Search

Highlights • Japanese encephalitis virus (JEV) remains a leading cause of neurological infection in Asia. • A systematic review identified 20,212 published human cases of laboratory-confirmed JEV infections from 205 studies. • 15,167 (75%) of cases were confirmed with the lowest confidence diagnostic test, i.e., level 3 or 4, or level 4. • Only 109 (53%) of the studies reported contemporaneous testing for dengue-specific antibodies. • A fundamental pre-requisite for the control of JE is lacking — that of a simple and specific diagnostic procedure that can be adapted for point-of-care tests and readily used throughout JE endemic regions of the world., Objective Japanese encephalitis virus infection (JE) remains a leading cause of neurological disease in Asia, mainly involving individuals living in remote areas with limited access to treatment centers and diagnostic facilities. Laboratory confirmation is fundamental for the justification and implementation of vaccination programs. We reviewed the literature on historical developments and current diagnostic capability worldwide, to identify knowledge gaps and instill urgency to address them. Methods Searches were performed in Web of Science and PubMed using the term 'Japanese encephalitis' up to 13th October 2019. Studies reporting laboratory-confirmed symptomatic JE cases in humans were included, and data on details of diagnostic tests were extracted. A JE case was classified according to confirmatory levels (Fischer et al., 2008; Campbell et al., 2011; Pearce et al., 2018; Heffelfinger et al., 2017), where level 1 represented the highest level of confidence. Findings 20,212 published JE cases were identified from 205 studies. 15,167 (75%) of these positive cases were confirmed with the lowest-confidence diagnostic tests (level 3 or 4, or level 4). Only 109 (53%) of the studies reported contemporaneous testing for dengue-specific antibodies. Conclusion A fundamental pre-requisite for the control of JEV is lacking — that of a simple and specific diagnostic procedure that can be adapted for point-of-care tests and readily used throughout JE-endemic regions of the world.

The recent global (re)emergence of arthropod-borne viruses (arboviruses), such as chikungunya and Zika virus, was widely reported in the media as though it was a new phenomenon. This is not the case. Arboviruses and other human microbial pathogens have been (re)emerging for centuries. The major difference today is that arbovirus emergence and dispersion are more rapid and geographically extensive, largely due to intensive growth of global transportation systems, arthropod adaptation to increasing urbanisation, our failure to contain mosquito population density increases and land perturbation. Here we select examples of (re)emerging pathogenic arboviruses and explain the reasons for their emergence and different patterns of dispersal, focusing particularly on the mosquito vectors which are important determinants of arbovirus emergence. We also attempt to identify arboviruses likely to (re)emerge in the future.

Tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are members of the tick-borne flaviviruses (TBFVs) in the family Flaviviridae which cause encephalomeningitis and encephalitis in humans and other animals. Although vaccines against TBEV and LIV are available, infection rates are rising due to the low vaccination coverage. To date, no specific therapeutics have been licensed. Several neutralizing monoclonal antibodies (MAbs) show promising effectiveness in the control of TBFVs, but the underlying molecular mechanisms are yet to be characterized. Here, we determined the crystal structures of the LIV envelope (E) protein and report the comparative structural analysis of a TBFV broadly neutralizing murine MAb (MAb 4.2) in complex with either the LIV or TBEV E protein. The structures reveal that MAb 4.2 binds to the lateral ridge of domain III of the E protein (EDIII) of LIV or TBEV, an epitope also reported for other potently neutralizing MAbs against mosquito-borne flaviviruses (MBFVs), but adopts a unique binding orientation. Further structural analysis suggested that MAb 4.2 may neutralize flavivirus infection by preventing the structural rearrangement required for membrane fusion during virus entry. These findings extend our understanding of the vulnerability of TBFVs and other flaviviruses (including MBFVs) and provide an avenue for antibody-based TBFV antiviral development. IMPORTANCE Understanding the mechanism of antibody neutralization/protection against a virus is crucial for antiviral countermeasure development. Tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are tick-borne flaviviruses (TBFVs) in the family Flaviviridae. They cause encephalomeningitis and encephalitis in humans and other animals. Although vaccines for both viruses are available, infection rates are rising due to low vaccination coverage. In this study, we solved the crystal structures of the LIV envelope protein (E) and a broadly neutralizing/protective TBFV MAb, MAb 4.2, in complex with E from either TBEV or LIV. Key structural features shared by TBFV E proteins were analyzed. The structures of E-antibody complexes showed that MAb 4.2 targets the lateral ridge of both the TBEV and LIV E proteins, a vulnerable site in flaviviruses for other potent neutralizing MAbs. Thus, this site represents a promising target for TBFV antiviral development. Further, these structures provide important information for understanding TBFV antigenicity.

The flavivirus, tick-borne encephalitis virus (TBEV) is transmitted by Ixodes spp. ticks and may cause severe and potentially lethal neurological tick-borne encephalitis (TBE) in humans. Studying TBEV requires the use of secondary methodologies to detect the virus in infected cells. To overcome this problem, we rationally designed and constructed a recombinant reporter TBEV that stably expressed the mCherry reporter protein. The resulting TBEV reporter virus (named mCherry-TBEV) and wild-type parental TBEV exhibited similar growth kinetics in cultured cells; however, the mCherry-TBEV virus produced smaller plaques. The magnitude of mCherry expression correlated well with progeny virus production but remained stable over

As revealed by the recent resurgence of yellow fever virus (YFV) activity in the tropical regions of Africa and South America, YFV control measures need urgent rethinking. Over the last decade, most reported outbreaks occurred in, or eventually reached, areas with low vaccination coverage but that are suitable for virus transmission, with an unprecedented risk of expansion to densely populated territories in Africa, South America and Asia. As reflected in the World Health Organization’s initiative launched in 2017, it is high time to strengthen epidemiological surveillance to monitor accurately viral dissemination, and redefine vaccination recommendation areas. Vector-control and immunisation measures need to be adapted and vaccine manufacturing must be reconciled with an increasing demand. We will have to face more yellow fever (YF) cases in the upcoming years. Hence, improving disease management through the development of efficient treatments will prove most beneficial. Undoubtedly, these developments will require in-depth descriptions of YFV biology at molecular, physiological and ecological levels. This second section of a two-part review describes the current state of knowledge and gaps regarding the molecular biology of YFV, along with an overview of the tools that can be used to manage the disease at the individual, local and global levels.

International audience; As revealed by the recent resurgence of yellow fever virus (YFV) activity in the tropical regions of Africa and South America, YFV control measures need urgent rethinking. Over the last decade, most reported outbreaks occurred in, or eventually reached, areas with low vaccination coverage but that are suitable for virus transmission, with an unprecedented risk of expansion to densely populated territories in Africa, South America and Asia. As reflected in the World Health Organization’s initiative launched in 2017, it is high time to strengthen epidemiological surveillance to monitor accurately viral dissemination, and redefine vaccination recommendation areas. Vector-control and immunisation measures need to be adapted and vaccine manufacturing must be reconciled with an increasing demand. We will have to face more yellow fever (YF) cases in the upcoming years. Hence, improving disease management through the development of efficient treatments will prove most beneficial. Undoubtedly, these developments will require in-depth descriptions of YFV biology at molecular, physiological and ecological levels. This second section of a two-part review describes the current state of knowledge and gaps regarding the molecular biology of YFV, along with an overview of the tools that can be used to manage the disease at the individual, local and global levels.

Sandfly-borne phleboviruses may cause a transient febrile illness (sandfly fever) or more severe neuroinvasive disease. In the Old World, they are vectored by phlebotomine flies, which are widely distributed in the Mediterranean basin, North Africa, the Indian subcontinent, the Middle East and central Asia. High seroprevalence rates have been recorded in humans and domestic animals in areas where sandflies are present. Most published studies have focused on phlebovirus infections of travelers and of soldiers stationed in endemic areas, but the health impact on local populations should not be underestimated, as seroprevalence studies indicate massive circulation of these viruses, even if disease is seldom documented. Except for Toscana virus, which shows a marked neurotropism and is a leading cause of aseptic meningitis in endemic regions, phlebovirus infections are inadequately considered by physicians and are generally underestimated. However, several properties of these viruses suggest that they will extend their geographic range. First, changes in the areas occupied by sandflies as a result of climate change have a direct impact on the epidemiology of associated human and animal diseases. Second, phleboviruses exhibit a high mutation rate, and their tri-segmented genome is prone to reassortment and recombination. Third, distinct virus strains can be transmitted by the same arthropod species. Recent studies have documented the distribution of sandfly-borne phleboviruses in Western Europe, but data for Eastern Europe, the Middle East and Africa are very limited. With the goal of filling knowledge gaps and planning new research programs, we have examined available information and present it as a comprehensive review, with a specific focus on understudied regions. We also discuss the need to conduct studies aimed at developing new antiviral drugs and vaccines.

The unexpected emergence of Zika virus (ZIKV) in the Pacific Islands and Latin America and its association with congenital Zika virus syndrome (CZVS) (which includes microcephaly) and Guillain-Barré syndrome (GBS) have stimulated wide-ranging research. High densities of susceptible Aedes spp., immunologically naive human populations, global population growth with increased urbanization, and escalation of global transportation of humans and commercial goods carrying vectors and ZIKV undoubtedly enhanced the emergence of ZIKV. However, flavivirus mutations accumulate with time, increasing the likelihood that genetic viral differences are determinants of change in viral phenotype. Based on comparative ZIKV complete genome phylogenetic analyses and temporal estimates, we identify amino acid substitutions that may be associated with increased viral epidemicity, CZVS, and GBS. Reverse genetics, vector competence, and seroepidemiological studies will test our hypothesis that these amino acid substitutions are determinants of epidemic and neurotropic ZIKV emergence.

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.

Tick-borne encephalitis virus (TBEV) induces acute central nervous system (CNS) disease in humans. In this study, we investigate the pathogenetic mechanisms that correlate with fatal infection with TBEV in a mouse model. Following subcutaneous infection with high challenge doses (>10(7) PFU), mice started to die early (8 days) and mortality rates reached >80%. These doses induced acute and widespread infection of the CNS. On the other hand, following subcutaneous infection with low challenge doses (10(2)-10(6) PFU), mice started to die late (11 days) and approximately one half of the mice survived but exhibited degrees of encephalitis similar to dying mice. However, low dose dying mice exhibited severe systemic stress response, and increased levels of TNF-alpha compared with recovering mice. We therefore conclude that in addition to the development of CNS disease, systemic inflammatory and stress responses contribute to induce a fatal infection following subcutaneous infection of mice with TBEV.

While some skeptics remain unconvinced that global climate change is a reality, there is no doubt that during the past 50 years or so, patterns of emerging arbovirus diseases have changed significantly. Can this be attributed to climate change? Climate is a major factor in determining: (1) the geographic and temporal distribution of arthropods; (2) characteristics of arthropod life cycles; (3) dispersal patterns of associated arboviruses; (4) the evolution of arboviruses; and (5) the efficiency with which they are transmitted from arthropods to vertebrate hosts. Thus, under the influence of increasing temperatures and rainfall through warming of the oceans, and alteration of the natural cycles that stabilise climate, one is inevitably drawn to the conclusion that arboviruses will continue to emerge in new regions. For example, we cannot ignore the unexpected but successful establishment of chikungunya fever in northern Italy, the sudden appearance of West Nile virus in North America, the increasing frequency of Rift Valley fever epidemics in the Arabian Peninsula, and very recently, the emergence of Bluetongue virus in northern Europe. In this brief review we ask the question, are these diseases emerging because of climate change or do other factors play an equal or even more important role in their emergence?

Approximately 2 billion people live in countries where Japanese encephalitis (JE) presents a significant risk to humans and animals, particularly in China and India, with at least 700 million potentially susceptible children. The combined effects of climate change, altered bird migratory patterns, increasing movement of humans, animals and goods, increasing deforestation and development of irrigation projects will inevitably lead to further geographic dispersal of the virus and an enhanced threat. Although most human infections are mild or asymptomatic, some 50% of patients who develop encephalitis suffer permanent neurologic defects, and 25% die. Vaccines have reduced the incidence of JE in some countries. No specific antiviral therapy is currently available. Interferon alpha-2a was tested in a double-blind placebo-controlled trial on children with Japanese encephalitis, but with negative results. There is thus a real need for antivirals that can reduce the toll of death and neurological sequelae resulting from infection with JE virus. Here we briefly review the epidemiological problems presented by this virus, the present state of drug development and the contributory role that antiviral therapy might play in developing future control strategies for JE.

The genus Flavivirus comprises more than 50 RNA virus species that include Yellow fever virus, Dengue virus, Japanese encephalitis virus, and the Tick-borne encephalitis virus complex.

A new flavivirus, Ecuador Paraiso Escondido virus (EPEV), named after the village where it was discovered, was isolated from sand flies ( Psathyromyia abonnenci , formerly Lutzomyia abonnenci ) that are unique to the New World. This represents the first sand fly-borne flavivirus identified in the New World. EPEV exhibited a typical flavivirus genome organization. Nevertheless, the maximum pairwise amino acid sequence identity with currently recognized flaviviruses was 52.8%. Phylogenetic analysis of the complete coding sequence showed that EPEV represents a distinct clade which diverged from a lineage that was ancestral to the nonvectored flaviviruses Entebbe bat virus, Yokose virus, and Sokoluk virus and also the Aedes -associated mosquito-borne flaviviruses, which include yellow fever virus, Sepik virus, Saboya virus, and others. EPEV replicated in C6/36 mosquito cells, yielding high infectious titers, but failed to reproduce either in vertebrate cell lines (Vero, BHK, SW13, and XTC cells) or in suckling mouse brains. This surprising result, which appears to eliminate an association with vertebrate hosts in the life cycle of EPEV, is discussed in the context of the evolutionary origins of EPEV in the New World. IMPORTANCE The flaviviruses are rarely (if ever) vectored by sand fly species, at least in the Old World. We have identified the first representative of a sand fly-associated flavivirus, Ecuador Paraiso Escondido virus (EPEV), in the New World. EPEV constitutes a novel clade according to current knowledge of the flaviviruses. Phylogenetic analysis of the virus genome showed that EPEV roots the Aedes -associated mosquito-borne flaviviruses, including yellow fever virus. In light of this new discovery, the New World origin of EPEV is discussed together with that of the other flaviviruses.

International audience; Slave trading of Africans to the Americas, during the 16th to the 19th century was responsible for the first recorded emergence in the New World of two arthropod-borne viruses (arboviruses), yellow fever virus and dengue virus. Many other arboviruses have since emerged from their sylvatic reservoirs and dispersed globally due to evolving factors that include anthropological behaviour, commercial transportation and land-remediation. Here, we outline some characteristics of these highly divergent arboviruses, including the variety of life cycles they have developed and the mechanisms by which they have adapted to evolving changes in habitat and host availability. We cite recent examples of virus emergence that exemplify how arboviruses have exploited the consequences of the modern human lifestyle. Using our current understanding of these viruses, we also attempt to demonstrate some of the limitations encountered in developing control strategies to reduce the impact of future emerging arbovirus diseases. Finally, we present recommendations for development by an international panel of experts reporting directly to World Health Organization, with the intention of providing internationally acceptable guidelines for improving emerging arbovirus disease control strategies. Success in these aims should alleviate the suffering and costs encountered during recent decades when arboviruses have emerged from their sylvatic environment.

The identification of Louping ill virus (LIV) in clinical specimens has been routinely achieved by virus isolation using susceptible pig kidney cells and subsequent serological analysis. While this method is sensitive and detects infectious virus, it is relatively labour intensive and time-consuming. In view of the veterinary and potential medical importance of LIV, a rapid and precise detection method for routine use that employs the TaqMan reverse transcription polymerase chain reaction (RT-PCR) has been developed to detect LIV RNA extracted from field samples. The TaqMan assay was evaluated against virus isolation using 22 cell culture grown LIV isolates, which had previously been partially characterised by sequencing, and material from 63 suspect field cases. Histopathological and/or serological reports were available for 39 of the suspect cases, providing additional diagnostic information to evaluate the results obtained from the TaqMan RT-PCR assay. The TaqMan assay was as sensitive as the cell culture infectious virus assay currently used and had the advantage that it was able to detect LIV in clinical specimens from which infectious virus could not be isolated possibly due to the presence of high levels of LIV antibody.

Climate change can cause arthropodborne diseases to emerge., Arboviruses have evolved a number of strategies to survive environmental challenges. This review examines the factors that may determine arbovirus emergence, provides examples of arboviruses that have emerged into new habitats, reviews the arbovirus situation in western Europe in detail, discusses potential arthropod vectors, and attempts to predict the risk for arbovirus emergence in the United Kingdom. We conclude that climate change is probably the most important requirement for the emergence of arthropodborne diseases such as dengue fever, yellow fever, Rift Valley fever, Japanese encephalitis, Crimean-Congo hemorrhagic fever, bluetongue, and African horse sickness in the United Kingdom. While other arboviruses, such as West Nile virus, Sindbis virus, Tahyna virus, and Louping ill virus, apparently circulate in the United Kingdom, they do not appear to present an imminent threat to humans or animals.

West Nile virus (WNV) is now the predominant circulating arthropod-borne virus in the United States with >15,000 human cases and >600 fatalities since 1999. Conventionally, mosquitoes become infected when feeding on viremic birds and subsequently transmit the virus to susceptible hosts. Here, we demonstrate nonviremic transmission of WNV between cofeeding mosquitoes. Donor, Culex pipiens quinquefasciatus mosquitoes infected with WNV were fed simultaneously with uninfected “recipient” mosquitoes on naïve mice. At all times, donor and recipient mosquitoes were housed in separate sealed containers, precluding the possibility of mixing. Recipients became infected in all five trials, with infection rates as high as 5.8% and no detectable viremia in the hosts. Remarkably, a 2.3% infection rate was observed when 87 uninfected mosquitoes fed adjacent to a single infected mosquito. This phenomenon could potentially enhance virus survival, transmission, and dispersion and obviate the requirement for viremia. All vertebrates, including immune and insusceptible animals, might therefore facilitate mosquito infection. Our findings question the status of dead-end hosts in the WNV transmission cycle and may partly explain the success with which WNV established and rapidly dispersed throughout North America.

Here we describe the complete genome sequences of two strains of Usutu virus (USUV), a mosquito-borne member of the genus Flavivirus in the Japanese encephalitis virus (JEV) serogroup. USUV was detected in Austria in 2001 causing a high mortality rate in blackbirds; the reference strain (SAAR-1776) was isolated in 1958 from mosquitoes in South Africa and has never been associated with avian mortality. The Austrian and South African isolates exhibited 97% nucleotide and 99% amino acid identity. Phylogenetic trees were constructed displaying the genetic relationships of USUV with other members of the genus Flavivirus. When comparing USUV with other JEV serogroup viruses, the closest lineage was Murray Valley encephalitis virus (nt: 73%, aa: 82%) followed by JEV (nt: 71%, aa: 81%) and West Nile virus (nt: 68%, aa: 75%). Comparison of the genomes showed that the conserved structural elements and putative enzyme motifs were homologous in the two USUV strains and the JEV serogroup. The factors that determine the severe clinical symptoms caused by the Austrian USUV strain in Eurasian blackbirds are discussed. We also offer a possible explanation for the origins and dispersal of USUV, JEV, and MVEV out of Africa.

Tick-borne encephalitis (TBE) is one of the most dangerous human infections occurring in Europe and many parts of Asia. The etiological agent Tick-borne encephalitis virus (TBEV), is a member of the virus genus Flavivirus, of the family Flaviviridae. TBEV is believed to cause at least 11,000 human cases of encephalitis in Russia and about 3000 cases in the rest of Europe annually. Related viruses within the same group, Louping ill virus (LIV), Langat virus (LGTV) and Powassan virus (POWV), also cause human encephalitis but rarely on an epidemic scale. Three other viruses within the same group, Omsk hemorrhagic fever virus (OHFV), Kyasanur Forest disease virus (KFDV) and Alkhurma virus (ALKV), are closely related to the TBEV complex viruses and tend to cause fatal hemorrhagic fevers rather than encephalitis. This review describes the clinical manifestations associated with TBEV infections, the main molecular-biological properties of these viruses, and the different factors that define the incidence and severity of disease. The role of ticks and their local hosts in the emergence of new virus variants with different pathogenic characteristics is also discussed. This review also contains a brief history of vaccination against TBE including trials with live attenuated vaccine and modern tendencies in developing of vaccine virus strains.

Arboviruses are transmitted by distantly related arthropod vectors such as mosquitoes (class Insecta) and ticks (class Arachnida). RNA interference (RNAi) is the major antiviral mechanism in arthropods against arboviruses. Unlike in mosquitoes, tick antiviral RNAi is not understood, although this information is important to compare arbovirus/host interactions in different classes of arbovirus vectos. Using an Ixodes scapularis-derived cell line, key Argonaute proteins involved in RNAi and the response against tick-borne Langat virus (Flaviviridae) replication were identified and phylogenetic relationships characterized. Analysis of small RNAs in infected cells showed the production of virus-derived small interfering RNAs (viRNAs), which are key molecules of the antiviral RNAi response. Importantly, viRNAs were longer (22 nucleotides) than those from other arbovirus vectors and mapped at highest frequency to the termini of the viral genome, as opposed to mosquito-borne flaviviruses. Moreover, tick-borne flaviviruses expressed subgenomic flavivirus RNAs that interfere with tick RNAi. Our results characterize the antiviral RNAi response in tick cells including phylogenetic analysis of genes encoding antiviral proteins, and viral interference with this pathway. This shows important differences in antiviral RNAi between the two major classes of arbovirus vectors, and our data broadens our understanding of arthropod antiviral RNAi.

Biotic factors contributing to the survival of tick-borne viruses in nature are poorly understood. Using tick-borne encephalitis virus (TBEV) and its principal European vector, Ixodes ricinus, we examined the relative roles of salivary gland infection, co-feeding transmission, and moulting in virus survival. Virus titres in the salivary glands increased after blood-feeding in a time- and dose-dependent manner. This was observed in ticks infected by inoculation but not in ticks infected by the natural route of co-feeding. Amplification of infection prevalence occurred via co-feeding. However, when larvae or nymphs subsequently moulted, the infection prevalence dramatically declined although this was not observed when ticks were infected by inoculation. Trans-stadial survival is a hitherto overlooked parameter that may contribute to the low incidence of TBEV infection in field-collected I. ricinus ticks.

Ixodes ricinus ticks were collected from dragging vegetation and from shot roe deer in the province of Trento and Belluno in northern Italy. Ticks were pooled for analyses and from 1060 pools of ticks collected in the province of Belluno and 12 390 tick samples collected in Trentino, four proved positive by immunofluorescence microscopy using a tick-borne encephalitis (TBE)-specific antiserum. The identity of the virus isolates was determined by RT-PCR cycle sequencing and they were all found to be closely similar (> 98% nucleotide identity) to typical western European TBE complex viruses as found in Austria. The isolates from Trentino differed from the Neudorfl strain of western European TBE virus at eight nucleotide positions but as these nucleotide substitutions were all synonymous, there were no amino acid changes. These results imply that the virus isolates in Trentino have changed slightly from the typical European strains isolated in nearby Austria. The abundance of questing ticks and ticks feeding on roe deer was greater in TBE positive hunting districts than in hunting districts where TBE complex viruses were only probable or believed to be absent. In TBE positive and probable districts synchrony in the seasonal dynamics of larvae and nymphs of I. ricinus was observed. This study provides evidence to suggest that roe deer may have an important role to play in the maintenance of tick density and in the persistence of TBE virus.

To date, tick-borne flaviviruses responsible for hemorrhagic fever in humans have been isolated in Siberia (Omsk hemorrhagic fever virus), India (Kyasanur Forest disease virus, KFDV), and in Saudi Arabia (Alkhurma virus, ALKV). Prior to this study, only partial coding sequences of these severe pathogens had been determined. We report here the complete coding sequence of ALK virus, which was determined to be 10,248 nucleotides (nt) long, and to encode a single 3,416 amino acid polyprotein. Independent analyses of the complete polyprotein and the envelope protein provided genetic and phylogenetic evidence that ALKV belongs to the tick-borne flavivirus group, within which it is most closely related to KFDV. Analysis of structural genes, genetic distances, and evolutionary relationship indicate that ALKV and KFDV derived from a common phylogenetic ancestor and constitute two genetic subtypes of the same virus species according to current genetic criteria of classification.

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_

Arthropod-borne viruses, that is, arboviruses, belong to different virus families and genera and are maintained through transmission between vertebrate hosts by virus-infected blood feeding arthropods (mosquitoes, sandflies, biting midges, and ticks). Several factors have contributed to the emergence and reemergence of arthropod-to-human infections in the last decade, also in nonendemic areas: climate changes which are believed to have contributed to increased dispersion of the virus vectors; the international trading system that transports an enormous range of goods, animals, and human beings all over the world, inadvertently increasing the possibility for vectors and viruses to spread; and the global increase of human population density, particularly in areas densely populated by arthropods with transmission capability for several different pathogenic viruses. As a consequence, the incidence of arboviral infections and diseases is increasing worldwide and therefore placing a heavier burden on public health agencies and requiring the need to ensure timely recognition and treatment of virus infections. This special issue is aimed at the presentation of recent results obtained by prominent colleagues in the field of human infections caused by vector-borne viruses. Faced with a limited number of manuscripts presented, the high quality of these papers will contribute significantly to our knowledge of these important issues. Pathogens, such as dengue virus, Japanese encephalitis virus, and alphaviruses are addressed by the authors. G. Anez and M. Rios described the epidemiology of what is likely to be the most widespread mosquito transmitted Flavivirus infection, that is, dengue fever, in the United States of America where this infection is currently generating huge concerns. In particular, the authors emphasize how, despite the current economic difficulties and restrictions, strict mosquito control policies and activities must be implemented and maintained in localities that have the potential to become the route of entry for viruses and the focus of epidemics. A second paper by C.-F. Liu and coworkers reported on the complementary role of clinical practice-based laboratory data in facilitating suspicion and diagnosis of dengue in clinical settings, thus ameliorating the overall diagnostic capabilities for this infection. Although the authors state that further studies are needed to confirm the validity of their approach, we believe that the study is important to define which procedures will have the most significant impact on clinical practice in tropical countries, where medical resources are limited. R. Rodriguez-Roche and E. A. Gould contributed a comprehensive review about the complex epidemiology and pathogenesis of the dengue viruses. They underlined the need for more effort to understand and accelerate the development of suitable vaccines and/or antiviral therapies for the control of this “scourge” of the tropical and subtropical world. Another paper by M. L. Munoz and coworkers is dedicated to the investigation of the interplay between dengue virus proteins and the receptor polypeptides located on the surface of mosquito cells, since this interaction is the fundamental basis for viral entry into susceptible cells, followed by development and transmission in the vectors. The paper by I.-K. Lee and coworkers provides an insight into the immunopathogenesis of dengue virus in patients with type 2 diabetes mellitus and reports on the ability of mononuclear cells from these patients to be infected by dengue virus. The aim of the paper is to fill a gap in our knowlwdge that concerns epidemiological data and immunological findings of frequent dengue haemorrhagic fever and Th2 cytokines in patients with diabetes mellitus. The authors demonstrated that in vitro infection of mononuclear cells from diabetes mellitus patients induces higher levels of both IL-4 and the anti-inflammatory Th2-cytokine IL-10, in addition to increased levels of GM-CSF, and they speculated that these findings might result from a counterbalance to the comparatively highly activated proinflammatory cytokines/chemokines in the diabetic hosts. Two papers describe different aspects of Japanese encephalitis virus (JEV) related diseases. The paper by G. Kakoti and coworkers reports on the clinical profile and the outcome of Japanese encephalitis virus infection in children up to 12 years old. This is a prospective study based on serological assays and it shows some points of interest from a clinical and epidemiological point of view as it investigates the presence of clinical or technical factors likely to be significantly correlated with increased mortality by JEV in children with acute encephalitis syndrome in North East India. B. Bandyopadhyay and collaborators reported the epidemiology of JEV in West Bengal (India) showing that 22.76% and 5% of the acute encephalitis syndrome cases were positive for JE IgM in 2011 and 2012, respectively, and that JE is mainly prevalent in children and adolescents below 20 years of age with no gender predilection. This is a significant observation from an epidemiological point of view, which shows how active surveillance of JE cases is still warranted. It also emphasizes the need for vigilance to identify the introduction of new genotypes in the endemic areas or to detect evidence of dispersion into newer geographical districts. This series of papers is completed by a paper by I. Assuncao-Miranda that describes the molecular mechanisms involved in the pathogenesis of alphavirus-induced arthritis, the paper by A. Burgueno and coworkers that investigated the seroprevalence of Saint Louis encephalitis related antibodies among horses in Uruguay, where no sharp epidemiological data about this virus were available so far, and the article by G. Sautto and collaborators that speculated about the possible use of candidate monoclonal antibodies to be possibly used in a future passive immunotherapy for arthropod-borne infections. This approach seems of particular importance, given the current absence of specific antiviral drugs as well as effective vaccines for the most diffused arbovirus infections.

Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. The flavivirus genus is characterized by a positive-sense single-stranded RNA genome. The unique open reading frame of the viral RNA is transcribed and translated as a single polyprotein which is post-translationally cleaved to yield three structural and seven nonstructural proteins, one of which is the NS3 gene that encodes a C-terminal helicase domain consisting of 431 amino acids. Helicase inhibitors are potential antiviral drugs as the helicase is essential to viral replication. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P3(1)21 (or P3(2)21), with unit-cell parameters a = 88.6, c = 138.6 A, and exhibit a diffraction limit of 2.3 A.

Rapid and precise virus detection procedures are an important component of any epizootiological study. An automated one tube reverse transcriptase and nested primer polymerase chain reaction (RT-PCR) followed by nucleotide sequencing of the cDNA product, was used for the rapid detection and identification of louping ill (LI) virus in field caught Ixodes ricinus and compared with a classical isolation method i.e. infectivity in cell culture. The results establish the genetic identity of LI virus on the Lochindorb Estate. There was a high correlation between the results obtained by RT-PCR and infectivity assays. RT-PCR and sequencing proved to be a rapid and accurate system for identifying LI virus in field specimens. Development of this system should improve the capacity to undertake detailed epizootiological studies of LI virus.

Most of the data on oral infection of ticks by louping ill virus have been obtained from experiments in which animals were infected by syringe inoculation with infectious material. Using infected ticks to mimic the natural situation, we have demonstrated that louping ill (LI) virus transmission can occur from infected to uninfected Ixodes ricinus feeding in close proximity on mountain hares (Lepus timidus). Under these conditions the hares developed either low or undetectable viraemias. Highest prevalence of LI virus infection was observed in recipient nymphs which had fed to repletion between days 3 and 7 post-attachment of virus-infected adults; following engorgement, 56% of nymphs acquired virus. These results demonstrate the efficient transmission of LI virus between co-feeding ticks on naive mountain hares. However, when ticks were allowed to co-feed on virus-immune hares a significant reduction in the frequency of infection was observed. Neither red deer (Cervus elaphus) nor New Zealand White rabbits supported transmission of LI virus. The significance of virus transmission between cofeeding ticks on LI virus epidemiology is discussed.

Highlights • Chikungunya fever is caused by a mosquito-borne alphavirus originating in East Africa. • During the past 7 years, the disease has spread to islands of the Indian Ocean, Asia and Europe. • Its spread has been facilitated by a mutation favouring replication in the mosquito Ae. albopictus. • No vaccines or antiviral drugs are available to prevent or treat chikungunya fever. • This paper provides an extensive review of the virus and disease, including Supplementary Tables., Chikungunya virus (CHIKV) is the aetiological agent of the mosquito-borne disease chikungunya fever, a debilitating arthritic disease that, during the past 7 years, 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.

Traditionally, the four dengue virus serotypes have been associated with fever, rash, and the more severe forms, haemorrhagic fever and shock syndrome. As our knowledge as well as understanding of these viruses increases, we now recognise not only that they are causing increasing numbers of human infections but also that they may cause neurological and other clinical complications, with sequelae or fatal consequences. In this review we attempt to highlight some of these features in the context of dengue virus pathogenesis. We also examine some of the efforts currently underway to control this “scourge” of the tropical and subtropical world.

West Nile virus (WNV), a mosquito-borne flavivirus in the Japanese encephalitis antigenic group, has caused sporadic outbreaks in humans, horses and birds throughout many of the warmer regions of Europe for at least 20 years. Occasional cases of West Nile encephalitis have also been associated with infected blood transfusions and organ donations. Currently, WNV appears to be expanding its geographical range in Europe and causing increasing numbers of epidemics/outbreaks associated with human morbidity and mortality. This brief review reports on the current epidemic situation regarding WNV in Europe, highlighting the clinical, diagnostic and preventive measures available for controlling this apparently emerging human pathogen.

The mode and tempo of arbovirus evolution and dispersal can help to explain the dynamics of pandemics, viral outbreaks, and emerging viruses. By comparing nucleotide and deduced amino acid sequences of their envelope proteins, we describe the continuous distribution of the tick-borne encephalitis (TBE) complex viruses, the most important flaviviruses in Europe, across major geographical areas and the conditions under which mutations occur The analyses reveal a correlation between the geographical and genetic distances of these viruses, The arthropod host appears to be a key factor for the formation and maintenance of this cline by constraining TBE dispersal and evolution. This is also illustrated by comparisons with mosquito-borne flaviviruses.

Duck egg-drop syndrome virus (DEDSV) is a newly emerging pathogenic flavivirus causing avian diseases in China. The infection occurs in laying ducks characterized by a severe drop in egg production with a fatality rate of 5–15 %. The virus was found to be most closely related to Tembusu virus (TMUV), an isolate from mosquitoes in South-east Asia. Here, we have sequenced and characterized the full-length genomes of seven DEDSV strains, including the 5′- and 3′-non-coding regions (NCRs). We also report for the first time the ORF sequences of TMUV and Sitiawan virus (STWV), another closely related flavivirus isolated from diseased chickens. We analysed the phylogenetic and antigenic relationships of DEDSV in relation to the Asian viruses TMUV and STWV, and other representative flaviviruses. Our results confirm the close relationship between DEDSV and TMUV/STWV and we discuss their probable evolutionary origins. We have also characterized the cleavage sites, potential glycosylation sites and unique motifs/modules of these viruses. Additionally, conserved sequences in both 5′- and 3′-NCRs were identified and the predicted secondary structures of the terminal sequences were studied. Antigenic cross-reactivity comparisons of DEDSV with related pathogenic flaviviruses identified a surprisingly close relationship with dengue virus (DENV) and raised the question of whether or not DEDSV may have a potential infectious threat to man. Importantly, DEDSV can be efficiently recognized by a broadly cross-reactive flavivirus mAb, 2A10G6, derived against DENV. The significance of these studies is discussed in the context of the emergence, evolution, epidemiology, antigenicity and pathogenicity of the newly emergent DEDSV.

Louping ill virus isolates from Great Britain, Ireland and Norway were compared antigenically by indirect immunofluorescence, haemagglutination-inhibition and neutralization tests using a panel of five envelope-specific and five non-structural protein NS1-specific monoclonal antibodies raised against louping ill virus. The viruses were grouped according to their reactivities with the antibodies. Group 1, members of which were isolated between 1931 and 1987, consisted of 13 viruses that reacted with all antibodies, whereas group 2, members of which were isolated after 1980, consisted of five viruses that were positive with only eight of the 10 monoclonal antibodies. The two monoclonal antibodies that did not react with the group 2 viruses are known to be neutralizing antibodies and the amino acids that they recognize in the viral envelope protein have been identified. We therefore refer to the group 2 viruses as naturally occurring monoclonal antibody escape variants. When compared with group 1 viruses, the escape variants showed reduced virulence for mice in terms of the time taken to kill and/or the proportion that died, following intraperitoneal inoculation. The nucleotide and deduced amino acid sequences of the envelope gene of one escape variant were compared with those of several group 1 viruses. A single amino acid substitution at residue 308 was detected in the envelope protein of the escape variant which corresponds precisely to the position in experimentally selected attenuated monoclonal antibody escape mutants. The importance and potential implications of these naturally occurring variants in louping ill epizootiology and vaccine-based control are discussed.

We have carried out an antigenic analysis and nucleotide sequence comparison of the envelope glycoprotein of recognized louping ill virus strains isolated from Scotland with that of a Norwegian virus known to cause encephalomyelitis in sheep. Monoclonal antibodies with defined specificity for the louping ill virus envelope glycoprotein failed to distinguish between the Norwegian virus and prototype louping ill virus in indirect immunofluorescence, haemagglutination inhibition and neutralization tests. Nucleotide sequencing of the envelope glycoprotein and alignment of the deduced amino acid sequence with other known sequences revealed that the Norwegian virus closely resembles (> 95% identity for nucleotide and > 98% identity for amino acid sequences) louping ill virus. Maximum variation in identities among four strains of louping ill virus were 4.4% and 1.8% respectively for nucleotide and amino acid alignments. We conclude that sheep encephalomyelitis in Norway is caused by louping ill virus. These results imply that other viruses present in Europe and known to cause encephalitis/encephalomyelitis of sheep could be caused by louping ill virus.

The alphaviruses were amongst the first arboviruses to be isolated, characterized and assigned a taxonomic status. They are globally very widespread, infecting a large variety of terrestrial animals, insects and even fish, and circulate both in the sylvatic and urban/peri-urban environment, causing considerable human morbidity and mortality. Nevertheless, despite their obvious importance as pathogens, there are currently no effective antiviral drugs with which to treat humans or animals infected by any of these viruses. The EU-supported project VIZIER (Comparative Structural Genomics of Viral Enzymes Involved in Replication, FP6 Project: 2004-511960) was instigated with an ultimate view of contributing to the development of antiviral therapies for RNA viruses, including the alphaviruses [Coutard, B., Gorbalenya, A.E., Snijder, E.J., Leontovich, A.M., Poupon, A., De Lamballerie, X., Charrel, R., Gould, E.A., Gunther, S., Norder, H., Klempa, B., Bourhy, H., Rohayemj, J., L'hermite, E., Nordlund, P., Stuart, D.I., Owens, R.J., Grimes, J.M., Tuckerm, PA., Bolognesi, M., Mattevi, A., Coll, M., Jones, TA., Aqvist, J., Unger, T., Hilgenfeld, R., Bricogne, G., Neyts, J., La Colla, P., Puerstinger, G., Gonzalez, J.P., Leroy, E., Cambillau, C., Romette, J.L., Canard, B., 2008. The VIZIER project: preparedness against pathogenic RNA viruses. Antiviral Res. 78, 37-46]. This review highlights some of the major features of alphaviruses that have been investigated during recent years. After describing their classification, epidemiology and evolutionary history and the expanding geographic distribution of Chikungunya virus, we review progress in understanding the structure and function of alphavirus replicative enzymes achieved under the VIZIER programme and the development of new disease control strategies. (C) 2009 Elsevier B.V. All rights reserved.

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.

Louping ill virus, a tick-borne arbovirus readily established a persistent infection in porcine kidney (PS) cells after initially inducing minor cytopathic changes. Nucleotide sequence analysis of the envelope glycoprotein of the viral RNA recovered from the persistently infected cells showed no changes as compared with the virus used to establish persistent infections. More than 80 per cent of the cells contained virus specific antigen when analysed by indirect immunofluorescence microscopy. This persistently infected cell line resisted superinfection with either homologous or most heterologous flaviviruses. However, the yellow fever French neurotropic virus (YF FNV) multiplied in the persistently infected cells and evidence of dual infections in these cells was obtained using specific monoclonal antibodies in double labelling immunofluorescence tests. The relevance of these observations is discussed in the light of other evidence that tick-borne viruses can survive for long periods in wild animal species.

Tick-borne encephalitis virus (TBEV) causes human epidemics across Eurasia. Clinical manifestations range from inapparent infections and fevers to fatal encephalitis but the factors that determine disease severity are currently undefined. TBEV is characteristically a hemagglutinating (HA) virus; the ability to agglutinate erythrocytes tentatively reflects virion receptor/fusion activity. However, for the past few years many atypical HA-deficient strains have been isolated from patients and also from the natural European host tick, Ixodes persulcatus. By analysing the sequences of HA-deficient strains we have identified 3 unique amino acid substitutions (D67G, E122G or D277A) in the envelope protein, each of which increases the net charge and hydrophobicity of the virion surface. Therefore, we genetically engineered virus mutants each containing one of these 3 substitutions; they all exhibited HA-deficiency. Unexpectedly, each genetically modified non-HA virus demonstrated increased TBEV reproduction in feeding Ixodes ricinus, not the recognised tick host for these strains. Moreover, virus transmission efficiency between infected and uninfected ticks co-feeding on mice was also intensified by each substitution. Retrospectively, the mutation D67G was identified in viruses isolated from patients with encephalitis. We propose that the emergence of atypical Siberian HA-deficient TBEV strains in Europe is linked to their molecular adaptation to local ticks. This process appears to be driven by the selection of single mutations that change the virion surface thus enhancing receptor/fusion function essential for TBEV entry into the unfamiliar tick species. As the consequence of this adaptive mutagenesis, some of these mutations also appear to enhance the ability of TBEV to cross the human blood-brain barrier, a likely explanation for fatal encephalitis. Future research will reveal if these emerging Siberian TBEV strains continue to disperse westwards across Europe by adaptation to the indigenous tick species and if they are associated with severe forms of TBE.

We analysed the complete coding sequences of all recognized species of Aedes-borne flavivirus, including previously uncharacterized viruses within the yellow fever virus (YFV), Spondweni virus (SPOV) and dengue virus (DENV) groups. Two major phylogenetic lineages were revealed: one included the YFV and Entebbe bat virus groups, and the other included the DENV, SPOV and Culex-borne flavivirus groups. This analysis supported previous evidence that Culex-borne flaviviruses have evolved from ancestral Aedes-borne viruses. However, the topology at the junction between these lineages remains complex and may be refined by the discovery of viruses related to the Kedougou virus. Additionally, viral evolution was found to be associated with the appearance of new biological characteristics; mutations that may modify the envelope protein structure were identified for seven viruses within the YFV group, and an expansion of host–vector range was identified in the two major evolutionary lineages, which in turn may facilitate the emergence of mosquito-borne flaviviruses.

Here we present a real-time one-step reverse transcriptase-polymerase chain reaction SYBR Green assay derived from the method reported by van Elden and collaborators ( 2001 ) that ensures the rapid, sensitive, and cost-effective detection of both seasonal influenza A virus and emerging (H1N1) swine-origin influenza A virus (S-OIV). In addition to this screening test, which successfully detected both seasonal influenza A virus and S-OIV in human clinical samples, we showed that the probe initially designed by van Elden and collaborators could detect seasonal influenza A virus, but not S-OIV; a new probe was designed and tested that specifically detects S-OIV, but not seasonal influenza A. Both probe-based assays were validated by testing human clinical samples and specifically detected either seasonal influenza virus or S-OIV. Finally, in silico analysis of databases predicted that minor modifications of the van Elden primers would facilitate the use of this assay for the broad spectrum detection of all currently characterized variants of influenza A virus, including avian strains.

Tick-borne encephalitis virus (TBEV; family Flaviviridae , genus Flavivirus ) is a complex of closely related arboviruses transmitted to humans and other vertebrates by infected Ixodes spp. ticks. The virions contain positive polarity single-stranded RNA (ssRNA) (∼ 11 kbp) enclosed in a core consisting of capsid protein surrounded by a lipid membrane containing envelope and membrane glycoproteins. Three-dimensional structural analysis has revealed the mechanism of the pH-dependent fusion between viral and endosomal membranes during entry into cells. After fusion, viral RNA is translated, producing a polyprotein of ∼ 3400 amino acids that is co-translationally processed into three structural and seven nonstructural proteins, the latter providing viral proteolytic and replicase functions. The mode of RNA replication is asymmetric and semiconservative and mediated by the interaction of 5′-and 3′-untranslated regions. Details of virion maturation have been resolved at the atomic level. These viruses circulate in the forests and on the sheep-rearing and goat-rearing hillsides of the Northern Hemisphere, using both viremic and nonviremic transmission strategies. The specific biological characteristics of different TBEVs and related viruses and their evolution and dispersal directly reflect the ecological requirements of the tick and its protracted life cycle. Human disease control in endemic regions relies primarily on the use of vaccines.

Severity of disease is markedly increased when infection with dengue virus type 2 follows infection with dengue virus type 2 by an interval of 20 years., Severity of disease is markedly increased when infection with dengue virus type 2 (DENV-2) follows infection with DENV-1. Studies have shown that heterologous neutralizing antibody titers are inversely correlated with severity of a second infection. If this mechanism controlled disease severity in Cuba, heterotypic antibody titers should have declined over time. To determine whether phenotypic changes in dengue antibodies occur over time, we analyzed serum samples collected 4–8 and 20–22 years after DENV-1 infection. We found a significant increase in mean titer of homologous DENV-1 neutralizing antibodies and a significant decrease in heterologous antibodies to 1 of 2 genotypes of DENV-2 virus (the American genotype). Asian DENV-2 viruses were not neutralized during either interval; however, the American genotype underwent phenotypic changes in heterotypic viral neutralizing antibodies in the predicted direction. This finding may be related to the time-dependent changes in severity of disease found with secondary dengue infection.

Over 40 cell lines are currently available from 13 ixodid and one argasid tick species. The successful isolation and propagation of several economically important tick-borne pathogens in tick cell lines has created a useful model to study interactions between tick cells and these viral and bacterial disease agents. Tick cell lines have already proved to be a useful tool in helping to define the complex nature of the host–vector–pathogen relationship. With the availability of genomics tools, tick cell lines will become increasingly important as a complement to tick and tick-borne disease research in vivo once genetic transformation and gene silencing using RNA interference become routine.

Here, we analyze the complete coding sequences of all recognized tick-borne flavivirus species, including Gadgets Gully, Royal Farm and Karshi virus, seabird-associated flaviviruses, Kadam virus and previously uncharacterized isolates of Kyasanur Forest disease virus and Omsk hemorrhagic fever virus. Significant taxonomic improvements are proposed, e.g. the identification of three major groups (mammalian, seabird and Kadam tick-borne flavivirus groups), the creation of a new species (Karshi virus) and the assignment of Tick-borne encephalitis and Louping ill viruses to a unique species (Tick-borne encephalitis virus) including four viral types (i.e. Western Tick-borne encephalitis virus, Eastern Tick-borne encephalitis virus, Turkish sheep Tick-borne encephalitis virus and Louping ill Tick-borne encephalitis virus). The analyses also suggest a complex relationship between viruses infecting birds and those infecting mammals. Ticks that feed on both categories of vertebrates may constitute the evolutionary bridge between the three distinct identified lineages.

To the Editor: In 1994, dengue virus type 3 (DENV-3) was reintroduced into Latin America after an absence of 17 years, and was isolated almost simultaneously in Nicaragua and Panama. In September 2000, DENV-3 was isolated for the first time in Cuba, producing a small outbreak that affected 3 areas of Havana City. In total, 138 cases of dengue fever were confirmed, with either DENV-3 or DENV-4 as the etiologic agents. No dengue hemorrhagic fever cases were observed, and the outbreak was brought under control within 6 weeks (1). At the end of June 2001, dengue was again reported in Havana. Through the end of February 2002, a total of 14,443 dengue cases were reported for the entire country, including 81 cases of dengue hemorrhagic fever and 3 fatalities (2). To understand the molecular epidemiology of DENV-3 in Cuba, and particularly to determine whether the 2000 and 2001 outbreaks were caused by the same viral genotype, the complete envelope (E) gene sequences of isolates from both outbreaks were determined. To assist in this analysis, we also sequenced a DENV-3 strain representing the 1994 Nicaraguan epidemic, the first one isolated in Latin America in 1994 (3). Three DENV-3 viruses isolated during the 2000–2002 Cuban outbreaks were studied. Two were obtained from acute-phase sera and the other from a spleen section sample from a patient who died. Serum samples and macerated spleen fragments were spread onto C6/36 mosquito cells (grown at 33°C) by using the rapid centrifugation assay (4). Viral RNA was extracted from 200 μL of supernatant medium of virus-infected cells by using the RNAgents Total RNA Isolation system (Promega, Madison, WI, USA). The E gene was amplified by using reverse transcription–polymerase chain reaction (RT-PCR) as described previously (5). Double-stranded sequencing of the E gene was conducted on an ABI sequencer according to the manufacture's protocol (Applied Biosystems, Foster City, CA, USA). All sequences determined in this study have been deposited in GenBank (accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"AY702030-AY702033","start_term":"AY702030","end_term":"AY702033","start_term_id":"56808993","end_term_id":"56808999"}}AY702030-AY702033). The E gene sequence of the 3 Cuban DENV-3 isolates and the single strain from Nicaragua isolated in 1994 were aligned with the E gene sequences (1,479 bp in length) of 60 DENV-3 isolates deposited in GenBank, representing the global genetic diversity of DENV-3. Phylogenetic trees were produced with a maximum likelihood method incorporating the GTR+ G+I model of nucleotide substitution, with the general time-reversible (GTR) substitution matrix, the base composition, the gamma (Γ) distribution of among-site rate variation, and the proportion of invariant sites (I) all estimated from the data. To explore the robustness of particular phylogenetic groupings, a bootstrap resampling analysis was undertaken. All analyses were performed with the PAUP* package (6). The tree (Figure) showed 5 major groups or genotypes of DENV-3: 1) a Pacific/Asian group (genotype I); 2) an Asian group, containing a large array of viruses sampled from Thailand and Malaysia (genotype II); 3) a Latin American group that includes the Cuban viruses and isolates from Venezuela, Martinique, and Nicaragua, as well as those from Samoa, India, and Sri Lanka (genotype III); 4) a small group of Asian viruses (genotype IV); and 5) a final, most divergent group containing virus samples from Puerto Rico (genotype V). A similar genotype structure of DENV-3 has been observed in previous studies of this virus (5,7–9). The 3 Cuban viruses clearly fall into genotype III. Figure Maximum likelihood (ML) phylogenetic tree showing the evolutionary relationships among the E gene sequences of 64 strains of DENV-3. All branch lengths are drawn to scale, and tree was rooted using strains from the Puerto Rico 1960s epidemic, which always ... Indeed, strain D3.Nicaragua.24/94 falls at the base of the Latin American clade within genotype III, as would be expected if it was the first DENV-3 strain isolated from this region in 1994. Moreover, the most divergent strains from genotype III (with the exception of a single strain from Samoa) come from Asia, which suggests that this group of viruses was exported from Asia to Latin America in the early 1990s. Finally, the Cuban viruses fall into 2 distinct groups: the 116/00 strain, isolated during the 2000 outbreak, is very closely related to the Brazilian/Martinique viruses isolated in the same year, whereas strains 580/01 and 21/02, isolated in 2001–2002, appear to have diverged distinctly to form a separate cluster (99% bootstrap support). A total of 15 nucleotide differences were distributed throughout the E gene. Of these, 12 were synonymous and 3 were nonsynonymous substitutions resulting in amino acid changes (E19 Thr/Pro, E226 Ile/Thr, and E329 Ala/Val). Moreover, 2 different nucleotide substitutions were found for the 2 isolates obtained during the major outbreak (2001–2002). These substitutions resulted in a nonconservative amino acid change (E22 Asp/Val). The ABI chromatograms of these sequences showed populations in this position, but the predominant population for each strain differed. By using appropriate phylogenetic methods, deducing the most likely dispersal pattern for closely related dengue virus strains with different patterns of spatial and temporal sampling is possible. The current study strongly suggests that an Asian DENV-3 virus assigned to genotype III has evolved in situ in the Caribbean region since 1994. The nucleotide differences observed in the Cuban isolates support the hypothesis that DENV-3 was introduced twice into Cuba from the wider Latin American region. Further evidence to support this hypothesis is that during the interepidemic period, from January to June 2001, no immunoglobulin (Ig) M–positive samples from patients with fever and suspected dengue cases were observed, which suggests that the virus was not circulating. Therefore, these data are consistent with the idea that, rather than in situ evolution, a second introduction of the virus occurred in 2001. By comparing the amino acid sequences of the Cuban isolates with other DENV-3 strains assigned to genotype III, we confirmed that several distinct amino acid replacements had occurred. In particular, the nonconservative substitution Ala/Val was seen in the Nicaragua 1994, Mexico 1996, Cuba 2001–2002, and Venezuela 2001–2002 isolates. However, the Cuba 2000, Brazil 2000, and Martinique 1999–2001 isolates preserved the Ala at this position, as was also the case for viruses sampled from the putative Asian source (from India and Sri Lanka). The functional significance of amino acids at this position has not been determined.

Several human diseases in Europe are caused by viruses transmitted by tick bite. These viruses belong to the genus Flavivirus, and include tick-borne encephalitis virus, Omsk haemorrhagic fever virus, louping ill virus, Powassan virus, Nairovirus (Crimean-Congo haemorrhagic fever virus) and Coltivirus (Eyach virus). All of these viruses cause more or less severe neurological diseases, and some are also responsible for haemorrhagic fever. The epidemiology, clinical picture and methods for diagnosis are detailed in this review. Most of these viral pathogens are classified as Biosafety Level 3 or 4 agents, and therefore some of them have been classified in Categories A–C of potential bioterrorism agents by the Centers for Disease Control and Prevention. Their ability to cause severe disease in man means that these viruses, as well as any clinical samples suspected of containing them, must be handled with specific and stringent precautions.

It was recently reported that disease severity increased during the 1997 Cuban dengue 2 virus epidemic and it was suggested that this might be explained by the appearance of neutralization resistant escape mutants. We investigated these observations and ideas by sequencing 20 dengue 2 virus isolates obtained during the early (low case fatality rate) and the late (high case fatality rate) phases of the outbreak. Our results showed total conservation of the E gene sequence for these isolates suggesting that the selection of envelope gene escape mutants was not the determinant of increased disease severity. Alignment of these sequences with those available in GenBank, followed by Maximum likelihood phylogenetic analysis generated a tree, which indicated that our isolates are closely related to the virus that circulated in Venezuela in 1997/98 and subsequently in Martinique in 1998. This "American/Asian" genotype has therefore gradually dispersed across the Caribbean region during the past 5 years.

Context Dengue, Japanese encephalitis, tick-borne encephalitis, yellow fever, and West Nile viruses cause substantial morbidity and mortality each year. Modern transportation and the relaxation of mosquito-control measures are largely responsible for the increase of disease caused by flaviviruses. Without effective antiviral drugs, vaccination offers the best chance of decreasing the incidence of these diseases, and live virus vaccines are the most promising and cost effective. However, flaviviruses can recombine, which raises the possibility of recombination between a vaccine strain and wild-type virus resulting in a new virus with potentially undesirable properties. Starting point Recently, Arunee Sabchareon and colleagues reported up to 90% seroconversion rates in a phase I trial of live-attenuated dengue-virus vaccines in children (Pediatr Infect Dis J 2004; 23: 99-109). Other live flavivirus vaccines have also been tested against dengue, Japanese encephalitis, and West Nile viruses. Thus far, efficacy seems promising. Where next Safety issues with the live flavivirus vaccines need to be recognised and addressed. The theoretical possibility of untoward recombination events can never be entirely dismissed, but steps can be taken to minimise risk. The development of non-live flavivirus vaccines should be encouraged.