Chikungunya Virus and Aedes Mosquitoes: Saliva Is Infectious as soon as Two Days after Oral Infection

Background

Aedes aegypti and Aedes albopictus are potential vectors of chikungunya virus (CHIKV). The recent CHIKV outbreaks were caused by a new variant characterized by a mutation in the E1 glycoprotein gene (E1-226V) which has favored a better transmissibility by Ae. albopictus. As Ae. albopictus tends to replace Ae. aegypti in many regions, one question remained: is Ae. albopictus as efficient as Ae. aegypti to transmit the variant E1-226V of CHIKV?

Methodology and Findings

We infected orally both species with the variant E1-226V and estimated the infection, the viral dissemination, and the transmission rate by real time RT-PCR. Additionally, we used an in vitro assay to determine the amount of virus delivered by mosquitoes in their saliva. We found that Ae. aegypti as well as Ae. albopictus ensured a high replication of the virus which underwent an efficient dissemination as detectable in the salivary glands at day 2 post-infection (pi). Infectious CHIKV particles were delivered by salivary glands from day 2 with a maximum at day 6 pi for Ae. albopictus (10^3.3 PFU) and day 7 pi for Ae. aegypti (10^2.5 PFU).

Conclusions

Ae. albopictus is slightly more efficient than Ae. aegypti to transmit the variant E1-226V of CHIKV. These results will help to design an efficient vector control to limit transmission as soon as the first human cases are diagnosed.     

Chikungunya Virus Arthritis in Adult Wild-Type Mice

Chikungunya virus is a mosquito-borne arthrogenic alphavirus that has recently reemerged to produce the largest epidemic ever documented for this virus. Here we describe a new adult wild-type mouse model of chikungunya virus arthritis, which recapitulates the self-limiting arthritis, tenosynovitis, and myositis seen in humans. Rheumatic disease was associated with a prolific infiltrate of monocytes, macrophages, and NK cells and the production of monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ). Infection with a virus isolate from the recent Reunion Island epidemic induced significantly more mononuclear infiltrates, proinflammatory mediators, and foot swelling than did an Asian isolate from the 1960s. Primary mouse macrophages were shown to be productively infected with chikungunya virus; however, the depletion of macrophages ameliorated rheumatic disease and prolonged the viremia. Only 1 μg of an unadjuvanted, inactivated, whole-virus vaccine derived from the Asian isolate completely protected against viremia and arthritis induced by the Reunion Island isolate, illustrating that protection is not strain specific and that low levels of immunity are sufficient to mediate protection. IFN-α treatment was able to prevent arthritis only if given before infection, suggesting that IFN-α is not a viable therapy. Prior infection with Ross River virus, a related arthrogenic alphavirus, and anti-Ross River virus antibodies protected mice against chikungunya virus disease, suggesting that individuals previously exposed to Ross River virus should be protected from chikungunya virus disease. This new mouse model of chikungunya virus disease thus provides insights into pathogenesis and a simple and convenient system to test potential new interventions.Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has caused periodic outbreaks of predominantly rheumatic disease in Africa and Asia (69). The disease usually involves weeks to months of arthralgia/arthritis and can involve myalgia, fever, and/or a rash (6). During 2004 to 2007 the largest documented outbreak of CHIKV disease occurred in Indian Ocean islands and India. Over 260,000 cases (about one-third of the population) were reported in Reunion Island (France) (56), with 1.39 million cases in India (42) and a small outbreak of ∼200 cases also occurring in Italy (56, 74). The recent outbreak was associated with the emergence of a new clade of CHIKV viruses within the large East, Central, and South African phylogroup, which is distinct from the more distantly related Asian phylogroup (52, 55, 62). A key mutation in the E1 gene (A226V) is believed to have allowed efficient CHIKV transmission by Aedes albopictus mosquitoes (13, 76, 80), which were the main vector in the outbreak in Reunion Island and in some parts of India (31). The recent epidemic was associated with a low level of asymptomatic infections and appeared to result in an increase in disease severity compared with that of previous epidemics (8, 51). A small percentage of cases resulted in death (42, 72), although in such cases other underlying medical conditions may have contributed to mortality (14). CHIKV has been declared a high-priority pathogen by the U.S. NIH (60). No licensed vaccine or particularly effective drug is available for human use for any alphavirus (60), although analgesics and nonsteroidal anti-inflammatory drug treatment can provide relief from rheumatic symptoms (48, 68).The development and testing of new interventions are greatly facilitated by the use of mouse models, which can also provide insights into disease pathogenesis (59). Mouse models of CHIKV disease have recently been developed and involve lethal infections of neonatal mice (89) or adult mice defective in the alpha/beta interferon (IFN-α/β) receptor (9). Such models have been used to illustrate the potential utility of treatment with adoptively transferred anti-CHIKV antibodies (10). A third model used intranasal inoculation of CHIKV but showed no rheumatic signs or symptoms (82). All these models used lethality rather than rheumatic manifestations as disease measures. In humans, arthritis/arthralgia is the main manifestation of CHIKV disease, and disease is only rarely fatal (14). The requirement for young mice makes the testing of prophylactic vaccines difficult, as there is insufficient time for vaccination. Human infants also tend not to develop arthritic symptoms following CHIKV infection (78). The use of mice lacking IFN-α/β responses complicates the testing of vaccines and other immunological interventions, as the absence of IFN-α/β signaling can affect both vaccine (26, 77) and virus (60) behaviors.Here we describe the behaviors of two virus isolates of CHIKV, an Asian isolate and a Reunion Island isolate, in a new adult wild-type mouse model of CHIKV arthritis. The Asian isolate was collected in the 1960s in Thailand, and the Reunion Island isolate was collected during the recent outbreak (55). The model produced a measurable self-limiting perimetatarsal foot swelling with clear histological signs of acute and persistent inflammatory disease. We also characterize the cells and inflammatory mediators associated with infection and disease and illustrate the use of the model for studying vaccines, IFN-α therapy, and cross-protection with Ross River virus (RRV), an Australiasian arthrogenic alphavirus related to CHIKV (16, 69).     

A Novel MVA Vectored Chikungunya Virus Vaccine Elicits Protective Immunity in Mice

Background

Chikungunya virus (CHIKV) is a re-emerging arbovirus associated with febrile illness often accompanied by rash and arthralgia that may persist for several years. Outbreaks are associated with high morbidity and create a public health challenge for countries affected. Recent outbreaks have occurred in both Europe and the Americas, suggesting CHIKV may continue to spread. Despite the sustained threat of the virus, there is no approved vaccine or antiviral therapy against CHIKV. Therefore, it is critical to develop a vaccine that is both well tolerated and highly protective.

Methodology/Principal Findings

In this study, we describe the construction and characterization of a modified Vaccinia virus Ankara (MVA) virus expressing CHIKV E3 and E2 proteins (MVA-CHIK) that protected several mouse models from challenge with CHIKV. In particular, BALB/c mice were completely protected against viremia upon challenge with CHIKV after two doses of MVA-CHIK. Additionally, A129 mice (deficient in IFNα/β) were protected from viremia, footpad swelling, and mortality. While high anti-virus antibodies were elicited, low or undetectable levels of neutralizing antibodies were produced in both mouse models. However, passive transfer of MVA-CHIK immune serum to naïve mice did not protect against mortality, suggesting that antibodies may not be the main effectors of protection afforded by MVA-CHIK. Furthermore, depletion of CD4⁺, but not CD8⁺ T-cells from vaccinated mice resulted in 100% mortality, implicating the indispensable role of CD4⁺ T-cells in the protection afforded by MVA-CHIK.

Conclusions/Significance

The results presented herein demonstrate the potential of MVA to effectively express CHIKV E3-E2 proteins and generate protective immune responses. Our findings challenge the assumption that only neutralizing antibodies are effective in providing protection against CHIKV, and provides a framework for the development of novel, more effective vaccine strategies to combat CHIKV.     

Orally Co-Infected Aedes albopictus from La Reunion Island,Indian Ocean,Can Deliver Both Dengue and Chikungunya Infectious Viral Particles in Their Saliva

Background

First described in humans in 1964, reports of co-infections with dengue (DENV) and chikungunya (CHIKV) viruses are increasing, particularly after the emergence of chikungunya (CHIK) in the Indian Ocean in 2005–2006 due to a new variant highly transmitted by Aedes albopictus. In this geographic area, a dengue (DEN) outbreak transmitted by Ae. albopictus took place shortly before the emergence of CHIK and co-infections were reported in patients. A co-infection in humans can occur following the bite of two mosquitoes infected with one virus or to the bite of a mosquito infected with two viruses. Co-infections in mosquitoes have never been demonstrated in the field or in the laboratory. Thus, we question about the ability of a mosquito to deliver infectious particles of two different viruses through the female saliva.

Methodology/Principal Findings

We orally exposed Ae. albopictus from La Reunion Island with DENV-1 and CHIKV isolated respectively during the 2004–2005 and the 2005–2006 outbreaks on this same island. We were able to show that Ae. albopictus could disseminate both viruses and deliver both infectious viral particles concomitantly in its saliva. We also succeeded in inducing a secondary infection with CHIKV in mosquitoes previously inoculated with DENV-1.

Conclusions/Significance

In this study, we underline the ability of Ae. albopictus to be orally co-infected with two different arboviruses and furthermore, its capacity to deliver concomitantly infectious particles of CHIKV and DENV in saliva. This finding is of particular concern as Ae. albopictus is still expanding its geographical range in the tropical as well as in the temperate regions. Further studies are needed to try to elucidate the molecular/cellular basis of this phenomenon.     

Reemergence of Chikungunya Virus

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes acute fever and acute and chronic musculoskeletal pain in humans. Since 2004, CHIKV has caused millions of cases of disease in the Indian Ocean region and has emerged in new areas, including Europe, the Middle East, and the Pacific region. The mosquito vectors for this virus are globally distributed in tropical and temperate zones, providing the opportunity for CHIKV to continue to expand into new geographic regions. In October 2013, locally acquired cases of CHIKV infection were identified on the Caribbean island of Saint Martin, signaling the arrival of the virus in the Western Hemisphere. In just 9 months, CHIKV has spread to 22 countries in the Caribbean and Central and South America, resulting in hundreds of thousands of cases. CHIKV disease can be highly debilitating, and large epidemics have severe economic consequences. Thus, there is an urgent need for continued research into the epidemiology, pathogenesis, prevention, and treatment of these infections.     

基孔肯亚假病毒模型的建立及其在模拟检测中的应用

目的:构建含基孔肯亚病毒核酸序列的重组慢病毒载体,以之转染细胞以分泌含基孔肯亚病毒核酸序列的假病毒,利用该病毒建立基孔肯亚病毒的模拟检测方法。方法:人工合成基孔肯亚病毒部分保守性核酸序列,连入慢病毒载体,构建含基孔肯亚病毒核酸序列的重组慢病毒质粒pLenti-chik,该质粒连同辅助质粒pLP1、pLP2、pLP/VSVG共转染293FT细胞;72 h后收集上清,用特异引物进行荧光定量RT-PCR检测。结果:建立了一个仅能进行一次复制、高度安全的基孔肯亚假病毒模型;同时采用荧光定量RT-PCR建立了较灵敏的检测病毒分泌的方法。结论:假病毒可以模拟基孔肯亚病毒分泌,但较真病毒安全性更高;用荧光定量的方法可以很灵敏地检测病毒的分泌,为突发基孔肯亚病毒感染的检测做好准备。     

正常猕猴与人视网膜血管的比较

目的比较正常猕猴与人视网膜血管的异同,为进一步利用猕猴建立动物模型来研究视网膜血管打下基础。方法取健康成年猕猴眼球6只和人角膜移植供体剩余眼杯8只的视网膜,用ADP酶法进行血管染色,对两者视网膜血管的走行、血管分级、毛细血管分层以及黄斑区血管拱环等进行比较,测量结果进行统计学检验。结果猕猴与人的视网膜铺片经ADP酶法染色后见视网膜血管自穿出视盘后的一级血管逐渐分支变细,直至五级血管即毛细血管;在视盘旁、赤道部、周边部两者血管面积百分比没有差异;视盘旁血管分为多层,赤道部有两层,且深浅层间相互交通,周边部仅见一层毛细血管且较稀疏;两者黄斑区毛细血管均较密集,有形态完整呈不规则状的血管拱环,血管面积百分比以及血管拱环的面积、周长和直径没有差异。结论猕猴与人在视网膜血管走行、分级、毛细血管分层以及黄斑区血管拱环等多方面有良好的相似性,可用作人类视网膜血管、尤其是黄斑区视网膜血管研究的良好动物模型。     

Administration of E2 and NS1 siRNAs Inhibit Chikungunya Virus Replication In Vitro and Protects Mice Infected with the Virus

Background

Chikungunya virus (CHIKV) has reemerged as a life threatening pathogen and caused large epidemics in several countries. So far, no licensed vaccine or effective antivirals are available and the treatment remains symptomatic. In this context, development of effective and safe prophylactics and therapeutics assumes priority.

Methods

We evaluated the efficacy of the siRNAs against ns1 and E2 genes of CHIKV both in vitro and in vivo. Four siRNAs each, targeting the E2 (Chik-1 to Chik-4) and ns1 (Chik-5 to Chik-8) genes were designed and evaluated for efficiency in inhibiting CHIKV growth in vitro and in vivo. Chik-1 and Chik-5 siRNAs were effective in controlling CHIKV replication in vitro as assessed by real time PCR, IFA and plaque assay.

Conclusions

CHIKV replication was completely inhibited in the virus-infected mice when administered 72 hours post infection. The combination of Chik-1 and Chik-5 siRNAs exhibited additive effect leading to early and complete inhibition of virus replication. These findings suggest that RNAi capable of inhibiting CHIKV growth might constitute a new therapeutic strategy for controlling CHIKV infection and transmission.     

Nowcasting the Spread of Chikungunya Virus in the Americas

Background

In December 2013, the first locally-acquired chikungunya virus (CHIKV) infections in the Americas were reported in the Caribbean. As of May 16, 55,992 cases had been reported and the outbreak was still spreading. Identification of newly affected locations is paramount to intervention activities, but challenging due to limitations of current data on the outbreak and on CHIKV transmission. We developed models to make probabilistic predictions of spread based on current data considering these limitations.

Methods and Findings

Branching process models capturing travel patterns, local infection prevalence, climate dependent transmission factors, and associated uncertainty estimates were developed to predict probable locations for the arrival of CHIKV-infected travelers and for the initiation of local transmission. Many international cities and areas close to where transmission has already occurred were likely to have received infected travelers. Of the ten locations predicted to be the most likely locations for introduced CHIKV transmission in the first four months of the outbreak, eight had reported local cases by the end of April. Eight additional locations were likely to have had introduction leading to local transmission in April, but with substantial uncertainty.

Conclusions

Branching process models can characterize the risk of CHIKV introduction and spread during the ongoing outbreak. Local transmission of CHIKV is currently likely in several Caribbean locations and possible, though uncertain, for other locations in the continental United States, Central America, and South America. This modeling framework may also be useful for other outbreaks where the risk of pathogen spread over heterogeneous transportation networks must be rapidly assessed on the basis of limited information.     

Recombinant Modified Vaccinia Virus Ankara Expressing Glycoprotein E2 of Chikungunya Virus Protects AG129 Mice against Lethal Challenge

Chikungunya virus (CHIKV) infection is characterized by rash, acute high fever, chills, headache, nausea, photophobia, vomiting, and severe polyarthralgia. There is evidence that arthralgia can persist for years and result in long-term discomfort. Neurologic disease with fatal outcome has been documented, although at low incidences. The CHIKV RNA genome encodes five structural proteins (C, E1, E2, E3 and 6K). The E1 spike protein drives the fusion process within the cytoplasm, while the E2 protein is believed to interact with cellular receptors and therefore most probably constitutes the target of neutralizing antibodies. We have constructed recombinant Modified Vaccinia Ankara (MVA) expressing E3E2, 6KE1, or the entire CHIKV envelope polyprotein cassette E3E26KE1. MVA is an appropriate platform because of its demonstrated clinical safety and its suitability for expression of various heterologous proteins. After completing the immunization scheme, animals were challenged with CHIV-S27. Immunization of AG129 mice with MVAs expressing E2 or E3E26KE1 elicited neutralizing antibodies in all animals and provided 100% protection against lethal disease. In contrast, 75% of the animals immunized with 6KE1 were protected against lethal infection. In conclusion, MVA expressing the glycoprotein E2 of CHIKV represents as an immunogenic and effective candidate vaccine against CHIKV infections.     

Evidence for Endemic Chikungunya Virus Infections in Bandung,Indonesia

Chikungunya virus (CHIKV) is known to cause sporadic or explosive outbreaks. However, little is known about the endemic transmission of CHIKV. To ascertain the endemic occurrence of CHIKV transmission, we tested blood samples from patients with a non-dengue febrile illness who participated in a prospective cohort study of factory workers in Bandung, Indonesia. From August 2000 to June 2004, and September 2006 to April 2008, 1901 febrile episodes occurred and 231 (12.2%) dengue cases were identified. The remaining febrile cases were evaluated for possible CHIKV infection by measuring anti-CHIKV IgM and IgG antibodies in acute and convalescent samples. Acute samples of serologically positive cases were subsequently tested for the presence of CHIKV RNA by RT-PCR and/or virus isolation. A total of 135 (7.1%) CHIKV infections were identified, providing an incidence rate of 10.1/1,000 person years. CHIKV infections were identified all year round and tended to increase during the rainy season (January to March). Severe illness was not found and severe arthralgia was not a prominently reported symptom. Serial post-illness samples from nine cases were tested to obtain a kinetic picture of IgM and IgG anti-CHIKV antibodies. Anti-CHIKV IgM antibodies were persistently detected in high titers for approximately one year. Three patients demonstrated evidence of possible sequential CHIKV infections. The high incidence rate and continuous chikungunya cases in this adult cohort suggests that CHIKV is endemically transmitted in Bandung. Further characterization of the circulating strains and surveillance in larger areas are needed to better understand CHIKV epidemiology in Indonesia.     

Olfactory Discrimination Ability for Aliphatic Esters in Squirrel Monkeys and Humans

Using a behavioral paradigm designed to simulate olfactory-guidedforaging, the ability of five squirrel monkeys to distinguishiso-amyl acetate from n-and iso-forms of other acetic esters(ethyl acetate to decyl acetate) and from other esters carryingthe iso-amyl group (iso-amyl propionate to iso-amyl capronate)was investigated. We found (i) that all five animals were clearlyable to discriminate between all odor pairs tested; (ii) a significantnegative correlation between discrimination performance andstructural similarity of odorants in terms of differences incarbon chain length of both the aliphatic alcohol group andthe aliphatic acid group of the esters; and (iii) that iso-and n-amyl acetate were perceived as qualitatively similar despitedifferent steric conformation. Using a triple-forced choiceprocedure, 20 human subjects were tested on the same tasks inparallel and showed a very similar pattern of discriminationperformance compared with the squirrel monkeys. Thus, the resultsof this study provide evidence of well-developed olfactory discriminationability in squirrel monkeys for aliphatic esters and supportthe assumption that human and non-human primates may share commonprinciples of odor quality perception. Chem. Senses 22: 457–465,1997.     

基孔肯雅病毒和辛德毕斯病毒可视化基因芯片与荧光基因芯片的建立

根据GenBank中收录的基孔肯雅病毒和辛德毕斯病毒E蛋白基因序列,设计及筛选针对2种病毒的寡核苷酸探针及引物,制备基孔肯雅病毒与辛德毕斯病毒可视化基因芯片与荧光基因芯片,对芯片的灵敏性、特异性进行了验证,并将可视化基因芯片、荧光基因芯片进行灵敏性比较.结果显示,制备的两种基因芯片都能检测到基孔肯雅病毒和辛德毕斯病毒特异性杂交信号.可视化基因芯片、荧光基因芯片检测两种病毒质粒的灵敏度达到9.1×103 copies/mL, 6.8×101 copies/mL和9.1×104 copies/mL, 6.8×103 copies/mL,与普通PCR比较差异显著. 荧光基因芯片灵敏度是PCR方法的10倍,可视化基因芯片是荧光基因芯片灵敏度的100倍. 模拟病毒检测过程特异性检验证明,可视化基因芯片都具有良好的特异性.本试验建立了基孔肯雅病毒与辛德毕斯病毒两种特异的可视化和荧光基因芯片检测方法,两种方法灵敏度高、特异性强,适用于基孔肯雅病毒与辛德毕斯病毒的流行病学调查和种特异性鉴定.     

Modeling Dynamic Introduction of Chikungunya Virus in the United States

Chikungunya is a mosquito-borne viral infection of humans that previously was confined to regions in central Africa. However, during this century, the virus has shown surprising potential for geographic expansion as it invaded other countries including more temperate regions. With no vaccine and no specific treatment, the main control strategy for Chikungunya remains preventive control of mosquito populations. In consideration for the risk of Chikungunya introduction to the US, we developed a model for disease introduction based on virus introduction by one individual. Our study combines a climate-based mosquito population dynamics stochastic model with an epidemiological model to identify temporal windows that have epidemic risk. We ran this model with temperature data from different locations to study the geographic sensitivity of epidemic potential. We found that in locations with marked seasonal variation in temperature there also was a season of epidemic risk matching the period of the year in which mosquito populations survive and grow. In these locations controlling mosquito population sizes might be an efficient strategy. But, in other locations where the temperature supports mosquito development all year the epidemic risk is high and (practically) constant. In these locations, mosquito population control alone might not be an efficient disease control strategy and other approaches should be implemented to complement it. Our results strongly suggest that, in the event of an introduction and establishment of Chikungunya in the US, endemic and epidemic regions would emerge initially, primarily defined by environmental factors controlling annual mosquito population cycles. These regions should be identified to plan different intervention measures. In addition, reducing vector: human ratios can lower the probability and magnitude of outbreaks for regions with strong seasonal temperature patterns. This is the first model to consider Chikungunya risk in the US and can be applied to other vector borne diseases.