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Tamana bat virus (TABV, isolated from the batPteronotus parnellii) is currently classified as a tentative species in the genusFlavivirus. We report here the determination and analysis of its complete coding sequence. Low but significant similarity scores between TABV and member-viruses of the genusFlaviviruswere identified in the amino acid sequences of the structural, NS3 and NS5 genes. A series of cysteines located in the envelope protein and the most important enzymatic domains of the virus helicase/NTPase, methyltransferase and RNA-dependent RNA polymerase were found to be highly conserved. In the serine-protease domain, the catalytic sites were conserved, but variations in sequence were found in the putative substrate-binding sites, implying possible differences in the protease specificity. In accordance with this finding, the putative cleavage sites of the TABV polyprotein by the virus protease are substantially different from those of flaviviruses. The phylogenetic position of TABV could not be determined precisely, probably due to the extremely significant genetic divergence from other member-viruses of the familyFlaviviridae. However, analysis based on both genetic distances and maximum-likelihood confirmed that TABV is more closely related to the flaviviruses than to the other genera. These findings have implications for the evolutionary history and taxonomic classification of the family as a whole: (i) the possibility that flaviviruses were derived from viruses infecting mammals rather than from mosquito viruses cannot be excluded; (ii) using the current criteria for the definition of genera in the familyFlaviviridae, TABV should be assigned to a new genus.

Millions of domestic and wild European rabbits (Oryctolagus cuniculus) have died in Europe, Asia, Australia and New Zealand during the past 17 years following infection by Rabbit haemorrhagic disease virus (RHDV). This highly contagious and deadly disease was first identified in China in 1984. Epidemics of RHDV then radiated across Europe until the virus apparently appeared in Britain in 1992. However, this concept of radiation of a new and virulent virus from China is not entirely consistent with serological and molecular evidence. This study shows, using RT–PCR and nucleotide sequencing of RNA obtained from the serum of healthy rabbits stored at 4 °C for nearly 50 years, that, contrary to previous opinions, RHDV circulated as an apparently avirulent virus throughout Britain more than 50 years ago and more than 30 years before the disease itself was identified. Based on molecular phylogenetic analysis of British and European RHDV sequences, it is concluded that RHDV has almost certainly circulated harmlessly in Britain and Europe for centuries rather than decades. Moreover, analysis of partial capsid sequences did not reveal significant differences between RHDV isolates that came from either healthy rabbits or animals that had died with typical haemorrhagic disease. The high stability of RHDV RNA is also demonstrated by showing that it can be amplified and sequenced from rabbit bone marrow samples collected at least 7 weeks after the animal has died.

The Flavivirus genus in the family Flaviviridae, is composed of approximately 70 antigenically related, positive-stranded RNA viruses (Chambers et al. 1990; Kuno et al. 1998). Kunjin (KUN) and West Nile (WN) viruses are mosquito-borne viruses that belong to the Japanese encephalitis (JE) antigenic complex of the Flavivirus genus (Heinz et al. 2000). Clinical symptoms most commonly associated with human infection with KUN (Mackenzie et al. 1993; Phillips et al. 1992; see chapter by Hall et al., this volume) and WN viruses (Monath and Heinz 1996; Cernescu et al. 2000) include febrile illness, often with myalgia, or mild to moderately severe encephalitis. However, WN virus has also been associated with fatal cases of acute meningoencephalitis and fulminant hepatitis (Monath and Heinz 1996; Cernescu et al. 2000). Both viruses are maintained in a natural transmission cycle involving mosquito vectors and bird reservoir hosts with humans and horses believed to be incidental hosts (Hayes 1988; Marshall 1988; see chapters by Hall et al. and McLean et al., this volume). There are several comprehensive reviews on the ecology and epidemiology of WN and KUN viruses (see Hayes 1988; Marshall 1988; Hubalek and Halouzka 1999; see also chapters by Hall et al., Malkinson and Banet, and Roehrig et al., this volume).

Viruses in the Japanese encephalitis (JE) serocomplex, are geographically widely dispersed. They display a variety of epidemiological and ecological characteristics that depend largely on the vertebrate and invertebrate hosts with which they have become associated following divergence and dispersal from their presumed African or Asian ancestors. As will be shown, molecular phylogenetic analysis of these viruses indicates that they are monophyletic and strongly suggests that their evolutionary lineage appeared after the first divergence of the Aedes spp.-associated mosquito-borne flaviviruses. In this sense therefore, they can be considered recent descendants of the Aedes spp.-associated viruses, and descriptions of their dissemination worldwide need to take this into account. Moreover, separate phylogenies based on different genes of a single species are not always congruent implying the possibility of genetic recombination within these species. This paper will explore the evolutionary relationships of these viruses, their origins and their possible dispersal patterns after diverging from the Aedes spp.-associated mosquito-borne flaviviruses.

Japanese encephalitis (JE) is arguably one of the most serious viral encephalitis diseases worldwide. China has a long history of high prevalence of Japanese encephalitis, with thousands of cases reported annually and incidence rates often exceeding 15/100,000. In global terms, the scale of outbreaks and high incidence of these pandemics has almost been unique, placing a heavy burden on the Chinese health authorities. However, the introduction of vaccines, developed in China, combined with an intensive vaccination program initiated during the 1970s, as well as other public health interventions, has dramatically decreased the incidence from 20.92/100,000 in 1971, to 0.12/100,000 in 2011. Moreover, in less readily accessible areas of China, changes to agricultural practices designed to reduce chances of mosquito bites as well as mosquito population densities have also been proven effective in reducing local JE incidence. This unprecedented public health achievement has saved many lives and provided valuable experience that could be directly applicable to the control of vector-borne diseases around the world. Here, we review and discuss strategies for promotion and expansion of vaccination programs to reduce the incidence of JE even further, for the benefit of health authorities throughout Asia and, potentially, worldwide.

Louping-ill (LI) is a tick-borne viral disease of red grouse, Lagopus lagopus scoticus Lath. (Tetraonidae: Galliformes), and sheep, Ovis aries L. (Bovidae: Artiodactyla), that causes economic loss to upland farms and sporting estates. Unvaccinated sheep, grouse and mountain hares, Lepus timidus L. (Leporidae: Lagomorpha), are known to transmit LI virus, whereas red deer, Cenrus elaphus L. (Cervidae: Artiodactyla), and rabbits, Oryctolagus cuniculus L. (Leporidae: Lagomorpha), do not. However, the role of small mammals is unknown. Here, we determine the role of small mammals, in particular field voles, Microtus agrestis L. (Muridae: Rodentia), in the persistence of LI virus on upland farms and sporting estates in Scotland, using field sampling and non-viraemic transmission trials. Small mammals were not abundant on the upland sites studied, few ticks were found per animal and none of the caught animals tested seropositive to LI virus. Laboratory trials provided no evidence that small mammals (field voles, bank voles, Clethrionomys glareolus L. (Muridae: Rodentia), and wood mice, Apodemus sylvaticus L. (Muridae: Rodentia), can transmit LI virus between cofeeding ticks and, in the field, LI virus was prevalent only in areas with known LI virus competent hosts (grouse, mountain hares or unvaccinated sheep) and absent elsewhere. In contrast to the case of tick-borne encephalitis (TBE) virus in Europe, it is concluded that small mammals seem to be relatively unimportant in LI virus persistence.

Viruses exhibit a wide variety of structural and chemical differences, but, in general, their infectivity may be destroyed by degradative enzymes that destroy nucleic acids, by detergents that solubilize the lipid-containing envelopes thus exposing the nucleic acid, by temperatures higher than about 50 degrees C, or by chemicals that breakdown capsid proteins. Preserving the viruses at low or ultra-low temperatures, and/or in the absence of water, slows down these destructive processes sufficiently to increase significantly the length of time that the virus can be stored as infectious material. Supplements such as serum are presumed to stabilize the environmental conditions and to block degradative processes. The methods by which viruses may be preserved for long periods of time are similar to those employed for other microorganisms and are relatively simple. Nevertheless, attention to detail, good laboratory practice, aseptic technique, meticulous recordkeeping, and regular monitoring of the stored materials will increase the success rate and reduce problems of contamination or loss in the storage containers, where many different viruses may be stored for posterity! This article describes some of the simplest and most reliable storage procedures for viruses, but the author recognizes that everyone will have a favorite method to suit his or her own particular virus.

A genetic algorithm-based RNA secondary structure prediction was combined with comparative sequence analysis to construct models of folding for the distal 380 nucleotides of the 3'-untranslated region (3'-UTR) of yellow fever virus (YFV). A number of structural elements that are thermodynamically stable, conserved in shape, and confirmed by compensatory mutations were revealed. At the same time structural polymorphisms were observed among strains of YFV. These polymorphisms showed an association with virulence: all wild and pathogenic strains were likely to be folded in a significantly different way from vaccine strains with reduced virulence. Structural divergence was also found among vaccine strains, with 17DD, the most virulent in the mouse model, exhibiting an intermediate pattern of folding, combining structural features of both wild and vaccine strains. The observation of a strong association between secondary structure of the 3'-UTR and virulence of YFV may help elucidate the molecular mechanisms of virus attenuation and lead to new strategies of vaccine development directed towards rational modification of secondary structure of the 3'-UTR.

Genetic algorithm-based RNA secondary structure prediction was used in combination with comparative sequence analysis to construct models of folding for the distal part of the 3'-untranslated region of flaviviruses belonging to four serological groups. Elements of RNA secondary structure that are preserved among all the flaviviruses studied were revealed, despite the high degree of sequence divergence between them. At the same time, structural elements were observed that distinguish members of different serological groups and, in particular, a region of remarkable structural divergence between the tick-borne and mosquito-borne flaviviruses was found. Application of the genetic algorithm also revealed that the 3'-terminus of flaviviral genomic RNA may take on alternative conformations, which are not observed in the 3'-terminus of complementary minus strand RNA. These alternative folding patterns may have roles in the regulation of transcription and translation initiation and in the switch between them.

Usutu virus (USUV) is a mosquito-borne flavivirus, belonging to the Japanese encephalitis antigenic complex, that circulates among mosquitoes and birds. We describe and analyze the complete genome sequence of the first USUV strain isolated from an immunocompromised patient with neuroinvasive disease. This USUV isolate showed an overall nucleotide identity of 99% and 96%, respectively, with the genomes of isolates from Europe and Africa. Comparison of the human USUV complete polyprotein sequence with bird-derived strains, showed two unique amino acid substitutions. In particular, one substitution (S595G) was situated in the DIII domain of the viral Envelope protein that is recognized by flavivirus neutralizing antibodies. An additional amino acid substitution (D3425E) was identified in the RNA-dependent RNA polymerase (RdRp) domain of the NS5 protein. This substitution is remarkable since E3425 is highly conserved among the other USUV isolates that were not associated with human infection. However, a similar substitution was observed in Japanese encephalitis and in West Nile viruses isolated from humans. Phylogenetic analysis of the human USUV strain revealed a close relationship with an Italian strain isolated in 2009. Analysis of synonymous nucleotide substitutions (SNSs) among the different USUV genomes showed a specific evolutionary divergence among different countries. In addition, 15 SNSs were identified as unique in the human isolate. We also identified four specific nucleotide substitutions in the 5′ and 3′ untranslated regions (UTRs) in the human isolate that were not present in the other USUV sequences. Our analyses provide the basis for further experimental studies aimed at defining the effective role of these mutations in the USUV genome, their potential role in the development of viral variants pathogenic for humans and their evolution and dispersal out of Africa.

The phylogeny of 123 complete envelope gene sequences was reconstructed in order to understand the evolution of tick- and mosquito-borne flaviviruses. An analysis of phylogenetic tree structure reveals a continual and asymmetric branching process in the tick-borne flaviviruses, compared with an explosive radiation in the last 200 years in viruses transmitted by mosquitoes. The distinction between these two viral groups probably reflects differences in modes of dispersal, propagation, and changes in the size of host populations. The most serious implication of this work is that growing human populations are being exposed to an expanding range of increasingly diverse viral strains.

Construction of infectious clones of flaviviruses can be problematic owing to instability, toxicity, and recombination events occurring while cloning cDNA in the bacterial vectors. To overcome these difficulties we have devised a rapid and simple method for producing an infectious genetically engineered tick-borne encephalitis virus in less than 10 days using viral RNA from an unpurified virus suspension. The experimental protocol utilized the high fidelity reverse transcription-polymerase chain reaction to produce two long (5.7 and 5.2 kb) overlapping cDNA segments. To produce full-length cDNA the two overlapping segments were either ligated or fused by polymerase chain reaction. The cDNA was then transcribed and the derived full-length RNA was injected intracerebrally into young mice which reproduced the infectious virus within 8–20 days. To differentiate the engineered virus from parent virus, aSunI restriction site was introduced by substituting nucleotides at positions 5688 and 5691 of the viral genome. This restriction site was present in the engineered virus recovered from infected mice. Antigenic and electrophoretic analysis of the proteins recovered from the engineered virus confirmed that it was indistinguishable from parent virus. In addition to its applicability as a rapid method of producing infectious engineered virus, this protocol offers the opportunity to introduce changes by site-directed mutagenesis without needing to clone the viral DNA. The method should be applicable to most viruses possessing an infectious RNA molecule and reduces the time required to produce a genetically engineered virus from years to days. When appropriate, the choice of mice for transfection of RNA has the advantage of being extremely simple, very sensitive, and producing high titers of stable virus.

We have constructed a recombinant baculovirus containing the gene encoding the structural protein VP60 from the Spanish field isolate AST/89 of rabbit haemorrhagic disease virus (RHDV). Infection of cultured Spodoptera frugiperda Sf9 cells with this recombinant virus resulted in the production of high yields of VP60 protein which did not seem to assemble to form virus like particles, but was antigenically similar to the corresponding viral protein obtained from purified virions. A VP60-dose study showed that the recombinant protein was able to elicit a protective response in rabbits against a nasal challenge with 100 LD 50 of RHDV. The effective dose able to protect 50% of the animals in the absence of adjuvant was found to be 10–25 μg of recombinant VP60.

Studies on the molecular basis of flavivirus neutralisation, attenuation and tropism indicate that amino acid substitutions, in different parts of the envelope gene, may be responsible for the altered phenotypes. However, the association of particular substitutions with individual characteristics has proven difficult. Comparative analysis of all known tick-borne flavivirus envelope proteins through sequence alignment and a sliding window, reveals clusters of amino acid variation distributed throughout the envelope protein coding region. Further comparison with mosquito-borne flaviviruses reveals essentially the same profile of variability throughout the envelope protein sequence although there is a major difference within the postulated B domain of these viruses which may reflect their different evolutionary development. Most phenotypically variant properties, such as serotypic differences, variants characteristic of vaccine strains, altered tropisms and neutralisation escape mutants, map within the variable clusters. Thus, we propose that natural mutagenesis and selection may occur at specific sites that do not destroy the secondary and tertiary E protein structure and that the variable clusters represent the exposed surface amino acids of the envelope protein defining antigenicity, tropicity and pathogenesis.

The nucleotide and deduced amino acid sequence of the envelope (E) gene of Tyuleniy (TYU) and Saumarez Reef (SRE) virus have been determined and the data used to classify these viruses in relation to the other tick- and mosquito-borne viruses in the genus Flavivirus. The phylogenetic trees obtained by maximum parsimony and distance methods for 22 flavivirus E genes showed that TYU and SRE virus are a sister group of the TBE virus complex. The trees were consistent with the Flavivirus serological classification and are compatible with the proposition that Cell Fusing Agent could be another genus in the family Flaviviridae. Comparison of the phylogeny and mutational regime of the E gene with the RNA-dependent RNA polymerase (NS5) gene shows the validity of the E gene as a phylogenetic marker and suggests that the mosquito-borne flaviviruses are evolving twice as fast as the tick-borne flaviviruses. The implications of these observations are discussed.

The nucleotide and deduced primary amino acid sequence of the envelope gene of two virus isolates from the brains of Spanish sheep with encephalomyelitis, were determined and compared with those of other flaviviruses. The amino acid alignments showed that the Spanish viruses shared 95 to 96 per cent homology with the envelope protein of louping ill virus and western European tick-borne encephalitis virus. In comparison, the maximum variation in amino acid identities among strains of louping ill virus from the British Isles is 1·8 per cent. The Spanish isolates were distinguishable from all other known flaviviruses by the presence of a unique tripeptide sequence ( AQR ) at amino acid positions 232 to 234 in the E protein, the position at which a genetic marker for distinct flavivirus species has been identified. Other genetic markers, viz DSGHD (amino acids 320 to 324) and EHLPTA (amino acids 207 to 212), which identify the tick-borne encephalitis group within the genus Flavivirus , were present in the amino acid sequences of the Spanish virus. It is concluded that the cause of sheep encephalomyelitis in Spain is a distinct species in the tick-borne encephalitis virus group.

SUMMARYThe population dynamics of tick-borne disease agents and in particular the mechanisms which influence their persistence are examined with reference to the flavivirus that causes louping-ill in red grouse and sheep. Pockets of infection cause heavy mortality and the infection probably persists as a consequence of immigration of susceptible hosts. Seroprevalence is positively associated with temporal variations in vectors per host, although variation between areas is associated with the abundance of mountain hares. The presence of alternative tick hosts, particularly large mammals, provides additional hosts for increasing tick abundance. Grouse alone can not support the vectors and the pathogen but both can persist when a non-viraemic mammalian host supports the tick population and a sufficiently high number of nymphs bite grouse. These alternative hosts may also amplify virus through non-viraemic transmission by the process of co-feeding, although the relative significance of this has yet to be determined. Another possible route of infection is through the ingestion of vectors when feeding or preening. Trans-ovarial transmission is a potentially important mechanism for virus persistence but has not been recorded with louping-ill and Ixodes ricinus. The influence of non-viraemic hosts, both in the multiplication of vectors and the amplification of virus through non-viraemic transmission are considered significant for virus persistence.

Recombinant vaccinia viruses expressing either the premembrane/truncated envelope (PrM/TrE) or truncated envelope (TrE) protein of louping ill virus were constructed. Both constructs expressed authentic E proteins as determined by their size and antigenic reactivity with a panel of monoclonal antibodies. The deletion of the C-terminal hydrophobic domain of the envelope glycoprotein resulted in the secretion of E protein into the supernatant culture medium. The immunisation of mice with these recombinant viruses showed that the recombinant expressing PrM/TrE proteins induced neutralising and protective antibodies against challenge with louping ill or tick-borne encephalitis virus, but that the recombinant expressing the E or the TrE protein alone failed to induce any detectable immune responses against homologous or heterologous virus challenge.

Flavivirus diseases have caused great public health concern for over three centuries, with diseases like yellow fever, dengue, Japanese encephalitis and tick-borne encephalitis causing thousands of deaths. Although yellow fever epidemics can be brought under control by the use of vaccine or mosquito-control measures, there have been many examples of its re-emergence as an epidemic disease. Similarly, the use of vaccines or arthropod-control measures have failed to prevent the spread of other flaviviruses such as Japanese encephalitis virus. There has been rapid growth in the knowledge of molecular information on flaviviruses in the last decade, and on the basis of this information several potential recombinant subunit vaccines are being developed and appear to be effective experimentally. Moreover, the assumption that humoral immunity induced by virus structural envelope glycoproteins is the only effective means of providing protection against flavivirus infection can be questioned. This review attempts to summarize recent thinking in this field and to evaluate the different systems available as potential future flavivirus vaccines in inducing protective immunity.

Serial passage of an uncloned tick-borne encephalitis virus (strain 4387 isolated from the liver and lungs of a bank vole) in Ixodes ricinus ticks, was accompanied by gradual reduction in virulence of the virus, as indicated by transmission of virus by infected ticks feeding on laboratory mice. After the 7th serial passage in ticks (strain 4387/7), 95% of mice survived the bite of infected ticks. The surviving infected mice showed either no or only low viraemia although virus could be isolated from the brains of some mice 14 and 30 days after commencement of tick feeding, implying that the tick passaged virus might have established a persistent infection in the mice. Tests for haemagglutinating capacity were positive with TBE strain 4387 but strain 4387/7 exhibited no heamagglutinating activity over a wide pH range, suggesting that phenotypic changes, resulting from selection, had affected the site on the viral envelope protein that binds red blood cell receptors. Sequencing of the envelope protein gene of the virulent TBE strain 4387 showed 3 amino acid codon differences from western European TBE virus strain Neudorfl, which is also virulent for mice. The attenuated virus 4387/7, had an amino acid substitution that was different from 4387 and Neudorfl TBE virus (amino acid 84, E to K) and a second substitution different from 4387 but identical to Neudorfl virus (amino acid 319, I to T). Thus, the phenotypic change from virulence to attenuation was associated with a single amino acid codon change in the viral envelope gene of TBE virus. It is recognised, however, that amino acid substitutions in other parts of the viral genome have not been ruled out.

The nucleotide sequence of the NS1 gene of louping ill (LI) virus has been determined. The sequence shows a high degree of homology with other members of the tick-borne serocomplex of flaviviruses and a lower homology with the mosquito-borne flaviviruses. Alignment of the deduced NS1 amino acid sequences with all tick-borne flavivirus NS1 sequences, identified four peptide regions which were conserved for all tick-borne flaviviruses, but were variable amongst mosquito-borne flaviviruses. A dendrogram, derived from the alignment of the NS1 protein sequences, indicated an evolutionary relationship that quite closely reflects the recognised serological classification. The LI virus NS1 protein expressed in Escherichia coli and baculoviruses showed similar antigenic reactivity to the authentic virus-coded protein when tested with NS1-specific monoclonal antibodies, but did not form high molecular weight complexes and was not secreted from cells.

Kyasanur Forest disease (KFD) virus is a highly pathogenic member of the family Flaviviridae producing a haemorrhagic disease in infected human beings. Despite this high pathogenicity and potential epidemiological importance, there have been relatively few detailed antigenic or molecular studies on KFD virus. The nucleotide sequences of the genes encoding the structural proteins of the virus have now been determined. From these data we conclude that KFD virus is a distinct member in the tick-borne flavivirus complex with characteristic protease cleavage sites, fusion peptide, signal sequences and hydrophobic transmembrane domains. Comparison of the deduced amino acid sequences of KFD virus showed close relationships with other tick-borne flaviviruses. Among the structural proteins, the E protein showed maximum similarity (77.4% to 81.3%) to tick-borne flaviviruses. Alignment of the amino acid sequence with those of other known tick-borne flaviviruses revealed many conserved regions confirming its identity as a member of the tick-borne encephalitis group, although the genetic marker EHLPTA showed a T → K substitution in KFD virus. The proposed genetic marker at amino acid positions 232 to 234 (AQE) was unique for KFD virus. A dendrogram derived from the amino acid alignment showed a phylogenetic relationship similar to those obtained on the basis of serological studies. The question of the sudden emergence of KFD virus in India and the possibilities of developing recombinant virus vaccines are discussed.

The gene encoding the envelope glycoprotein of Omsk haemorrhagic fever (OHF) virus was cloned and sequenced. A freeze-dried preparation of infected suckling mouse brain suspension was used as the source material for viral RNA. The derived cDNA was amplified using the polymerase chain reaction and the cloned DNA sequenced by dideoxynucleotide sequencing. Alignment of the OHF virus sequence with those of other known tick-borne flaviviruses showed that they shared N-glycosylation sites, cysteine residues, the fusion peptide and a hexapeptide (EHLPTA) that identifies tick-borne flaviviruses. OHF virus was distinguishable from the other viruses but shared closest amino acid identity (93.0%) with the tick-borne encephalitis viruses. A sequence of three amino acids (AQN; amino acids 282 to 234), which was previously shown to be specific for the tick-borne encephalitis viruses, was altered to MVG in OHF virus. This is predicted to have a higher hydrophobicity than the AQN sequence and may therefore have significant implications for the phenotypic characteristics of OHF virus. The results demonstrate close phylogenetic relationships between these viruses but also show their distinct evolutionary development. Sequence changes within the envelope glycoprotein of OHF virus have been identified that may be responsible for the distinct tropism of this flavivirus. These results support and enlarge upon previous data obtained from serological analysis.

In view of a previous report on significant antigenic and biophysical differences between the purified soluble complement-fixing antigens of dengue-1 virus strains Hawaii and TH-Sman, the NS 1 genes of both virus isolates were cloned, sequenced, and compared in an attempt to define the genetic basis for the observed differences. Sequence comparison revealed ten encoded amino acid differences between the NS 1 genes of both viruses. Three of these amino acid differences, which are associated with a change in charge distribution, are clustered within the major antigenic region previously defined by studies of recombinant dengue-1 NS 1 protein expressed in E. coli. In parallel, the NS 1 sequences of both Hawaii and TH-Sman isolates were also aligned and compared with two other published dengue-1 NS 1 protein sequences to determine the intratypic variation of dengue-1 NS 1 antigen. Pairwise comparisons between the encoded amino acid sequences revealed a variability of 1.1% to 3.1% difference in the NS 1 protein among dengue-1 strains, which is comparable to that reported for dengue-1 envelope protein (0.2% to 3.6% difference) but less than that of dengue-2 NS 1 protein (0.6% to 7.4% difference).

Complementary DNAs were synthesized from the envelope protein genes of two isolates of dengue virus (TH-36 and TH-Sman, previously suggested as possible dengue virus type 5 and dengue virus type 6 respectively) and amplified by the polymerase chain reaction using sense and antisense primers designed from conserved dengue virus gene sequences. The amplified cDNA clones were sequenced in both directions by double-stranded dideoxynucleotide sequencing. Alignment with published dengue virus sequences enabled us to assign these viruses accurately to classified serotypes, confirming that TH-36 and TH-Sman are strains of dengue virus type 2 and dengue virus type 1 respectively. Amino acid changes between the proteins encoded by these two isolates and strains of their respective serotypes may account for the significant antigenic differences observed during previous serological typing of these viruses. Moreover, sequence alignment of flavivirus envelope proteins revealed a hypervariable region, within which members of the dengue and tick-borne virus antigenic complexes show unique peptide sequences. This type-specific hypervariable domain may be useful as a genetic marker for typing dengue and tick-borne flaviviruses.

To the editor: Following the recent emergence of an Influenza virus variant in Mexico and the threat of its pandemic potential, both the media and scientific experts spontaneously referred to ‘Mexican flu’. However, the name secondarily used by WHO to designate this virus was ‘Influenza A(H1N1) virus’. This refers to the scientific designation of the taxonomic species Influenza A virus combined with the associated haemagglutinin/neuraminidase subtypes. Unfortunately, this is wholly inappropriate for identifying the current variant and novel virus. Following the same logic the virus that caused the 1918–1919 Spanish influenza pandemic, as well as the dominant virus that caused seasonal flu last winter in the USA, would also be called ‘Influenza A(H1N1) virus’. Just imagine the confusion that this could cause. To designate a virus, a vernacular or a scientific name can be used. Regarding the relevant taxonomic designation, the International Committee on Taxonomy of Viruses has recommended for years that geographical information should be included in the designation of virus strains and even virus species. For example, the designation of influenza virus strains commonly includes the location of the virus isolation {e.g. Influenza A virus [A/Mexico/4603/2009(H1N1)]}. Indeed, by analogy with West Nile virus, Kedougou virus, Rift Valley fever virus and many others, the designation of the influenza virus variant should evoke the location of initial discovery of the disease. Accordingly, referring to Mexico, the country where the disease first appeared as a Public Health problem and, by extension, using the vernacular name ‘Mexican flu virus’ is not in contradiction with the logic of taxonomic nomenclature. Why was the name Mexican flu virus disallowed by health authorities? This seems to be linked with the ‘politically correct’ fear of stigmatising Mexican people. However, when the term ‘Spanish flu’ is used, we do not blame the Spanish people. We simply recognise the subject of discussion and newsworthiness. Similarly, the name ‘Mexican flu’ evokes the influenza outbreak that emerged in the Mexican population, without blaming the Mexican people. It precisely designates the current episode, reminds us that it’s first recognised victims were Mexicans, and enables to distinguish the new virus from seasonal H1N1 variants that may circulate simultaneously. Globally, this is much more efficient than the recommended ‘official’ name: if it does not appear to satisfy the questionable needs of political correctness, the vernacular name is perfectly acceptable for the purpose of informing the world in a simple, scientifically relevant and informative manner. Why is it incorrect to speak about the Mexican flu?

We have studied the structure and morphogenesis of Dugbe (DUG) virus (Bunyaviridae, Nairovirus) in cultured porcine kidney (PS) cells and a tick cell line (Ra 243) using immunogold electron microscopy. DUG virus is a tickborne arbovirus, considered to be a low health hazard, that is antigenically and genetically related to Crimean Congo haemorrhagic fever (CCHF) virus (Marriott et al., 1990). We have investigated the maturation and intracellular transport of DUG virus particles as a model for other more pathogenic nairoviruses using monoclonal antibodies for immunogold labelling of ultrathin cryosections and immunofluorescence techniques. The spherical DUG virus particle measures about 90 nm in diameter, with a 5 nm thick membrane covered by 5-7 nm long projections or "spikes". These projections form hollow cylindrical morphological units, about 5 nm in diameter. DUG virus infection caused only a slight cytopathogenic effect in mammalian cells and none in tick cells. DUG virus particles assembled by budding from the Golgi complex, where the DUG virus glycoprotein G1 accumulated in vesicles originating from Golgi cisternae. The nucleocapsid protein N accumulated in scattered foci throughout the cytoplasm, and this appears to be related to the limited maturation of DUG virus particles that occurred. The reduced number of budding virus particles observed in tick cells was correlated with the reduced cytopathology observed.

The Sarawak strain of Japanese encephalitis virus (JE-Sar) is virulent in 3-week-old mice when inoculated intraperitoneally. The nucleotide sequence for the envelope glycoprotein (E) of this virus was determined and compared with the published sequences of four other strains. There were several silent nucleotide differences and five codon changes. Monoclonal antibodies (MAbs) against the E protein of JE-Sar virus were prepared and characterized. MAb-resistant mutants of JE-Sar were selected to determine if mutations in the E protein gene could affect its virulence for mice. Eight mutants were isolated using five different MAbs that identified virus-specific or group-reactive epitopes on the E protein. The mutants lost either complete or partial reactivity with selecting MAb. Several showed decreased virulence in 3-week-old mice after intraperitoneal inoculation. Two (r27 and r30) also showed reduced virulence in 2-week-old mice. JE-Sar and the derived mutants were comparable in their virulence for mice, when inoculated intracranially. Mutant r30 but not r27 induced protective immunity in adult mice against intracranial challenge with parent virus. However, r27-2 did induce protective immunity against itself. Nucleotide sequencing of the E coding region for the mutants revealed single base changes in both r30 and r27 resulting in a predicted change from isoleucine to serine at position 270 in r30 and from glycine to aspartic acid at position 333 in r27. The altered capacity of the mutants to induce protective immunity is consistent with the immunogenicity changes predicted by computer analysis using the Protean II program.

The genomic RNA of louping ill virus coding for capsid, premembrane, membrane, and envelope proteins was cloned and sequenced. Hydrophilicity profiles of the deduced amino acid sequence shared homologous functional domains with other flaviviruses. The premembrane and envelope proteins contain N-glycosylation sites and conserved cysteine residues which are important for maintaining the secondary structures of the proteins. Sequence comparisons of louping ill envelope protein showed greater homology with tick-borne than mosquito-borne flaviviruses and greater homology with the western than the far eastern subtype of tick-borne encephalitis virus. With the capsid and membrane proteins, the degree of homology between louping ill and the western subtype was greater than that between the two subtypes, indicating very close evolutionary relationships between louping ill and the western subtype of tick-borne encephalitis. Thus, louping ill and tick-borne encephalitis may be varieties of a common tick-borne ancestral virus. The average amino acid sequence diversity between members of the tick-borne serogroup was significantly lower than that of mosquito-borne serogroups, suggesting that tick-borne flaviviruses have been subjected to different evolutionary immune selection pressure from the mosquito-borne viruses. Using the published model of tick-borne encephalitis envelope protein and our sequence data on louping ill virus, we have identified three discontinuous peptides (amino acids 81–88, 207–212, and 230–234) which may represent critical molecular determinants within the receptor binding site of tick-borne flaviviruses and may provide a specific genetic marker for these viruses.

The genomic RNA encoding the structural proteins of louping ill, a tickborne flavivirus, was cloned and sequenced. Sequence comparisons of louping ill envelope protein showed greater homology with tick-borne than mosquito-borne flaviviruses and greater homology with the western than the far eastern subtype of tick-borne encephalitis virus. Louping ill and tick-borne encephalitis viruses are probably varieties of a common tick-borne ancestral virus. The average amino acid sequence diversity between members of the tick-borne serogroup was significantly lower than that of mosquito-borne serogroups, suggesting that tick-borne flaviviruses have been subjected to different evolutionary immune selection pressure from the mosquito-borne viruses. Using the published model of tick-borne encephalitis envelope protein and the derived sequence data on louping ill virus, three discontinuous peptides (amino acids 81-88, 207-212 and 230-234) which may represent critical molecular determinants within the receptor binding site of tick-borne flaviviruses, were identified. These peptides may provide a specific genetic marker for these viruses. Recombinant baculoviruses and vaccinia viruses containing cloned DNA, encoding either the envelope protein or the structural proteins of louping ill virus, were constructed. Glycosylated envelope protein, presented both inside and on the surface of insect and mammalian cells, was expressed by all four recombinant viruses. Differences in antigenic presentation of envelope protein were observed between envelope protein and structural protein constructs as well as between insect cell and mammalian cell expression systems. Despite the expression of epitopes known to elicit neutralizing and protective antibodies when present in authentic antigen, the recombinant envelope protein expressed by either baculovirus or vaccinia virus failed to induce, under the experimental conditions employed, either neutralizing or protective antibodies in both mice and rabbits against louping ill virus. Hence, louping ill envelope protein expressed by baculoviruses and vaccinia viruses was antigenically reactive but immunogenically inert.

Since 2004, several million indigenous cases of Chikungunya virus disease occurred in Africa, the Indian Ocean, India, Asia and, recently, Europe. The virus, usually transmitted by Aedes aegypti mosquitoes, has now repeatedly been associated with a new vector, Ae. Albopictus. Analysis of full-length viral sequences reveals three independent events of virus exposure to Ae. Albopictus, each followed by the acquisition of a single adaptive mutation providing selective advantage for transmission by this mosquito. This disconcerting and current unique example of "evolutionary convergence" occurring in nature illustrates rapid pathogen adaptation to ecological perturbation, driven directly as a consequence of human activities.

The importance of viral structural and non-structural antigens in flavivirus pathogenesis has not been satisfactorily assessed. This review examines the antigenic interrelationships of the flaviviruses, considers some of the implications of antigenic cross reactivity in virus virulence and highlights potential consequences of virus-antibody interactions.

Dugbe virus is assigned to the family Bunyaviridae, genus Nairovirus and is related to the tick-borne Crimean-Congo hemorrhagic fever and Nairobi sheep disease viruses. The proteins of Dugbe virus were studied biochemically and immunochemically using monoclonal and poly-clonal antibodies. The G1, N and G2 proteins were detected by PAGE at 73, 49, and 35 kDa, respectively. On a Western blot, polyclonal antisera to Dugbe virus reacted with the G1, N and G2 proteins and with several additional proteins (210, 45, 40, 33, and 30 kDa). The Gl and G2 proteins were shown to be located on the surface of virus particles and to be glycosylated while the N protein was internal and non-glycosylated. The 40 kDa protein also was found to be glycosylated. This 40 kDa glycoprotein may represent an additional glycoprotein, as found in Hazara virus, or may be a breakdown product of G1. A monoclonal antibody (McAb H28.89) against the N protein specifically labelled purified nucleocapsids in an immunogold EM reaction. This McAb did not neutralise viral infectivity in an in vitro assay and did not label whole virus particles with colloidal gold. Another McAb (H28.17) against the G1 protein neutralised virus infectivity and labelled whole virus particles by immunogold EM.

A panel of monoclonal antibodies (MAb), reactive with the nairovirus Dugbe, was prepared and characterized. When used to compare seven strains of Dugbe virus by indirect immunofluorescence, they were antigenically indistinguishable. On the basis of in vitro plaque reduction neutralization and in vivo mouse protection tests by passive immunization, these strains could be divided into two groups. Five of the seven strains were neutralized by most or all of the neutralizing MAb and mice were protected from lethal infection by these viruses in the presence of passively administered MAb. The other two strains were not neutralized by most of the MAb and most of the MAb failed to protect mice from lethal infection by them. The binding capacity of the antibodies with these viruses in ELISA tests showed no correlation with their neutralization or protective capacity, therefore differences in avidity of antibody binding did not account for the differences in neutralization or protection. On the other hand, there was a direct relationship between neurovirulence of these strains for mice and their readiness to be neutralized or protected.

A corrigendum to the paper by Mastrangelo et al. (2006), Acta Cryst. F62, 768–770., A correction is made to the names of two of the authors in Mastrangelo et al. (2006), Acta Cryst. F62, 768–770.

We previously reported evidence of West Nile virus (WNV) circulation in UK birds, probably introduced by migratory birds from overseas. We now demonstrate WNV-specific seroconversion in sentinel chickens raised on an English farm. Maternal neutralizing antibodies to WNV in hatchlings declined within three weeks. During the following months, healthy chickens developed WNV neutralizing antibodies that were confirmed by immunoblotting and indirect immunofluorescence tests using WNV antigens. The proportion of seropositive chickens was higher for WNV than for Usutu virus or Sindbis virus. Attempts to isolate infectious virus or to detect viral RNA in the sera, failed.

We propose a novel method for direct cloning of foreign genes into baculoviruses which avoids the use of bacterial transfer vectors. The foreign gene to be inserted is derived by PCR using appropriate primers each of which contains an additional 50 nt of baculovirus sequence for homologous recombination between the PCR-derived DNA and the baculovirus DNA, thus accomplishing insertion of the foreign gene into the baculovirus. The direct cloning of green fluorescent protein and beta-glucuronidase in different baculovirus loci is described. The method is simple and avoids the use of cumbersome techniques associated with enzymatic treatment and DNA purification.

♦Research on which this article was based was completed while the senior author was temporarily assigned as Bullard Fellow at Harvard Univ., Cambridge, Mass. tury, perhaps 80 per cent of the land had been cleared at one time or another for farming.2 Beginning about 100 years ago, much of this farmland began to revert to forest, and this process has continued up to the present time. About 65 per cent of the state is now forested. Did streamflow increase as forests were cleared for farming? Has streamflow decreased as trees reclaimed unused farmland?

Previously, direct repeats (DRs) of 20-70 nucleotides were identified in the 3' untranslated regions (3'UTR) of flavivirus sequences. To address their functional significance, we have manually generated a pan-flavivirus 3'UTR alignment and correlated it with the corresponding predicted RNA secondary structures. This approach revealed that intra-group-conserved DRs evolved from six long repeated sequences (LRSs) which, as approximately 200-nucleotide domains were preserved only in the genomes of the slowly evolving tick-borne flaviviruses. We propose that short DRs represent the evolutionary remnants of LRSs rather than distinct molecular duplications. The relevance of DRs to virus replication enhancer function, and thus survival, is discussed.

Epidemics of tick-borne encephalitis involving thousands of humans occur annually in the forested regions of Europe and Asia. Despite the importance of this disease, the underlying basis for the development of encephalitis remains undefined. Here, we prove the key role of CD8+ T-cells in the immunopathology of tick-borne encephalitis, as demonstrated by prolonged survival of SCID or CD8−/− mice, following infection, when compared with immunocompetent mice or mice with adoptively transferred CD8+ T-cells. The results imply that tick-borne encephalitis is an immunopathological disease and that the inflammatory reaction significantly contributes to the fatal outcome of the infection.

An attenuated strain (263) of the tick-borne encephalitis virus, isolated from field ticks, was either serially subcultured, 5 times in mice, or at 40 °C in PS cells, producing 2 independent strains, 263-m5 and 263-TR with identical genomes; both strains exhibited increased plaque size, neuroinvasiveness and temperature-resistance. Sequencing revealed two unique amino acid substitutions, one mapping close to the catalytic site of the viral protease. These observations imply that virus adaptation from ticks to mammals occurs by selection of pre-existing virulent variants from the quasispecies population rather than by the emergence of new random mutations. The significance of these observations is discussed.