Attempted Transmission of Marburg Virus by Bat-Associated Fleas Thaumapsylla breviceps breviceps (Ischnopsyllidae: Thaumapsyllinae) to the Egyptian Rousette Bat (Rousettus aegyptiacus).

Egyptian rousette bats (ERBs) are implicated as reservoir hosts for Marburg virus (MARV), but natural mechanisms involved in maintenance of MARV in ERB populations remain undefined. A number of hematophagous ectoparasites, including fleas, parasitize bats. Subcutaneous (SC) inoculation of ERBs with MARV consistently results in viremia, suggesting that infectious MARV could be ingested by blood-sucking ectoparasites during feeding. In our study, MARV RNA was detected in fleas that took a blood meal during feeding on viremic bats on days 3, 7, and 11 after SC inoculation. Virus concentration in individual ectoparasites was consistent with detectable levels of viremia in the blood of infected host bats. There was neither seroconversion nor viremia in control bats kept in close contact with MARV-infected bats infested with fleas for up to 40 days post-exposure. In fleas inoculated intracoelomically, MARV was detected up to 14 days after intracoelomic (IC) inoculation, but the virus concentration was lower than that delivered in the inoculum. All bats that had been infested with inoculated, viremic fleas remained virologically and serologically negative up to 38 days after infestation. Of 493 fleas collected from a wild ERB colony in Matlapitsi Cave, South Africa, where the enzootic transmission of MARV occurs, all tested negative for MARV RNA. While our findings seem to demonstrate that bat fleas lack vectorial capacity to transmit MARV biologically, their role in mechanical transmission should not be discounted. Regular blood-feeds, intra- and interhost mobility, direct feeding on blood vessels resulting in venous damage, and roosting behaviour of ERBs provide a potential physical bridge for MARV dissemination in densely populated cave-dwelling bats by fleas. The virus transfer might take place through inoculation of skin, mucosal membranes, and wounds when contaminated fleas are squashed during auto- and allogrooming, eating, biting, or fighting.

Detection of Dengue Virus 1 and Mammalian Orthoreovirus 3, with Novel Reassortments, in a South African Family Returning from Thailand, 2017.

In July 2017, a family of three members, a 46-year-old male, a 45-year-old female and their 8-year-old daughter, returned to South Africa from Thailand. They presented symptoms consistent with mosquito-borne diseases, including fever, headache, severe body aches and nausea. Mosquito bites in all family members suggested recent exposure to arthropod-borne viruses. Dengue virus 1 (Genus Orthoflavivirus) was isolated (isolate no. SA397) from the serum of the 45-year-old female via intracerebral injection in neonatal mice and subsequent passage in VeroE6 cells. Phylogenetic analysis of this strain indicated close genetic identity with cosmopolitan genotype 1 DENV1 strains from Southeast Asia, assigned to major lineage K, minor lineage 1 (DENV1I_K.1), such as GZ8H (99.92%) collected in November 2018 from China, and DV1I-TM19-74 isolate (99.72%) identified in Bangkok, Thailand, in 2019. Serum samples from the 46-year-old male yielded a virus isolate that could not be confirmed as DENV1, prompting unbiased metagenomic sequencing for virus identification and characterization. Illumina sequencing identified multiple segments of a mammalian orthoreovirus (MRV), designated as Human/SA395/SA/2017. Genomic and phylogenetic analyses classified Human/SA395/SA/2017 as MRV-3 and assigned a tentative genotype, MRV-3d, based on the S1 segment. Genomic analyses suggested that Human/SA395/SA/2017 may have originated from reassortments of segments among swine, bat, and human MRVs. The closest identity of the viral attachment protein σ1 (S1) was related to a human isolate identified from Tahiti, French Polynesia, in 1960. This indicates ongoing circulation and co-circulation of Southeast Asian and Polynesian strains, but detailed knowledge is hampered by the limited availability of genomic surveillance. This case represents the rare concurrent detection of two distinct viruses with different transmission routes in the same family with similar clinical presentations. It highlights the complexity of diagnosing diseases with similar sequelae in travelers returning from tropical areas.

Use of Human Lung Tissue Models for Screening of Drugs against SARS-CoV-2 Infection.

The repurposing of licenced drugs for use against COVID-19 is one of the most rapid ways to develop new and alternative therapeutic options to manage the ongoing pandemic. Given circa 7817 licenced compounds available from Compounds Australia that can be screened, this paper demonstrates the utility of commercially available ex vivo/3D airway and alveolar tissue models. These models are a closer representation of in vivo studies than in vitro models, but retain the benefits of rapid in vitro screening for drug efficacy. We demonstrate that several existing drugs appear to show anti-SARS-CoV-2 activity against both SARS-CoV-2 Delta and Omicron Variants of Concern in the airway model. In particular, fluvoxamine, as well as aprepitant, everolimus, and sirolimus, has virus reduction efficacy comparable to the current standard of care (remdesivir, molnupiravir, nirmatrelvir). Whilst these results are encouraging, further testing and efficacy studies are required before clinical use can be considered.

Epidemiological and Genomic Characterisation of Middelburg and Sindbis Alphaviruses Identified in Horses with Febrile and Neurological Infections, South Africa (2014-2018).

Although Old World alphaviruses, Middelburg- (MIDV) and Sindbis virus (SINV), have previously been detected in horses and wildlife with neurologic disease in South Africa, the pathogenesis and clinical presentation of MIDV and SINV infections in animals are not well documented. Clinical samples from horses across South Africa with acute or fatal neurologic and febrile infections submitted between 2014-2018 were investigated. In total, 69/1084 (6.36%) and 11/1084 (1.01%) horses tested positive for MIDV and SINV, respectively, by real-time reverse transcription (RT) PCR. Main signs/outcomes for MIDV (n = 69): 73.91% neurological, 75.36% fever, 28.99% icterus and anorexia, respectively, 8.70% fatalities; SINV (n = 11): 54.54% neurological, 72.73% fever, 36.36% anorexia and 18.18% fatalities. MIDV cases peaked in the late summer/autumn across most South African provinces while SINV cases did not show a clear seasonality and were detected in fewer South African provinces. MIDV could still be detected in blood samples via RT-PCR for up to 71,417 and 21 days after onset of signs in 4 horses respectively, suggesting prolonged replication relative to SINV which could only be detected in the initial sample. Phylogenetic analyses based on partial sequences of the nsP4 (MIDV n = 59 and SINV n = 7) and E1 (MIDV n = 45) genes, as well as full genome sequences (MIDV n = 6), clustered the MIDV and SINV strains from the present study with previously detected strains. MIDV infection appears to be more prevalent in horses than SINV infection based on RT-PCR results, however, prevalence estimates might be different when also considering serological surveillance data.

Chemical inactivation of foot-and-mouth disease virus in bovine tongue epithelium for safe transport and downstream processing.

Epithelial tissue or vesicular fluid from an unruptured or recently ruptured vesicle is the sample of choice for confirmatory laboratory diagnosis of foot-and-mouth disease (FMD). However, in 'FMD-free' countries the transport and downstream processing of such samples from potentially infected animals present a biosafety risk, particularly during heightened surveillance, potentially involving decentralised testing in laboratories without adequate biocontainment facilities. In such circumstances, rapid inactivation of virus, if present, prior to transport becomes a necessity, while still maintaining the integrity of diagnostic analytes. Tongue epithelium collected from cattle infected with FMD virus (FMDV) of serotype O (O/ALG/3/2014 - Lineage O/ME-SA/Ind-2001d) or A (A/IRN/22/2015 - Lineage A/ASIA/G-VII) was incubated in the PAXGene Tissue System Fixative (pH 4) and Stabiliser (pH 6.5) components respectively, in McIlvaine's citrate-phosphate buffer (pH 2.6) or in phosphate-buffered saline (PBS, pH 7.4) at room temperature for 2, 6, 24 or 48 h. Following incubation, tissues were homogenised and tested by virus isolation and titration using LFBKαVß6 cells. The integrity of FMD viral RNA was assessed by RT-qPCR (3Dpol coding region), Sanger sequencing of the VP1 region and transfection of LFBKαVß6 cells to recover infectious virus. Viable virus could be recovered from samples incubated in PBS for at least 48 h. The PAXgene Tissue System Stabiliser component yielded variable results dependent on virus serotype, requiring at least 6 h of incubation to inactivate A/IRN/22/2015 in most samples, whereas the Fixative component required up to 2 h in some samples. McIlvaine's citrate-phosphate buffer rapidly inactivated both viruses within 2 h of incubation. There was no demonstrable degradation of FMD viral RNA resulting from incubation in any of the buffers for up to 48 h, as assessed by RT-qPCR, and 24 h by sequencing and transfection to recover infectious virus. McIlvaine's citrate-phosphate buffer (pH 2.6) is easy to prepare, inexpensive and inactivates serotype A and O FMDV in epithelial tissue within 2 h, while maintaining RNA integrity for downstream diagnostic processes and virus characterisation.

Highly Thermotolerant SARS-CoV-2 Vaccine Elicits Neutralising Antibodies against Delta and Omicron in Mice.

As existing vaccines fail to completely prevent COVID-19 infections or community transmission, there is an unmet need for vaccines that can better combat SARS-CoV-2 variants of concern (VOC). We previously developed highly thermo-tolerant monomeric and trimeric receptor-binding domain derivatives that can withstand 100 °C for 90 min and 37 °C for four weeks and help eliminate cold-chain requirements. We show that mice immunised with these vaccine formulations elicit high titres of antibodies that neutralise SARS-CoV-2 variants VIC31 (with Spike: D614G mutation), Delta and Omicron (BA.1.1) VOC. Compared to VIC31, there was an average 14.4-fold reduction in neutralisation against BA.1.1 for the three monomeric antigen-adjuvant combinations and a 16.5-fold reduction for the three trimeric antigen-adjuvant combinations; the corresponding values against Delta were 2.5 and 3.0. Our findings suggest that monomeric formulations are suitable for upcoming Phase I human clinical trials and that there is potential for increasing the efficacy with vaccine matching to improve the responses against emerging variants. These findings are consistent with in silico modelling and AlphaFold predictions, which show that, while oligomeric presentation can be generally beneficial, it can make important epitopes inaccessible and also carries the risk of eliciting unwanted antibodies against the oligomerisation domain.

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.

Detection and genome characterization of Middelburg virus strains isolated from CSF and whole blood samples of humans with neurological manifestations in South Africa.

BACKGROUND: The Old world Alphavirus, Middelburg virus (MIDV), is not well known and although a few cases associated with animal illness have previously been described from Southern Africa, there has been no investigation into the association of the virus with human illness. The current study aimed to investigate possible association of MIDV infection with febrile or neurological manifestations in hospitalized or symptomatic patients fromGauteng, South Africa. METHODS: This study is a descriptive retrospective and prospective laboratory based study. Archived cerebrospinal fluid (CSF) samples submitted to the National Health Laboratory Service (NHLS), Tshwane Academic division for viral investigation from public sector hospitals in Gauteng as well as EDTA (ethylenediaminetetraacetic acid) whole blood samples from ad hoc cases of veterinary students, presenting with neurological and febrile illness, were selected and screened for the presence of alphaviruses using real-time reverse transcription(rtRT) PCR.Virus isolations from rtRT-PCR positive samples were conducted in Vero cell culture and used to obtain full genome sequences. Basic descriptive statistical analysis was conducted using EpiInfo. RESULTS: MIDV was detected by rtRT-PCR in 3/187 retrospective CSF specimens obtained from the NHLS from hospitalised patients in the Tshwane region of Gauteng and 1/2 EDTA samples submitted in the same year (2017) from ad hoc query arbovirus cases from veterinary students from the Faculty of Veterinary Science University of Pretoria.Full genome sequences were obtained for virus isolates from two cases; one from an EDTA whole blood sample (ad hoc case) and another from a CSF sample (NHLS sample).Two of the four Middelburg virus positive cases,for which clinical information was available, had other comorbidities or infections at the time of infection. CONCLUSION: Detection of MIDV in CSF of patients with neurological manifestations suggests that the virus should be investigated as a human pathogen with the potential of causing or contributing to neurological signs in children and adults.

Rift Valley Fever Virus Seroprevalence among Humans, Northern KwaZulu-Natal Province, South Africa, 2018-2019.

We detected Rift Valley fever virus (RVFV) IgM and IgG in human serum samples collected during 2018-2019 in northern KwaZulu-Natal Province, South Africa. Our results show recent RVFV circulation and likely RVFV endemicity in this tropical coastal plain region of South Africa in the absence of apparent clinical disease.

Vector Competence of Eucampsipoda africana (Diptera: Nycteribiidae) for Marburg Virus Transmission in Rousettus aegyptiacus (Chiroptera: Pteropodidae).

This study aimed to determine the vector competence of bat-associated nycteribiid flies (Eucamsipoda africana) for Marburg virus (MARV) in the Egyptian Rousette Bat (ERB), Rousettus aegyptiacus. In flies fed on subcutaneously infected ERBs and tested from 3 to 43 days post infection (dpi), MARV was detected only in those that took blood during the peak of viremia, 5-7 dpi. Seroconversion did not occur in control bats in contact with MARV-infected bats infested with bat flies up to 43 days post exposure. In flies inoculated intra-coelomically with MARV and tested on days 0-29 post inoculation, only those assayed on day 0 and day 7 after inoculation were positive by q-RT-PCR, but the virus concentration was consistent with that of the inoculum. Bats remained MARV-seronegative up to 38 days after infestation and exposure to inoculated flies. The first filial generation pupae and flies collected at different times during the experiments were all negative by q-RT-PCR. Of 1693 nycteribiid flies collected from a wild ERB colony in Mahune Cave, South Africa where the enzootic transmission of MARV occurs, only one (0.06%) tested positive for the presence of MARV RNA. Our findings seem to demonstrate that bat flies do not play a significant role in the transmission and enzootic maintenance of MARV. However, ERBs eat nycteribiid flies; thus, the mechanical transmission of the virus through the exposure of damaged mucous membranes and/or skin to flies engorged with contaminated blood cannot be ruled out.

A 1958 Isolate of Kedougou Virus (KEDV) from Ndumu, South Africa, Expands the Geographic and Temporal Range of KEDV in Africa.

The mosquito-borne flavivirus, Kedougou virus (KEDV), first isolated in Senegal in 1972, is genetically related to dengue, Zika (ZIKV) and Spondweni viruses (SPOV). Serological surveillance studies in Senegal and isolation of KEDV in the Central African Republic indicate occurrence of KEDV infections in humans, but to date, no disease has been reported. Here, we assembled the coding-complete genome of a 1958 isolate of KEDV from a pool of Aedes circumluteolus mosquitoes collected in Ndumu, KwaZulu-Natal, South Africa. The AR1071 Ndumu KEDV isolate bears 80.51% pairwise nucleotide identity and 93.34% amino acid identity with the prototype DakAar-D1470 strain and was co-isolated with SPOV through intracerebral inoculation of suckling mice and passage on VeroE6 cells. This historical isolate expands the known geographic and temporal range of this relatively unknown flavivirus, aiding future temporal phylogenetic calibration and diagnostic assay refinement.

Large-Scale International Validation of an Indirect ELISA Based on Recombinant Nucleocapsid Protein of Rift Valley Fever Virus for the Detection of IgG Antibody in Domestic Ruminants.

Diagnostic performance of an indirect enzyme-linked immunosorbent assay (I-ELISA) based on a recombinant nucleocapsid protein (rNP) of the Rift Valley fever virus (RVFV) was validated for the detection of the IgG antibody in sheep (n = 3367), goat (n = 2632), and cattle (n = 3819) sera. Validation data sets were dichotomized according to the results of a virus neutralization test in sera obtained from RVF-endemic (Burkina Faso, Democratic Republic of Congo, Mozambique, Senegal, Uganda, and Yemen) and RVF-free countries (France, Poland, and the USA). Cut-off values were defined using the two-graph receiver operating characteristic analysis. Estimates of the diagnostic specificity of the RVFV rNP I-ELISA in animals from RVF-endemic countries ranged from 98.6% (cattle) to 99.5% (sheep) while in those originating from RVF-free countries, they ranged from 97.7% (sheep) to 98.1% (goats). Estimates of the diagnostic sensitivity in ruminants from RVF-endemic countries ranged from 90.7% (cattle) to 100% (goats). The results of this large-scale international validation study demonstrate the high diagnostic accuracy of the RVFV rNP I-ELISA. Standard incubation and inactivation procedures evaluated did not have an adverse effect on the detectable levels of the anti-RVFV IgG in ruminant sera and thus, together with recombinant antigen-based I-ELISA, provide a simple, safe, and robust diagnostic platform that can be automated and carried out outside expensive bio-containment facilities. These advantages are particularly important for less-resourced countries where there is a need to accelerate and improve RVF surveillance and research on epidemiology as well as to advance disease control measures.

Lithium inhibits NF-κB nuclear translocation and modulate inflammation profiles in Rift valley fever virus-infected Raw 264.7 macrophages.

INTRODUCTION: Rift Valley fever virus (RVFV) is a zoonotic life-threatening viral infection endemic across sub-Saharan African countries and the Arabian Peninsula; however, there is a growing panic of its spread to non-endemic regions. This viral infection triggers a wide spectrum of symptoms that span from fibril illnesses to more severe symptoms such as haemorrhagic fever and encephalitis. These severe symptoms have been associated with dysregulated immune response propagated by the virulence factor, non-structural protein (NSs). Thus, this study investigates the effects of lithium on NF-κB translocation and RFVF-induced inflammation in Raw 264.7 macrophages. METHODS: The supernatant from RVFV-infected Raw 264.7 cells, treated with lithium, was examined using an ELISA assay kit to measure levels of cytokines and chemokines. The H2DCF-DA and DAF-2 DA florigenic assays were used to determine the levels of ROS and RNS by measuring the cellular fluorescence intensity post RVFV-infection and lithium treatment. Western blot and immunocytochemistry assays were used to measure expression levels of the inflammatory proteins and cellular location of the NF-κB, respectively. RESULTS: Lithium was shown to stimulate interferon-gamma (IFN-γ) production as early as 3 h pi. Production of the secondary pro-inflammatory cytokine and chemokine, interleukin-6 (IL-6) and regulated on activation, normal T cell expressed and secreted (RANTES), were elevated as early as 12 h pi. Treatment with lithium stimulated increase of production of tumor necrosis factor-alpha (TNF-α) and Interleukin-10 (IL-10) in RVFV-infected and uninfected macrophages as early as 3 h pi. The RVFV-infected cells treated with lithium displayed lower ROS and RNS production as opposed to lithium-free RVFV-infected control cells. Western blot analyses demonstrated that lithium inhibited iNOS expression while stimulating expression of heme oxygenase (HO) and IκB in RVFV-infected Raw 264.7 macrophages. Results from immunocytochemistry and Western blot assays revealed that lithium inhibits NF-κB nuclear translocation in RVFV-infected cells compared to lithium-free RVFV-infected cells and 5 mg/mL LPS controls. CONCLUSION: This study demonstrates that lithium inhibits NF-kB nuclear translocation and modulate inflammation profiles in RVFV-infected Raw 264.7 macrophage cells.

Epidemiology and Genomic Analysis of Equine Encephalosis Virus Detected in Horses with Clinical Signs in South Africa, 2010-2017.

Equine encephalosis virus (EEV) is a neglected virus endemic to South Africa and is considered to generally result in mild disease in equines. Specimens were analyzed from live horses that presented with undefined neurological, febrile, or respiratory signs, or sudden and unexpected death. Between 2010 and 2017, 111 of 1523 (7.3%) horse samples tested positive for EEV using a nested real-time reverse transcriptase polymerase chain reaction (rRT-PCR). Clinical signs were reported in 106 (7.2%) EEV positive and 1360 negative horses and included pyrexia (77/106, 72.6%), icterus (20/106, 18.9%) and dyspnea (12/106, 11.3%). Neurological signs were inversely associated with EEV infection (OR < 1, p < 0.05) relative to EEV negative cases despite a high percentage of animals presenting with neurological abnormalities (51/106, 48.1%). Seventeen of the EEV positive horses also had coinfections with either West Nile (5/106, 4.7%), Middelburg (4/106, 3.8%) or African Horse sickness virus (8/106, 7.6%). To investigate a possible genetic link between EEV strains causing the observed clinical signs in horses, the full genomes of six isolates were compared to the reference strains. Based on the outer capsid protein (VP2), serotype 1 and 4 were identified as the predominant serotypes with widespread reassortment between the seven different serotypes.

Ribosome-Profiling Reveals Restricted Post Transcriptional Expression of Antiviral Cytokines and Transcription Factors during SARS-CoV-2 Infection.

The global COVID-19 pandemic caused by SARS-CoV-2 has resulted in over 2.2 million deaths. Disease outcomes range from asymptomatic to severe with, so far, minimal genotypic change to the virus so understanding the host response is paramount. Transcriptomics has become incredibly important in understanding host-pathogen interactions; however, post-transcriptional regulation plays an important role in infection and immunity through translation and mRNA stability, allowing tight control over potent host responses by both the host and the invading virus. Here, we apply ribosome profiling to assess post-transcriptional regulation of host genes during SARS-CoV-2 infection of a human lung epithelial cell line (Calu-3). We have identified numerous transcription factors (JUN, ZBTB20, ATF3, HIVEP2 and EGR1) as well as select antiviral cytokine genes, namely IFNB1, IFNL1,2 and 3, IL-6 and CCL5, that are restricted at the post-transcriptional level by SARS-CoV-2 infection and discuss the impact this would have on the host response to infection. This early phase restriction of antiviral transcripts in the lungs may allow high viral load and consequent immune dysregulation typically seen in SARS-CoV-2 infection.

Shedding of Marburg Virus in Naturally Infected Egyptian Rousette Bats, South Africa, 2017.

We detected Marburg virus RNA in rectal swab samples from Egyptian rousette bats in South Africa in 2017. This finding signifies that fecal contamination of natural bat habitats is a potential source of infection for humans. Identified genetic sequences are closely related to Ravn virus, implying wider distribution of Marburg virus in Africa.

Patterns of Rift Valley fever virus seropositivity in domestic ruminants in central South Africa four years after a large outbreak.

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis showing complex epidemiological patterns that are poorly understood in South Africa. Large outbreaks occur in the central interior at long, irregular intervals, most recently in 2010-2011; however, the level of herd immunity of ruminant livestock, a key determinant of outbreaks, is unknown. During 2015-2016 a cross-sectional study on 234 randomly-selected farms investigated the prevalence, patterns of, and factors associated with, antibodies to RVF virus (RVFV) in livestock in an area heavily affected by that outbreak. A RVFV inhibition ELISA was used to screen 977 cattle, 1,549 sheep and 523 goats and information on potential risk factors was collected using a comprehensive questionnaire. The estimated RVFV seroprevalence, adjusted for survey design, was 42.9% in cattle, 28.0% in sheep and 9.3% in goats, showing a high degree of farm-level clustering. Seroprevalence increased with age and was higher on private vs. communal land, on farms with seasonal pans (temporary, shallow wetlands) and perennial rivers and in recently vaccinated animals. Seropositivity amongst unvaccinated animals born after the last outbreak indicates likely viral circulation during the post-epidemic period. The current level of herd immunity in livestock may be insufficient to prevent another large outbreak, should suitable conditions recur.

Development and validation of a pen side test for Rift Valley fever.

BACKGROUND: Rift Valley fever (RVF) is one of the main vector borne zoonotic diseases that affects a wide range of ruminants and human beings in Africa and the Arabian Peninsula. A rapid and specific test for RVF diagnosis at the site of a suspected outbreak is crucial for the implementation of control measures. METHODOLOGY/PRINCIPAL FINDINGS: A first-line lateral flow immunochromatographic strip test (LFT) was developed for the detection of the nucleoprotein (N) of the RVF virus (RVFV). Its diagnostic performance characteristics were evaluated using reference stocks isolates recovered from different hosts and in geographic regions mimicking clinical specimens and from known RVF negative serum samples. A high level of diagnostic accuracy (DSe (35/35), DSp (167/169)) was observed, including the absence of cross-reactivity with viruses belonging to different genera. CONCLUSION/SIGNIFICANCE: The fact no specialized reagents and laboratory equipment are needed, make this assay a valuable, first-line diagnostic tool in resource-poor diagnostic territories for on-site RVFV detection, however the staff require training.

Paramyxo- and Coronaviruses in Rwandan Bats.

A high diversity of corona- and paramyxoviruses have been detected in different bat species at study sites worldwide, including Africa, however no biosurveillance studies from Rwanda have been reported. In this study, samples from bats collected from caves in Ruhengeri, Rwanda, were tested for the presence of corona- and paramyxoviral RNA using reverse transcription PCR assays. Positive results were further characterized by DNA sequencing and phylogenetic analysis. In addition to morphological identification of bat species, we also did molecular confirmation of species identities, contributing to the known genetic database available for African bat species. We detected a novel Betacoronavirus in two Geoffroy's horseshoe bats (Rhinolophus clivosus) bats. We also detected several different paramyxoviral species from various insectivorous bats. One of these viral species was found to be homologous to the genomes of viruses belonging to the Jeilongvirus genus. Additionally, a Henipavirus-related sequence was detected in an Egyptian rousette fruit bat (Rousettus aegyptiacus). These results expand on the known diversity of corona- and paramyxoviruses and their geographical distribution in Africa.

Multiplex real-time RT-PCR for detection and distinction of Spondweni and Zika virus.

Zika (ZIKV) and Spondweni viruses (SPOV) are closely related mosquito borne flaviviruses in the Spondweni serogroup. The co-circulation and similar disease presentation following ZIKV and SPOV infection necessitates the development of a diagnostic tool for their simultaneous detection and distinction. We developed a one-step multiplex real-time RT-PCR (ZIKSPOV) to detect and distinguish between SPOV and ZIKV by utilizing a single primer set combined with virus specific hydrolysis probes. The ZIKSPOV assay was compared to published virus specific real-time RT-PCR assays and the limit of detection was comparable. The SPOV reference strain AR94 was detectable to 0.001 TCID50 per PCR reaction, while African lineage ZIKV (MR 766) was detectable to 0.002 TCID50 per reaction and Asian lineage ZIKV (H/PF/2013) to 0.05 TCID50 per reaction. The ZIKSPOV assay did not detect other flaviviruses, indicative of its specificity for Spondweni serogroup. The ZIKSPOV assay is a useful addition to arbovirus diagnostic and surveillance tools in areas where ZIKV and SPOV are expected to co-circulate. Further evaluation is required to demonstrate the application of the assay for detection of ZIKV and SPOV in mosquito and human clinical samples.