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Crimean-Congo haemorrhagic fever virus (CCHFV) is a member of the Orthonairovirus genus of the Nairoviridae family and is associated with haemorrhagic fever in humans. Although T lymphocyte responses are known to play a role in protection from and clearance of viral infections, specific T cell epitopes have yet to be identified for CCHFV following infection. A panel of overlapping peptides covering the CCHFV nucleoprotein and the structural glycoproteins, GN and GC, were screened by ELISpot assay to detect interferon gamma (IFN-γ) production in vitro by peripheral blood mononuclear cells from eleven survivors with previous laboratory confirmed CCHFV infection. Reactive peptides were located predominantly on the nucleoprotein, with only one survivor reacting to two peptides from the glycoprotein GC. No single epitope was immunodominant, however all but one survivor showed reactivity to at least one T cell epitope. The responses were present at high frequency and detectable several years after the acute infection despite the absence of continued antigenic stimulation. T cell depletion studies confirmed that IFN-γ production as detected using the ELISpot assay was mediated chiefly by CD8+ T cells. This is the first description of CD8+ T cell epitopic regions for CCHFV and provides confirmation of long-lived T cell responses in survivors of CCHFV infection. [ABSTRACT FROM AUTHOR]

Antibodies specific for Rift Valley fever virus (RVFV) can be detected by diverse methods, including ezyme-linked immunosortbent assay (ELISA) and virus neutralization test (VNT). The VNT is superior in sensitivity and specificity and is therefore considered the gold standard serological assay. Classical VNTs make use of virulent RVFV and therefore have to be performed in biosafety level 3 laboratories. Here, we report the development of a novel VNT that is based on an avirulent RVFV expressing the enhanced green fluorescent protein (eGFP), which can be performed safely outside level 3 biocontainment facilities. Evaluation with a broad panel of experimental sera and field sera demonstrated that this novel VNT is faster and more sensitive than the classical VNT. [ABSTRACT FROM AUTHOR]

Background: In 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 findings: The 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. Conclusions: The 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Rift Valley fever (RVF) is a viral zoonosis that primarily affects animals resulting in considerable economic losses due to death and abortions among infected livestock. RVF also affects humans with clinical symptoms ranging from an influenza-like illness to a hemorrhagic fever. Over the past years, RVF virus (RVFV) has caused severe outbreaks in livestock and humans throughout Africa and regions of the world previously regarded as free of the virus. This situation prompts the need to evaluate the diagnostic capacity and performance of laboratories worldwide. Diagnostic methods for RVFV detection include virus isolation, antigen and antibody detection methods, and nucleic acid amplification techniques. Molecular methods such as reverse-transcriptase polymerase chain reaction and other newly developed techniques allow for a rapid and accurate detection of RVFV. This study aims to assess the efficiency and accurateness of RVFV molecular diagnostic methods used by expert laboratories worldwide. Thirty expert laboratories from 16 countries received a panel of 14 samples which included RVFV preparations representing several genetic lineages, a specificity control and negative controls. In this study we present the results of the first international external quality assessment (EQA) for the molecular diagnosis of RVF. Optimal results were reported by 64% of the analyses, 21% of the analyses achieved acceptable results and 15% of the results revealed that there is need for improvement. Evenly good performances were achieved by specific protocols which can therefore be recommended as an accurate molecular protocol for the diagnosis of RVF. Other protocols showed uneven performances revealing the need for improved optimization and standardization of these protocols. Author Summary: Rift Valley fever (RVF) is a zoonotic viral disease posing an increasing threat to animals and humans worldwide. Recent severe outbreaks of the disease in animal and human populations in endemic regions and outside the disease's traditional geographic boundaries necessitate the need for evaluating the diagnostic performance of RVF expert laboratories. Molecular methods are increasingly used for a rapid and accurate detection of viral nucleic acid. In this study we present the results of the first international external quality assessment (EQA) for the molecular diagnosis of RVF. Such EQA studies allow participating laboratories to monitor the quality and identify possible weaknesses of current diagnostic methods. Participants to this RVF EQA were 30 expert laboratories from 16 different countries worldwide. The study demonstrated that optimal results could be achieved by the majority of laboratories. Specific protocols showed evenly good performances and can therefore be recommended to all expert laboratories. However, other methods showed uneven performances suggesting the need for improved optimization and standardization of these protocols. [ABSTRACT FROM AUTHOR]

Rift Valley fever (RVF) is a viral zoonosis that primarily affects animals resulting in considerable economic losses due to death and abortions among infected livestock. RVF also affects humans with clinical symptoms ranging from an influenza-like illness to a hemorrhagic fever. Over the past years, RVF virus (RVFV) has caused severe outbreaks in livestock and humans throughout Africa and regions of the world previously regarded as free of the virus. This situation prompts the need to evaluate the diagnostic capacity and performance of laboratories worldwide. Diagnostic methods for RVFV detection include virus isolation, antigen and antibody detection methods, and nucleic acid amplification techniques. Molecular methods such as reverse-transcriptase polymerase chain reaction and other newly developed techniques allow for a rapid and accurate detection of RVFV. This study aims to assess the efficiency and accurateness of RVFV molecular diagnostic methods used by expert laboratories worldwide. Thirty expert laboratories from 16 countries received a panel of 14 samples which included RVFV preparations representing several genetic lineages, a specificity control and negative controls. In this study we present the results of the first international external quality assessment (EQA) for the molecular diagnosis of RVF. Optimal results were reported by 64% of the analyses, 21% of the analyses achieved acceptable results and 15% of the results revealed that there is need for improvement. Evenly good performances were achieved by specific protocols which can therefore be recommended as an accurate molecular protocol for the diagnosis of RVF. Other protocols showed uneven performances revealing the need for improved optimization and standardization of these protocols. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A nosocomial outbreak of disease involving 5 patients, 4 of whom died, occurred in South Africa during September-October 2008. The first patient had been transferred from Zambia to South Africa for medical management. Three cases involved secondary spread of infection from the first patient, and 1 was a tertiary infection. A novel arenavirus was identified. The source of the first patient's infection remains undetermined. [ABSTRACT FROM AUTHOR]

Lujo virus (LUJV), a new member of the family Arenaviridae and the first hemorrhagic fever-associated arenavirus from the Old World discovered in three decades, was isolated in South Africa during an outbreak of human disease characterized by nosocomial transmission and an unprecedented high case fatality rate of 80% (4/5 cases). Unbiased pyrosequencing of RNA extracts from serum and tissues of outbreak victims enabled identification and detailed phylogenetic characterization within 72 hours of sample receipt. Full genome analyses of LUJV showed it to be unique and branching off the ancestral node of the Old World arenaviruses. The virus G1 glycoprotein sequence was highly diverse and almost equidistant from that of other Old World and New World arenaviruses, consistent with a potential distinctive receptor tropism. LUJV is a novel, genetically distinct, highly pathogenic arenavirus. [ABSTRACT FROM AUTHOR]

Abstract: Wild ruminants are thought to serve as natural hosts for Rift Valley fever virus (RVFV) but the role of these animals as reservoirs for RVFV during inter-epidemic periods and as amplifiers during epidemics is not well understood. An indirect enzyme-linked immunoassay (I-ELISA) based on the recombinant nucleocapsid protein (rNp) of RVFV was validated for the detection of specific IgG antibodies in African buffalo. Data sets derived from testing buffalo sera from Kenya (n =405) and South Africa (n =618) were dichotomised according to the results of a virus neutralisation test. The assay characteristic performance was analysed using threshold values optimised by the two-graph receiver operating characteristics (TG-ROC) analysis, and by mean plus two, as well as by mean plus three standard deviations derived from I-ELISA PP values in uninfected animals. Among 1023 buffalo sera tested, 77 (7.5%) had detectable virus neutralising antibodies. The assay had high intra- and inter-plate repeatability in routine runs. At a cut-off optimised by the TG-ROC at 95% accuracy level, the diagnostic sensitivity of the I-ELISA was 98.7% and diagnostic specificity 99.36% while estimates for the Youden''s index (J) and efficiency (Ef) were 0.98 and 99.31%. When cut-off values determined by traditional statistical approaches were used, the diagnostic sensitivity was 100% but estimates of J, Ef and other combined measures of diagnostic accuracy were lower compared to those based on cut-off value derived from the TG-ROC. Results of the study indicate that the I-ELISA based on the rNp would be useful for seroepidemiological studies of RVFV infections in African buffalo. [Copyright &y& Elsevier]

Abstract: An indirect enzyme-linked immunoassay (I-ELISA) based on the recombinant nucleocapsid protein (rNp) of Rift Valley fever virus was validated for the detection of specific IgG antibody in human sera. Validation data sets derived from testing sera collected in Africa (n =2967) were categorized according to the results of a virus neutralisation test. The assay had high intra- and inter-plate repeatability in routine runs. No detectable cross-reactions between IgG antibodies generated from mice experimentally infected with viruses representing genus Phlebovirus, Nairovirus, Orthobunyavirus and Bhanja virus of the family Bunyaviridae were observed. At a cut-off optimised by the two-graph receiver operating characteristics analysis at 95% accuracy level, the diagnostic sensitivity of the I-ELISA was 99.72% and diagnostic specificity 99.62% while estimates for the Youden''s index (J) and efficiency (Ef) were 0.993 and 99.62%. When cut-off values determined by mean plus two and by mean plus three standard deviations derived from I-ELISA readings in an uninfected reference population were used, the diagnostic sensitivity was 100% but estimates of Y, Ef and other combined measures of diagnostic accuracy were lower. The I-ELISA based on rNp is highly sensitive, specific and robust and can be applied for diagnosis of infection of Rift Valley fever and disease-surveillance studies in humans. [Copyright &y& Elsevier]

Asian bats have been identified as potential reservoir hosts of coronaviruses associated with severe acute respiratory syndrome (SARS-CoV). We detected antibody reactive with SARS-CoV antigen in 47 (6.7%) of 705 bat serum specimens comprising 26 species collected in Africa; thus, African bats may harbor agents related to putative group 4 CoV. [ABSTRACT FROM AUTHOR]

We developed a real-time reverse transcription--PCR that detected 1,164 copies/mL of Crimean-Congo hemorrhagic fever virus per milliliter of serum at 95% probability (probit analysis) and was 100% concordant with nested PCR on 63 samples from 31 patients with confirmed infection. Infected patients who died appeared to have higher viral loads; low viral loads correlated with IgG detection. [ABSTRACT FROM AUTHOR]

Abstract: This paper describes the development and validation of an inhibition ELISA based on gamma-irradiated tissue culture-derived antigen for the detection of antibody to Rift Valley fever virus (RVFV) in humans, domestic and wild ruminants. Validation data sets derived from field-collected sera in Africa (humans=1367, cattle=649, goats=806, sheep=493, buffalo=258, camels=156) were categorized according to the results of a virus neutralisation test. In addition, individual sera from 93 laboratory workers immunized with inactivated RVF vaccine, 136 serial bleeds from eight sheep experimentally infected with wild-type of RVFV, and 200 serial bleeds from 10 sheep vaccinated with the live-attenuated strain of the virus, were used to study the kinetics of RVFV antibody production under controlled conditions. At cut-off values selected at 95% accuracy level by the two-graph receiver operating characteristic analysis the ELISA sensitivity ranged from 99.47% (humans) to 100% (sheep, buffalo, camels). The specificity ranged from 99.29% (sheep) to 100% (camels). Compared to virus neutralisation and haemagglutination-inhibition tests, the ELISA was more sensitive in detection of the earliest immunological responses in experimentally infected and vaccinated sheep. Our results demonstrate that the ELISA format reported here can be used as a safe, robust and highly accurate diagnostic tool in disease-surveillance and control programmes, import/export veterinary certification, and for monitoring of the immune response in vaccinees. [Copyright &y& Elsevier]

Abstract: This paper describes the production and purification of a group-specific recombinant protein VP7 of African horse sickness virus serotype 3 (AHSV-3) and validation of an I-ELISA for the detection of IgG-antibodies to VP7 in horse sera. Baculovirus-expressed VP7 crystals were purified from infected insect cells. Analytical accuracy of the I-ELISA was examined using sera (n =38) from an experimentally infected horse, from foals born to vaccinated mares, from guinea-pigs immunized with nine serotypes of AHSV, and from sera of animals infected with other orbiviruses. Compared to traditional serological assays, the I-ELISA was more sensitive in detection of the earliest immunological response in an infected horse and declining levels of maternal immunity in foals. Antibodies to all nine serotypes of AHSV could be detected. Cross-reactivity to related orbiviruses was not observed. Diagnostic accuracy of the I-ELISA was assessed by testing sera from vaccinated horses (n =358) residing in AHS-enzootic areas and from unvaccinated horses (n =481) residing in an AHS-free area. Sera were categorised as positive or negative for antibodies to AHSV using virus neutralisation tests. The TG-ROC analysis was used for the selection of the cut-off value. At a cut-off of 11.9 of the high positive control serum (percentage positivity), the I-ELISA specificity was 100%, sensitivity 99.4%, and the Jouden index was 0.99. [Copyright &y& Elsevier]

Abstract: Rift Valley fever (RVF) virus is an important zoonotic and a potential biothreat agent. This paper describes validation of sandwich and capture enzyme-linked immunoassays (ELISA) based on gamma-irradiated antigens for the detection of RVFV-specific IgG and IgM antibody in humans. Validation data sets derived from testing field-collected sera from Africa (n =2400) were dichotomised according to the results of a virus neutralisation test. In addition, sera from laboratory workers immunized with inactivated RVF vaccine (n =93) and serial sera (n =3) from a single RVF case were used. ELISA data were expressed as percentage of high-positive control serum (PP). Cut-off values at 95% accuracy level were optimised using the misclassification cost term option of the two-graph receiver operating characteristics analysis. During the routine use of assays there was no evidence for excessive intra- and inter-plate variations within and between runs of assays. At a cut-off of 13.2 PP the sensitivity of the IgG-sandwich ELISA was 100% and specificity 99.95%, while for the IgM-capture ELISA the values were 96.47 and 99.44%, respectively, at a cut-off of 7.1 PP. Compared to the virus neutralisation test, the IgG-sandwich ELISA was more sensitive in detection of immunological responses in vaccines. Following natural infection class-specific antibodies were detected in serum taken 6 days after onset of symptoms. The results demonstrate that both assays will be useful for early diagnosis of infection, epidemiological surveillance and for monitoring of immune response after vaccination. As highly accurate, robust and safe tests, they have the potential to replace traditional diagnostic methods which are unable to distinguish between different classes of immunoglobulins, and pose health risks necessitating their use being restricted to high containment facilities outside RVF endemic areas. [Copyright &y& Elsevier]

The recent occurrence of the first confirmed outbreaks of Rift Valley fever in humans and livestock outside the African region, namely in the Kingdom of Saudi Arabia and Yemen, is of global medical and veterinary concern. Disadvantages of classical techniques for serological diagnosis of Rift Valley fever include health risk to laboratory personnel, restrictions for their use outside endemic areas and inability to distinguish between different classes of immunoglobulins. We report on the development and validation of sandwich and capture ELISAs (both based on inactivated antigen) for detection of IgG and IgM antibody to Rift Valley fever virus in bovine, caprine and ovine sera. Compared to virus neutralisation and haemagglutination-inhibition tests, the IgG sandwich ELISA was more sensitive in detection of the earliest immunological responses to infection or vaccination with Rift Valley fever virus. Its sensitivity and specificity derived from field data sets ranged in different ruminant species from 99.05 to 100% and from 99.1 to 99.9%, respectively. The specificity of IgM-capture ELISA varied between different species from 97.4 to 99.4%; its sensitivity was 100% in sheep tested 5–42 days post-infection. Our results in field-collected, experimental and post-vaccination sera demonstrate that these assays will be useful for epidemiological surveillance and control programmes, import/export veterinary certification, early diagnosis of infection, and for monitoring of immune response in vaccinated animals. As highly accurate and safe tests, they have the potential to replace traditional diagnostic methods, which pose biohazard risks limiting their use outside of endemic areas to high containment facilities. [Copyright &y& Elsevier]

Duvenhage virus was isolated from a patient who died of a rabieslike disease after being scratched by a bat early in 2006. This occurred ≈80 km from the site where the only other known human infection with the virus had occurred 36 years earlier. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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 TCID 50 per PCR reaction, while African lineage ZIKV (MR 766) was detectable to 0.002 TCID 50 per reaction and Asian lineage ZIKV (H/PF/2013) to 0.05 TCID 50 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. [ABSTRACT FROM AUTHOR]

Rift Valley fever (RVF) is a re-emerging zoonotic disease of domestic ruminants and humans. While neighbouring countries have reported outbreaks of RVF, Ghana has not yet identified any cases. The aim of this study was to determine whether RVF virus (RVFV) was circulating in livestock and herders in the southern part of Ghana, to estimate its seroprevalence, and to identify associated risk factors. The study surveyed 165 livestock farms randomly selected from two districts in southern Ghana. Serum samples of 253 goats, 246 sheep, 220 cattle, and 157 herdsmen were tested to detect IgG and IgM antibodies against RVFV. The overall seroprevalence of anti-RVF antibodies in livestock was 13.1% and 30.9% of farms had RVFV seropositive animals. The species-specific prevalence was 24.1% in cattle, 8.5% in sheep, and 7.9% in goats. A RVFV IgG seroprevalence of 17.8% was found among the ruminant herders, with 8.3% of all herders being IgM positive. RVFV was shown, for the first time, to have been circulating in southern Ghana, with evidence of a recent outbreak in Kwahu East; however, it was clinically undetected despite significant recent human exposure. A One Health approach is recommended to better understand RVF epidemiology and socio-economic impact in Ghana. [ABSTRACT FROM AUTHOR]

The capacity of Rift Valley fever virus (RVFV) to spread into new territories by crossing significant natural geographic barriers, re-emerge in endemic regions after long periods of silence to cause large outbreaks in human and animal populations constitute a formidable challenge for public and veterinary health authorities as well as for scientific communities worldwide. In spite of recent advances in research on RVFV pathogenesis, molecular epidemiology, outbreak prediction, development of new diagnostic tools and vaccines, some fundamental aspects of the epidemiology and ecology of the virus remain elusive. Large outbreaks of RVF are associated with anomalous high rainfalls leading to massive flooding and the resultant swarms of competent mosquito vectors transmitting the virus to a wide range of susceptible vertebrate species. However, the exact mechanism of RVFV natural transmission during interepizootic periods remains largely unknown, including the postulated long-term virus persistence in transovarially infected eggs of floodwater Aedes mosquito species, and the role of wild mammals as reservoirs. The presence of competent mosquito vectors in countries free of RVF, the wide range of mammals susceptible to the virus, the global changes in climate, and increased animal trade and travel are some of the factors which might contribute to international spread. This presentation provides the background to the major outbreaks, molecular biology and epidemiology of RVFV, and overviews aspects of ecology, host and vector range which make the virus a potential global emerging threat. [ABSTRACT FROM AUTHOR]

Biosecurity measures have been introduced to limit economic losses and zoonotic exposures to humans by preventing and controlling animal diseases. However, they are implemented on individual farms with varying frequency. The goal of this study was to evaluate which biosecurity measures were used by farmers to prevent infectious diseases in ruminant livestock and to identify factors that influenced these decisions. We conducted a survey in 264 ruminant livestock farmers in a 40,000 km2 area in the Free State and Northern Cape provinces of South Africa. We used descriptive statistics, to characterize biosecurity measures and farm attributes, then multivariable binomial regression to assess the strength of the association between the attributes and the implementation of biosecurity measures including property fencing, separate equipment use on different species, separate rearing of species, isolation of sick animals, isolation of pregnant animals, quarantine of new animals, animal transport cleaning, vaccination, tick control and insect control. Ninety‐nine percent of farmers reported using at least one of the 10 biosecurity measures investigated (median [M]: 6; range: 0–10). The most frequently used biosecurity measures were tick control (81%, 214 out of 264), vaccination (80%, 211 out of 264) and isolation of sick animals (72%, 190 out of 264). More biosecurity measures were used on farms with 65–282 animals (M: 6; odds ratio [OR]: 1.52) or farms with 283–12,030 animals (M: 7; OR: 1.87) than on farms with fewer than 65 animals (M: 4). Furthermore, farmers who kept two animal species (M: 7; OR: 1.41) or three or more species (M: 7) used more biosecurity measures than single‐species operations (M: 4). Farmers with privately owned land used more biosecurity measures (M: 6; OR: 1.51) than those grazing their animals on communal land (M: 3.5). Farms that reported previous Rift Valley fever (RVF) outbreaks used more biosecurity measures (M: 7; OR: 1.25) compared with farms without RVF reports (M: 6) and those that purchased animals in the 12 months prior to the survey (M: 7; OR: 1.19) compared with those that did not (M: 6). When introducing new animals into their herds (n = 122), most farmers used fewer biosecurity measures than they did for their existing herd: 34% (41 out of 122) used multiple biosecurity measures like those of vaccination, tick control, quarantine or antibiotic use, whereas 36% (44 out of 122) used only one and 30% (37 out of 122) used none. Certain farm features, primarily those related to size and commercialization, were associated with more frequent use of biosecurity measures. Given the variation in the application of biosecurity measures, more awareness and technical assistance are needed to support the implementation of a biosecurity management plan appropriate for the type of farm operation and available resources. [ABSTRACT FROM AUTHOR]

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

Background: Sierra Leone has the most cases of Ebola virus disease (EVD) ever reported. Trends in laboratoryconfirmed EVD, symptom presentation, and risk factors have not been fully described. Methods: EVD cases occurring from 23 May 2014 to 31 January 2015 are presented by geography, demographics, and risk factors for all persons who had laboratory-confirmed EVD, which was identified by Ebola virus-specific reverse-transcription polymerase chain reaction-based testing. Results: During the study period, 8056 persons had laboratory-confirmed EVD. Their median age was 28 years; 51.7% were female. Common symptoms included fever (90.4%), fatigue (88.3%), loss of appetite (87.0%), headache (77.9%), joint pain (73.7%), vomiting (71.2%), and diarrhea (70.6%). Among persons with confirmed cases, 47.9% reported having had contact with someone with suspected EVD or any sick person, and 25.5% reported having attended a funeral, of whom 66.2% reported touching the body. The incidence of EVD was highest during 1-30 November 2014, at 7.5 per 100 000 population per week, and decreased to 2.1 per week during 1-31 January 2015. Between 23 May and 30 August 2014, two districts had the highest incidence of 3.8 and 7.0 per 100 000 population per week which decreased >97% by 1-31 January 2015. In comparison, the districts that include the capital city reported a 10-fold increase in incidence per week during the same time periods. Conclusions: Almost half of patients with EVD in Sierra Leone reported physical contact with a person ill with EVD or a dead body, highlighting prevention opportunities. [ABSTRACT FROM AUTHOR]

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

In 2008, a suspected outbreak of Rift Valley fever (RVF) was reported on a farm in the Bela-Bela area, Limpopo Province, South Africa. Seven calves died on the affected dairy farm, where no RVF vaccination programme was practised. No apparent clinical disease was reported in the other 300 cattle (33 calves included) or 200 sheep on the farm. During the outbreak, blood samples from 77.7% (233/300) of the cattle and 36.5% (73/200) of the sheep were collected on the affected farm and 55 blood samples were taken from cattle on a neighbouring farm. Eight weeks later, 78% of the cattle (234/300) and 42.5% of the sheep (85/200) were bled on the affected farm only. All sera were tested by an Immunoglobulin M (IgM)-capture Enzymelinked immunosorbent assay (ELISA) and by an indirect Immunoglobulin G (IgG) ELISA. Selected IgM-positive (n = 14), IgG-positive (n = 23) and samples negative for both IgM and IgG-specific antibodies against RVF virus (n = 19) were tested using the serum neutralisation test (SNT). Sera from IgM-positive (n = 14) and negative (n = 20) animals were also tested by a TaqMan polymerase chain reaction (PCR). On the affected farm, 7% (16/233) of the cattle were IgM-positive and 13.7% (32/233) IgG-positive at the first bleed and 2% were IgM-positive at the second bleed, whilst the number of cattle positive for IgG-specific antibodies increased by 21.3% compared with the first bleed. Only 1.4% of sheep were positive for both IgM and IgG antibodies at the first collection; at the second bleed, IgM-positive cases decreased to 1.2%, whilst IgG-positive cases increased to 2.4%. Whilst no IgM-positive cattle were found on the neighbouring farm, 5.5% of cattle were IgG-positive. The SNT confirmed most of the ELISA results, whilst PCR results were all negative. Although serology results indicated virus circulation on both farms, the negative PCR results demonstrated that the animals were not viraemic at the time they were sampled. The movement of infected mosquito vectors by wind over long distances into a low-lying area that favoured their breeding on the Bela-Bela farm may have led to an outbreak of the disease there, but the reason for the low level of virus circulation amongst susceptible animals remains unclear. [ABSTRACT FROM AUTHOR]

Abstract: Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne viral zoonosis distributed widely in Africa, Asia, Russia and the Balkans. The emergence and re-emergence of CCHFV emphasize the importance of increasing both human and veterinary surveillance and developing diagnostic capacity. Recombinant CCHFV nucleocapsid protein (NP) has been expressed using insect cells and mammalian cells and used as a diagnostic tool but bacterial expression has not been described previously. The S gene of CCHFV was codon optimized and the NP expressed in Escherichia coli from the synthetic gene. The protein was reacted against serum samples collected from confirmed CCHFV patients at varying intervals after the onset of illness from acute to convalescent stages using both an ELISA and a Western blot. To confirm that the protein was able to induce a humoral antibody response that could be detected using CCHFV antigen derived from live virus, mice were immunized and serum samples were tested using IF slides prepared from CCHFV infected Vero cells. The recombinant antigen was able to detect IgG antibody in acute and convalescent sera. In addition, a detectable IgG antibody response was induced in mice immunized using NP. The results suggest that proteins expressed in a bacterial system lacking post-translational modifications can be used in ELISA to detect IgG antibody against CCHFV in human sera which may be used for routine diagnosis and seroepidemiology. [Copyright &y& Elsevier]

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 IFNb 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 proapoptotic 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. [ABSTRACT FROM AUTHOR]

Abstract: Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral zoonosis distributed widely in Africa, Asia, Russia and the Balkans. Occurrence of segment reassortment has been established and may be associated with pathogenicity. The ability to distinguish between an infection with a reassortant and a non-reassortant variant may have prognostic value. In this study the use of Simple-Probe technology and real-time PCR was investigated to detect rapidly CCHF viral nucleic acid and to distinguish between reassorted and non-reassorted variants of southern African CCHF virus (CCHFV) isolates. A Simple-Probe® was designed based on multiple alignments of 19 CCHFV partial M segment sequences that included reassorted and non-reassorted isolates from two phylogenies. Real-time PCR followed by probe-specific melting-curve analysis allowed differentiation of three selected isolates representative of two phylogenetically distinct groups, SPU 431/85 (group IV), 383/87 (group IV) and 103/87 (group III) based on melting temperatures. Within group IV there are two distinct lineages which were represented in the optimization. A further 16 clinical isolates could be genotyped into groups III and IV using the Simple-Probe real-time PCR assay developed in this study. This assay provides a reliable and sensitive tool for the differentiation between reassorted and non-reassorted variants of CCHFV which finds application for diagnosis and epidemiologic and surveillance studies. [ABSTRACT FROM AUTHOR]

Abstract: Serodiagnosis of Rift Valley fever (RVF) currently relies on the use of live or inactivated whole virus as antigens. The recombinant nucleocapsid (N) protein of RVF virus was tested for diagnostic applicability in an indirect enzyme-linked immunosorbent assay (I-ELISA), using sera from experimentally infected sheep (n =128), vaccinated sheep (n =240), and field-collected sera from sheep (n =251), goats (n =362) and cattle (n =100). The N-protein based I-ELISA performed at least as good as VN and HI tests. In goat the diagnostic sensitivity (D-Sn) and specificity (D-Sp) of the I-ELISA was 100% when using the anti-species IgG conjugate. Using protein G as a detection system, the D-Sn and D-Sp in goats were 99.4% and 99.5%, in sheep field sera both 100%, in cattle 100% and 98.3%, respectively. The I-ELISA based on recombinant N-protein has the potential to complement the traditional assays for serodiagnosis of RVF. Advantages of the N-protein are its safety, stability and cost-effectiveness in use and production. [Copyright &y& Elsevier]

Abstract: This paper describes the cloning, sequencing and bacterial expression of the N protein of the Rift Valley fever virus (RVFV) ZIM688/78 isolate and its evaluation in indirect ELISAs (I-ELISA) for the detection of IgM and IgG antibodies in human and sheep sera. Sera used for the evaluation were from 106 laboratory workers immunised with an inactivated RVF vaccine, 16 RVF patients, 168 serial bleeds from 8 sheep experimentally infected with wild type RVFV and 210 serial bleeds from 10 sheep vaccinated with the live attenuated Smithburn RVFV strain. All human and animal sera that tested positive in the virus neutralisation test were also positive in the IgG I-ELISA. There was a high correlation (R 2 =0.8571) between virus neutralising titres and IgG I-ELISA readings in human vaccinees. In experimentally infected sheep IgG antibodies were detected from day 4 to 5 post-infection onwards and IgM antibodies from day 3 to 4. The IgG I-ELISA was more sensitive than virus neutralisation and haemagglutination-inhibition tests in detecting the early immune response in experimentally infected sheep. The I-ELISAs demonstrated that the IgG and IgM response to the Smithburn vaccine strain was slower and the levels of antibodies induced markedly lower than to wild type RVFV infection. [Copyright &y& Elsevier]

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 FROM AUTHOR]

Crimean-Congo haemorrhagic fever (CCHF) is a severe tick-borne viral zoonosis endemic to parts of Africa, Europe, the Middle East and Central Asia. Human cases are reported annually in South Africa, with a 25% case fatality rate since the first case was recognized in 1981. We investigated CCHF virus (CCHFV) seroprevalence and risk factors associated with infection in cattle and humans, and the presence of CCHFV in Hyalomma spp. ticks in central South Africa in 2017–18. CCHFV IgG seroprevalence was 74.2% (95%CI: 64.2–82.1%) in 700 cattle and 3.9% (95%CI: 2.6–5.8%) in 541 farm and wildlife workers. No veterinary personnel (117) or abattoir workers (382) were seropositive. The prevalence of CCHFV RNA was significantly higher in Hyalomma truncatum (1.6%) than in H. rufipes (0.2%) (P = 0.002). Seroprevalence in cattle increased with age and was greater in animals on which ticks were found. Seroprevalence in cattle also showed significant geographic variation. Seroprevalence in humans increased with age and was greater in workers who handled livestock for injection and collection of samples. Our findings support previous evidence of widespread high CCHFV seroprevalence in cattle and show significant occupational exposure amongst farm and wildlife workers. Our seroprevalence estimate suggests that CCHFV infections are five times more frequent than the 215 confirmed CCHF cases diagnosed in South Africa in the last four decades (1981–2019). With many cases undiagnosed, the potential seriousness of CCHF in people, and the lack of an effective vaccine or treatment, there is a need to improve public health awareness, prevention and disease control. Author summary: Crimean-Congo haemorrhagic fever (CCHF) is an infection caused by a tick-borne virus endemic to a large geographic area, that is asymptomatic in animals but presents a significant health risk to humans with no effective vaccine. Serological surveys were carried out in rural populations and animals shortly after the first reported case in South Africa in 1981, but little is known about the current seroprevalence of CCHFV in humans and animals, and tick infection rates. We conducted a survey in 2017–18 to obtain recent estimates and examined exposure factors in an area of South Africa where cases are known to occur. We observed a high seroprevalence in cattle of 74.2%. Farm and wildlife workers at risk of occupational exposure to CCHFV had a seroprevalence of 3.9%, which was higher than previously reported. No evidence of CCHFV infection was found in abattoir workers or veterinary professionals in that area. CCHFV RNA was detected in both species of Hyalomma ticks but with a higher prevalence (1.6%) in H. truncatum. This study suggests that CCHFV is widespread in South Africa, that exposure to CCHFV is more common than generally believed in animal-related occupations and that increased public health awareness of risk factors associated with CCHFV infection are required to reduce morbidity and mortality from CCHF. [ABSTRACT FROM AUTHOR]

We detected 3 lyssaviruses in insectivorous bats sampled in South Africa during 2003-2018. We used phylogenetic analysis to identify Duvenhage lyssavirus and a potentially new lyssavirus, provisionally named Matlo bat lyssavirus, that is related to West Caucasian bat virus. These new detections highlight that much about lyssaviruses remains unknown. [ABSTRACT FROM AUTHOR]

We evaluated the prevalence of Rift Valley fever virus IgG and IgM in human serum samples (n = 1,276) collected in 2013-2014 in northern Botswana. Our findings provide evidence of active circulation of this virus in humans in the absence of clinical disease in this region. [ABSTRACT FROM AUTHOR]

To investigate West Nile virus (WNV) circulation in rural populations in Gabon, we undertook a large serological survey focusing on human rural populations, using two different ELISA assays. A sample was considered positive when it reacted in both tests. A total of 2320 villagers from 115 villages were interviewed and sampled. Surprisingly, the WNVspecific IgG prevalence was high overall (27.2%) and varied according to the ecosystem: 23.7% in forested regions, 21.8% in savanna, and 64.9% in the lakes region. The WNV-specific IgG prevalence rate was 30% in males and 24.6% in females, and increased with age. Although serological cross-reactions between flaviviruses are likely and may be frequent, these findings strongly suggest that WNV is widespread in Gabon. The difference in WNV prevalence among ecosystems suggests preferential circulation in the lakes region. The linear increase with age suggests continuous exposure of Gabonese populations to WNV. Further investigations are needed to determine the WNV cycle and transmission patterns in Gabon. [ABSTRACT FROM AUTHOR]

Outbreaks of Rift Valley fever have devastating impacts on ruminants, humans, as well as on regional and national economies. Although numerous studies on the impact and outbreak of Rift Valley fever exist, relatively little is known about the role of environmental factors, especially soil, on the aestivation of the virus. This study thus selected 22 sites for study in central South Africa, known to be the recurrent epicenter of widespread Rift Valley fever outbreaks in Southern Africa. Soils were described, sampled and analyzed in detail at each site. Of all the soil variables analyzed for, only eight (cation exchange capacity, exchangeable Ca2+, exchangeable K+, exchangeable Mg2+, soluble Ca2+, medium sand, As, and Br) were statistically identified to be potential indicators of sites with reported Rift Valley fever mortalities, as reported for the 2009–2010 Rift Valley fever outbreak. Four soil characteristics (exchangeable K+, exchangeable Mg2+, medium sand, and Br) were subsequently included in a discriminant function that could potentially be used to predict sites that had reported Rift Valley fever-associated mortalities in livestock. This study therefore constitutes an initial attempt to predict sites prone to Rift Valley fever livestock mortality from soil properties and thus serves as a basis for broader research on the interaction between soil, mosquitoes and Rift Valley fever virus. Future research should include other environmental components such as vegetation, climate, and water properties as well as correlating soil properties with floodwater Aedes spp. abundance and Rift Valley fever virus prevalence. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In South Africa, RABV circulates both in domestic animals and wildlife cycles, involving the canid and mongoose variants of the virus (Nel, Thomson & Von Teichman [17]). Sylvatic cycles of the canid variant RABV in bat-eared foxes and black-backed jackal (Zulu et al. [23]) and the mongoose variant RABV in certain species of mongoose occur in South Africa (Van Zyl, Markotter & Nel [21]). Apart from the reservoir species, canid and mongoose RABV infections are reported in an array of domestic and wildlife species in the country, with these animals primarily serving as dead-end hosts (Sabeta et al. [20]). [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The NSs protein encoded by the S segment of Rift Valley fever virus (RVFV) is the major virulence factor, counteracting the host innate antiviral defence. It contains five highly conserved cysteine residues at positions 39, 40, 149, 178 and 194, which are thought to stabilize the tertiary and quaternary structure of the protein. Here, we report significant differences between clinical, virological, histopathological and host gene responses in BALB/c mice infected with wild-type RVFV (wtRVFV) or a genetic mutant having a double cysteine-to-serine substitution at residues 39 and 40 of the NSs protein (RVFV-C39S/C40S). Mice infected with the wtRVFV developed a fatal acute disease; characterized by high levels of viral replication, severe hepatocellular necrosis, and massive up-regulation of transcription of genes encoding type I and −II interferons (IFN) as well as pro-apoptotic and pro-inflammatory cytokines. The RVFV-C39S/C40S mutant did not cause clinical disease and its attenuated virulence was consistent with virological, histopathological and host gene expression findings in BALB/c mice. Clinical signs in mice infected with viruses containing cysteine-to-serine substitutions at positions 178 or 194 were similar to those occurring in mice infected with the wtRVFV, while a mutant containing a substitution at position 149 caused mild, non-fatal disease in mice. As mutant RVFV-C39S/C40S showed an attenuated phenotype in mice, the molecular mechanisms behind this attenuation were further investigated. The results show that two mechanisms are responsible for the attenuation; (1) loss of the IFN antagonistic propriety characteristic of the wtRVFV NSs and (2) the inability of the attenuated mutant to degrade Proteine Kinase R (PKR). [ABSTRACT FROM AUTHOR]

Rift Valley fever (RVF) is endemic in Africa and parts of the Middle East. It is an emerging zoonotic disease threat to veterinary and public health. Outbreaks of the disease have severe socio-economic impacts. RVF virus emergence is closely associated with specific endorheic wetlands that are utilized by the virus’ mosquito vectors. Limited botanical vegetation surveys had been published with regard to RVF virus (RVFV) ecology. We report on a phytosociological classification, analysis and description of wetland vegetation and related abiotic parameters to elucidate factors possibly associated with the 2010–2011 RVFV disease outbreak in South Africa. The study sites were located in the western Free State and adjacent Northern Cape covering an area of ~40,000 km2 with wetlands associated with high RVF mortality rates in livestock. Other study sites included areas where no RVF activity was reported during the 2010–11 RVF outbreak. A total of 129 plots (30 m2) were selected where a visible difference could be seen in the wetland and upland vegetation. The Braun-Blanquet method was used for plant sampling. Classification was done using modified Two-Way Indicator Species Analysis. The vegetation analysis resulted in the identification of eight plant communities, seven sub-communities and two variants. Indirect ordination was carried out using CANOCO to investigate the relationship between species and wetland ecology. The study also identified 5 categories of wetlands including anthropogenic wetlands. Locations of reported RVF cases overlapped sites characterized by high clay-content soils and specific wetland vegetation. These findings indicate ecological and environmental parameters that represent preferred breeding habitat for RVFV competent mosquito vectors. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A suspicion on West Nile virus (WNV) in Serbia was first reported in 1972 by a seroprevalence study, after which no data were available for four decades. We report full sequence of the isolate obtained for the first time from a human sample in Serbia. The closest clustering was obtained with lineage 2 WNV identified in Greece in 2010. Since WNV lineage 2 emerged in Europe in 2004, a cocirculation of lineages 1 and 2-as observed in Hungary and Italy-cannot be excluded. The reinforcement of surveillance will be required to investigate the possible cocirculation of the two lineages and the burden of WNV in the local population. [ABSTRACT FROM AUTHOR]

In contrast to animals, Crimean-Congo hemorrhagic fever (CCHF) causes a severe disease in humans with a high mortality rate. The etiological agent, CCHF virus (CCHFV), can be transmitted by argasid and ixodid ticks, but arachnids of the genus Hyalomma, followed by Rhipicephalus and Dermacentor serve as the major vectors of this virus. The goal of the study was to assess the epidemiological situation of CCHFV infection in cattle in south-east Poland, and survey for potential tick vector species. A total of 592 bovine blood samples from animals located in the southernmost region in Poland were tested by IgG sandwich enzyme-linked immunosorbent assay. Ticks ( n = 993) from south-east Poland were collected from dogs, cats, cattle, and horses and tested by RT-PCR. All 592 serum samples were negative for IgG antibodies to CCHFV. Of the ticks collected, 125 were Dermacentor reticulatus and 868 represented Ixodes ricinus, both species are regarded as potential vectors of CCHFV. All tick samples were negative for the presence of CCHFV. Considering the zoonotic nature, public health importance, and the virus increasing spread, it was prudent to assess the seroprevalence of CCHFV in the south-east area of Poland, bordering with CCHFV endemic areas. It seems unlikely that CCHFV infection will suddenly spread in Poland, but considering the multiple possibilities of the virus introduction, serosurveys and vector biosurveillance should be conducted at regular intervals. [ABSTRACT FROM AUTHOR]

Crimean-Congo hemorrhagic fever virus (CCHFV) is a bunyavirus causing severe hemorrhagic fever disease in humans, with high mortality rates. The requirement of a high-containment laboratory and the lack of an animal model hampered the study of the immune response and protection of vaccine candidates. Using the recently developed interferon alpha receptor knockout (IFNAR-/-) mouse model, which replicates human disease, we investigated the immunogenicity and protection of two novel CCHFV vaccine candidates: a DNA vaccine encoding a ubiquitin-linked version of CCHFV Gc, Gn, and N and one using transcriptionally competent virus-like particles (tc-VLPs). In contrast to most studies that focus on neutralizing antibodies, we measured both humoral and cellular immune responses. We demonstrated a clear and 100% efficient preventive immunity against lethal CCHFV challenge with the DNA vaccine. Interestingly, there was no correlation with the neutralizing antibody titers alone, which were higher in the tc-VLP-vaccinated mice. However, the animals with a lower neutralizing titer, but a dominant cell-mediated Th1 response and a balanced Th2 response, resisted the CCHFV challenge. Moreover, we found that in challenged mice with a Th1 response (immunized by DNA/DNA and boosted by tc-VLPs), the immune response changed to Th2 at day 9 postchallenge. In addition, we were able to identify new linear B-cell epitope regions that are highly conserved between CCHFV strains. Altogether, our results suggest that a predominantly Th1-type immune response provides the most efficient protective immunity against CCHFV challenge. However, we cannot exclude the importance of the neutralizing antibodies as the surviving immunized mice exhibited substantial amounts of them. [ABSTRACT FROM AUTHOR]

The mononegaviral family Filoviridae has eight members assigned to three genera and seven species. Until now, genus and species demarcation were based on arbitrarily chosen filovirus genome sequence divergence values ( 50% for genera, 30% for species) and arbitrarily chosen phenotypic virus or virion characteristics. Here we report filovirus genome sequence-based taxon demarcation criteria using the publicly accessible PAirwise Sequencing Comparison (PASC) tool of the US National Center for Biotechnology Information (Bethesda, MD, USA). Comparison of all available filovirus genomes in GenBank using PASC revealed optimal genus demarcation at the 55-58% sequence diversity threshold range for genera and at the 23-36% sequence diversity threshold range for species. Because these thresholds do not change the current official filovirus classification, these values are now implemented as filovirus taxon demarcation criteria that may solely be used for filovirus classification in case additional data are absent. A near-complete, coding-complete, or complete filovirus genome sequence will now be required to allow official classification of any novel "filovirus." Classification of filoviruses into existing taxa or determining the need for novel taxa is now straightforward and could even become automated using a presented algorithm/flowchart rooted in RefSeq (type) sequences. [ABSTRACT FROM AUTHOR]

Many bats, including some small-bodied tropical and subtropical Pteropodidae, use torpor to offset energetic constraints. We tested the hypothesis that medium-sized (110-160 g) cave-roosting Egyptian rousette bats (Rousettus aegyptiacus) at the southern extent of their range are able to employ torpor. We measured daytime body temperatures (Tb) of 9 wild individuals using implanted temperature-sensitive radiotransmitters. The bats roosted in a cave on Table Mountain, Cape Town, South Africa (a typically cool and wet area). Daily mean cave temperature (Tc) ranged between 7°C and 12°C (mean 9.3°C). All wild individuals exhibited a circadian cycle in Tb, with an average of 37.7°C upon return from foraging, decreasing to 35.5°C by mid-day. Before emergence for feeding, Tb increased to about 37°C. No individual allowed Tb to drop below 34°C indicating (assuming a threshold temperature of 30°C) that individuals in this population do not use torpor. Bats were active throughout the day within the roost and metabolic heat production may have contributed to the maintenance of rest-phase Tb. Ten individuals implanted with temperature-sensitive passive integrated transponder tags were held in captivity at temperatures of 25-30°C and subjected to food deprivation for 2-3 days. The lowest Tb recorded for any captive individual was 33.4°C despite losing an average of 10% of their initial body mass. Despite exposure to cool Ta in the cave, and often cold, wet, and windy conditions while foraging, or substantial food restriction in captive bats, R. aegyptiacus did not resort to using torpor. [ABSTRACT FROM AUTHOR]