Evaluation of a recombinant rift valley fever virus nucleocapsid protein as a vaccine and an immunodiagnostic reagent

The serodiagnosis of Rift Valley fever (RVF) relies on the use of inactivated whole virus based reagents which present biosafety, financial and operational constraints. There are no vaccines for humans, the availability of animal vaccines is limited and they have several drawbacks. The aim of this study was to evaluate a bacterially expressed recombinant RVF virus (RVFV) nucleocapsid protein (recNP) as a safe immunodiagnostic reagent, and an immunogen in a mouse and host animal model. Several enzyme-linked immunosorbent assays (ELISAs) were developed in this study, enabling sensitive and specific detection of antibodies and RVFV antigen in human and animal specimens. The recNP was combined with different adjuvants and used to immunize mice and sheep subsequently challenged with a virulent wild type RVFV strain. Depending on the recNP/adjuvant combination, protection against disease in mice ranged between 17 and 100%, with sterilizing immunity elicited in some experimental groups, compared to 100% morbidity/mortality and excessive viral replication in adjuvant and PBS control mice. Immunization with recNP combined with Alhydrogel, an adjuvant that biases immunity towards Th2 humoral immunity, that yielded 100% protection, induced an earlier and stronger type I interferon response in mice after challenge, compared to repression of the same gene in adjuvant and PBS control mice. There was massive activation of pro-inflammatory responses and genes with pro-apoptotic effects in the livers of control mice at the acute phase of infection, accompanied by high viral replication, possibly contributing to the pathology of the liver. There was also evidence of activation and repression of several genes involved in activation of B- and T-cell immunity in control mice, some indicating possible immune evasion by the challenge virus. Immunization of sheep with the same recNP/adjuvant combinations were, however, not able to decrease replication of challenge virus. The recNP based ELISAs are an important addition to and improvement of the currently available serodiagnostic tests for RVF. The mechanism by which recNP immunization protects mice from developing severe disease during the acute phase of infection is now better understood, but the mechanism for earlier clearance of the virus needs further investigation

Anti-Nucleocapsid Protein Immune Responses Counteract Pathogenic Effects of Rift Valley Fever Virus Infection in Mice

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

Antibody responses to Marburg virus in Egyptian rousette bats and their role in protection against infection

Egyptian rousette bats (ERBs) are reservoir hosts for the Marburg virus (MARV). The immune dynamics and responses to MARV infection in ERBs are poorly understood, and limited information exists on the role of antibodies in protection of ERBs against MARV infection. Here, we determine the duration of maternal immunity to MARV in juvenile ERBs, and evaluate the duration of the antibody response to MARV in bats naturally or experimentally infected with the virus. We further explore whether antibodies in previously naturally exposed bats is fully protective against experimental reinfection with MARV. Maternal immunity was lost in juvenile ERBs by 5 months of age. Antibodies to MARV remained detectable in 67% of experimentally infected bats approximately 4 months post inoculation (p.i.), while antibodies to MARV remained present in 84% of naturally exposed bats at least 11 months after capture. Reinfection of seropositive ERBs with MARV produced an anamnestic response from day 5 p.i. Although PCR-defined viremia was present in 73.3% of reinfected ERBs, replicating virus was recovered from the serum of only one bat on day 3 p.i. The negative PCR results in the salivary glands, intestines, bladders and reproductive tracts of reinfected bats, and the apparent absence of MARV in the majority of swabs collected from these bats suggest that reinfection may only play a minor role in the transmission and maintenance of MARV amongst ERBs in nature.The National Institute for Communicable Diseases, the National Research Foundation of South Africa (86228), the Poliomyelitis Research Foundation (13/54), and the University of Pretoria (postgraduate bursary).http://www.mdpi.com/journal/virusesam2018Medical VirologyMicrobiology and Plant Patholog

Rift Valley fever virus exposure amongst farmers, farm workers, and veterinary professionals in central South 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.U.S. Department of Defense, Defense Threat Reduction Agencyhttps://www.mdpi.com/journal/virusespm2020Animal and Wildlife Science

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.http://www.mdpi.com/journal/virusespm2022Medical Virolog

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 from Gauteng, 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 Epi Info. 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.The G7 Global Health Fund program, the National Research Foundation, Poliomyelitis Research Foundation and the German Federal Ministry of Education and Research.https://journals.plos.org/plosntdsdm2022Paraclinical Science

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.The Division of Global Migration and Quarantine, National Center for Emerging and Zoonotic Infectious Diseases, US Centers for Disease Control and Prevention Division .http://www.cdc.gov/eidam2022Medical Virolog

Epidemiological and genomic characterisation of Middelburg and Sindbis alphaviruses identified in horses with febrile and neurological infections, South Africa (2014–2018)

SUPPLEMENTARY MATERIALS : TABLE S1: GenBank accession numbers for members of the Alphavirus genus used for phylogenetic analysis in this study. Sequences obtained in the current study are in bold font.; TABLE S2: Comparison of percentage identity of individual proteins and Middelburg virus full genome isolated from horses in the present study to previously identified Middelburg virus strains. Nucleotide and amino acid identities are shown for complete (concatenated) genomes with amino acids in the lower left matrix and nucleotides in the upper right matrix. Only amino acid identities are shown for individual proteins.DATA AVAILABILITY STATEMENT : All sequence data can be obtained from https://www.ncbi.nlm.nih. gov/genbank using the Genbank accession numbers indicated in Supplementary Table S1.Although OldWorld 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.Prof M. Venter’s University of Pretoria Development Fund and a grant from the EU/NRF LEAP AGRI LEARN project; A Long term EU- Africa research and innovation Partnership on food and nutrition security and sustainable Agriculture, LEARN project: LEARN: Long-term Europe-Africa Research Network on neglected arboviral zoonotic diseases). The APC was funded by G7 Global Health Fund program.https://www.mdpi.com/journal/virusesam2023Medical VirologyParaclinical Science

Serum neutralising antibody response of seronegative horses against lineage 1 and lineage 2 West Nile virus following vaccination with an inactivated lineage 1 West Nile virus vaccine

Lineage 2 West Nile virus (WNV) strains are endemic in South Africa and cause severe neurological disease in horses. An inactivated lineage 1 vaccine, Duvaxyn WNV, protects mice against challenge with a neuroinvasive South African lineage 2 strain of WNV. To evaluate the potential of Duvaxyn WNV to protect horses against lineage 2 strains of WNV, serum neutralising antibody responses of horses against lineage 1 WNV strain NY385/99 and lineage 2 WNV strain SPU93/01, isolated from a human with meningo-encephalitis in South Africa, were compared following vaccination with two doses of Duvaxyn WNV, 28 days apart, and a third dose one year later. Twenty-two seronegative horses were randomly assigned to two treatment groups: 16 to a vaccinated group and six retained as unvaccinated controls. Blood samples were taken from all horses on study days 0, 28, 35, 42, 49, 91, 141, 182, 231, 274, 322, 364 and 413. Primovaccination with Duvaxyn WNV resulted in high titres of serum neutralising antibodies against both strains. Following a single dose of Duvaxyn WNV on day 399, one year after primovaccination, there was a strong anamnestic response with a log25-fold rise in the titres of neutralising antibodies against strains NY385/99 and SPU93/01. These results provide further evidence that Duvaxyn WNV is likely to protect horses against infection with lineage 2 strains of WNV and that a single annual booster may be sufficient to maintain immunity against lineage 2 WNV infection in horses.Pfizer Animal Health.http://www.jsava.co.zaam2014ay201