Combining hydrology and mosquito population models to identify the drivers of rift valley Fever emergence in semi-arid regions of west Africa

Background

Rift Valley fever (RVF) is a vector-borne viral zoonosis of increasing global importance. RVF virus (RVFV) is transmitted either through exposure to infected animals or through bites from different species of infected mosquitoes, mainly of Aedes and Culex genera. These mosquitoes are very sensitive to environmental conditions, which may determine their presence, biology, and abundance. In East Africa, RVF outbreaks are known to be closely associated with heavy rainfall events, unlike in the semi-arid regions of West Africa where the drivers of RVF emergence remain poorly understood. The assumed importance of temporary ponds and rainfall temporal distribution therefore needs to be investigated.

Methodology/Principal Findings

A hydrological model is combined with a mosquito population model to predict the abundance of the two main mosquito species (Aedes vexans and Culex poicilipes) involved in RVFV transmission in Senegal. The study area is an agropastoral zone located in the Ferlo Valley, characterized by a dense network of temporary water ponds which constitute mosquito breeding sites.The hydrological model uses daily rainfall as input to simulate variations of pond surface areas. The mosquito population model is mechanistic, considers both aquatic and adult stages and is driven by pond dynamics. Once validated using hydrological and entomological field data, the model was used to simulate the abundance dynamics of the two mosquito species over a 43-year period (1961–2003). We analysed the predicted dynamics of mosquito populations with regards to the years of main outbreaks. The results showed that the main RVF outbreaks occurred during years with simultaneous high abundances of both species.

Conclusion/Significance

Our study provides for the first time a mechanistic insight on RVFV transmission in West Africa. It highlights the complementary roles of Aedes vexans and Culex poicilipes mosquitoes in virus transmission, and recommends the identification of rainfall patterns favourable for RVFV amplification.     

Seroprevalence of Alphavirus Antibodies in a Cross-Sectional Study in Southwestern Tanzania Suggests Endemic Circulation of Chikungunya

Background

To date, Alphavirus infections and their most prominent member, chikungunya fever, a viral disease which first became apparent in Tanzania in 1953, have been very little investigated in regions without epidemic occurrence. Few data exist on burden of disease and socio-economic and environmental covariates disposing to infection.

Methods

A cross-sectional seroprevalence study was undertaken in 1,215 persons from Mbeya region, South-Western Tanzania, to determine the seroprevalence of anti-Alphavirus IgG antibodies, and to investigate associated risk factors.

Results

18% of 1,215 samples were positive for Alphavirus IgG. Seropositivity was associated with participant age, low to intermediate elevation, flat terrain and with IgG positivity for Rift Valley fever, Flaviviridae, and rickettsiae of the spotted fever group. When comparing the geographical distribution of Alphavirus seropositivity to that of Rift Valley fever, it was obvious that Alphaviruses had spread more widely throughout the study area, while Rift Valley fever was concentrated along the shore of Lake Malawi.

Conclusion

Alphavirus infections may contribute significantly to the febrile disease burden in the study area, and are associated with several arthropod-borne infections. Their spread seems only limited by factors affecting mosquitoes, and seems less restricted than that of Rift Valley fever.     

Seroprevalence of Sheep and Goat Pox,Peste Des Petits Ruminants and Rift Valley Fever in Saudi Arabia

Sheep and goat pox, peste des petits ruminants and Rift Valley fever are important diseases of small ruminant livestock. Sheep and goat pox, along with peste des petits ruminants, are endemic throughout most of Africa, Asia and the Middle East. Whereas Rift Valley fever is endemic in Africa, outbreaks in the Middle East have been reported over the past decade, including the Arabian Peninsula. Saudi Arabia is a major importer of livestock, and understanding the prevalence of these viral infections would be useful for disease control. In this study, sera from sheep and goats were collected from 3 regions in Saudi Arabia. They were evaluated for antibodies specific to sheep and goat pox, peste des petits ruminants and Rift Valley fever by virus neutralization assays. To the best of our knowledge, this is the first study to evaluate the seroprevalence of these viruses in sheep and goats.     

A Single Immunization with MVA Expressing GnGc Glycoproteins Promotes Epitope-specific CD8+-T Cell Activation and Protects Immune-competent Mice against a Lethal RVFV Infection

Background

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen causing an important disease in ruminants often transmitted to humans after epizootic outbreaks in African and Arabian countries. To help combat the spread of the disease, prophylactic measures need to be developed and/or improved.

Methodology/Principal Findings

In this work, we evaluated the immunogenicity and protective efficacy of recombinant plasmid DNA and modified vaccinia virus Ankara (rMVA) vectored vaccines against Rift Valley fever in mice. These recombinant vaccines encoded either of two components of the Rift Valley fever virus: the viral glycoproteins (Gn/Gc) or the nucleoprotein (N). Following lethal challenge with live RVFV, mice immunized with a single dose of the rMVA-Gn/Gc vaccine showed no viraemia or clinical manifestation of disease, but mounted RVFV neutralizing antibodies and glycoprotein specific CD8+ T-cell responses. Neither DNA-Gn/Gc alone nor a heterologous prime-boost immunization schedule (DNA-Gn/Gc followed by rMVAGn/Gc) was better than the single rMVA-Gn/Gc immunization schedule with regards to protective efficacy. However, the rMVA-Gn/Gc vaccine failed to protect IFNAR^−/− mice upon lethal RVFV challenge suggesting a role for innate responses in protection against RVFV. Despite induction of high titer antibodies against the RVFV nucleoprotein, the rMVA-N vaccine, whether in homologous or heterologous prime-boost schedules with the corresponding recombinant DNA vaccine, only conferred partial protection to RVFV challenge.

Conclusions/Significance

Given the excellent safety profile of rMVA based vaccines in humans and animals, our data supports further development of rMVA-Gn/Gc as a vaccine strategy that can be used for the prevention of Rift Valley fever in both humans and livestock.     

High seroprevalence of Rift Valley FEVER AND EVIDENCE FOR ENDEMIC circulation in Mbeya region, Tanzania, in a cross-sectional study

Background

The Rift Valley fever virus (RVFV) is an arthropod-borne phlebovirus. RVFV mostly causes outbreaks among domestic ruminants with a major economic impact. Human infections are associated with these events, with a fatality rate of 0.5–2%. Since the virus is able to use many mosquito species of temperate climates as vectors, it has a high potential to spread to outside Africa.

Methodology/Principal Findings

We conducted a stratified, cross-sectional sero-prevalence survey in 1228 participants from Mbeya region, southwestern Tanzania. Samples were selected from 17,872 persons who took part in a cohort study in 2007 and 2008. RVFV IgG status was determined by indirect immunofluorescence. Possible risk factors were analyzed using uni- and multi-variable Poisson regression models. We found a unique local maximum of RVFV IgG prevalence of 29.3% in a study site close to Lake Malawi (N = 150). The overall seroprevalence was 5.2%. Seropositivity was significantly associated with higher age, lower socio-economic status, ownership of cattle and decreased with distance to Lake Malawi. A high vegetation density, higher minimum and lower maximum temperatures were found to be associated with RVFV IgG positivity. Altitude of residence, especially on a small scale in the high-prevalence area was strongly correlated (PR 0.87 per meter, 95% CI = 0.80–0.94). Abundant surface water collections are present in the lower areas of the high-prevalence site. RVF has not been diagnosed clinically, nor an outbreak detected in the high-prevalence area.

Conclusions

RVFV is probably circulating endemically in the region. The presence of cattle, dense vegetation and temperate conditions favour mosquito propagation and virus replication in the vector and seem to play major roles in virus transmission and circulation. The environmental risk-factors that we identified could serve to more exactly determine areas at risk for RVFV endemicity.     

Rift Valley fever virus

Rift Valley fever is considered to be one of the most important viral zoonoses in Africa. In 2000, the Rift valley fever virus spread to the Arabian Peninsula and caused two simultaneous outbreaks in Yemen and Saudi Arabia. It is transmitted to ruminants and to humans by mosquitoes. The viral agent is an arbovirus, which belongs to the Phlebovirus genus in the Bunyaviridae family. This family of viruses comprises more than 300 members grouped into five genera: Orthobunyavirus, Phlebovirus, Hantavirus, Nairovirus, and Tospovirus. Several members of the Bunyaviridae family are responsible for fatal hemorrhagic fevers: Rift Valley fever virus (Phlebovirus), Crimean-Congo hemorrhagic fever virus (Nairovirus), Hantaan, Sin Nombre and related viruses (Hantavirus), and recently Garissa, now identified as Ngari virus (Orthobunyavirus). Here are reviewed recent advances in Rift Valley fever virus, its epidemiology, molecular biology and focus on recent data on the interactions between viral and cellular proteins, which help to understand the molecular mechanisms utilized by the virus to circumvent the host cellular response.     

A statistical model of Rift Valley fever activity in Egypt

Rift Valley fever (RVF) is a viral disease of animals and humans and a global public health concern due to its ecological plasticity, adaptivity, and potential for spread to countries with a temperate climate. In many places, outbreaks are episodic and linked to climatic, hydrologic, and socioeconomic factors. Although outbreaks of RVF have occurred in Egypt since 1977, attempts to identify risk factors have been limited. Using a statistical learning approach (lasso‐regularized generalized linear model), we tested the hypotheses that outbreaks in Egypt are linked to (1) River Nile conditions that create a mosquito vector habitat, (2) entomologic conditions favorable to transmission, (3) socio‐economic factors (Islamic festival of Greater Bairam), and (4) recent history of transmission activity. Evidence was found for effects of rainfall and river discharge and recent history of transmission activity. There was no evidence for an effect of Greater Bairam. The model predicted RVF activity correctly in 351 of 358 months (98.0%). This is the first study to statistically identify risk factors for RVF outbreaks in a region of unstable transmission.     

Multidisciplinary prospective study of mother-to-child chikungunya virus infections on the island of La Réunion

Background

An outbreak of chikungunya virus affected over one-third of the population of La Réunion Island between March 2005 and December 2006. In June 2005, we identified the first case of mother-to-child chikungunya virus transmission at the Groupe Hospitalier Sud-Réunion level-3 maternity department. The goal of this prospective study was to characterize the epidemiological, clinical, biological, and radiological features and outcomes of all the cases of vertically transmitted chikungunya infections recorded at our institution during this outbreak.

Methods and Findings

Over 22 mo, 7,504 women delivered 7,629 viable neonates; 678 (9.0%) of these parturient women were infected (positive RT-PCR or IgM serology) during antepartum, and 61 (0.8%) in pre- or intrapartum. With the exception of three early fetal deaths, vertical transmission was exclusively observed in near-term deliveries (median duration of gestation: 38 wk, range 35–40 wk) in the context of intrapartum viremia (19 cases of vertical transmission out of 39 women with intrapartum viremia, prevalence rate 0.25%, vertical transmission rate 48.7%). Cesarean section had no protective effect on transmission. All infected neonates were asymptomatic at birth, and median onset of neonatal disease was 4 d (range 3–7 d). Pain, prostration, and fever were present in 100% of cases and thrombocytopenia in 89%. Severe illness was observed in ten cases (52.6%) and mainly consisted of encephalopathy (n = 9; 90%). These nine children had pathologic MRI findings (brain swelling, n = 9; cerebral hemorrhages, n = 2), and four evolved towards persistent disabilities.

Conclusions

Mother-to-child chikungunya virus transmission is frequent in the context of intrapartum maternal viremia, and often leads to severe neonatal infection. Chikungunya represents a substantial risk for neonates born to viremic parturients that should be taken into account by clinicians and public health authorities in the event of a chikungunya outbreak.     

Modelling the Effects of Seasonality and Socioeconomic Impact on the Transmission of Rift Valley Fever Virus

Rift Valley fever (RVF) is an important mosquito-borne viral zoonosis in Africa and the Middle East that causes human deaths and significant economic losses due to huge incidences of death and abortion among infected livestock. Outbreaks of RVF are sporadic and associated with both seasonal and socioeconomic effects. Here we propose an almost periodic three-patch model to investigate the transmission dynamics of RVF virus (RVFV) among ruminants with spatial movements. Our findings indicate that, in Northeastern Africa, human activities, including those associated with the Eid al Adha feast, along with a combination of climatic factors such as rainfall level and hydrological variations, contribute to the transmission and dispersal of the disease pathogen. Moreover, sporadic outbreaks may occur when the two events occur together: 1) abundant livestock are recruited into areas at risk from RVF due to the demand for the religious festival and 2) abundant numbers of mosquitoes emerge. These two factors have been shown to have impacts on the severity of RVF outbreaks. Our numerical results present the transmission dynamics of the disease pathogen over both short and long periods of time, particularly during the festival time. Further, we investigate the impact on patterns of disease outbreaks in each patch brought by festival- and seasonal-driven factors, such as the number of livestock imported daily, the animal transportation speed from patch to patch, and the death rate induced by ceremonial sacrifices. In addition, our simulations show that when the time for festival preparation starts earlier than usual, the risk of massive disease outbreaks rises, particularly in patch 3 (the place where the religious ceremony will be held).     

Clone 13-infected Aedes aegypti salivary components inhibit Rift Valley fever virus pathogenicity

Rift Valley fever virus (RVFV) continues to cause large outbreaks among humans and domestic animals in Africa. RVFV Clone 13, a naturally attenuated clone, is a promising vaccine which was used during the 2009–2010 outbreak in South Africa and played a key role in the control of the disease. In this work, we infected Aedes aegypti mosquitoes with RVFV Clone 13 and prepared salivary gland extracts (SGE). C57BL/6-NRJ male mice were infected with a mixture of SGE infected by Clone 13 and the ZH548 RVFV strain. With the injection of increasing doses of Clone 13-infected SGE, all mice were protected. Our results suggest Clone 13 infected SGE contain unique antiviral components able to counteract the replication of RVFV when injected into vertebrates.     

Infection and transmission of Rift Valley fever viruses lacking the NSs and/or NSm genes in mosquitoes: potential role for NSm in mosquito infection

Background

Rift Valley fever virus is an arthropod-borne human and animal pathogen responsible for large outbreaks of acute and febrile illness throughout Africa and the Arabian Peninsula. Reverse genetics technology has been used to develop deletion mutants of the virus that lack the NSs and/or NSm virulence genes and have been shown to be stable, immunogenic and protective against Rift Valley fever virus infection in animals. We assessed the potential for these deletion mutant viruses to infect and be transmitted by Aedes mosquitoes, which are the principal vectors for maintenance of the virus in nature and emergence of virus initiating disease outbreaks, and by Culex mosquitoes which are important amplification vectors.

Methodology and Principal Findings

Aedes aegypti and Culex quinquefasciatus mosquitoes were fed bloodmeals containing the deletion mutant viruses. Two weeks post-exposure mosquitoes were assayed for infection, dissemination, and transmission. In Ae. aegypti, infection and transmission rates of the NSs deletion virus were similar to wild type virus while dissemination rates were significantly reduced. Infection and dissemination rates for the NSm deletion virus were lower compared to wild type. Virus lacking both NSs and NSm failed to infect Ae. aegypti. In Cx. quinquefasciatus, infection rates for viruses lacking NSm or both NSs and NSm were lower than for wild type virus.

Conclusions/Significance

In both species, deletion of NSm or both NSs and NSm reduced the infection and transmission potential of the virus. Deletion of both NSs and NSm resulted in the highest level of attenuation of virus replication. Deletion of NSm alone was sufficient to nearly abolish infection in Aedes aegypti mosquitoes, indicating an important role for this protein. The double deleted viruses represent an ideal vaccine profile in terms of environmental containment due to lack of ability to efficiently infect and be transmitted by mosquitoes.     

Seasonal rainfall and runoff promote coral disease on an inshore reef

Background

Declining water quality coupled with the effects of climate change are rapidly increasing coral diseases on reefs worldwide, although links between coral diseases and environmental parameters remain poorly understood. This is the first study to document a correlation between coral disease and water quality on an inshore reef.

Methodology/Principal Findings

The temporal dynamics of the coral disease atramentous necrosis (AN) was investigated over two years within inshore populations of Montipora aequituberculata in the central Great Barrier Reef, in relation to rainfall, salinity, temperature, water column chlorophyll a, suspended solids, sedimentation, dissolved organic carbon, and particulate nitrogen, phosphorus and organic carbon. Overall, mean AN prevalence was 10-fold greater during summer wet seasons than winter dry seasons. A 2.5-fold greater mean disease abundance was detected during the summer of 2009 (44 ± SE 6.7 diseased colonies per 25 m²), when rainfall was 1.6-fold greater than in the summer of 2008. Two water quality parameters explained 67% of the variance in monthly disease prevalence in a Partial Least Squares regression analysis; disease abundance was negatively correlated with salinity (R2 = −0.6) but positively correlated with water column particulate organic carbon concentration (R2 = 0.32). Seasonal temperature patterns were also positively correlated with disease abundance, but explained only a small portion of the variance.

Conclusions/Significance

The results suggest that rainfall and associated runoff may facilitate seasonal disease outbreaks, potentially by reducing host fitness or by increasing pathogen virulence due to higher availability of nutrients and organic matter. In the future, rainfall and seawater temperatures are likely to increase due to climate change which may lead to decreased health of inshore reefs.     

Predictive Factors and Risk Mapping for Rift Valley Fever Epidemics in Kenya

Background

To-date, Rift Valley fever (RVF) outbreaks have occurred in 38 of the 69 administrative districts in Kenya. Using surveillance records collected between 1951 and 2007, we determined the risk of exposure and outcome of an RVF outbreak, examined the ecological and climatic factors associated with the outbreaks, and used these data to develop an RVF risk map for Kenya.

Methods

Exposure to RVF was evaluated as the proportion of the total outbreak years that each district was involved in prior epizootics, whereas risk of outcome was assessed as severity of observed disease in humans and animals for each district. A probability-impact weighted score (1 to 9) of the combined exposure and outcome risks was used to classify a district as high (score ≥ 5) or medium (score ≥2 - <5) risk, a classification that was subsequently subjected to expert group analysis for final risk level determination at the division levels (total = 391 divisions). Divisions that never reported RVF disease (score < 2) were classified as low risk. Using data from the 2006/07 RVF outbreak, the predictive risk factors for an RVF outbreak were identified. The predictive probabilities from the model were further used to develop an RVF risk map for Kenya.

Results

The final output was a RVF risk map that classified 101 of 391 divisions (26%) located in 21 districts as high risk, and 100 of 391 divisions (26%) located in 35 districts as medium risk and 190 divisions (48%) as low risk, including all 97 divisions in Nyanza and Western provinces. The risk of RVF was positively associated with Normalized Difference Vegetation Index (NDVI), low altitude below 1000m and high precipitation in areas with solonertz, luvisols and vertisols soil types (p <0.05).

Conclusion

RVF risk map serves as an important tool for developing and deploying prevention and control measures against the disease.     

Climate drives the meningitis epidemics onset in west Africa

Background

Every year West African countries within the Sahelo-Sudanian band are afflicted with major meningococcal meningitis (MCM) disease outbreaks, which affect up to 200,000 people, mainly young children, in one of the world''s poorest regions. The timing of the epidemic year, which starts in February and ends in late May, and the spatial distribution of disease cases throughout the “Meningitis Belt” strongly indicate a close linkage between the life cycle of the causative agent of MCM and climate variability. However, mechanisms responsible for the observed patterns are still not clearly identified.

Methods and Findings

By comparing the information on cases and deaths of MCM from World Health Organization weekly reports with atmospheric datasets, we quantified the relationship between the seasonal occurrence of MCM in Mali, a West African country, and large-scale atmospheric circulation. Regional atmospheric indexes based on surface wind speed show a clear link between population dynamics of the disease and climate: the onset of epidemics and the winter maximum defined by the atmospheric index share the same mean week (sixth week of the year; standard deviation, 2 wk) and are highly correlated.

Conclusions

This study is the first that provides a clear, quantitative demonstration of the connections that exist between MCM epidemics and regional climate variability in Africa. Moreover, this statistically robust explanation of the MCM dynamics enables the development of an Early Warning Index for meningitis epidemic onset in West Africa. The development of such an index will undoubtedly help nationwide and international public health institutions and policy makers to better control MCM disease within the so-called westward–eastward pan-African Meningitis Belt.     

Satellite Based Assessment of Hydroclimatic Conditions Related to Cholera in Zimbabwe

Introduction

Cholera, an infectious diarrheal disease, has been shown to be associated with large scale hydroclimatic processes. The sudden and sporadic occurrence of epidemic cholera is linked with high mortality rates, in part, due to uncertainty in timing and location of outbreaks. Improved understanding of the relationship between pathogenic abundance and climatic processes allows prediction of disease outbreak to be an achievable goal. In this study, we show association of large scale hydroclimatic processes with the cholera epidemic in Zimbabwe reported to have begun in Chitungwiza, a city in Mashonaland East province, in August, 2008.

Principal Findings

Climatic factors in the region were found to be associated with triggering cholera outbreak and are shown to be related to anomalies of temperature and precipitation, validating the hypothesis that poor conditions of sanitation, coupled with elevated temperatures, and followed by heavy rainfall can initiate outbreaks of cholera. Spatial estimation by satellite of precipitation and global gridded air temperature captured sensitivities in hydroclimatic conditions that permitted identification of the location in the region where the disease outbreak began.

Discussion

Satellite derived hydroclimatic processes can be used to capture environmental conditions related to epidemic cholera, as occurred in Zimbabwe, thereby providing an early warning system. Since cholera cannot be eradicated because the causative agent, Vibrio cholerae, is autochthonous to the aquatic environment, prediction of conditions favorable for its growth and estimation of risks of triggering the disease in a given population can be used to alert responders, potentially decreasing infection and saving lives.     

Tectonics, orbital forcing, global climate change, and human evolution in Africa: introduction to the African paleoclimate special volume

The late Cenozoic climate of Africa is a critical component for understanding human evolution. African climate is controlled by major tectonic changes, global climate transitions, and local variations in orbital forcing. We introduce the special African Paleoclimate Issue of the Journal of Human Evolution by providing a background for and synthesis of the latest work relating to the environmental context for human evolution. Records presented in this special issue suggest that the regional tectonics, appearance of C(4) plants in East Africa, and late Cenozoic global cooling combined to produce a long-term drying trend in East Africa. Of particular importance is the uplift associated with the East African Rift Valley formation, which altered wind flow patterns from a more zonal to more meridinal direction. Results in this volume suggest a marked difference in the climate history of southern and eastern Africa, though both are clearly influenced by the major global climate thresholds crossed in the last 3 million years. Papers in this volume present lake, speleothem, and marine paleoclimate records showing that the East African long-term drying trend is punctuated by episodes of short, alternating periods of extreme wetness and aridity. These periods of extreme climate variability are characterized by the precession-forced appearance and disappearance of large, deep lakes in the East African Rift Valley and paralleled by low and high wind-driven dust loads reaching the adjacent ocean basins. Dating of these records show that over the last 3 million years such periods only occur at the times of major global climatic transitions, such as the intensification of Northern Hemisphere Glaciation (2.7-2.5 Ma), intensification of the Walker Circulation (1.9-1.7 Ma), and the Mid-Pleistocene Revolution (1-0.7 Ma). Authors in this volume suggest this onset occurs as high latitude forcing in both Hemispheres compresses the Intertropical Convergence Zone so that East Africa becomes locally sensitive to precessional forcing, resulting in rapid shifts from wet to dry conditions. These periods of extreme climate variability may have provided a catalyst for evolutionary change and driven key speciation and dispersal events amongst mammals and hominins in Africa. In particular, hominin species seem to differentially originate and go extinct during periods of extreme climate variability. Results presented in this volume may represent the basis of a new theory of early human evolution in Africa.     

Rift valley fever: recent insights into pathogenesis and prevention

Rift Valley fever virus (RVFV) is a zoonotic pathogen that primarily affects ruminants but can also be lethal in humans. A negative-stranded RNA virus of the family Bunyaviridae, this pathogen is transmitted mainly via mosquito vectors. RVFV has shown the ability to inflict significant damage to livestock and is also a threat to public health. While outbreaks have traditionally occurred in sub-Saharan Africa, recent outbreaks in the Middle East have raised awareness of the potential of this virus to spread to Europe, Asia, and the Americas. Although the virus was initially characterized almost 80 years ago, the only vaccine approved for widespread veterinary use is an attenuated strain that has been associated with significant pathogenic side effects. However, increased understanding of the molecular biology of the virus over the last few years has led to recent advances in vaccine design and has enabled the development of more-potent prophylactic measures to combat infection. In this review, we discuss several aspects of RVFV, with particular emphasis on the molecular components of the virus and their respective roles in pathogenesis and an overview of current vaccine candidates. Progress in understanding the epidemiology of Rift Valley fever has also enabled prediction of potential outbreaks well in advance, thus providing another tool to combat the physical and economic impact of this disease.     

Multiple Assessment and Serum Neutralization of Arbovirus Mixtures

Mixtures of Venezuelan equine encephalomyelitis, Rift Valley fever, and chikungunya viruses may be assayed by selective immunofluorescence staining of infected cell monolayers. A multiple serum neutralization test is described for quantifying reactions of these viruses with mixtures of serum antibodies.