Novel flaviviruses from mosquitoes: mosquito-specific evolutionary lineages within the phylogenetic group of mosquito-borne flaviviruses.

Novel flaviviruses that are genetically related to pathogenic mosquito-borne flaviviruses (MBFV) have been isolated from mosquitoes in various geographical locations, including Finland. We isolated and characterized another novel virus of this group from Finnish mosquitoes collected in 2007, designated as Ilomantsi virus (ILOV). Unlike the MBFV that infect both vertebrates and mosquitoes, the MBFV-related viruses appear to be specific to mosquitoes similar to the insect-specific flaviviruses (ISFs). In this overview of MBFV-related viruses we conclude that they differ from the ISFs genetically and antigenically. Phylogenetic analyses separated the MBFV-related viruses isolated in Africa, the Middle East and South America from those isolated in Europe and Asia. Serological cross-reactions of MBFV-related viruses with other flaviviruses and their potential for vector-borne transmission require further characterization. The divergent MBFV-related viruses are probably significantly under sampled to date and provide new information on the variety, properties and evolution of vector-borne flaviviruses.

Novel insect-specific flavivirus isolated from northern Europe.

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

Rate of evolution and molecular epidemiology of tick-borne encephalitis virus in Europe, including two isolations from the same focus 44 years apart.

Tick-borne encephalitis virus (TBEV) is a member of the family Flaviviridae. It is transmitted by Ixodes spp. ticks in a cycle involving rodents and small mammals. TBEV has three subtypes: European, Siberian and Far Eastern. The virus causes thousands of cases of meningoencephalitis in Europe annually, with an increasing trend. The increase may be attributed to a complex network of elements, including climatic, environmental and socio-economic factors. In an attempt to understand the evolutionary history and dispersal of TBEV, to existing genetic data we add two novel complete ORF sequences of TBEV strains from northern Europe and the completion of the genome of four others. Moreover, we provide a unique measure for the natural rate of evolution of TBEV by studying two isolations from the same forest on an island in Åland archipelago 44 years apart. For all isolates, we analysed the phylogeny, rate of evolution and probable time of radiation of the different TBEV strains. The results show that the two lineages of TBEV in different Ixodes species have evolved independently for approximately 3300 years. Notably, rapid radiation of TBEV-Eur occurred approximately 300 years ago, without the large-scale geographical clustering observed previously for the Siberian subtype. The measurements from the natural rate of evolution correlated with the estimates done by phylogenetic programs, demonstrating their robustness.

Early diagnosis of dengue in travelers: comparison of a novel real-time RT-PCR, NS1 antigen detection and serology.

BACKGROUND: The increased traveling to dengue endemic regions and the numerous epidemics have led to a rise in imported dengue. The laboratory diagnosis of acute dengue requires several types of tests and often paired samples are needed for obtaining reliable results. Although several diagnostic methods are available, proper comparative data on their performance are lacking. OBJECTIVES: To compare the performance of novel methods including a novel pan-DENV real-time RT-PCR and a commercially available NS1 capture-EIA in regard to IgM detection for optimizing the early diagnosis of DENV in travelers. STUDY DESIGN: A panel of 99 selected early phase serum samples of dengue patients was studied by real-time RT-PCR, NS1 antigen ELISA, IgM-EIA, IgG-IFA and cell culture virus isolation. RESULTS: The novel real-time RT-PCR was shown specific and sensitive for detection of DENV-1-4 RNA and suitable for diagnostic use. The diagnostic rate using combination of RNA and IgM detection was 99% and using NS1 and IgM detection 95.9%. The results of RNA and NS1 antigen detection disagreed in 15.5% of samples that had only RNA or NS1 antigen detected. CONCLUSIONS: The diagnostic rates of early samples are higher when either RNA or NS1 antigen detection is combined with IgM detection. Besides the differences in the RNA and NS1 detection assays, the observed discrepancy of results could suggest individual variation or differences in timing of these markers in patient serum.

Characterization of a novel flavivirus from mosquitoes in northern europe that is related to mosquito-borne flaviviruses of the tropics.

A novel flavivirus was isolated from mosquitoes in Finland, representing the first mosquito-borne flavivirus from Northern Europe. The isolate, designated Lammi virus (LAMV), was antigenically cross-reactive with other flaviviruses and exhibited typical flavivirus morphology as determined by electron microscopy. The genomic sequence of LAMV was highly divergent from the recognized flaviviruses, and yet the polyprotein properties resembled those of mosquito-borne flaviviruses. Phylogenetic analysis of the complete coding sequence showed that LAMV represented a distinct lineage related to the Aedes sp.-transmitted human pathogenic flaviviruses, similarly to the newly described Nounané virus (NOUV), a flavivirus from Africa (S. Junglen et al., J. Virol. 83:4462-4468, 2009). Despite the low sequence homology, LAMV and NOUV were phylogenetically grouped closely, likely representing separate species of a novel group of flaviviruses. Despite the biological properties preferring replication in mosquito cells, the genetic relatedness of LAMV to viruses associated with vertebrate hosts warrants a search for disease associations.

Sindbis virus infection in resident birds, migratory birds, and humans, Finland.

Sindbis virus (SINV), a mosquito-borne virus that causes rash and arthritis, has been causing outbreaks in humans every seventh year in northern Europe. To gain a better understanding of SINV epidemiology in Finland, we searched for SINV antibodies in 621 resident grouse, whose population declines have coincided with human SINV outbreaks, and in 836 migratory birds. We used hemagglutination-inhibition and neutralization tests for the bird samples and enzyme immunoassays and hemagglutination-inhibition for the human samples. SINV antibodies were first found in 3 birds (red-backed shrike, robin, song thrush) during their spring migration to northern Europe. Of the grouse, 27.4% were seropositive in 2003 (1 year after a human outbreak), but only 1.4% were seropositive in 2004. Among 2,529 persons, the age-standardized seroprevalence (1999-2003) was 5.2%; seroprevalence and incidence (1995-2003) were highest in North Karelia (eastern Finland). Grouse may contribute to the epidemiology of SINV in humans.

Molecular epidemiology of dengue virus strains from Finnish travelers.

We characterized 11 dengue virus (DENV) isolates obtained from Finnish travelers during 2000-2005 using monoclonal antibodies and phylogenetic analysis. The analysis of DENV isolated from travelers contributes to the global picture of strain distribution and circulation. The isolates included all serotypes, including a DENV-2 isolate from Ghana.

Siberian subtype tickborne encephalitis virus, Finland.

We isolated 11 Siberian subtype tickborne encephalitis virus (TBEV) strains from Ixodes persulcatus ticks from a TBEV-endemic focus in the Kokkola Archipelago, western Finland. Thus I. persulcatus and the Siberian TBEV are reported in a focus considerably northwest of their previously known range in eastern Europe and Siberia.

Molecular epidemiology of tick-borne encephalitis virus in Ixodes ricinus ticks in Lithuania.

In Lithuania, 171-645 serologically confirmed cases of tick-borne encephalitis occurred annually [Mickiene et al. (2001): Eur J Clin Microbiol Infect Dis 20:886-888] in 1993-1999, and the tick-borne encephalitis virus (TBEV) seroprevalence in the general population was found previously to be 3.0% [Juceviciene et al. (2002): J Clin Virol 25:23-27]. To assess the risk for TBEV virus infection in Lithuania and to characterize the agent a panel of 3,234 ticks combined into 436 pools [Juceviciene et al., 2005] were tested for presence of TBEV RNA by a nested RT-PCR targeting at the NS5 gene. Six pools were confirmed positive and the prevalence of the infected ticks was 0.2% (if one tick per pool [Juceviciene et al., 2005] was considered positive) and the proportion of positive tick pools was 1.4%. The prevalence of the infected ticks in the Panevezys, Siauliai, and Radviliskis regions (in central Lithuania) was 0.1%, 0.4%, and 1.7% corresponding with a higher TBE disease burden in these regions. The 252-nucleotide NS5-region amplicons, and a longer sequence (737 nucleotides) obtained from one sample from the PrM-E gene region, were sequenced. Phylogenetic analysis of the latter showed that all western type TBEV PrM-E sequences, including the Lithuanian strains, were monophyletic, showed no clustering and had very little variation. The NS5 sequences, although identical within one locality, did not show any mutations common to strains from the two Lithuanian regions, nor could any geographical clustering be found among western type TBEV strains from other areas.