emerged in north-western Europe in 2011. Shuni virus (SHUV) is an orthobunyavirus related to SBV that is associated with neurological disease in horses in southern Africa and recently caused an outbreak manifesting with neurological disease and birth defects among ruminants in Israel. The zoonotic potential of SHUV was recently underscored by its association with neurological disease in humans. We here report a reverse genetics system for SHUV and provide first evidence that the non-structural (NSs) protein of SHUV functions as an antagonist of host innate immune responses. We furthermore report the rescue of a reassortant containing the L and S segments of SBV and the M segment of SHUV. This novel reverse genetics system can now be used to study SHUV virulence and tropism, and to elucidate the molecular mechanisms that drive reassortment events.

The Simbu serogroup of orthobunyaviruses includes several pathogens of veterinary importance, among them Schmallenberg virus (SBV), Akabane virus (AKAV) and Shuni virus (SHUV). They infect predominantly ruminants and induce severe congenital malformation. In adult animals, the intra vitam diagnostics by direct virus detection is limited to only a few days due to a short-lived viremia. For surveillance purposes the testing for specific antibodies is a superior approach. However, the serological differentiation is hampered by a considerable extent of cross-reactivity, as viruses were assigned into this serogroup based on antigenic relatedness. Here, we established a glycoprotein Gc-based triplex enzyme-linked immunosorbent assay (ELISA) for the detection and differentiation of antibodies against SBV, AKAV, and SHUV. A total of 477 negative samples of various ruminant species, 238 samples positive for SBV-antibodies, 36 positive for AKAV-antibodies and 53 SHUV antibody-positive samples were tested in comparison to neutralization tests. For the newly developed ELISA, overall diagnostic specificities of 84.56%, 94.68% and 89.39% and sensitivities of 89.08%, 69.44% and 84.91% were calculated for SBV, AKAV and SHUV, respectively, with only slight effects of serological cross-reactivity on the diagnostic specificity. Thus, this test system could be used for serological screening in suspected populations or as additional tool during outbreak investigations.

Shuni virus (SHUV) is a neglected teratogenic and neurotropic orthobunyavirus that was discovered in the 1960s in Nigeria and was subsequently detected in South Africa, Zimbabwe, and Israel. The virus was isolated from field-collected biting midges and mosquitoes and shown to disseminate efficiently in laboratory-reared biting midges, suggesting that members of the families Culicidae and Ceratopogonidae may function as vectors. SHUV infections have been associated with severe neurological disease in horses, a variety of wildlife species, and domesticated ruminants. SHUV infection of ruminants is additionally associated with abortion, stillbirth, and congenital malformations. The detection of antibodies in human sera also suggests that the virus may have zoonotic potential. To understand how SHUV crosses the ruminant placenta, we here infected pregnant ewes and subsequently performed detailed clinical- and histopathological examination of placental tissue. We found that SHUV targets both maternal epithelial cells and fetal trophoblasts, that together form the maternal-fetal interface of the ovine placenta. Experiments with human placental explants, furthermore, revealed replication of SHUV in syncytiotrophoblasts, which are generally highly resistant to virus infections. Our findings provide novel insights into vertical transmission of SHUV in sheep and call for research on the potential risk of SHUV infection during human pregnancies.