Functional analysis of microRNA-122 binding sequences of hepatitis C virus and identification of variants with high resistance against a specific antagomir.

MicroRNA 122 (miR-122) stimulates the replication and translation of hepatitis C virus (HCV) RNA by binding to two adjacent sites, S1 and S2, within the HCV 5'UTR. We demonstrated previously that the miR-122 antagomir miravirsen (SPC3649) suppresses the infection of HCV strain JFH1-based recombinants with HCV genotypes 1-6 5'UTR-NS2 in human hepatoma Huh7.5 cells. However, specific S1 mutations were permitted and conferred virus resistance to miravirsen treatment. Here, using the J6 (genotype 2a) 5'UTR-NS2 JFH1-based recombinant, we performed reverse-genetics analysis of S1 (ACACUCCG, corresponding to miR-122 seed nucleotide positions 8-1), S2 (CACUCC, positions 7-2), and ACCC (positions 1-4) at the 5' end of the HCV genome (5'E); the CC at positions 2-3 of 5'E is involved in miR-122 binding. We demonstrated that the 5'E required four nucleotides for optimal function, and that G or A at position 3 or combined GA at positions 2-3 of 5'E was permitted. In S1 and S2, several single mutations were allowed at specific positions. A UCC → CGA change at positions 4-3-2 of S1, S2, or both S1 and S2 (S1/S2), as well as a C → G change at position 2 of S1/S2 were permitted. We found that 5'E mutations did not confer virus resistance to miravirsen treatment. However, mutations in S1 and S2 induced virus resistance, and combined S1 and/or S2 mutations conferred higher resistance than single mutations. Identification of miR-122 antagomir resistance-associated mutations will facilitate the study of additional functions of miR-122 in the HCV life cycle and the mechanism of virus escape to host-targeting antiviral approaches.

Hydrogen Peroxide Vapor Decontamination in a Patient Room Using Feline Calicivirus and Murine Norovirus as Surrogate Markers for Human Norovirus.

OBJECTIVE: To determine whether hydrogen peroxide vapor (HPV) could be used to decontaminate caliciviruses from surfaces in a patient room. DESIGN: Feline calicivirus (FCV) and murine norovirus (MNV) were used as surrogate viability markers to mimic the noncultivable human norovirus. Cell culture supernatants of FCV and MNV were dried in triplicate 35-mm wells of 6-well plastic plates. These plates were placed in various positions in a nonoccupied patient room that was subsequently exposed to HPV. Control plates were positioned in a similar room but were never exposed to HPV. METHODS: Virucidal activity was measured in cell culture by reduction in 50% tissue culture infective dose titer for FCV and by both 50% tissue culture infective dose titer and plaque reduction for MNV. RESULTS: Neither viable FCV nor viable MNV could be detected in the test room after HPV treatment. At least 3.65 log reduction for FCV and at least 3.67 log reduction for MNV were found by 50% tissue culture infective dose. With plaque assay, measurable reduction for MNV was at least 2.85 log units. CONCLUSIONS: The successful inactivation of both surrogate viruses indicates that HPV could be a useful tool for surface decontamination of a patient room contaminated by norovirus. Hence nosocomial spread to subsequent patients can be avoided.

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.

Adapted J6/JFH1-based Hepatitis C virus recombinants with genotype-specific NS4A show similar efficacies against lead protease inhibitors, alpha interferon, and a putative NS4A inhibitor.

To facilitate studies of hepatitis C virus (HCV) NS4A, we aimed at developing J6/JFH1-based recombinants with genotype 1- to 7-specific NS4A proteins. We developed efficient culture systems expressing NS4A proteins of genotypes (isolates) 1a (H77 and TN), 1b (J4), 2a (J6), 4a (ED43), 5a (SA13), 6a (HK6a), and 7a (QC69), with peak infectivity titers of ∼3.5 to 4.5 log10 focus-forming units per ml. Except for genotype 2a (J6), growth depended on adaptive mutations identified in long-term culture. Genotype 1a, 1b, and 4a recombinants were adapted by amino acid substitutions F772S (p7) and V1663A (NS4A), while 5a, 6a, and 7a recombinants required additional substitutions in the NS3 protease and/or NS4A. We demonstrated applicability of the developed recombinants for study of antivirals. Genotype 1 to 7 NS4A recombinants showed similar responses to the protease inhibitors telaprevir (VX-950), boceprevir (Sch503034), simeprevir (TMC435350), danoprevir (ITMN-191), and vaniprevir (MK-7009), to alpha interferon 2b, and to the putative NS4A inhibitor ACH-806. The efficacy of ACH-806 was lower than that of protease inhibitors and was not influenced by changes at amino acids 1042 and 1065 (in the NS3 protease), which have been suggested to mediate resistance to ACH-806 in replicons. Genotype 1a, 1b, and 2a recombinants showed viral spread under long-term treatment with ACH-806, without acquisition of resistance mutations in the NS3-NS4A region. Relatively high concentrations of ACH-806 inhibited viral assembly, but not replication, in a single-cycle production assay. The developed HCV culture systems will facilitate studies benefitting from expression of genotype-specific NS4A in a constant backbone in the context of the complete viral replication cycle, including functional studies and evaluations of the efficacy of antivirals.

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.

Molecular epidemiology of Aleutian mink disease virus in Finland.

Aleutian mink disease virus (AMDV) is a parvovirus that causes an immune complex-mediated disease in minks. To gain a more detailed view of the molecular epidemiology of mink AMDV in Finland, we phylogenetically analysed 14 new Finnish strains from 5 farms and all 40 strains with corresponding sequences available in GenBank. A part of the major non-structural (NS1) protein gene was amplified and analysed phylogenetically. A rooted nucleotide tree was constructed using the maximum parsimony method. The strains described in this study showed 86-100% nucleotide identity and were nearly identical on each farm. The ratio of synonymous to non-synonymous substitutions was approximately 2.7, indicating a mild purifying selection. Phylogenetic analysis confirmed that AMDV strains form three groups (I-III), all of which contained Finnish strains. The tree inferred that the three lineages of AMDV have been introduced to Finland independently. The analysis suggested that AMDV strains do not cluster into genotypes based on geographical origin, year of isolation or pathogenicity. Based on these data, the molecular clock is not applicable to AMDV, and within this gene area no recombination was detected.

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.

Viral zoonoses in Europe.

A number of new virus infections have emerged or re-emerged during the past 15 years. Some viruses are spreading to new areas along with climate and environmental changes. The majority of these infections are transmitted from animals to humans, and thus called zoonoses. Zoonotic viruses are, as compared to human-only viruses, much more difficult to eradicate. Infections by several of these viruses may lead to high mortality and also attract attention because they are potential bio-weapons. This review will focus on zoonotic virus infections occurring in Europe.

Ixodid and argasid tick species and west nile virus.

Control of West Nile virus (WNV) can only be effective if the vectors and reservoirs of the virus are identified and controlled. Although mosquitoes are the primary vectors, WNV has repeatedly been isolated from ticks. Therefore, tick-borne transmission studies were performed with an ixodid (Ixodes ricinus) and an argasid tick species (Ornithodoros moubata). Both species became infected after feeding upon viremic hosts, but I. ricinus ticks were unable to maintain the virus. In contrast, O. moubata ticks were infected for at least 132 days, and the infection was maintained through molting and a second bloodmeal. Infected O. moubata ticks transmitted the virus to rodent hosts, albeit at a low level. Moreover, the virus was nonsystemically transmitted between infected and uninfected O. moubata ticks co-fed upon uninfected hosts. Although ticks are unlikely to play a major role in WNV transmission, our findings suggest that some species have the potential to act as reservoirs for the virus.

Desarrollo de un ensayo enzimático para la determinación de anticuerpos IgM anti-cápside del virus de la hepatitis B / An enzyme immunoassay for detection of hepatitis B virus IgM anti-core antibodies

La determinación de anticuerpos IgM anti-cápside (anti-AgcHB) del Virus de la Hepatitis B (VHB) es de gran utilidad para la identificación de infecciones agudas por este virus. El objeto de este estudio fue el desarrollar un sistema inmunodiagnóstico para la detección de anticuerpos IgM anti-AgcHB) Para la realización del ensayo fue necesaria la producción del AgcHB recombinante expresado en Escherichia coli. Una vez obtenido y purificado el antígeno se procedió a estandarizar y evaluar el ensayo. Un ELISA sandwich de captura resultó ser el último para la detección de anticuerpos IgM anti-AgcHB. Se realizó entonces la evaluación de 110 sueros humanos a través de dos ensayos inmunodiagnósticos, el inmunoensayo diagnóstico desarrollado y un estuche comercial. El inmunoensayo desarrollado arrojó una alta sensibilidad (99 por ciento) y especificidad (93 por ciento) al compararlo con el estuche comercial. Los resultados obtenidos permitieron evidenciar la utilidad del sistema de determinación de anticuerpos IgM anti-AgcHB como marcador serológico de una infección causada por el VHB, en particular en áreas endémicas de Sur América