Large-scale codon re-encoding represents a powerful method of attenuating viruses to generate safe and cost-effective vaccines. In contrast to specific approaches of codon re-encoding which modify genome-scale properties, we evaluated the effects of random codon re-encoding on the re-emerging human pathogen Chikungunya virus (CHIKV), and assessed the stability of the resultant viruses during serial in cellulo passage. Using different combinations of three 1.4 kb randomly re-encoded regions located throughout the CHIKV genome six codon re-encoded viruses were obtained. Introducing a large number of slightly deleterious synonymous mutations reduced the replicative fitness of CHIKV in both primate and arthropod cells, demonstrating the impact of synonymous mutations on fitness. Decrease of replicative fitness correlated with the extent of re-encoding, an observation that may assist in the modulation of viral attenuation. The wild-type and two re-encoded viruses were passaged 50 times either in primate or insect cells, or in each cell line alternately. These viruses were analyzed using detailed fitness assays, complete genome sequences and the analysis of intra-population genetic diversity. The response to codon re-encoding and adaptation to culture conditions occurred simultaneously, resulting in significant replicative fitness increases for both re-encoded and wild type viruses. Importantly, however, the most re-encoded virus failed to recover its replicative fitness. Evolution of these viruses in response to codon re-encoding was largely characterized by the emergence of both synonymous and non-synonymous mutations, sometimes located in genomic regions other than those involving re-encoding, and multiple convergent and compensatory mutations. However, there was a striking absence of codon reversion (<0.4%). Finally, multiple mutations were rapidly fixed in primate cells, whereas mosquito cells acted as a brake on evolution. In conclusion, random codon re-encoding provides important information on the evolution and genetic stability of CHIKV viruses and could be exploited to develop a safe, live attenuated CHIKV vaccine.

Author Summary

Emerging arthropod-borne viruses (arboviruses) are a major cause of human and animal morbidity and mortality. Climatic and anthropological activities are responsible for the dispersal of arbovirus transmission vectors into new territories. Chikungunya virus (CHIKV) is an important example of a re-emerging pathogen for which no licensed vaccine exists. One of the vectors of CHIKV, the mosquito Aedes albopictus, has dispersed into new temperate regions resulting in outbreaks where they had not been previously observed. Here, we demonstrate that random codon re-encoding, a method that modifies the nucleic acid composition of large coding regions without modifying the encoded proteins, can significantly decrease the replicative fitness of CHIKV. This powerful method of attenuating viruses has several potential advantages for vaccine development, including the possibility to modulate precisely the degree of replicative fitness loss and to generate safe, live-attenuated vaccines that confer long-term protection, in a cost effective manner. Our studies also demonstrate that these re-encoded viruses exhibit a stable phenotype, and that the response to codon re-encoding was largely compensatory in nature, with little reversion of mutations. Finally, we provide further evidence that many synonymous sites in RNA viruses are not neutral and clearly impact viral fitness.

Japanese encephalitis virus (JEV) (Flaviviridae, Flavivirus) is an arthropod-borne flavivirus transmitted by Culex species mosquitoes. We report here the complete genome of the JEV genotype I strain JEV_CNS769_Laos_2009 isolated from an infected patient in Vientiane, Lao People’s Democratic Republic (PDR) (Laos).

Background

Chikungunya virus (CHIKV) is responsible for acute febrile polyarthralgia and, in a proportion of cases, severe complications including chronic arthritis. CHIKV has spread recently in East Africa, South-West Indian Ocean, South-Asia and autochthonous cases have been reported in Europe. Although almost all patients are outpatients, medical investigations mainly focused on hospitalised patients.

Methodology/Principal Findings

Here, we detail clinico-biological characteristics of Chikungunya (CHIK) outpatients in Reunion Island (2006). 76 outpatients with febrile arthralgia diagnosed within less than 48 hours were included by general practitioners during the CuraChik clinical trial. CHIK was confirmed in 54 patients and excluded in 22. A detailed clinical and biological follow-up was organised, that included analysis of viral intrahost diversity and telephone survey until day 300. The evolution of acute CHIK included 2 stages: the ‘viral stage’ (day 1–day 4) was associated with rapid decrease of viraemia and improvement of clinical presentation; the ‘convalescent stage’ (day 5–day 14) was associated with no detectable viraemia but a slower clinical improvement. Women and elderly had a significantly higher number of arthralgia at inclusion and at day 300. Based on the study clinico-biological dataset, scores for CHIK diagnosis in patients with recent febrile acute polyarthralgia were elaborated using arthralgia on hands and wrists, a minor or absent myalgia and the presence of lymphopenia (<1G/L) as major orientation criteria. Finally, we observed that CHIKV intra-host genetic diversity increased over time and that a higher viral amino-acid complexity at the acute stage was associated with increased number of arthralgia and intensity of sequelae at day 300.

Conclusions/Significance

This study provided a detailed picture of clinico-biological CHIK evolution at the acute phase of the disease, allowed the elaboration of scores to assist CHIK diagnosis and investigated for the first time the impact of viral intra-host genetic diversity on the disease course.

Author Summary

The mosquito-transmitted chikungunya virus is responsible for acute febrile polyarthralgia and, in a proportion of cases, complications including chronic arthritis. Since 2005, it has massively re-emerged in the Old World. Although the large majority of patients are outpatients, the most detailed studies have focused previously on hospitalised patients (i.e., severe cases). Here, we report the detailed clinico-biological characteristics of ‘standard’ clinical presentations in patients followed-up by general practitioners in Reunion Island (2006) during the CuraChik clinical trial. At the onset of the disease, two stages were observed: (i) a ‘viral stage’ during the first 4 days, associated with an acute febrile polyarthralgic syndrome and a subsequent rapid clinical improvement; the main clinico-biological characteristics during that period were used to elaborate supportive chikungunya diagnostic scores, (ii) a ‘convalescent stage’ (days 5–14) with no detectable viraemia but a slower clinical improvement. Woman and elderly patients were found at risk for more symptomatic forms of the disease at both the acute and late stages (day 300) and we observed that the viral intra-host genetic diversity increased over time and that a higher viral amino-acid complexity at the acute stage was associated with more symptomatic illness at the late stage of the disease.

Five persons in France were infected with Orf virus after skin wounds were exposed to infected sheep tissues during Eid al-Adha, the Muslim Feast of Sacrifice. Infections were confirmed by electron microscopy, PCR, and sequence analysis. Prevention and control of this underdiagnosed disease can be achieved by educating physicians, slaughterhouse workers, and persons participating in Eid al-Adha.

Yellow fever (YF) is a serious public health problem in Bolivia since at least the 19th century. Surprisingly, very limited information has been made available to date regarding the genetic characterisation and epidemiology of Bolivian YF virus (YFV) strains. Here, we conducted the genetic characterization of 12 human isolates of YFV collected in Bolivia between 1999 and 2008, by sequencing and analysis of two regions of the viral genome: a fragment encoding structural proteins “PrM” (premembrane and envelope) and a distal region “EMF,” spanning the end of the virus genome. Our study reveals a high genetic diversity of YFV strains circulating in Bolivia during the last decade: we identified not only “Peruvian-like” genotype II viruses (related to previously characterized Bolivian strains), but also, for the fist time, “Brazilian-like” genotype I viruses. During the complete period of the study, only cases of “jungle” YF were detected (i.e., circulation of YFV via a sylvatic cycle) with no cluster of urban cases. However, the very significant spread of the Aedes aegypti mosquito across Bolivian cities threatens the country with the reappearance of an urban YFV transmission cycle and thus is required a sustained epidemiological surveillance.

Dengue fever was first recognized in Bolivia in 1931. However, very limited information was available to date regarding the genetic characterization and epidemiology of Bolivian dengue virus strains. Here, we performed genetic characterization of the full-length envelope gene of 64 Bolivian isolates from 1998 to 2008 and investigated their origin and evolution to determine whether strains circulated simultaneously or alternatively, and whether or not multiple introductions of distinct viral variants had occurred during the period studied. We determined that, during the last decade, closely related viruses circulated during several consecutive years (5, 6, and 6 years for DENV-1, DENV-2, and DENV-3, respectively) and the co-circulation of two or even three serotypes was observed. Emergence of new variants (distinct from those identified during the previous episodes) was identified in the case of DENV-1 (2007 outbreak) and DENV-2 (2001 outbreak). In all cases, it is likely that the viruses originated from neighboring countries.

Sandfly-transmitted phleboviruses, such as Toscana, sandfly fever Sicilian, and sandfly fever Naples, can cause human disease and circulate at high rates in Mediterranean countries. Previous studies have also established that viruses other than phleboviruses may be detected in and isolated from sand flies. The recent detection and isolation (in a large variety of mosquito species) of insect-only flaviviruses related to cell fusing agent virus has indicated that the latter is not an evolutionary remnant but the first discovered member of a group of viruses, larger than initially assumed, that has high genetic heterogeneity. Insect-only flaviviruses have been detected in and/or isolated from various species of mosquitoes, but nevertheless only from mosquitoes to date; other dipterans have not been screened for the presence of insect-only flaviviruses. The possible presence of flaviviruses, including insect-only flaviviruses, was investigated in sand flies collected around the Mediterranean during a trapping campaign already underway. Accordingly, a total of 1508 sand flies trapped in France and Algeria, between August 2006 and July 2007, were tested for the presence of flaviviruses using a PCR assay previously demonstrated experimentally to amplify all recognized members of the genus Flavivirus, including insect-only flaviviruses. Two of 67 pools consisting of male Phlebotomus perniciosus trapped in Algeria were positive. The two resulting sequences formed a monophyletic group and appeared more closely related to insect-only flaviviruses associated with Culex mosquitoes than with Aedes mosquitoes, and more closely related to insect-only flaviviruses than to arthropod-borne or to no-known-vector vertebrate flaviviruses. This is the first description of insect-only flaviviruses in dipterans distinct from those belonging to the family Culicidae (including Aedes, Culex, Mansonia, Culiseta, and Anopheles mosquito genera), namely sand flies within the family Psychodidae. Accordingly, we propose their designation as phlebotomine-associated flaviviruses.

Sandflies are widely distributed around the Mediterranean Basin. Therefore, human populations in this area are potentially exposed to sandfly-transmitted diseases, including those caused by phleboviruses. Whilst there are substantial data in countries located in the northern part of the Mediterranean basin, few data are available for North Africa. In this study, a total of 1489 sandflies were collected in 2008 in Tunisia from two sites, bioclimatically distinct, located 235 km apart, and identified morphologically. Sandfly species comprised Phlebotomus perniciosus (52.2 %), Phlebotomus longicuspis (30.1 %), Phlebotomus papatasi (12 .0%), Phlebotomus perfiliewi (4.6 %), Phlebotomus langeroni (0.4 %) and Sergentomyia minuta (0.5 %). PCR screening, using generic primers for the genus Phlebovirus, resulted in the detection of ten positive pools. Sequence analysis revealed that two pools contained viral RNA corresponding to a novel virus closely related to sandfly fever Naples virus. Virus isolation in Vero cells was achieved from one pool. Genetic and phylogenetic characterization based on sequences in the three genomic segments showed that it was a novel virus distinct from other recognized members of the species. This novel virus was provisionally named Punique virus. Viral sequences in the polymerase gene corresponding to another phlebovirus closely related to but distinct from sandfly fever Sicilian virus were obtained from the eight remaining positive pools.

During 2009, pandemic influenza A(H1N1)pdm09 virus affected humans on Réunion Island. Since then, the virus has sustained circulation among local swine herds, raising concerns about the potential for genetic evolution of the virus and possible retransmission back to humans of variants with increased virulence. Continuous surveillance of A(H1N1)pdm09 infection in pigs is recommended.

Background

The risk of influenza infection depends on biological characteristics, individual or collective behaviors and the environmental context. The Cohorts for Pandemic Influenza (CoPanFlu) France study was set up in 2009 after the identification of the novel swine-origin A/H1N1 pandemic influenza virus. This cohort of 601 households (1450 subjects) representative for the general population aims at using an integrative approach to study the risk and characteristics of influenza infection as a complex combination of data collected from questionnaires regarding sociodemographic, medical, behavioral characteristics of subjects and indoor environment, using biological samples or environmental databases.

Methods/Design

Households were included between December 2009 and July 2010. The design of this study relies on systematic follow-up visits between influenza seasons and additional visits during influenza seasons, when an influenza-like illness is detected in a household via an active surveillance system. During systematic visits, a nurse collects individual and environmental data on questionnaires and obtains blood samples from all members of the household. When an influenza-like-illness is detected, a nurse visits the household three times during the 12 following days, and collects data on questionnaires regarding exposure and symptoms, and biological samples (including nasal swabs) from all subjects in the household. The end of the follow-up period is expected in fall 2012.

Discussion

The large amount of data collected throughout the follow-up will permit a multidisciplinary study of influenza infections. Additional data is being collected and analyzed in this ongoing cohort. The longitudinal analysis of these households will permit integrative analyses of complex phenomena such as individual, collective and environmental risk factors of infection, routes of transmission, or determinants of the immune response to infection or vaccination.

Abstract

Yellow fever (YF) is a serious public health problem in Bolivia since at least the 19th century. Surprisingly, very limited information has been made available to date regarding the genetic characterisation and epidemiology of Bolivian YF virus (YFV) strains. Here, we conducted the genetic characterization of 12 human isolates of YFV collected in Bolivia between 1999 and 2008, by sequencing and analysis of two regions of the viral genome: a fragment encoding structural proteins “PrM” (premembrane and envelope) and a distal region “EMF,” spanning the end of the virus genome. Our study reveals a high genetic diversity of YFV strains circulating in Bolivia during the last decade: we identified not only “Peruvian-like” genotype II viruses (related to previously characterized Bolivian strains), but also, for the fist time, “Brazilian-like” genotype I viruses. During the complete period of the study, only cases of “jungle” YF were detected (i.e., circulation of YFV via a sylvatic cycle) with no cluster of urban cases. However, the very significant spread of the Aedes aegypti mosquito across Bolivian cities threatens the country with the reappearance of an urban YFV transmission cycle and thus is required a sustained epidemiological surveillance.

Background

Dengue dynamics are driven by complex interactions between human-hosts, mosquito-vectors and viruses that are influenced by environmental and climatic factors. The objectives of this study were to analyze and model the relationships between climate, Aedes aegypti vectors and dengue outbreaks in Noumea (New Caledonia), and to provide an early warning system.

Methodology/Principal Findings

Epidemiological and meteorological data were analyzed from 1971 to 2010 in Noumea. Entomological surveillance indices were available from March 2000 to December 2009. During epidemic years, the distribution of dengue cases was highly seasonal. The epidemic peak (March–April) lagged the warmest temperature by 1–2 months and was in phase with maximum precipitations, relative humidity and entomological indices. Significant inter-annual correlations were observed between the risk of outbreak and summertime temperature, precipitations or relative humidity but not ENSO. Climate-based multivariate non-linear models were developed to estimate the yearly risk of dengue outbreak in Noumea. The best explicative meteorological variables were the number of days with maximal temperature exceeding 32°C during January–February–March and the number of days with maximal relative humidity exceeding 95% during January. The best predictive variables were the maximal temperature in December and maximal relative humidity during October–November–December of the previous year. For a probability of dengue outbreak above 65% in leave-one-out cross validation, the explicative model predicted 94% of the epidemic years and 79% of the non epidemic years, and the predictive model 79% and 65%, respectively.

Conclusions/Significance

The epidemic dynamics of dengue in Noumea were essentially driven by climate during the last forty years. Specific conditions based on maximal temperature and relative humidity thresholds were determinant in outbreaks occurrence. Their persistence was also crucial. An operational model that will enable health authorities to anticipate the outbreak risk was successfully developed. Similar models may be developed to improve dengue management in other countries.

Author Summary

Dengue fever is a major public health problem in the tropics and subtropics. Since no vaccine exists, understanding and predicting outbreaks remain of crucial interest. Climate influences the mosquito-vector biology and the viral transmission cycle. Its impact on dengue dynamics is of growing interest. We analyzed the epidemiology of dengue in Noumea (New Caledonia) from 1971 to 2010 and its relationships with local and remote climate conditions using an original approach combining a comparison of epidemic and non epidemic years, bivariate and multivariate analyses. We found that the occurrence of outbreaks in Noumea was strongly influenced by climate during the last forty years. Efficient models were developed to estimate the yearly risk of outbreak as a function of two meteorological variables that were contemporaneous (explicative model) or prior (predictive model) to the outbreak onset. Local threshold values of maximal temperature and relative humidity were identified. Our results provide new insights to understand the link between climate and dengue outbreaks, and have a substantial impact on dengue management in New Caledonia since the health authorities have integrated these models into their decision making process and vector control policies. This raises the possibility to provide similar early warning systems in other countries.

There has been an explosion in the discovery of ‘insect-specific’ flaviviruses and/or their related sequences in natural mosquito populations. Herein we review all ‘insect-specific’ flavivirus sequences currently available and conduct phylogenetic analyses of both the ‘insect-specific’ flaviviruses and available sequences of the entire genus Flavivirus. We show that there is no statistical support for virus–mosquito co-divergence, suggesting that the ‘insect-specific’ flaviviruses may have undergone multiple introductions with frequent host switching. We discuss potential implications for the evolution of vectoring within the family Flaviviridae. We also provide preliminary evidence for potential recombination events in the history of cell fusing agent virus. Finally, we consider priorities and guidelines for future research on ‘insect-specific’ flaviviruses, including the vast potential that exists for the study of biodiversity within a range of potential hosts and vectors, and its effect on the emergence and maintenance of the flaviviruses.

A serosurvey conducted in a sample of first quarter pregnant women in France at week 48-49 of 2009 exhibit a seroprevalence level of 10.6%. It has been extrapolated in male and female population living in France mainland, aged 20-39 yr, that 1,712,000, 95%CI (1,112,700 – 2,311,300) people were recently infected by H1N1pdm (recently vaccinated women were excluded from analysis). From week 36 to 46-47 of 2009, 336,288, 95%CI (207,303-421,299) patients visited their general practitioners with clinical influenza in France, mainland. We then extrapolated the proportion of symptomatic H1N1pdm influenza in both males and females aged 20-39 yr who visited their GP to be 19.6%.

Background

The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4). Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle.

Results

We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation.

Conclusions

We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy.

A doxorubicin derivate, SA-17, that carries a squaric acid amide ester moiety at the carbohydrate (α-l-daunosaminyl) group was identified as a selective inhibitor of in vitro dengue virus (DENV) serotype 2 replication (50% effective concentration [EC50] = 0.34 ± 0.20 μg/ml [0.52 ± 0.31 μM]). SA-17 is markedly less cytostatic than the parent compound, resulting in a selectivity index value of ∼100. SA-17 also inhibits yellow fever virus 17D (YFV-17D) replication (EC50 = 3.1 ± 1.0 μg/ml [4.8 ± 1.5 μM]), although less efficiently than DENV replication, but proved inactive against a variety of enveloped and nonenveloped viruses. SA-17 inhibits in vitro flavivirus replication in a dose-dependent manner, as was assessed by virus yield reduction assays and quantification of viral RNA by means of real-time quantitative reverse transcriptase PCR (RT-qPCR) (∼2 to 3 log reduction). The anti-DENV activity was confirmed using a Renilla luciferase-expressing dengue reporter virus. Time-of-drug-addition studies revealed that SA-17 acts at the very early stages of the viral replication cycle (i.e., virus attachment and/or virus entry). This observation was corroborated by the observation that SA-17, unlike the nucleoside analogue ribavirin, does not inhibit the replication of DENV subgenomic replicons. Preincubation of high-titer stocks of DENV or YFV-17D with ≥5 μg/ml SA-17 resulted in 100% inhibition of viral infectivity (≥3 log reduction). SA-17, however, did not prove virucidal.

Abstract

A new virus was isolated from three independent pools of Phlebotomus perniciosus sandflies (Diptera; Psychodidae) trapped in two regions of southeastern France, located 90 miles apart. Microscopic, antigenic and genetic analyses indicate that this novel virus belongs to the genus Phlebovirus in the family Bunyaviridae. The new virus is designated Massilia virus since the first isolate was obtained from sandflies collected in the suburban area of Marseille. The complete genome sequence was determined and used to compare the genetic and phylogenetic relationships of Massilia virus with other phleboviruses. Genetic and antigenic properties were employed to address whether or not Massilia virus should be considered a new species within the genus, or a member of a previously recognized species. Cerebrospinal fluid specimens, collected from local patients with central nervous system infections during the previous four-year period were tested for the presence of Massilia virus RNA, but gave negative results. In conclusion, Massilia virus is proposed as a member of the Sand-fly fever Naples virus complex; its public health importance has yet to be determined.

Arenaviridae synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a ‘cap-snatching’ mechanism. Here, we report the crystal structure and functional characterization of the N-terminal 196 residues (NL1) of the L protein from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/β architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures, mutagenesis and reverse genetics studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. We show that this endonuclease domain is conserved and active across the virus families Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease.

Author Summary

The Arenaviridae virus family includes several life-threatening human pathogens that cause meningitis or hemorrhagic fever. These RNA viruses replicate and transcribe their genome using an RNA synthesis machinery for which no structural data currently exist. They synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a ‘cap-snatching’ mechanism thought to involve the large L protein, which carries RNA-dependent RNA polymerase signature sequences. Here, we report the crystal structure and functional characterization of an isolated N-terminal domain of the L protein (NL1) from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/β architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures and mutagenesis studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. Reverse genetic studies show that mutation of active site residues selectively abolish transcription, not replication. We show that this endonuclease domain is conserved and active across the virus families: Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease.

Background

Cross-immunity between seasonal and pandemic A/H1N1 influenza viruses remains uncertain. In particular, the extent that previous infection or vaccination by seasonal A/H1N1 viruses can elicit protective immunity against pandemic A/H1N1 is unclear.

Methodology/Principal Findings

Neutralizing titers against seasonal A/H1N1 (A/Brisbane/59/2007) and against pandemic A/H1N1 (A/California/04/2009) were measured using an HIV-1-based pseudovirus neutralization assay. Using this highly sensitive assay, we found that a large fraction of subjects who had never been exposed to pandemic A/H1N1 express high levels of pandemic A/H1N1 neutralizing titers. A significant correlation was seen between neutralization of pandemic A/H1N1 and neutralization of a standard seasonal A/H1N1 strain. Significantly higher pandemic A/H1N1 neutralizing titers were measured in subjects who had received vaccination against seasonal influenza in 2008–2009. Higher pandemic neutralizing titers were also measured in subjects over 60 years of age.

Conclusions/Significance

Our findings reveal that the extent of protective cross-immunity between seasonal and pandemic A/H1N1 influenza viruses may be more important than previously estimated. This cross-immunity could provide a possible explanation of the relatively mild profile of the recent influenza pandemic.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that induces in humans a disease characterized by fever, rash, and pain in muscles and joints. The recent emergence or reemergence of CHIKV in the Indian Ocean Islands and India has stressed the need to better understand the pathogenesis of this disease. Previous CHIKV disease models have used young or immunodeficient mice, but these do not recapitulate human disease patterns and are unsuitable for testing immune-based therapies. Herein, we describe what we believe to be a new model for CHIKV infection in adult, immunocompetent cynomolgus macaques. CHIKV infection in these animals recapitulated the viral, clinical, and pathological features observed in human disease. In the macaques, long-term CHIKV infection was observed in joints, muscles, lymphoid organs, and liver, which could explain the long-lasting CHIKV disease symptoms observed in humans. In addition, the study identified macrophages as the main cellular reservoirs during the late stages of CHIKV infection in vivo. This model of CHIKV physiopathology should allow the development of new therapeutic and/or prophylactic strategies.

Background

Within months of the emergence of the novel A/H1N1 pandemic influenza virus (nA/H1N1v), systematic screening for the surveillance of the pandemic was abandoned in France and in some other countries. At the end of June 2009, we implemented, for the public hospitals of Marseille, a Point Of Care (POC) strategy for rapid diagnosis of the novel A/H1N1 influenza virus, in order to maintain local surveillance and to evaluate locally the kinetics of the pandemic.

Methodology/Principal Findings

Two POC laboratories, located in strategic places, were organized to receive and test samples 24 h/24. POC strategy consisted of receiving and processing naso-pharyngeal specimens in preparation for the rapid influenza diagnostic test (RIDT) and real-time RT-PCR assay (rtRT-PCR). This strategy had the theoretical capacity of processing up to 36 samples per 24 h. When the flow of samples was too high, the rtRT-PCR test was abandoned in the POC laboratories and transferred to the core virology laboratory. Confirmatory diagnosis was performed in the core virology laboratory twice a day using two distinct rtRT-PCR techniques that detect either influenza A virus or nA/N1N1v. Over a period of three months, 1974 samples were received in the POC laboratories, of which 111 were positive for nA/H1N1v. Specificity and sensitivity of RIDT were 100%, and 57.7% respectively. Positive results obtained using RIDT were transmitted to clinical practitioners in less than 2 hours. POC processed rtRT-PCR results were available within 7 hours, and rtRT-PCR confirmation within 24 hours.

Conclusions/Significance

The POC strategy is of benefit, in all cases (with or without rtRT-PCR assay), because it provides continuous reception/processing of samples and reduction of the time to provide consolidated results to the clinical practitioners. We believe that implementation of the POC strategy for the largest number of suspect cases may improve the quality of patient care and our knowledge of the epidemiology of the pandemic.

To better understand Zaire ebolavirus (ZEBOV) circulation and transmission to humans, we conducted a large serological survey of rural populations in Gabon, a country characterized by both epidemic and non epidemic regions. The survey lasted three years and covered 4,349 individuals from 220 randomly selected villages, representing 10.7% of all villages in Gabon. Using a sensitive and specific ELISA method, we found a ZEBOV-specific IgG seroprevalence of 15.3% overall, the highest ever reported. The seroprevalence rate was significantly higher in forested areas (19.4%) than in other ecosystems, namely grassland (12.4%), savannah (10.5%), and lakeland (2.7%). No other risk factors for seropositivity were found. The specificity of anti-ZEBOV IgG was confirmed by Western blot in 138 individuals, and CD8 T cells from seven IgG+ individuals were shown to produce IFN-γ after ZEBOV stimulation. Together, these findings show that a large fraction of the human population living in forested areas of Gabon has both humoral and cellular immunity to ZEBOV. In the absence of identified risk factors, the high prevalence of “immune” persons suggests a common source of human exposure such as fruits contaminated by bat saliva. These findings provide significant new insights into ZEBOV circulation and human exposure, and raise important questions as to the human pathogenicity of ZEBOV and the existence of natural protective immunization.