Host nutritional status affects alphavirus virulence, transmission, and evolution.

Malnourishment, specifically overweight/obesity and undernourishment, affects more than 2.5 billion people worldwide, with the number affected ever-increasing. Concurrently, emerging viral diseases, particularly those that are mosquito-borne, have spread dramatically in the past several decades, culminating in outbreaks of several viruses worldwide. Both forms of malnourishment are known to lead to an aberrant immune response, which can worsen disease outcomes and reduce vaccination efficacy for viral pathogens such as influenza and measles. Given the increasing rates of malnutrition and spread of arthropod-borne viruses (arboviruses), there is an urgent need to understand the role of host nutrition on the infection, virulence, and transmission of these viruses. To address this gap in knowledge, we infected lean, obese, and undernourished mice with arthritogenic arboviruses from the genus Alphavirus and assessed morbidity, virus replication, transmission, and evolution. Obesity and undernourishment did not consistently influence virus replication in the blood of infected animals except for reductions in virus in obese mice late in infection. However, morbidity was increased in obese mice under all conditions. Using Mayaro virus (MAYV) as a model arthritogenic alphavirus, we determined that both obese and undernourished mice transmit virus less efficiently to mosquitoes than control (lean) mice. In addition, viral genetic diversity and replicative fitness were reduced in virus isolated from obese compared to lean controls. Taken together, nutrition appears to alter the course of alphavirus infection and should be considered as a critical environmental factor during outbreaks.

Genetic control of alphavirus pathogenesis.

Alphaviruses, members of the positive-sense, single-stranded RNA virus family Togaviridae, represent a re-emerging public health concern worldwide as mosquito vectors expand into new geographic ranges. Members of the alphavirus genus tend to induce clinical disease characterized by rash, arthralgia, and arthritis (chikungunya virus, Ross River virus, and Semliki Forest virus) or encephalomyelitis (eastern equine encephalitis virus, western equine encephalitis virus, and Venezuelan equine encephalitis virus), though some patients who recover from the initial acute illness may develop long-term sequelae, regardless of the specific infecting virus. Studies examining the natural disease course in humans and experimental infection in cell culture and animal models reveal that host genetics play a major role in influencing susceptibility to infection and severity of clinical disease. Genome-wide genetic screens, including loss of function screens, microarrays, RNA-sequencing, and candidate gene studies, have further elucidated the role host genetics play in the response to virus infection, with the immune response being found in particular to majorly influence the outcome. This review describes the current knowledge of the mechanisms by which host genetic factors influence alphavirus pathogenesis and discusses emerging technologies that are poised to increase our understanding of the complex interplay between viral and host genetics on disease susceptibility and clinical outcome.

Epidemic sindbis virus infection in Finland: a population-based case-control study of risk factors.

BACKGROUND: Sindbis virus (SINV) is an arthropod-borne alphavirus that causes rash and arthritis. In Finland, epidemics occur cyclically, but factors associated with clinical SINV infection are largely unknown. We conducted a population-based case-control study during the epidemic year 2002. METHODS: SINV cases were serologically confirmed and reported to the National Infectious Disease Registry. Five control subjects, matched for age, sex, and residence, were selected from the National Population Information System. Data were collected using a self-administered mail survey. Conditional logistic regression models were used to identify independent risk factors; missing data were addressed using Bayesian full-likelihood modeling. RESULTS: A total of 337 case patients (58% female; age range, 1-94 y) and 934 control subjects were enrolled. Reported exposure to mosquito bites (matched odds ratio [mOR], 16.7; 95% confidence interval [CI], 9.1-33.4) and spending time in woods or marshland (mOR, 1.8; 95% CI, 1.3-2.5) were independently associated with SINV infection in the multivariable model. The population-attributable risk for mosquito bites was 87.2%. There were dose-response relations for increased number of insect bites (mOR, 23.8-72.5) and increased time spent in woods or marshland (mOR, 1.3-2.2). CONCLUSIONS: Educating the public in endemic areas to avoid mosquito exposure and use protective measures remain important prevention measures for SINV infection.

Environmental factors of households in five districts of Kerala affected by the epidemic of chikungunya fever in 2007.

BACKGROUND: Two epidemics of chikungunya fever were reported from Kerala in 2006 and 2007. We aimed to investigate the environmental factors of households affected by chikungunya fever and to estimate the proportion of population that suffered from the disease during the epidemic in 2007. METHODS: A cross-sectional survey was conducted in the 5 heavily affected districts of Kerala during October-November 2007. The 2-stage sampling technique was used to collect data from 10 clusters, each having 18 houses from every district, by interviewing the subjects using a structured questionnaire. The sample size was 900. RESULTS: The proportion of the population affected by chikungunya fever was 57.1% (95% CI: 52.8%-61.4%). There was a significant association between location of houses and disease status. Houses located near a public conveyance facility (within 250 m) were relatively protected from the disease (OR 0.19 [0.06-0.60]). About 69% of the households perceived mosquito infestation as a problem and 46.6% used fumes to avoid mosquitoes. More than 42% of households were not using any anti-mosquito measures at the time of the survey. Stored drinking water was the most common potential breeding source in the houses (23.5%). Households which did not store water inside were protected from the disease (OR 0.22 [0.08-0.65]). CONCLUSION: The study indicated the persistence of favourable domestic and environmental factors after the epidemic, reflecting the necessity to strengthen anti-mosquito campaigns.

Chikungunya.

Chikungunya virus is a zoonotic, vector-borne pathogen that has been responsible for numerous outbreaks of febrile arthralgia since its discovery in the early 1950s. In the past decade, the virus has re-emerged more frequently, causing massive epidemics that have moved from Africa throughout the Indian Ocean to India and Southeast Asia. A discussion of the virus, its epidemiology, diagnostic criteria, and immunity are presented in this article.

Chikungunya fever in two German tourists returning from the Maldives, September, 2009.

This report describes the first isolation and molecular characterisation of a chikungunya virus from two German tourists who became ill after a visit to the Maldives in September 2009. The virus contained the E1 A226V mutation, shown to be responsible for an adaptation to the Asian tiger mosquito Aedes albopictus. The E1 coding sequence was identical to chikungunya virus isolates from Sri Lanka and showed three nt-mismatches to the only available E1 nt sequence from the Maldives.

Differential incorporation of cholesterol by Sindbis virus grown in mammalian or insect cells.

Cholesterol has been shown to be essential for the fusion of alphaviruses with artificial membranes (liposomes). Cholesterol has also been implicated as playing an essential and critical role in the processes of entry and egress of alphaviruses in living cells. Paradoxically, insects, the alternate host for alphaviruses, are cholesterol auxotrophs and contain very low levels of this sterol. To further evaluate the role of cholesterol in the life cycle of alphaviruses, the cholesterol levels of the alphavirus Sindbis produced from three different mosquito (Aedes albopictus) cell lines; one other insect cell line, Sf21 from Spodoptera frugiperda; and BHK (mammalian) cells were measured. Sindbis virus was grown in insect cells under normal culture conditions and in cells depleted of cholesterol by growth in serum delipidated by using Cab-O-sil, medium treated with methyl-beta-cyclodextrin, or serum-free medium. The levels of cholesterol incorporated into the membranes of the cells and into the virus produced from these cells were determined. Virus produced from these treated and untreated cells was compared to virus grown in BHK cells under standard conditions. The ability of insect cells to produce Sindbis virus after delipidation was found to be highly cell specific and not dependent on the level of cholesterol in the cell membrane. A very low level of cholesterol was required for the generation of wild-type levels of infectious Sindbis virus from delipidated cells. The data show that one role of the virus membrane is structural, providing the stability required for infectivity that may not be provided by the delipidated membranes in some cells. These data show that the amount of cholesterol in the host cell membrane in and of itself has no effect on the process of virus assembly or on the ability of virus to infect cells. Rather, these data suggest that the cholesterol dependence reported for infectivity and assembly of Sindbis virus is a reflection of differences in the insect cell lines used and the methods of delipidation.

The role of environmental and individual factors in the social epidemiology of chikungunya disease on Mayotte Island.

This article examines the role of environmental and individual factors in the social epidemiology of chikungunya disease on the island of Mayotte (South-western Indian Ocean). In an epidemic setting, an interdisciplinary study combining interviews, observations, and serological tests was conducted to: (1) estimate the frequency and social distribution of chikungunya disease and (2) identify its principal cognitive, behavioral, and environmental determinants within a stratified random sample of the Mayotte population (n=888). Semi-parametric tests and multiple correspondence analyses were used to describe the statistical relationships between the different classes of variables examined in this study and the presence of antibodies attributable to chikungunya. These analyses highlighted differences between two main types of populations: one more autochthonous, more urban and better educated population, which shared 'legitimate' representations of the disease-from a biomedical viewpoint; and the other more migrant, more suburban, and more deprived, which is characterized by folk theories of chikungunya virus infection. Moreover, a series of logistic regression models revealed that social disparities in the distribution of virus infection were primarily structured by the housing conditions and cognitive representations of the disease held by the participants. These results suggest that environmental and individual factors are equally crucial in epidemic settings, and that they could explain, to a considerable extent, the social differences observed in morbidity associated with recent emerging infectious diseases in tropical countries.

The Chikungunya epidemic in Italy and its repercussion on the blood system.

BACKGROUND: The Chikungunya virus (CHIKV) is transmitted by Aedes mosquitoes and recently caused a massive epidemic on La Réunion Island, in the Indian Ocean. Between July and September 2007 it caused the first autochthonous epidemic outbreak in Europe, in the Region of Emilia-Romagna in the north-east of Italy. MATERIALS AND METHODS: After the first reports of an unusually high number of patients with a febrile illness of unknown origin in two contiguous villages, an outbreak investigation was carried out to identify the primary source of infection, the modes of transmission and the dynamics of the epidemic. An active surveillance system was also implemented. Laboratory diagnosis was performed through serology and polymerase chain reaction (PCR) analysis. Blood donation was discontinued in the areas involved from September to October 2007 and specific precautionary blood safety and self-sufficiency measures were adopted by the regional health and blood authorities and the National Blood Centre. An estimate method to early assess the risk of viraemic blood donations by asymptomatic donors was developed, as a tool for "pragmatic" risk assessment and management, aiming at providing a reliable order of magnitude of the mean risk of CHIKV transmission through blood transfusion. RESULTS: Two hundred and seventeen cases of CHIKV infection were identified between 4th July and 28th September. The disease was fairly mild in most of the cases. The precautionary measures adopted in the blood system caused a considerable reduction of the collection of blood components and of the delivery of plasma to the pharmaceutical industry for contract manufacturing. The estimated risk of CHIKV transmission through blood transfusion peaked in the third week of August. CONCLUSION: ACHIKV epidemic poses considerable problems for public health authorities, who not only need good routine programmes of vector control and epidemiological surveillance but also local and national emergency plans to sustain the blood supply, so as to promptly deal with the potentially severe effects of an epidemic outbreak, especially when affected areas locally require a significant blood inventory and at the same time represent a critical resource for other areas depending on external supplies of blood components.

Alphavirus production is inhibited in neurofibromin 1-deficient cells through activated RAS signalling.

Virus-host interactions essential for alphavirus pathogenesis are poorly understood. To address this shortcoming, we coupled retrovirus insertional mutagenesis and a cell survival selection strategy to generate clonal cell lines broadly resistant to Sindbis virus (SINV) and other alphaviruses. Resistant cells had significantly impaired SINV production relative to wild-type (WT) cells, although virus binding and fusion events were similar in both sets of cells. Analysis of the retroviral integration sites identified the neurofibromin 1 (NF1) gene as disrupted in alphavirus-resistant cell lines. Subsequent analysis indicated that expression of NF1 was significantly reduced in alphavirus-resistant cells. Importantly, independent down-regulation of NF1 expression in WT HEK 293 cells decreased virus production and increased cell viability during SINV infection, relative to infected WT cells. Additionally, we observed hyperactive RAS signalling in the resistant HEK 293 cells, which was anticipated because NF1 is a negative regulator of RAS. Expression of constitutively active RAS (HRAS-G12V) in a WT HEK 293 cell line resulted in a marked delay in virus production, compared with infected cells transfected with parental plasmid or dominant-negative RAS (HRAS-S17N). This work highlights novel host cell determinants required for alphavirus pathogenesis and suggests that RAS signalling may play an important role in neuronal susceptibility to SINV infection.

[Chikungunya: fever and joint pains after vacation in a tropical area]. / Chikungunya-Fieber und Gelenkschmerzen nach Tropenaufenthalt.

HISTORY: A 27-year-old man was admitted to our hospital, two days after returning from a vacation in Mauritius, with fever, chills, headache and myalgia, as well as arthritis of the legs. He had a temperature of 38.6 C and painful swellings of both talocalcanean joints. INVESTIGATIONS: Laboratory studies revealed leukopenia, lymphopenia and thrombopenia, as well as a slight elevation of transaminases and serum bilirubin. Diagnostic tests for malaria were negative, as were tests for antibodies against Epstein-Barr virus, cytomegalovirus, leptospirosis, dengue, chikungunya and hepatitis viruses, legionella, streptococcus and HIV. But PCR with chikungunya virus RNA was positive, establishing the diagnosis of acute chikungunya. DIAGNOSIS, TREATMENT AND COURSE: All symptoms disappeared on administration of analgesics and antipyretics. But after two days a maculopapular rash on the trunk and limbs was noted: it responded well to the application of clemastine. The patient was discharged from hospital three days after admission. CONCLUSION: Chikungunya virus belongs to the family of alphaviruses and is common in Southeast Asia and Africa. It can be transmitted to humans by bites of the Aedes mosquitoes. Symptoms are similar to those of dengue and consist of fever, headache, arthralgia, myalgia and conjunctivitis. After two or three days these symptoms subside and a maculopapular rash appears. The fever may return. Arthralgia can persist over weeks and even months. While the diagnosis is normally established by antibody tests, these may be negative in early stages of the disease. when the diagnosis can be made by PCR. Treatment is symptomatic with analgesics and antipyretics.

Estimated risk of Chikungunya viremic blood donation during an epidemic on Reunion Island in the Indian Ocean, 2005 to 2007.

BACKGROUND: Between 2005 and 2007, Chikungunya virus (CHIKV) caused a massive epidemic on Reunion Island with a major peak in the number of cases in February 2006. Blood donation was interrupted on the island in January 2006. STUDY DESIGN AND METHODS: Estimates of the mean risk of viremic blood donation on Reunion Island were computed for different phases of the epidemic. Calculations used CHIKV incidence estimates derived from sentinel surveillance, duration of viremia, and frequency of asymptomatic infection. Data on these two last parameters were initially based on hypotheses and subsequently obtained from studies carried out during the outbreak. The estimated risk was compared to the results of CHIKV nucleic acid testing (NAT) implemented for platelet (PLT) donations screening. RESULTS: Over the course of the outbreak, the mean risk was estimated at 132 per 100,000 donations. The risk peaked at 1,500 per 100,000 donations at the height of the outbreak in February 2006. In total, 47 blood donations would have been potentially viremic if blood collection had not been interrupted. During this period, an estimated 312,500 of 757,000 inhabitants had been infected by mosquito-borne transmission. From January to May 2006, the estimated mean risk (0.7%) and observed risk on PLT donations (0.4%) were of the same order of magnitude. CONCLUSION: During this large outbreak, the estimated risk of viremic blood donation was high, but low compared to the risk of mosquito-borne CHIKV transmission. The estimated risk was corroborated by the concordant results with the observed risk.

Entomologic investigations of a chikungunya virus epidemic in the Union of the Comoros, 2005.

From January to April 2005, an epidemic of chikungunya virus (CHIKV) illness occurred in the Union of Comoros. Entomological studies were undertaken during the peak of the outbreak, from March 11 to March 31, aimed at identifying the primary vector(s) involved in transmission so that appropriate public health measures could be implemented. Adult mosquitoes were collected by backpack aspiration and human landing collection in homes and neighborhoods of clinically ill patients. Water-holding containers were inspected for presence of mosquito larvae. Adult mosquitoes were analyzed by RT-PCR and cultivation in cells for the presence of CHIK virus and/or nucleic acid. A total of 2,326 mosquitoes were collected and processed in 199 pools. The collection consisted of 62.8% Aedes aegypti, 25.5% Culex species, and 10.7% Aedes simpsoni complex, Eretmapodites spp and Anopheles spp. Seven mosquito pools were found to be positive for CHIKV RNA and 1 isolate was obtained. The single CHIKV mosquito isolate was from a pool of Aedes aegypti and the minimum infection rate (MIR) for this species was 4.0, suggesting that Ae. aegypti was the principal vector responsible for the outbreak. This was supported by high container (31.1%), household (68%), and Breteau (126) indices, with discarded tires (58.8%) and small cooking and water storage vessels (31.1%) registering the highest container indices.

Drought-associated chikungunya emergence along coastal East Africa.

Epidemics of chikungunya fever, an Aedes spp.-borne viral disease, affected hundreds of thousands of people in western Indian Ocean islands and India during 2005-2006. The initial outbreaks occurred in coastal Kenya (Lamu, then Mombasa) in 2004. We investigated eco-climatic conditions associated with chikungunya fever emergence along coastal Kenya using epidemiologic investigations and satellite data. Unusually dry, warm conditions preceded the outbreaks, including the driest since 1998 for some of the coastal regions. Infrequent replenishment of domestic water stores and elevated temperatures may have facilitated Chikungunya virus transmission. These results suggest that drought-affected populations may be at heightened risk for chikungunya fever, and underscore the need for safe water storage during drought relief operations.

Chikungunya.

Chikungunya fever is a viral disease transmitted to humans by the bite of infected Aedes aegypti mosquito. Like malaria and dengue, this infection has almost become endemic in India, especially central and south India. Symptoms of sudden onset of fever, chills, headache, nausea, vomiting, joint pain with or without swelling, low back pain, and rash are very similar to those of dengue but, unlike dengue, there is no hemorrhagic or shock syndrome form. Chikungunya is a self-limiting illness with no specific treatment. Travellers visiting endemic areas should be careful and take precautions to see that they are not bitten by mosquitoes.

Heparin-binding and patterns of virulence for two recombinant strains of Sindbis virus.

E2 is an important determinant of Sindbis virus neurovirulence. Increased heparan sulfate (HS) binding is associated with rapid clearance of viremia and usually with decreased virulence. However, substitution of histidine for arginine at E2-157 (R157H) or glutamate for lysine at E2-159 (K159E) produces viruses with decreases in heparin-Sepharose binding and increases in viremia but different levels of binding to HS-expressing cells and virulence phenotypes in newborn CD-1 mice (Byrnes, A.P., Griffin, D.E., 2000. Large-plaque mutants of Sindbis virus show reduced binding to heparan sulfate, heightened viremia and slower clearance from the circulation. J. Virol. 74, 644-651). To identify mechanisms of virulence, R157H and K159E were studied in newborn CD-1 and BALB/c mice. Subcutaneous inoculation of R157H caused 100% and K159E 60% mortality in 2-day-old CD-1 mice. R157H caused 25% and K159E no mortality in 2-day-old BALB/c mice. R157H and K159E replicated similarly at the site of inoculation with the same level of viremia, but clearance was slower in CD-1 than BALB/c mice. R157H replicated better than K159E in the central nervous system (CNS) after subcutaneous and intracerebral inoculation and in undifferentiated neurons. These studies show a genetic restriction of replication in newborn BALB/c mice, and that amino acid substitutions affecting binding to proteoglycans may differ in importance for CNS infection and viremia.

Exacerbation of soft tissue lesions in lead exposed virus infected mice.

OBJECTIVE: To investigate the effect of Lead (Pb) acetate exposure on Semliki forest virus (SFV) pathogenesis in mice. METHODS: Different doses (62.5, 125, 250 and 500 mg/Kg body weight) of Pb dissolved in normal saline were given to mice by oral intubation in a sub-acute (28 days) and sub-chronic (90 days) regimen followed by SFV infection. Morbidity, mortality, clinical symptoms, mean survival time (MST), changes in body and organ weight, accumulation of lead in soft tissues, virus titre in brain and histopathological alterations were compared between lead exposed and infected groups. RESULTS: Early appearance of virus symptoms, increased mortality, decreased MST, enhanced SFV titre and greater tissue damage were observed in lead exposed-SFV-infected mice. CONCLUSION: Pre-exposure to lead increases the susceptibility of mice towards SFV infection. Further studies are suggested in view of the persistence of lead in the environment and the possibility of infection by microbial pathogens.

Pathogenesis of Semliki Forest virus encephalitis.

This article provides a review of the pathogenesis of Semliki Forest virus (SFV) encephalitis. In mice, outcome of infection varies according to age of the mouse and strain of the virus and can include acute encephalitis, subacute demyelinating meningoencephalomyelitis, and persistent subclinical central nervous system (CNS) infection. All strains of virus are virulent in mice infected <12 days of age. The L10 strain is also virulent in mice >14 days age, whereas the A7(74) strain is avirulent. The genetic difference between these strains maps to the nsp3 gene. For A7(74) virus, age-related virulence correlates with ability of CNS neurons to replicate virus and undergo apoptotic cell death. Immature developing neurons support complete virus replication but as neuronal populations and circuits mature in the postnatal brain, virus infection becomes progressively restricted and nonproductive. This restricted replication can be overcome by gold I compounds, which may function by inducing neuronal dedifferentiation to a state permissive for virus replication. Biochemical pathways associated with membrane biogenesis may be an important determinant of this effect. Infection of some developing neuronal populations results in apoptosis, whereas infection of mature neurons results in persistent infection. An active type-I interferon system prevents virus spread in extraneural tissues. An initial high-titer plasma viremia is controlled by immunoglobulin M (IgM) antibodies. Virus enters the brain across cerebral endothelial cells and initiates scattered foci of perivascular infection. The blood-brain barrier is disrupted. Neurons and oligodendrocytes are the cell types most frequently infected. Infectivity in the brain can be eliminated by IgG antibodies, though an active T-cell response is required for virus elimination. Lesions of inflammatory demyelination require the presence of CD8(+) T lymphocytes and probably result from destruction by these cells of virally infected oligodendrocytes.