Flaviviruses isolated from mosquitoes collected during the first recorded outbreak of Japanese encephalitis virus on Cape York Peninsula, Australia

In response to an outbreak of Japanese encephalitis (JE) virus on Cape York Peninsula, Australia, in 1998, mosquitoes were collected using CO2 and octenol-baited Centers for Disease Control and Prevention light traps. A total of 35,235 adult mosquitoes, comprising 31 species, were processed for virus isolation. No isolates of JE virus were recovered from these mosquitoes. However, 18 isolates of Kokobera virus, another flavivirus were obtained from Culex annulirostris. Twelve isolates were from western Cape York (minimum infection rate (MIR) of 0.61: 1,000 mosquitoes) and 6 were from the Northern Peninsula Area (MIR of 1.0:1,000). Potential explanations for the failure to detect JE virus in mosquitoes collected from Cape York Peninsula include the timing of collections, the presence of alternative bloodmeal hosts, differences in pig husbandry, asynchronous porcine seroconversion, and the presence of other flaviviruses.

Experimental infections of pigs with Japanese encephalitis virus and closely related Australian flaviviruses

The flavivirus Japanese encephalitis (JE) virus has recently emerged in the Australasian region. To investigate the involvement of infections with related enzootic flaviviruses, namely Murray Valley encephalitis (MVE) virus and Kunjin (KUN) virus, on immunity of pigs to JE virus and to provide a basis for interpretation of serologic data, experimental infections were conducted with combinations of these viruses. Antibody responses to primary and secondary infections were evaluated using panels of monoclonal antibody-based blocking enzyme-link-ed immuno-sorbent assays and microtiter scrum neutralization tests (mSNTs). Identification of the primary infecting virus was possible only using the mSNTs. Following challenge, unequivocal diagnosis was impossible due to variation in immune responses between animals and broadened and/or anamnestic responses. Viremia for JE virus was readily detected in pigs following primary infection, but was not detected following prior exposure to MVE or KUN viruses. Boosted levels of existing cross-neutralizing antibodies to JE virus suggested a role for this response in suppressing JE viremia.

Cross-protective and Infection-Enhancing Immunity in Mice Vaccinated Against Flaviviruses Belonging to the Japanese Encephalitis Virus Serocomplex

The Japanese encephalitis virus serocomplex is a group of mosquito-borne flaviviruses that cause severe encephalitic disease in humans. The recent emergence of several members of this serocomplex in geographic regions where other closely related flaviviruses are endemic has raised urgent human health issues. Thus, the impact of vaccination against one of these neurotropic virus on the outcome of infection with a second, serologically related virus is unknown. We show here that immunity against Murray Valley encephalitis virus in vaccinated mice can cross-protect but also augment disease severity following challenge with Japanese encephalitis virus. Immunepotentiation of heterologous flavivirus disease was apparent in animals immunized with a 'killed' virus preparation when humoral antiviral immunty of low magnitude was elicited. (C) 2002 Elsevier Science Ltd. All rights reserved.

ANTIGENIC RELATEDNESS OF SELECTED FLAVIVIRUSES: STUDY WITH HOMOLOGOUS AND HETEROLOGOUS IMMUNE MOUSE ASCITIC FLUIDS

The antigenic relationship of 9 flaviviruses, Yellow fever (YF) , Wesselsbron (WSL) , Uganda S (UGS) , Potiskum (POT), West Nile (WN) , Banzi (BAN) , Zika (ZK) , Dengue type 1 (DEN-1) and Dengue type 2 (DEN-2), was assessed by cross-haemagglutination-inhibition (Cross-HI) and cross-complement fixation (Cross-CF) reactions between each of the viruses and their homologous immune mouse ascitic fluids. Titre ratios were calculated using the heterologous and homologous titres. Cross-CF reactions revealed wider antigenic variations among viruses than Cross-HI reactions. There was no significant antigenic variation between WSL, POT and YF viruses using either of those methods. However, definite differences in antigenicity were observed between them and UGS, BAN and ZK viruses. There were no significant differences between UGS, BAN and ZK or between DEN-1 and DEN-2. The serological relationship among flaviviruses is important in establishing diagnosis and epidemiology of these infections in Africa.

Flaviviruses in mosquitoes from Southern Portugal, 2009-2010

Os flavivírus são vírus pertencentes à família Flaviviridae, género Flavivirus. Estes formam um grande grupo caraterizado pela sua ampla distribuição e diversidade genética. Os flavivírus são, na sua maioria, transmitidos por artrópodes vectores incluíndo agentes patogénicos para humanos e animais que podem potencialmente provocar grandes epidemias e causar elevadas taxas de mortalidade e morbidade. Nos últimos anos, tem-se registado uma grande expansão a nível da distribuição geográfica dos flavivirus e diversidade dos seus hospedeiros. O vírus do Nilo Ocidental tem sido continuamente detectado em toda a Europa recentemente, e também isolado de mosquitos colhidos no Sul de Portugal, onde já foram registados casos humanos e animais. O principal objectivo deste trabalho é o rastreio de flavivírus em mosquitos colhidos em duas regiões do Sul de Portugal, onde os mesmos foram anteriormente detectados. As colheitas de mosquitos foram realizadas em 24 locais em zonas húmidas nos districtos de Faro e Setúbal, através de armadilhas luminosas tipo CDC com CO2 e aspiradores mecânicos manuais para colheita de mosquitos em repouso em abrigos de animais. Os mosquitos colhidos foram agrupados por lotes contendo aproximadamente 50 espécimens cada, e rastreados para a presença de flavivírus por heminested RT-PCR, direccionado à amplificação de um pequeno fragmento do gene NS5 usando oligonucleótidos degenerados específicos para flavivírus. Entre Abril e Outubro de 2009 e 2010 foram colhidos no total 36273 mosquitos pertencentes às seguintes espécies: Anopheles algeriensis, An.atroparvus, Aedes berlandi, Ae.caspius, Ae.detritus, Coquillettidia richiardii, Culex laticinctus, Cx.pipiens, Cx.theileri, Cx.univittatus, Culiseta annulata, Cs.longiareolata, Cs.subochrea, e Uranotaenia unguiculata. As espécies mais abundantes foram Ae.caspius, Cx.theileri e Cx.pipiens, respectivamente. Contudo, as densidades de mosquitos foram variáveis de acordo com o método de colheita e área de amostragem. As densidades de mosquitos colhidos em 2010 foram quatro vezes superior às registadas no ano anterior. No total foram analisados 745 lotes dos quais 31% testaram positivos para a presença de sequências de flavivirus. As espécies que apresentaram taxas de positividade mais elevadas foram: An.algeriensis com uma Taxa Mínima de Infecção (TMI) de 56/1000 no Algarve em 2009, Cs.annulata TMI =22/1000 no Algarve em 2010, Cx.theileri e Cx.pipiens em Setúbal em 2010, TMI =20/1000. An. atroparvus, Ae. caspius, Ae. detritus e Cx. univittatus também produziram lotes positives. No geral, a positividade foi maior no Algarve. Análise das sequências virais obtidas revelou homologia das nossas sequências virais com sequências de referência de flavivírus específicos de mosquitos depositadas em bases de dados de acesso livre. A análise filogenética reflectiu a variabilidade genética dos flavivírus e revelou a relação genética das nossas sequências com as de outros flavivírus, especialmente os específicos de insectos. Tendo em consideração os anteriores isolamentos do vírus do Nilo Ocidental, o aumento acentuado nas densidades de mosquitos, o aumento de temperaturas que se tem vindo a registar, os casos recentes de transmissão de flavivírus por toda a Europa e o padrão desconhecido e imprevisível dos surtos destes vírus, os programas contínuos de vigilância epidemiológica têm-se revelado uma ferramenta indispensável para a Saúde Pública.

Cytokine and nitric oxide production by mouse macrophages infected with brazilian flaviviruses

The Flaviviridae family, Flavivirus genus includes viruses that are transmitted to vertebrates by infected mosquitoes or ticks. The genus Flavivirus includes a variety of viruses that cause diseases such as acute febrile illness, encephalitis, and hemorrhagic fever. Flaviviruses primarily infect blood monocytes and tissue macrophages, which have been shown to be permissive, supporting viral replication and serving as virus reservoirs. On the other hand, these cells may have an important antiviral activity related to modulation by cytokine production and by the capacity of these cells to synthesize reactive free radicals such as nitric oxide (NO) which can have a microbicidal effect. The present study was performed in order to determine the production of cytokines interleukin-1beta (IL-1β), tumor necrosis factor -alpha (TNF-α), transforming growth factor- beta (TGF-β) and interferon -alpha (IFN-α) and NO by macrophages infected with one of four Brazilian flaviviruses, Bussuquara virus (BUSV), Yellow Fever virus (YFV), Rocio virus (ROCV) and Encephalitis Saint Louis virus (SLEV), and to verify the possible antiviral effect of NO during macrophage infection with ROCV. Moreover, we asked if the different viruses were able to regulate bacterial lipopolysaccharide (LPS) induced cytokine production. Our results showed that YFV and SLEV reduced the production of IL-1β and TGF-β by LPS-stimulated macrophages, while ROCV only diminished LPS-stimulated TGF-β synthesis. On the other hand, BUSV more likely favored an enhancement of the LPS-induced production of IL-1β by macrophages. Additionally, while most of the viruses stimulated the production of IFN-α, none of them altered the production of TNF-α by murine macrophages. Interestingly, all viruses induced synthesis of NO that was not correlated with antiviral activity for ROCV.

Non-hemagglutinating flaviviruses: molecular mechanisms for the emergence of new strains via adaptation to European ticks

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.

Genetic characterization of tick-borne flaviviruses: new insights into evolution, pathogenetic determinants and taxonomy

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.

Origin and evolution of 3'UTR of flaviviruses: long direct repeats as a basis for the formation of secondary structures and their significance for virus transmission

The 3' untranslated regions (3'UTRs) of flaviviruses are reviewed and analyzed in relation to short sequences conserved as direct repeats (DRs). Previously, alignments of the 3'UTRs have been constructed for three of the four recognized flavivirus groups, namely mosquito-borne, tick-borne, and nonclassified flaviviruses (MBFV, TBFV, and NCFV, respectively). This revealed (1) six long repeat sequences (LRSs) in the 3'UTR and open-reading frame (ORF) of the TBFV, (2) duplication of the 3'UTR of the NCFV by intramolecular recombination, and (3) the possibility of a common origin for all DRs within the MBFV. We have now extended this analysis and review it in the context of all previous published analyses. This has been achieved by constructing a robust alignment between all flaviviruses using the published DRs and secondary RNA structures as "anchors" to reveal additional homologies along the 3'UTR. This approach identified nucleotide regions within the MBFV, NKV (no-known vector viruses), and NCFV 3'UTRs that are homologous to different LRSs in the TBFV 3'UTR and ORF. The analysis revealed that some of the DRs and secondary RNA structures described individually within each flavivirus group share common evolutionary origins. The 3'UTR of flaviviruses, and possibly the ORF, therefore probably evolved through multiple duplication of an RNA domain, homologous to the LRS previously identified only in the TBFV. The short DRs in all virus groups appear to represent the evolutionary remnants of these domains rather than resulting from new duplications. The relevance of these flavivirus DRs to evolution, diversity, 3'UTR enhancer function, and virus transmission is reviewed.

Molecular archaeology of Flaviviridae untranslated regions: duplicated RNA structures in the replication enhancer of flaviviruses and pestiviruses emerged via convergent evolution.

RNA secondary structures in the 3'untranslated regions (3'UTR) of the viruses of the family Flaviviridae, previously identified as essential (promoters) or beneficial (enhancers) for replication, have been analysed. Duplicated enhancer elements are revealed as a global feature in the evolution of the 3'UTR of distantly related viruses within the genera Flavivirus and Pestivirus. For the flaviviruses, duplicated structures occur in the 3'UTR of all four distantly related ecological virus subgroups (tick-borne, mosquito-borne, no known vector and insect-specific flaviviruses (ISFV). RNA structural differences distinguish tick-borne flaviviruses with discrete pathogenetic characteristics. For Aedes- and Culex-associated ISFV, secondary RNA structures with different conformations display numerous short ssRNA direct repeats, exposed as loops and bulges. Long quadruplicate regions comprise almost the entire 3'UTR of Culex-associated ISFV. Extended duplicated sequence and associated RNA structures were also discovered in the 3'UTR of pestiviruses. In both the Flavivirus and Pestivirus genera, duplicated RNA structures were localized to the enhancer regions of the 3'UTR suggesting an adaptive role predominantly in wild-type viruses. We propose sequence reiteration might act as a scaffold for dimerization of proteins involved in assembly of viral replicase complexes. Numerous nucleotide repeats exposed as loops/bulges might also interfere with host immune responses acting as a molecular sponge to sequester key host proteins or microRNAs.

Population dynamics of flaviviruses revealed by molecular phylogenies.

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.

Identification of new flaviviruses in the Kokobera virus complex

Novel flavivirus isolates from mosquitoes collected in northern Australia were analysed by partial genomic sequencing, monoclonal antibody-binding assays and polyclonal cross-neutralization tests. Two isolates were found to be antigenically distinct from, but related to, viruses of the Kokobera virus complex, which currently contains Kokobera (KOKV) and Stratford (STRV) viruses. Nucleotide sequence comparison of two separate regions of the genome revealed that an isolate from Saibai Island in the Torres Strait in 2000 (TS5273) was related closely to KOKV and STRV, with 74-80 and 75-76% nucleotide similarity, respectively. An isolate from mainland Cape York in 1998 (CY1014) was found to be more divergent from KOKV and STRV, with