Antoine Nougairede

Chikungunya in the Americas

514 www.thelancet.com Vol 383 February 8, 2014 circulated in New Caledonia, which harbours diff erent aminoacid deletion in the NSP3 gene. This episode represents the first evidence for the emergence of autochthonous chikungunya cases in the Americas. It is likely that the chikungunya epidemic will extend to other Caribbean islands, and it also has substantial potential for spreading from this region visited yearly by millions of tourists to the American mainland where A aegypti is endemic. Assuming that this strain will be transmitted effi ciently by A albopictus mosquitoes, its persistence in the Caribbean would also represent, as a consequence of seasonal synchronicity, a great threat for southern European countries where the mosquito has recently dispersed. This situation warrants reinforced epidemiological surveillance and specifi c preparedness.

Chikungunya fever: Epidemiology, clinical syndrome, pathogenesis and therapy

n Abstractn n Chikungunya virus (CHIKV) is the aetiological agent of the mosquito-borne disease chikungunya fever, a debilitating arthritic disease that, during the past 7years, has caused immeasurable morbidity and some mortality in humans, including newborn babies, following its emergence and dispersal out of Africa to the Indian Ocean islands and Asia. Since the first reports of its existence in Africa in the 1950s, more than 1500 scientific publications on the different aspects of the disease and its causative agent have been produced. Analysis of these publications shows that, following a number of studies in the 1960s and 1970s, and in the absence of autochthonous cases in developed countries, the interest of the scientific community remained low. However, in 2005 chikungunya fever unexpectedly re-emerged in the form of devastating epidemics in and around the Indian Ocean. These outbreaks were associated with mutations in the viral genome that facilitated the replication of the virus in Aedes albopictus mosquitoes. Since then, nearly 1000 publications on chikungunya fever have been referenced in the PubMed database. This article provides a comprehensive review of chikungunya fever and CHIKV, including clinical data, epidemiological reports, therapeutic aspects and data relating to animal models for in vivo laboratory studies. It includes Supplementary Tables of all WHO outbreak bulletins, ProMED Mail alerts, viral sequences available on GenBank, and PubMed reports of clinical cases and seroprevalence studies.n n

In vitro antiviral activity of arbidol against Chikungunya virus and characteristics of a selected resistant mutant.

Arbidol (ARB) is an antiviral drug originally licensed in Russia for use against influenza and other respiratory viral infections. Although a broad-spectrum antiviral activity has been reported for this drug, there is until now no data regarding its effects against alphavirus infection. Here, the in vitro antiviral effect of ARB on Chikungunya virus (CHIKV) replication was investigated and this compound was found to present potent inhibitory activity against the virus propagated onto immortalized Vero cells or primary human fibroblasts (MRC-5 lung cells) (IC(50)<10μg/ml). A CHIKV resistant mutant was then selected and adapted to growth in the presence of 30μg/ml ARB in MRC5 cells; its complete sequence analysis revealed a single amino acid substitution (G407R) localized in the E2 envelope protein. To confirm the G407R role in the molecular mechanism of ARB resistance, a CHIKV infectious clone harboring the same substitution was engineered, tested, and was found to display a similar level of resistance. Finally, our results demonstrated the effective in vitro antiviral activity of ARB against CHIKV and gave some tracks to understand the molecular basis of ARB activity.

Role of host cell factors in flavivirus infection: Implications for pathogenesis and development of antiviral drugs.

The genus Flavivirus contains approximately 70 arthropod-borne enveloped RNA viruses many of which cause severe human and in some cases, animal disease. They include dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and tick-borne encephalitis virus. Hundreds of thousands of deaths due to flavivirus infections occur each year, many of which are unpreventable due to lack of availability of appropriate vaccines and/or antiviral drugs. Flaviviruses exploit the cytoplasmic cellular machinery to facilitate propagation of infectious progeny virions. They engage in dynamic and antagonistic interactions with host cell membranes and biochemical processes. Following infection, the cells initiate various antiviral strategies to counteract viral invasion. In its defense, the virus has alternative strategies to suppress these host responses to infection. The fine balance between these interactions determines the outcome of the viral infection and disease progression. Published studies have revealed specific effects of flaviviruses on cellular processes, but the underlying mechanisms that determine the specific cytopathogenetic changes induced by different flaviviruses have not, as yet, been elucidated. Independently of the suppression of the type I IFN response which has been described in detail elsewhere, this review focuses on recent discoveries relating to alterations of host metabolism following viral infection. Such studies may contribute to new approaches to antiviral drug development. The role of host cellular factors will be examined in the context of protection and/or pathogenesis resulting from flavivirus infection, with particular emphasis on West Nile virus and dengue virus.

Lack of nasal carriage of novel corona virus (HCoV‐EMC) in French Hajj pilgrims returning from the Hajj 2012, despite a high rate of respiratory symptoms

n Abstractn n A cohort of 154 French Hajj pilgrims participating in the 2012 Hajj were systematically sampled with nasal swabs prior to returning to France, and screened for the novel HCoV-EMC coronavirus by two real-time RT-PCR assays. Despite a high rate of respiratory symptoms (83.4%), including 41.0% influenza-like illness, no case of HCoV-EMC infection was detected. Despite the fact that zoonotic transmission was suspected in the first few cases, a recent family cluster in the Kingdom of Saudi Arabia suggests that the virus might show at least limited spread from person to person, which justifies continuing epidemiological surveillance.n n

Circulation of Respiratory Viruses Among Pilgrims During the 2012 Hajj Pilgrimage

This study suggests a rapid acquisition of respiratory viruses among pilgrims during their stay in the Kingdom of Saudi Arabia and highlights the potential of the spread of these infections into the pilgrims home countries upon their return.

RNA and DNA Bacteriophages as Molecular Diagnosis Controls in Clinical Virology: A Comprehensive Study of More than 45,000 Routine PCR Tests

Real-time PCR techniques are now commonly used for the detection of viral genomes in various human specimens and require for validation both external and internal controls (ECs and ICs). In particular, ICs added to clinical samples are necessary to monitor the extraction, reverse transcription, and amplification steps in order to detect false-negative results resulting from PCR-inhibition or errors in the technical procedure. Here, we performed a large scale evaluation of the use of bacteriophages as ICs in routine molecular diagnosis. This allowed to propose simple standardized procedures (i) to design specific ECs for both DNA and RNA viruses and (ii) to use T4 (DNA) or MS2 (RNA) phages as ICs in routine diagnosis. Various technical formats for using phages as ICs were optimised and validated. Subsequently, T4 and MS2 ICs were evaluated in routine real-time PCR or RT-PCR virological diagnostic tests, using a series of 8,950 clinical samples (representing 36 distinct specimen types) sent to our laboratory for the detection of a variety of DNA and RNA viruses. The frequency of inefficient detection of ICs was analyzed according to the nature of the sample. Inhibitors of enzymatic reactions were detected at high frequency in specific sample types such as heparinized blood and bone marrow (>70%), broncho-alveolar liquid (41%) and stools (36%). The use of T4 and MS2 phages as ICs proved to be cost-effective, flexible and adaptable to various technical procedures of real-time PCR detection in virology. It represents a valuable strategy for enhancing the quality of routine molecular diagnosis in laboratories that use in-house designed diagnostic systems, which can conveniently be associated to the use of specific synthetic ECs. The high rate of inhibitors observed in a variety of specimen types should stimulate the elaboration of improved technical protocols for the extraction and amplification of nucleic acids.

Revolutionizing Clinical Microbiology Laboratory Organization in Hospitals with In Situ Point-of-Care

Background Clinical microbiology may direct decisions regarding hospitalization, isolation and anti-infective therapy, but it is not effective at the time of early care. Point-of-care (POC) tests have been developed for this purpose. Methods and Findings One pilot POC-lab was located close to the core laboratory and emergency ward to test the proof of concept. A second POC-lab was located inside the emergency ward of a distant hospital without a microbiology laboratory. Twenty-three molecular and immuno-detection tests, which were technically undemanding, were progressively implemented, with results obtained in less than four hours. From 2008 to 2010, 51,179 tests yielded 6,244 diagnoses. The second POC-lab detected contagious pathogens in 982 patients who benefited from targeted isolation measures, including those undertaken during the influenza outbreak. POC tests prevented unnecessary treatment of patients with non-streptococcal tonsillitis (nu200a=u200a1,844) and pregnant women negative for Streptococcus agalactiae carriage (nu200a=u200a763). The cerebrospinal fluid culture remained sterile in 50% of the 49 patients with bacterial meningitis, therefore antibiotic treatment was guided by the molecular tests performed in the POC-labs. With regard to enterovirus meningitis, the mean length-of-stay of infected patients over 15 years old significantly decreased from 2008 to 2010 compared with 2005 when the POC was not in place (1.43±1.09 versus 2.91±2.31 days; pu200a=u200a0.0009). Altogether, patients who received POC tests were immediately discharged nearly thrice as often as patients who underwent a conventional diagnostic procedure. Conclusions The on-site POC-lab met physicians needs and influenced the management of 8% of the patients that presented to emergency wards. This strategy might represent a major evolution of decision-making regarding the management of infectious diseases and patient care.

Lack of MERS coronavirus but prevalence of influenza virus in French pilgrims after 2013 Hajj.

To the Editor: Saudi Arabia has reported the highest number of Middle East respiratory syndrome coronavirus (MERS-CoV) cases since the virus first emerged in 2012, with >127 confirmed cases and a case-fatality rate of 42%, as of November 2013 (1). Global attention has focused on the potential for spread of MERS-CoV after the Hajj pilgrimage during which Muslims from 180 countries converge in Mecca, Saudi Arabia. Such pilgrims have a high risk for respiratory tract infections because of severe overcrowding. The International Health Regulations Emergency Committee advised all countries (particularly those with returning pilgrims) to strengthen their surveillance capacities and ensure robust reporting of any identified cases (2). n nWe report the results of a prospective cohort study conducted in Saudi Arabia in October 2013. Participants in the survey were adult Hajj pilgrims who traveled together in a group (through 1 travel agency in Marseille, France) from October 3 through October 24, 2013. Pilgrims were included in the study on a voluntary basis and were asked to sign a written consent form. All pilgrims received advice about individual prevention measures against respiratory tract infection before departing, and follow-up was conducted during the journey by a medical doctor who systematically documented travel-associated diseases. Nasal swab specimens were obtained just before the pilgrims left Saudi Arabia, frozen <48 hours after sampling, and processed (3,4). Each sample was tested for MERS-CoV (upE and ORF1a genes) (5,6) and influenza A, B (7), and A/2009/H1N1 viruses (8) by real-time reverse transcription PCR. The protocol was approved by our Institutional Review Board (July 23, 2013; reference no. 2013-{type:entrez-nucleotide,attrs:{text:A00961,term_id:14678}}A00961–44) and by the Saudi Ministry of Health ethics committee. n nOn departure from France, the study comprised 129 pilgrims. Their mean age was 61.7 years (range 34–85 years), and the male/female ratio was 0.7:1. Sixty-eight (52.7%) pilgrims reported having a chronic disease, including hypertension (43 [33.3%]), diabetes (34 [26.4%]), chronic cardiac disease (11 [8.5%]), and chronic respiratory disease (5 [3.9%]). Forty-six (35.7%) pilgrims reported receiving influenza vaccination in 2012; none had been vaccinated in 2013 before the Hajj because the vaccine was not yet available in France. n nClinical data were available for 129 persons: 117 (90.7%) had respiratory symptoms while in Saudi Arabia, including cough (112 [86.8%]) and sore throat (107 [82.9%]); 64 (49.6%) reported fever, and 61 (47.3%) had conditions that met the criteria for influenza-like illness (ILI; i.e., the association of cough, sore throat, and subjective fever) (Figure) (4). One patient was hospitalized during travel (undocumented pneumonia). Nasal swab specimens were obtained from 129 pilgrims on October 23, 2013 (week 43), 1 day before pilgrims left Saudi Arabia for France; 90 (69.8%) pilgrims were still symptomatic. All PCRs were negative for MERS-CoV. n n n nFigure n nOnset of respiratory symptoms by week, reported by 129 Hajj pilgrims from France during their stay in Saudi Arabia, October 2013. n n n nEight pilgrims tested positive for influenza A(H3N2), 1 for influenza A(H1N1), and 1 for influenza B virus. No dual infections were reported. 70 (54.3%) pilgrims were seen 3–5 weeks after they returned to France, and the remaining were lost to follow-up. Fifty-five (78.6%) had experienced respiratory symptoms since their return, including cough (50 [71.4%]) and sore throat (14 [20.0%]); 12 (17.1%) reported fever, and illness in 5 (7.1%) pilgrims met the criteria for ILI. The 10 pilgrims who had positive test results for influenza virus on return had cleared their infection; only 1 additional sample was positive (for influenza A[H1N1]). n nOur results support data obtained from a similar cohort in 2012 that showed a lack of nasal carriage of MERS-CoV among Hajj pilgrims from France (3). However, a higher prevalence of influenza virus (7.8%) was observed in nasal swab specimens in 2013 than in 2012 when 2 (3.2%) cases of influenza B virus infection were detected and no case of influenza A virus infection was detected among 162 pilgrims returning from the Hajj (4). n nThe estimated incidence of ILI in France during week 43 was 27 per 100,000 inhabitants, far below the epidemic threshold (126/100,000) with few sporadic cases of influenza A virus infection reported in some regions in France (www.grog.org/bullhebdo_pdf/bull_grog_43-2013.pdf). No case was reported in the Marseille area (http://websenti.u707.jussieu.fr/sentiweb). The high prevalence of respiratory symptoms in our cohort probably reflects the close surveillance performed and is consistent with 2012 results (3,4). n nIn Marseille, all patients with suspected MERS-CoV infection are referred to the Institut Hospitalo-Universitaire Mediterranee Infection. As of November 8, 2013, of the 14 first returning patients hospitalized for respiratory symptoms and screened for MERS-CoV and other pathogens, including influenza, 4 were infected with influenza A(H3N2), 4 with influenza A(H1N1), and 1 with influenza B virus. All samples tested negative for MERS-CoV. n nOur preliminary results indicate that pilgrims from France returning from the 2013 Hajj were free of MERS-CoV but that a proportion were infected with influenza viruses and may represent a potential for early introduction of influenza in southern France. This proportion may have been underestimated because screening was performed at the end of the study period when some infections had cleared. Influenza vaccination should be a priority for pilgrims attending the Hajj (9,10).

Chikungunya Virus Transmission Potential by Local Aedes Mosquitoes in the Americas and Europe

Background Chikungunya virus (CHIKV), mainly transmitted in urban areas by the mosquitoes Aedes aegypti and Aedes albopictus, constitutes a major public health problem. In late 2013, CHIKV emerged on Saint-Martin Island in the Caribbean and spread throughout the region reaching more than 40 countries. Thus far, Ae. aegypti mosquitoes have been implicated as the sole vector in the outbreaks, leading to the hypothesis that CHIKV spread could be limited only to regions where this mosquito species is dominant. Methodology/Principal Findings We determined the ability of local populations of Ae. aegypti and Ae. albopictus from the Americas and Europe to transmit the CHIKV strain of the Asian genotype isolated from Saint-Martin Island (CHIKV_SM) during the recent epidemic, and an East-Central-South African (ECSA) genotype CHIKV strain isolated from La Réunion Island (CHIKV_LR) as a well-characterized control virus. We also evaluated the effect of temperature on transmission of CHIKV_SM by European Ae. albopictus. We found that (i) Aedes aegypti from Saint-Martin Island transmit CHIKV_SM and CHIKV_LR with similar efficiency, (ii) Ae. aegypti from the Americas display similar transmission efficiency for CHIKV_SM, (iii) American and European populations of the alternative vector species Ae. albopictus were as competent as Ae. aegypti populations with respect to transmission of CHIKV_SM and (iv) exposure of European Ae. albopictus to low temperatures (20°C) significantly reduced the transmission potential for CHIKV_SM. Conclusions/Significance CHIKV strains belonging to the ECSA genotype could also have initiated local transmission in the new world. Additionally, the ongoing CHIKV outbreak in the Americas could potentially spread throughout Ae. aegypti- and Ae. albopictus-infested regions of the Americas with possible imported cases of CHIKV to Ae. albopictus-infested regions in Europe. Colder temperatures may decrease the local transmission of CHIKV_SM by European Ae. albopictus, potentially explaining the lack of autochthonous transmission of CHIKV_SM in Europe despite the hundreds of imported CHIKV cases returning from the Caribbean.