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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.

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.

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.

Abstract 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.

Abstract Vaccines against SARS-CoV-2 are likely to be critical in the management of the ongoing pandemic. A number of candidates are in Phase III human clinical trials, including ChAdOx1 nCoV-19 (AZD1222), a replication-deficient chimpanzee adenovirus-vectored vaccine candidate. In preclinical trials, the efficacy of ChAdOx1 nCoV-19 against SARS-CoV-2 challenge was evaluated in a ferret model of infection. Groups of ferrets received either prime-only or prime-boost administration of ChAdOx1 nCoV-19 via the intramuscular or intranasal route. All ChAdOx1 nCoV-19 administration combinations resulted in significant reductions in viral loads in nasal-wash and oral swab samples. No vaccine-associated adverse events were observed associated with the ChAdOx1 nCoV-19 candidate, with the data from this study suggesting it could be an effective and safe vaccine against COVID-19. Our study also indicates the potential for intranasal administration as a way to further improve the efficacy of this leading vaccine candidate.

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.

Plasma samples taken at different time points from donors who received either AstraZeneca (Vaxzevria) or Pfizer (Comirnaty) or Moderna (Spikevax) coronavirus disease-19 (COVID-19) vaccine were assessed in virus neutralization assays against Delta and Omicron variants of concern and a reference isolate (VIC31). With the Pfizer vaccine there was 6-8-fold reduction in 50% neutralizing antibody titres (NT50) against Delta and VIC31 at 6 months compared to 2 weeks after the second dose; followed by 25-fold increase at 2 weeks after the third dose. Neutralisation of Omicron was only consistently observed 2 weeks after the third dose, with most samples having titres below the limit of detection at earlier timepoints. Moderna results were similar to Pfizer at 2 weeks after the second dose, while the titres for AstraZeneca samples derived from older donors were 7-fold lower against VIC31 and below the limit of detection against Delta and Omicron. Age and gender were not found to significantly impact our results. These findings indicate that vaccine matching may be needed, and that at least a third dose of these vaccines is necessary to generate sufficient neutralising antibodies against emerging variants of concern, especially Omicron, amidst the challenges of ensuring vaccine equity worldwide.

Rift Valley fever (RVF) is a mosquito-borne viral disease, principally of ruminants, that is endemic to Africa. The causative Phlebovirus, Rift Valley fever virus (RVFV), has a broad host range and, as such, also infects humans to cause primarily a self-limiting febrile illness. A small number of human cases will also develop severe complications, including haemorrhagic fever, encephalitis and visual impairment. In parts of Africa, it is a major disease of domestic ruminants, causing epidemics of abortion and mortality. It infects and can be transmitted by a broad range of mosquitos, with those of the genus Aedes and Culex thought to be the major vectors. Therefore, the virus has the potential to become established beyond Africa, including in Australia, where competent vector hosts are endemic. Vaccines for humans have not yet been developed to the commercial stage. This review examines the threat of this virus, with particular reference to Australia, and assesses gaps in our knowledge that may benefit from research focus.

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. Author summary Old World alphaviruses are mainly associated with polyarthritis in humans. Global emergence of chikungunya virus raises concerns regarding disease potential of neglected African arboviruses.New World alphaviruses are associated with severe neurological disease in humans and horses in the Americas with a high mortality rate. Middelburg virus (MIDV) is an Old World alphavirus previously associated with neurological disease and fatalities in horses and wildlife in southern Africa but not yet linked to disease in humans. This study investigated MIDV in humans presenting with acute neurological symptoms in South Africa. MIDV infection was confirmed in 4/189 (2%) such cases using a generic Alphavirus real-time RT-PCR and phylogenetic analysis. MIDV full genomes were obtained from a cerebrospinal fluid sample of a 2-year-old child and a whole blood sample of a veterinary student, both presenting with neurological signs. This study suggests MIDV may be associated with unsolved neurological disease in humans in Africa.

Saturation suppressor mutagenesis was used to generate thermostable mutants of the SARS-CoV-2 spike receptor-binding domain (RBD). A triple mutant with an increase in thermal melting temperature of ~7°C with respect to the wild-type B.1 RBD and was expressed in high yield in both mammalian cells and the microbial host, Pichia pastoris, was downselected for immunogenicity studies. An additional derivative with three additional mutations from the B.1.351 (beta) isolate was also introduced into this background. Lyophilized proteins were resistant to high-temperature exposure and could be stored for over a month at 37°C. In mice and hamsters, squalene-in-water emulsion (SWE) adjuvanted formulations of the B.1-stabilized RBD were considerably more immunogenic than RBD lacking the stabilizing mutations and elicited antibodies that neutralized all four current variants of concern with similar neutralization titers. However, sera from mice immunized with the stabilized B.1.351 derivative showed significantly decreased neutralization titers exclusively against the B.1.617.2 (delta) VOC. A cocktail comprising stabilized B.1 and B.1.351 RBDs elicited antibodies with qualitatively improved neutralization titers and breadth relative to those immunized solely with either immunogen. Immunized hamsters were protected from high-dose viral challenge. Such vaccine formulations can be rapidly and cheaply produced, lack extraneous tags or additional components, and can be stored at room temperature. They are a useful modality to combat COVID-19, especially in remote and low-resource settings.

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.

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.

Phylogenetic analysis of Rift Valley fever virus partial genomic sequences from a patient infected in South Africa in May 2018 suggests reemergence of an endemic lineage different from that of the epidemic in South Africa during 2010–2011. Surveillance during interepidemic periods should be intensified to better predict future epidemics.

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.

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.

We detected a high seroprevalence of Marburg virus (MARV) antibodies in fruit bats in South Africa; 19.1% of recaptured bats seroconverted. The MARV RNA isolated closely resembled the 1975 Ozolin strain. These findings indicate endemic MARV circulation in bats in South Africa and should inform policies on MARV disease risk reduction.

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.

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.

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.

BackgroundRift 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 findingsA 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/significanceThe 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.

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.

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.

Introduction: Chikungunya virus (CHIKV) infection is an emerging mosquito-borne disease that has been associated with frequent epidemics in the world. However, there is a dearth of information on its magnitude and associated risk factors in Tanzania. Objective: A study was conducted to determine seroprevalence of CHIKV among febrile patients seeking medical care at health facilities in Karagwe, Sengerema, Kilombero and Kyela districts. Methods: Structured questionnaires were administered and 728 serum samples were collected between May and June, 2015 and tested for the presence of CHIKV-IgM and IgG-specific antibodies using Enzyme-linked immunosorbent assay. Results and discussion: The common clinical characteristics exhibited by outpatients were fever, headache and joint pains (100%, 70%, and 68.3% respectively). Out of 728 outpatients screened for CHIKV, 105 (14%) tested CHIKV IgG positive whilst 11 (1.5%) tested CHIKV IgM positive. Chikungunya seropositivity was significantly higher than previously reported in Tanzania. The most affected age group was 20–29 years. Our results indicate that CHIKV infection is prevalent and contributes to the burden of febrile illnesses in Tanzania. The seroprevalence varies between districts, reflecting variation in mosquito vector transmission dynamics in different parts of the country. Abbreviations: CHIKV: Chikungunya virus; EDTA: Ethylenediaminetetraacetic acid; ELISA: Enzyme-linked immunosorbent assay; IgG: Immunoglobulin G; IgM: Immunoglobulin M; NIMR: National Institute for Medical Research; RU: Relative Units; SACIDS: Southern African Centre for Infectious Disease Surveillance; USA: United States of America

BackgroundIn August 2014, the National Institute for Communicable Diseases (NICD) in South Africa established a modular high-biosafety field Ebola diagnostic laboratory (SA FEDL) near Freetown, Sierra Leone in response to the rapidly increasing number of Ebola virus disease (EVD) cases.Methods and findingsThe SA FEDL operated in the Western Area of Sierra Leone, which remained a "hotspot" of the EVD epidemic for months. The FEDL was the only diagnostic capacity available to respond to the overwhelming demand for rapid EVD laboratory diagnosis for several weeks in the initial stages of the EVD crisis in the capital of Sierra Leone. Furthermore, the NICD set out to establish local capacity amongst Sierra Leonean nationals in all aspects of the FEDL functions from the outset. This led to the successful hand-over of the FEDL to the Sierra Leone Ministry of Health and Sanitation in March 2015. Between 25 August 2014 and 22 June 2016, the laboratory tested 11,250 specimens mostly from the Western Urban and Western Rural regions of Sierra Leone, of which 2,379 (21.14%) tested positive for Ebola virus RNA.ConclusionsThe bio-safety standards and the portability of the SA FEDL, offered a cost-effective and practical alternative for the rapid deployment of a field-operated high biocontainment facility. The SA FEDL teams demonstrated that it is highly beneficial to train the national staff in the course of formidable disease outbreak and accomplished their full integration into all operational and diagnostic aspects of the laboratory. This initiative contributed to the international efforts in bringing the EVD outbreak under control in Sierra Leone, as well as capacitating local African scientists and technologists to respond to diagnostic needs that might be required in future outbreaks of highly contagious pathogens.

Filovirus serological diagnosis and epidemiological investigations are hampered due to the unavailability of validated immunoassays. Diagnostic performance of three indirect enzyme-linked immunosorbent assays (I-ELISA) was evaluated for the detection of IgG antibody to Ebola virus (EBOV) in human sera. One I-ELISA was based on a whole EBOV antigen (WAg) and two utilized recombinant nucleocapsid (NP) and glycoproteins (GP), respectively. Validation data sets derived from individual sera collected in South Africa (SA), representing an EBOV non-endemic country, and from sera collected during an Ebola disease (EBOD) outbreak in Sierra Leone (SL), were categorized according to the compounded results of the three I-ELISAs and real time reverse-transcription polymerase chain reaction (RT-PCR). At the cut-off values selected at 95% accuracy level by the two-graph receiver operating characteristic analysis, specificity in the SA EBOV negative serum panel (n = 273) ranged from 98.17% (GP ELISA) to 99.27% (WAg ELISA). Diagnostic specificity in the SL EBOV negative panel (n = 676) was 100% by the three ELISAs. The diagnostic sensitivity in 423 RT-PCR confirmed EBOD patients was dependent on the time when the serum was collected after onset of disease. It significantly increased 2 weeks post-onset, reaching 100% sensitivity by WAg and NP and 98.1% by GP I-ELISA.

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.

Rift Valley fever (RVF) is a re-emerging arboviral disease of public health and veterinary importance in Africa and the Arabian Peninsula. Major RVF epidemics were documented in South Africa in 1950⁻1951, 1974⁻1975, and 2010⁻2011. The number of individuals infected during these outbreaks has, however, not been accurately estimated. A total of 823 people in close occupational contact with livestock were interviewed and sampled over a six-month period in 2015⁻2016 within a 40,000 km2 study area encompassing parts of the Free State and Northern Cape provinces that were affected during the 2010⁻2011 outbreak. Seroprevalence of RVF virus (RVFV) was 9.1% (95% Confidence Interval (CI95%): 7.2⁻11.5%) in people working or residing on livestock or game farms and 8.0% in veterinary professionals. The highest seroprevalence (SP = 15.4%; CI95%: 11.4⁻20.3%) was detected in older age groups (≥40 years old) that had experienced more than one known large epidemic compared to the younger participants (SP = 4.3%; CI95%: 2.6⁻7.3%). The highest seroprevalence was in addition found in people who injected animals, collected blood samples (Odds ratio (OR) = 2.3; CI95%: 1.0⁻5.3), slaughtered animals (OR = 3.9; CI95%: 1.2⁻12.9) and consumed meat from an animal found dead (OR = 3.1; CI95%: 1.5⁻6.6), or worked on farms with dams for water storage (OR = 2.7; CI95%: 1.0⁻6.9). We estimated the number of historical RVFV infections of farm staff in the study area to be most likely 3849 and 95% credible interval between 2635 and 5374 based on seroprevalence of 9.1% and national census data. We conclude that human RVF cases were highly underdiagnosed and heterogeneously distributed. Improving precautions during injection, sample collection, slaughtering, and meat processing for consumption, and using personal protective equipment during outbreaks, could lower the risk of RVFV infection.

We generated genome sequences from 218 cases of Ebola virus disease (EVD) in Sierra Leone (SLE) during 2014–2015 to complement available datasets, particularly by including cases from a period of low sequence coverage during peak transmission of Ebola virus (EBOV) in the highly-affected Western Area division of SLE. The combined dataset was utilized to produce phylogenetic and phylodynamic inferences, to study sink–source dynamics and virus dispersal from highly-populated transmission hotspots. We identified four districts in SLE where EBOV was introduced and transmission occurred without onward exportation to other districts. We also identified six districts that substantially contributed to the dispersal of the virus and prolonged the EVD outbreak: five of these served as major hubs, with lots of movement in and out, and one acted primarily as a source, exporting the virus to other areas of the country. Positive correlations between case numbers, inter-district transition events, and district population sizes reaffirm that population size was a driver of EBOV transmission dynamics in SLE. The data presented here confirm the role of urban hubs in virus dispersal and of a delayed laboratory response in the expansion and perpetuation of the EVD outbreak in SLE.

Rift Valley fever (RVF) is an emerging zoonosis posing a public health threat to humans in Africa. During sporadic RVF outbreaks in 2008–2009 and widespread epidemics in 2010–2011, 302 laboratory-confirmed human infections, including 25 deaths (case-fatality rate, 8%) were identified. Incidence peaked in late summer to early autumn each year, which coincided with incidence rate patterns in livestock. Most case-patients were adults (median age 43 years), men (262; 87%), who worked in farming, animal health or meat-related industries (83%). Most case-patients reported direct contact with animal tissues, blood, or other body fluids before onset of illness (89%); mosquitoes likely played a limited role in transmission of disease to humans. Close partnership with animal health and agriculture sectors allowed early recognition of human cases and appropriate preventive health messaging.

West Nile virus (WNV) and Usutu virus (USUV) are members of the family Flaviviridae which, natural life cycles involve mosquito–bird–mosquito transmission. Both represent emerging viruses in Europe with potential to cause neuroinvasive disease in humans. This study investigates the seroprevalence of serum neutralizing antibodies to WNV and to USUV in birds and in horses in Poland. Antibodies against WNV and USUV were detected in 5 (35.7%) and in 1 (7.14%) of 14 birds and in 62 (15.08%) and in 115 (27.98%) of 411 horses, respectively. Twenty-one WNV serologically positive horses (33.87%) and 67 USUV serologically positive horses (58.26%) did not travel outside Polish borders. Given the high abundance of potentially competent mosquito species in Poland, high populations of horses and different bird species, our findings highlight implementation of active control programs, including monitoring of geographic spread and dynamics of WNV and USUV transmission in both primary and accidental hosts. It is also important to improve public health awareness about the disease these viruses may cause.

We report on the isolation of a novel fusogenic orthoreovirus from bat flies (Eucampsipoda africana) associated with Egyptian fruit bats (Rousettus aegyptiacus) collected in South Africa. Complete sequences of the ten dsRNA genome segments of the virus, tentatively named Mahlapitsi virus (MAHLV), were determined. Phylogenetic analysis places this virus into a distinct clade with Baboon orthoreovirus, Bush viper reovirus and the bat-associated Broome virus. All genome segments of MAHLV contain a 5' terminal sequence (5'-GGUCA) that is unique to all currently described viruses of the genus. The smallest genome segment is bicistronic encoding for a 14 kDa protein similar to p14 membrane fusion protein of Bush viper reovirus and an 18 kDa protein similar to p16 non-structural protein of Baboon orthoreovirus. This is the first report on isolation of an orthoreovirus from an arthropod host associated with bats, and phylogenetic and sequence data suggests that MAHLV constitutes a new species within the Orthoreovirus genus.

Colonized Egyptian fruit bats (Rousettus aegyptiacus), originating in South Africa, were inoculated subcutaneously with Ebola virus (EBOV). No overt signs of morbidity, mortality, or gross lesions were noted. Bats seroconverted by Day 10–16 post inoculation (p.i.), with the highest mean anti-EBOV IgG level on Day 28 p.i. EBOV RNA was detected in blood from one bat. In 16 other tissues tested, viral RNA distribution was limited and at very low levels. No seroconversion could be demonstrated in any of the control bats up to 28 days after in-contact exposure to subcutaneously-inoculated bats. The control bats were subsequently inoculated intraperitoneally, and intramuscularly with the same dose of EBOV. No mortality, morbidity or gross pathology was observed in these bats. Kinetics of immune response was similar to that in subcutaneously-inoculated bats. Viral RNA was more widely disseminated to multiple tissues and detectable in a higher proportion of individuals, but consistently at very low levels. Irrespective of the route of inoculation, no virus was isolated from tissues which tested positive for EBOV RNA. Viral RNA was not detected in oral, nasal, ocular, vaginal, penile and rectal swabs from any of the experimental groups.

The Egyptian fruit bat, Rousettus aegyptiacus, is currently regarded as a potential reservoir host for Marburg virus (MARV). However, the modes of transmission, the level of viral replication, tissue tropism and viral shedding pattern remains to be described. Captive-bred R. aegyptiacus, including adult males, females and pups were exposed to MARV by different inoculation routes. Blood, tissues, feces and urine from 9 bats inoculated by combination of nasal and oral routes were all negative for the virus and ELISA IgG antibody could not be demonstrated for up to 21 days post inoculation (p.i.). In 21 bats inoculated by a combination of intraperitoneal/subcutaneous route, viremia and the presence of MARV in different tissues was detected on days 2-9 p.i., and IgG antibody on days 9-21 p.i. In 3 bats inoculated subcutaneously, viremia was detected on days 5 and 8 (termination of experiment), with virus isolation from different organs. MARV could not be detected in urine, feces or oral swabs in any of the 3 experimental groups. However, it was detected in tissues which might contribute to horizontal or vertical transmission, e.g. lung, intestines, kidney, bladder, salivary glands, and female reproductive tract. Viremia lasting at least 5 days could also facilitate MARV mechanical transmission by blood sucking arthropods and infections of susceptible vertebrate hosts by direct contact with infected blood. All bats were clinically normal and no gross pathology was identified on post mortem examination. This work confirms the susceptibility of R. aegyptiacus to infection with MARV irrespective of sex and age and contributes to establishing a bat-filovirus experimental model. Further studies are required to uncover the mode of MARV transmission, and to investigate the putative role of R. aegyptiacus as a reservoir host.

The known virulence factor of Rift Valley fever virus (RVFV), the NSs protein, counteracts the antiviral effects of the type I interferon response. In this study we evaluated the expression of several genes in the liver and spleen involved in innate and adaptive immunity of mice immunized with a RVFV recombinant nucleocapsid protein (recNP) combined with Alhydrogel adjuvant and control animals after challenge with wild type RVFV. Mice immunized with recNP elicited an earlier IFNβ response after challenge compared to non-immunized controls. In the acute phase of liver infection in non-immunized mice there was a massive upregulation of type I and II interferon, accompanied by high viral titers, and the up- and downregulation of several genes involved in the activation of B- and T-cells, indicating that both humoral and cellular immunity is modulated during RVFV infection. Various genes involved in pro-inflammatory responses and with pro-apoptotic effects were strongly upregulated and anti-apoptotic genes were downregulated in liver of non-immunized mice. Expression of many genes involved in B- and T-cell immunity were downregulated in spleen of non-immunized mice but normal in immunized mice. A strong bias towards apoptosis and inflammation in non-immunized mice at an acute stage of liver infection associated with suppression of several genes involved in activation of humoral and cellular immunity in spleen, suggests that RVFV evades the host immune response in more ways than only by inhibition of type I interferon, and that immunopathology of the liver plays a crucial role in RVF disease progression.