Ernest A. Gould

First cases of autochthonous dengue fever and chikungunya fever in France: from bad dream to reality!

Following the outbreak of autochthonous chikungunya fever in humans in northern Italy, first reported 3 years ago (1) we have now witnessed two independent cases of autochthonous transmission of dengue fever and two cases of chikungunya fever in south-eastern France during September 2010. This worrying trend has raised serious concern among the health authorities, as Aedes albopictus, the Asian tiger mosquito, is the most likely transmission vector of both viruses, and this mosquito species is known to be spreading widely throughout many regions of southern Europe. The two cases of dengue fever occurred in patients (a 64-year-old male and an 18-year- old male) living in Nice in the same neighbourhood and know- ing each other. The two cases of chikungunya fever occurred in 12-year-old female patients also living in the vicinity of each other in Frejus, and attending the same high school. Both pre- sented with high fever, headache, lombalgia and arthralgia. These two patients lived in the same neighbourhood as the unique laboratory-documented imported case of the county, affecting a 7-year-old female patient returning from Asia. All four cases resulted in mild, self-resolving infections. The virological identification of these four cases of arbo- viral disease, with no travel history, and within the recorded area of dissemination of A. albopictus, along the 180-km region of the Mediterranean coast from Menton to Toulon (2): (i) demonstrates the first cases of autochthonous trans- mission of both viruses (dengue and chikungunya) in France; (ii) implies that A. albopictus, which is resident in this region, is the most likely competent vector for dengue virus (http:// www.sante-sports.gouv.fr/apparition-des-premiers-cas-auto chtones-de-dengue-en-france-metropolitaine.html); and (iii) raises important issues to be resolved in time for the next mosquito season, namely the adaptation of surveillance systems and the implementation of countermeasures to limit dispersal of the viruses and to limit the number of human cases of dengue or chikungunya fever. How alarmed should we be in Europe? Imported cases of dengue and chikungunya fever have long been reported in infected viraemic travellers (3) returning from tropical regions where the viruses are endemic or transiently epi- demic. Indeed, dengue virus ranks second only to the malar- ial parasite as an agent of systemic febrile illness in travellers to the tropics who are returning to Europe (4). However, during the past 5-10 years, the perceived risk of infection in Europe, particularly with these two arboviruses, has increased. This is because a competent mosquito vector was not previously present at a sufficient density in Europe to be able to initiate and generate secondary human cases of chikungunya or dengue fever among human populations. With the establishment and amplification of A. albopictus in Europe, and these cases of dengue and chikungunya fever in France, the perceived risk of epidemics caused by these viruses or other emerging arboviruses has increased dramati- cally. Consequently, in an attempt to prevent and monitor the appearance of autochthonous cases, the French authori- ties have elaborated a surveillance programme targeting both viruses in returning travellers during the summer period of vector activity (http://www.circulaires.gouv.fr/pdf/2010/05/cir_

Molecular evolution of the insect-specific flaviviruses.

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.

Phylogeny of the genus Flavivirus using complete coding sequences of arthropod-borne viruses and viruses with no known vector.

Attempts to define the evolutionary relationships and origins of viruses in the genus Flavivirus are hampered by the lack of genetic information particularly amongst the non-vectored flaviviruses. Using a novel protocol for sequence determination, the first complete coding sequence of St Louis encephalitis virus and those of two representative non-vectored flaviviruses, Rio Bravo (isolated from bat) and Apoi (isolated from rodent), are reported. The encoded polyproteins of Rio Bravo and Apoi virus are the smallest described to date within the genus Flavivirus. The highest similarities with other flaviviruses were found in the NS3 and NS5 genes. The proteolytic cleavage sites for the viral serine protease were highly conserved among the flaviviruses completely sequenced to date. Comparative genetic amino acid alignments revealed that p-distance cut-off values of 0·330–0·470 distinguished the arthropod-borne viruses according to their recognized serogroups and Rio Bravo and Apoi virus were assigned to two distinct non-vectored virus groups. Within these serogroups, cladogenesis based on the complete ORF sequence was similar to trees based on envelope and NS5 sequences. In contrast, branching patterns at the deeper nodes of the tree were different from those reported in the previous study of NS5 sequences. The significance of these observations is discussed.

Single amino acid codon changes detected in louping ill virus antibody-resistant mutants with reduced neurovirulence.

Seven mutant viruses were derived from a Scottish strain of louping ill virus using a virus envelope-specific neutralizing monoclonal antibody. None of the mutants was neutralized and immunofluorescence microscopy confirmed that they did not bind to this antibody. Four mutants showed reduced mouse neurovirulence compared with parent virus and two mutants failed to induce protective immune responses in mice challenged with virulent tick-borne encephalitis virus. The mutants with the lowest virulence showed poor or undetectable haemagglutinating activity. The nucleotide sequence of the envelope glycoprotein gene of each of the seven mutants was determined and the deduced amino acid sequence was compared with parent virus. For each mutant, only a single amino acid codon change was detected and all the amino acid substitutions occurred within amino acid positions 308 to 311. A change from the amino acid aspartate to asparagine at amino acid position 308, which represented a potential glycosylation site, was the most effective substitution in reducing mouse neurovirulence. The results demonstrate the importance of critical sites within the envelope glycoprotein as determinants of virus virulence.

Evolution of base composition and codon usage bias in the genus Flavivirus.

Abstract. The extent to which base composition and codon usage vary among RNA viruses, and the possible causes of this bias, is undetermined in most cases. A maximum-likelihood statistical method was used to test whether base composition and codon usage bias covary with arthropod association in the genus Flavivirus, a major source of disease in humans and animals. Flaviviruses are transmitted by mosquitoes, by ticks, or directly between vertebrate hosts. Those viruses associated with ticks were found to have a significantly lower G+C content than non-vector-borne flaviviruses and this difference was present throughout the genome at all amino acids and codon positions. In contrast, mosquito-borne viruses had an intermediate G+C content which was not significantly different from those of the other two groups. In addition, biases in dinucleotide and codon usage that were independent of base composition were detected in all flaviviruses, but these did not covary with arthropod association. However, the overall effect of these biases was slight, suggesting only weak selection at synonymous sites. A preliminary analysis of base composition, codon usage, and vector specificity in other RNA virus families also revealed a possible association between base composition and vector specificity, although with biases different from those seen in the Flavivirus genus.

Evolution, epidemiology, and dispersal of flaviviruses revealed by molecular phylogenies.

Publisher Summary This chapter reviews the current knowledge of flavivirus phylogeny and illustrates the significance of arthropods, habitats, and vertebrates, including humans, in their evolution, epidemiology, and dispersal. For a long time, it has been assumed that the opportunity for genetic variation among the arboviruses is significantly constrained by their need to replicate in both vertebrate and invertebrate hosts. In this sense, the flaviviruses present an interesting case for analysis, because the genus Flavivirus contains both arthropod-vectored and nonvectored viruses. The genus Flavivirus contains approximately 70 antigenically related viruses, many of which infect both vertebrate and invertebrate species. The flaviviruses that have no known vectors (NKV) are associated either with rodents or bats. The individual bat-associated NKV viruses are found either in the New World or in the Old World but none so far has been found in both regions. Despite the clear evidence that many of the flaviviruses are transmitted among vertebrate hosts by arthropods, it is known that they can also be transmitted orally and transplacentally.

Tracing the origins of louping ill virus by molecular phylogenetic analysis.

The nucleotide and deduced amino acid sequences of louping ill (LI) virus isolates, collected from representative regions of the British Isles and Norway, were determined for either the entire envelope gene (20 isolates) or for a portion of the envelope gene that spans a hypervariable region and includes an LI virus specific marker sequence (53 isolates). Phylogenetic analysis reveals the presence of three major geographical populations of LI virus in the British Isles, viz. Irish, Welsh and British LI viruses, which all cause encephalomyelitis in animals, predominantly sheep, and co-habit the same tick population. British LI virus occurs throughout Scotland, England, Ireland and Norway. Irish and Welsh LI viruses occur only in Ireland and Wales, respectively. Phylogenetic analysis also predicts that LI virus initially emerged in Ireland and that a descendant was introduced into Great Britain via Wales and was subsequently transported to the borders of Scotland, from where it was dispersed throughout Scotland, northern England and Norway. More recently, the British LI virus was reintroduced into Ireland and also into south-west England. Dates of lineage divergence, calculated from the synonymous substitution rate, indicate that LI virus emerged in the British Isles less than 800 years ago and most LI virus dispersal occurred during the last 300 years. By combining these data with historical records it appears that livestock movement can be implicated in the dispersal of LI virus.

Likely correlation between sources of information and acceptability of A/H1N1 swine-origin influenza virus vaccine in Marseille, France.

Background In France, there was a reluctance to accept vaccination against the A/H1N1 pandemic influenza virus despite government recommendation and investment in the vaccine programme. Methods and Findings We examined the willingness of different populations to accept A/H1N1vaccination (i) in a French hospital among 3315 employees immunized either by in-house medical personnel or mobile teams of MDs and (ii) in a shelter housing 250 homeless persons. Google was used to assess the volume of enquiries concerning incidence of influenza. We analyzed the information on vaccination provided by Google, the website of the major French newspapers, and PubMed. Two trust Surveys were used to assess public opinion on the trustworthiness of people in different professions. Paramedics were significantly more reluctant to accept immunisation than qualified medical staff. Acceptance was significantly increased when recommended directly by MDs. Anecdotal cases of directly observed severe infections were followed by enhanced acceptance of paramedical staff. Scientific literature was significantly more in favour of vaccination than Google and French newspaper websites. In the case of the newspaper websites, information correlated with their recognised political reputations, although they would presumably claim independence from political bias. The Trust Surveys showed that politicians were highly distrusted in contrast with doctors and pharmacists who were considered much more trustworthy. Conclusions The low uptake of the vaccine could reflect failure to convey high quality medical information and advice relating to the benefits of being vaccinated. We believe that the media and internet contributed to this problem by raising concerns within the general population and that failure to involve GPs in the control programme may have been a mistake. GPs are highly regarded by the public and can provide face-to-face professional advice and information. The top-down strategy of vaccine programme management and information delivered by the Ministry of Health could have aggravated the problem, because the general population does not always trust politicians.

Nucleotide sequence of the envelope glycoprotein of negishi virus shows very close homology to louping III virus

Negishi virus, a member of the family Flaviviridae, was originally isolated in Japan, during an outbreak of Japanese encephalitis. Antigenically, however, Negishi virus resembles the tick-borne rather than the mosquito-borne flaviviruses. Monoclonal antibodies that bind louping ill virus showed a close antigenic relationship between louping ill and Negishi virus. The genes encoding the envelope glycoprotein of Negishi virus (strain 3248/49/P10) and louping ill virus (strain SB526) were cloned and sequenced. They showed a very close homology at both the nucleotide and deduced amino acid levels. Comparison with the known sequence of another strain of louping ill virus (strain 369/T2) and with other tick-borne flaviviruses showed that Negishi virus was more closely related to louping ill virus than to the other tick-borne viruses. The significance of this observation for virus evolution, virus distribution in the environment, and the potential use of nucleotide sequencing for rapid and precise identification of flaviviruses are discussed.

Novel insect-specific flavivirus isolated from northern Europe

Mosquitoes collected in Finland were screened for flaviviral RNA leading to the discovery and isolation of a novel flavivirus designated Hanko virus (HANKV). Virus characterization, including phylogenetic analysis of the complete coding sequence, confirmed HANKV as a member of the “insect-specific” flavivirus (ISF) group. HANKV is the first member of this group isolated from northern Europe, and therefore the first northern European ISF for which the complete coding sequence has been determined. HANKV was not transcribed as DNA in mosquito cell culture, which appears atypical for an ISF. HANKV shared highest sequence homology with the partial NS5 sequence available for the recently discovered Spanish Ochlerotatus flavivirus (SOcFV). Retrospective analysis of mitochondrial sequences from the virus-positive mosquito pool suggested an Ochlerotatus mosquito species as the most likely host for HANKV. HANKV and SOcFV may therefore represent a novel group of Ochlerotatus-hosted insect-specific flaviviruses in Europe and further afield.