Niina Putkuri

Tula hantavirus triggers pro-apoptotic signals of ER stress in Vero E6 cells

Abstract Tula virus is a member of the Hantavirus genus of the family Bunyaviridae. Viruses of this family have an unusual pattern of intracellular maturation at the ER–Golgi compartment. We recently found that Tula virus, similar to several other hantaviruses, is able to induce apoptosis in cultured cells [Li, X.D., Kukkonen, S., Vapalahti, O., Plyusnin, A., Lankinen, H., Vaheri, A., 2004. Tula hantavirus infection of Vero E6 cells induces apoptosis involving caspase 8 activation. J. Gen. Virol. 85, 3261–3268.]. However, the cellular mechanisms remain to be clarified. In this study, we demonstrate that the progressive replication of Tula virus in Vero E6 cells initiates several death programs that are intimately associated with ER stress: (1) early activation of ER-resident caspase-12; (2) phosphorylation of Jun NH2-terminal kinase (JNK) and its downstream target transcriptional factor, c-jun; (3) induction of the pro-apoptotic transcriptional factor, growth arrest- and DNA damage-inducible gene 153, or C/EBP homologous protein (Gadd153/chop); and (4) changes in the ER-membrane protein BAP31 implying cross-talk with the mitochondrial apoptosis pathway. Furthermore, we confirmed that a sustained ER stress was induced marked by an increased expression of an ER chaperone Grp78/BiP. Taken together, we have identified involvement of ER stress-mediated death program in Tula virus-infected Vero E6 cells which provides a new approach to understand the mechanisms in hantavirus-induced apoptosis.

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

ABSTRACT 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.

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.

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.

Detection of human orthopoxvirus infections and differentiation of smallpox virus with real-time PCR.

We developed a real‐time PCR protocol to detect orthopoxviruses (OPVs) from different clinical specimens and to separate variola virus from other OPVs. In our protocol, we used automated nucleic acid extraction system together with real‐time PCR to create a simple, safe and fast procedure to obtain an initial result. The sensitivity was better by using designed hybridization probes as compared to SYBR green I for detection. The detection limit ranged from 13 to 1,300 copies per 20 µl reaction volume depending on the sample type. The PCR detected all OPVs pathogenic to human (variola, cowpox, monkeypox, vaccinia) as well as camelpox and ectromelia viruses. Amplification of variola virus sequences could be distinguished from other OPVs by melting curve analysis. We also demonstrated the applicability of the assay in human cases of cowpox and vaccinia virus infections. J. Med. Virol. 81:146–152, 2009.

Complete coding sequence and molecular epidemiological analysis of Sindbis virus isolates from mosquitoes and humans, Finland

Sindbis virus (SINV) is an arthropod-borne alphavirus, which causes rash-arthritis, particularly in Finland. SINV is transmitted by mosquitoes in Finland but thus far no virus has been isolated from mosquitoes. In this study, we report the isolation of the first SINV strain from mosquitoes in Finland and its full-length protein-coding sequence. We furthermore describe the full-length coding sequence of six SINV strains previously isolated from humans in Finland and from a mosquito in Russia. The strain isolated from mosquitoes (Ilomantsi-2005M) was very closely related to all the other Northern European SINV strains. We found 9 aa positions, of which five in the nsP3 protein C terminus, to be distinctive signatures for the Northern European strains that may be associated with vector or host species adaptation. Phylogenetic analyses further indicate that SINV has a local circulation in endemic regions in Northern Europe and no novel strains are frequently being introduced.

Isolation and characterization of a California encephalitis serogroup orthobunyavirus from Finnish mosquitoes

The mosquito-borne California encephalitis serogroup viruses of the genus Orthobunyavirus (family Bunyaviridae) include several causative agents of encephalitis in humans. Until recently, Inkoo virus (INKV) was the only orthobunyavirus isolated in Finland, showing high seroprevalence in the population. In this study, we recovered five orthobunyavirus isolates from mosquitoes collected in eastern Finland in the early autumns of 2007 and 2008 by inoculation of Vero cells. The isolates were determined by S, M and L segment sequences to represent the California encephalitis virus species but distinct from INKV (68% polyprotein amino acid (aa) identity). In genetic analyses, isolates clustered together with a number of westernmost Chatanga virus isolates (98% polyprotein aa identity) reported from Russia, forming a distinct phylogroup. However, the sequence homology of this phylogroup to the majority of Chatanga isolates, comprising three different geographically clustered phylogroups, was considerably lower (89-92% polyprotein aa identity). The five new isolates were designated as Möhkö isolates of Chatanga virus, according to the village of origin. The isolates were closely related to Snowshoe hare virus (SSHV) and La Crosse virus (LACV) with an aa identity of 87% and 82% within the M segment polyprotein, respectively. The genetic relatedness of Möhkö isolates to a number of human pathogenic orthobunyaviruses warrants further investigation on their potential disease associations and further serological analysis is needed to compare them to other Chatanga virus isolates and SSHV to determine their true antigenic relation.

Prevalence and Protein Specificity of Human Antibodies to Inkoo Virus Infection

ABSTRACT Inkoo virus (INKV), a member of the California serogroup orthobunyaviruses, is circulating widely in northern Europe. Although the virus was discovered over 40 years ago, the disease associations and immune responses in human infection are poorly characterized. We first developed an immunofluorescence assay (IFA) for the detection of INKV antibodies in humans, and then we studied a panel of 1,292 sera in patients with a febrile illness in Finland. We found four acute (immunoglobulin M [IgM] positive) INKV infections and an IgG seroprevalence of 51.3%. The data indicate that the infection has become more common than it was in the 1960s, especially in southern Finland. Two distinct IgG IFA fluorescence patterns were observed: a granular pattern in sera from patients with acute INKV infection and a diffuse pattern in those with long-standing immunity. Further analysis with a panel of INKV-positive sera (n = 18; verified by neutralization assay) of protein-specific responses, using immunoprecipitation and IFA based on baculovirus-expressed INK N, Gn, and Gc proteins, demonstrated a strong IgG response predominantly towards N protein in the acute phase. In contrast, in patients with long-standing immunity, the Gc response was more prominent and the N response was weaker. In conclusion, a diagnostic IgG IFA pattern distinguishing between acute infection and long-standing immunity was observed. N protein seems to be the optimal antigen for the serodiagnosis of acute infection, and the Gc protein could be appropriate for the serosurveillance of INKV.

Development and evaluation of a real-time EBOV-L-RT-qPCR for detection of Zaire ebolavirus

An RT-qPCR targeting EBOV-L including the preceding RNA extraction protocol were set up and evaluated.

Acute Human Inkoo and Chatanga Virus Infections, Finland

Most cases appeared to be subclinical, but a few patients, usually children, required hospitalization.