Jonas Näslund

Vaccination with virus-like particles protects mice from lethal infection of Rift Valley Fever Virus

Rift Valley Fever virus (RVFV) regularly accounts for severe and often lethal outbreaks among livestock and humans in Africa. Safe and effective veterinarian and human vaccines are highly needed. We present evidence that administration of RVF virus-like particles (VLPs) induces protective immunity in mice. In an accompanying paper, (Habjan, M., Penski, N., Wagner, V., Spiegel, M., Overby, A.K., Kochs, G., Huiskonen, J., Weber, F., 2009. Efficient production of Rift Valley fever virus-like particles: the antiviral protein MxA can inhibit primary transcription of Bunyaviruses. Virology 385, 400-408) we report the production of these VLPs in mammalian cells. After three subsequent immunizations with 1x10(6) VLPs/dose, high titers of virus-neutralizing antibodies were detected; 11 out of 12 mice were protected from challenge and only 1 out of 12 mice survived infection in the control groups. VLP vaccination efficiently suppressed replication of the challenge virus, whereas in the control animals high RNA levels and increasing antibody titers against the nucleocapsid protein indicated extensive viral replication. Our study demonstrates that the RVF VLPs are highly immunogenic and confer protection against RVFV infection in mice. In the test groups, the vaccinated mice did not exhibit any side effects, and the lack of anti-nucleocapsid protein antibodies serologically distinguished vaccinated animals from experimentally infected animals.

Characterisation of immune responses and protective efficacy in mice after immunisation with Rift Valley Fever virus cDNA constructs

BackgroundAffecting both livestock and humans, Rift Valley Fever is considered as one of the most important viral zoonoses in Africa. However, no licensed vaccines or effective treatments are yet available for human use. Naked DNA vaccines are an interesting approach since the virus is highly infectious and existing attenuated Rift Valley Fever virus vaccine strains display adverse effects in animal trials. In this study, gene-gun immunisations with cDNA encoding structural proteins of the Rift Valley Fever virus were evaluated in mice. The induced immune responses were analysed for the ability to protect mice against virus challenge.ResultsImmunisation with cDNA encoding the nucleocapsid protein induced strong humoral and lymphocyte proliferative immune responses, and virus neutralising antibodies were acquired after vaccination with cDNA encoding the glycoproteins. Even though complete protection was not achieved by genetic immunisation, four out of eight, and five out of eight mice vaccinated with cDNA encoding the nucleocapsid protein or the glycoproteins, respectively, displayed no clinical signs of infection after challenge. In contrast, all fourteen control animals displayed clinical manifestations of Rift Valley Fever after challenge.ConclusionThe appearance of Rift Valley Fever associated clinical signs were significantly decreased among the DNA vaccinated mice and further adjustment of this strategy may result in full protection against Rift Valley Fever.

Association of Rift Valley fever virus infection with miscarriage in Sudanese women: a cross-sectional study

BACKGROUND Rift Valley fever virus is an emerging mosquito-borne virus that causes infections in animals and human beings in Africa and the Arabian Peninsula. Outbreaks of Rift Valley fever lead to mass abortions in livestock, but such abortions have not been identified in human beings. Our aim was to investigate the cause of miscarriages in febrile pregnant women in an area endemic for Rift Valley fever. METHODS Pregnant women with fever of unknown origin who attended the governmental hospital of Port Sudan, Sudan, between June 30, 2011, and Nov 17, 2012, were sampled at admission and included in this cross-sectional study. Medical records were retrieved and haematological tests were done on patient samples. Presence of viral RNA as well as antibodies against a variety of viruses were analysed. Any association of viral infections, symptoms, and laboratory parameters to pregnancy outcome was investigated using Pearsons χ2 test. FINDINGS Of 130 pregnant women with febrile disease, 28 were infected with Rift Valley fever virus and 31 with chikungunya virus, with typical clinical and laboratory findings for the infection in question. 15 (54%) of 28 women with an acute Rift Valley fever virus infection had miscarriages compared with 12 (12%) of 102 women negative for Rift Valley fever virus (p<0·0001). In a multiple logistic regression analysis, adjusting for age, haemorrhagic disease, and chikungunya virus infection, an acute Rift Valley fever virus infection was an independent predictor of having a miscarriage (odds ratio 7·4, 95% CI 2·7-20·1; p<0·0001). INTERPRETATION This study is the first to show an association between infection with Rift Valley fever virus and miscarriage in pregnant women. Further studies are warranted to investigate the possible mechanisms. Our findings have implications for implementation of preventive measures, and evidence-based information to the public in endemic countries should be strongly recommended during Rift Valley fever outbreaks. FUNDING Schlumberger Faculty for the Future, CRDF Global (31141), the Swedish International Development Cooperation Agency, the County Council of Västerbotten, and the Faculty of Medicine, Umeå University.

Kinetics of Rift Valley Fever Virus in experimentally infected mice using quantitative real-time RT-PCR.

Rift Valley Fever (RVF) is an important viral zoonosis in Africa affecting animals and humans. Since no protective vaccines or effective treatments are available for human use, accurate and reliable diagnostic methods are essential for surveillance of the disease in order to implement adequate public health actions. To study the kinetics of the RVF Virus (RVFV) infection, a SYBR Green-based quantitative real-time RT-PCR assay was developed. By using primers targeting the S-segment of RVFV, the detection limit of this assay was estimated to 30 RNA templates. Blood and organs of experimentally infected mice were sampled at different time points and RVFV RNA was quantified. High amounts of RVFV RNA were found in blood, brain, and liver samples shortly after infection with a 1-4 days post infection window for viral RNA detection. Mice developed symptoms after the appearance of serum antibodies, indicating that the host response plays an important role in the outcome of the disease. The RVFV quantitative RT-PCR proved to be a valuable diagnostic tool during the first days of infection, before detectable antibody levels and visual symptoms of RVF were observed.

Identification of Swedish mosquitoes based on molecular barcoding of the COI gene and SNP analysis

Mosquito‐borne infectious diseases are emerging in many regions of the world. Consequently, surveillance of mosquitoes and concomitant infectious agents is of great importance for prediction and prevention of mosquito‐borne infectious diseases. Currently, morphological identification of mosquitoes is the traditional procedure. However, sequencing of specified genes or standard genomic regions, DNA barcoding, has recently been suggested as a global standard for identification and classification of many different species. Our aim was to develop a genetic method to identify mosquitoes and to study their relationship. Mosquitoes were captured at collection sites in northern Sweden and identified morphologically before the cytochrome c oxidase subunit I (COI) gene sequences of 14 of the most common mosquito species were determined. The sequences obtained were then used for phylogenetic placement, for validation and benchmarking of phenetic classifications and finally to develop a hierarchical PCR‐based typing scheme based on single nucleotide polymorphism sites (SNPs) to enable rapid genetic identification, circumventing the need for morphological characterization. The results showed that exact phylogenetic relationships between mosquito taxa were preserved at shorter evolutionary distances, but at deeper levels, they could not be inferred with confidence using COI gene sequence data alone. Fourteen of the most common mosquito species in Sweden were identified by the SNP/PCR‐based typing scheme, demonstrating that genetic typing using SNPs of the COI gene is a useful method for identification of mosquitoes with potential for worldwide application.

Cross-reactive and serospecific epitopes of nucleocapsid proteins of three hantaviruses: Prospects for new diagnostic tools

The diagnosis of infectious diseases is sometimes difficult because of extensive immunological cross-reactivity between related viral antigens. On the path of constructing sero-specific antigens, we have identified residues involved in sero-specific and cross-reactive recognition of the nucleocapsid proteins (NPs) of Puumala virus (PUUV), Seoul virus (SEOV), and Sin Nombre virus (SNV) using serum samples from 17 Nephropathia epidemica patients. The mapping was performed by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis on a panel of N protein derivatives and alanine-substitution mutants in the three different hantavirus backgrounds. Four regions with different serological profiles were identified encompassing the amino acids (aa) 14-17, 22-24, 26, and 35-38. One of the regions showed strong cross-reactivity and was important for the recognition of SEOV and SNV antigens, but not the PUUV antigen (aa 35-38). Two regions displayed perceivable SEOV characteristics (aa 14-17 and aa 22-24 and 26) and the combined result of the alanine replacements resulted in a synergetic effect against the PUUV antigen (aa 14-17, 22-24, 26).

Detection of Puumala and Rift Valley Fever virus by quantitative RT-PCR and virus viability tests in samples of blood dried and stored on filter paper

Haemorrhagic fever viruses cause emerging infections worldwide, and blood or serum is the main sample used for diagnosis. However, storage and transportation of such samples from remote areas to regional laboratories may be complicated and expensive. In this study, a novel approach was evaluated for the detection of Puumala hantavirus (PUUV) RNA and Rift Valley fever virus (RVFV) RNA. Whole-blood samples spiked with viable virus particles were tested in parallel with clinical samples from patients with acute haemorrhagic fever with renal syndrome (nephropathia epidemica). Individual blood samples were spotted on filter paper, dried, and used for RNA extraction at later time points. PUUV RNA was detected by RT-PCR after storage at room temperature for up to six weeks. In contrast, only low copy numbers of RVFV RNA were detected after 1-2 days even though viable RVFV was eluted from the dried filter papers after the same time. The use of filter paper to collect and store blood samples for PUUV RNA detection is therefore a simple and reliable procedure. This approach might facilitate sampling and analysis of other RNA viruses from human or animal sources and could be used for field studies in remote areas or in developing countries.

The Rift Valley Fever virus protein NSm and putative cellular protein interactions

Rift Valley Fever is an infectious viral disease and an emerging problem in many countries of Africa and on the Arabian Peninsula. The causative virus is predominantly transmitted by mosquitoes and high mortality and abortion rates characterize outbreaks in animals while symptoms ranging from mild to life-threatening encephalitis and hemorrhagic fever are noticed among infected humans. For a better prevention and treatment of the infection, an increased knowledge of the infectious process of the virus is required.The focus of this work was to identify protein-protein interactions between the non-structural protein (NSm), encoded by the M-segment of the virus, and host cell proteins. This study was initiated by screening approximately 26 million cDNA clones of a mouse embryonic cDNA library for interactions with the NSm protein using a yeast two-hybrid system.We have identified nine murine proteins that interact with NSm protein of Rift Valley Fever virus, and the putative protein-protein interactions were confirmed by growth selection procedures and β-gal activity measurements. Our results suggest that the cleavage and polyadenylation specificity factor subunit 2 (Cpsf2), the peptidyl-prolyl cis-trans isomerase (cyclophilin)-like 2 protein (Ppil2), and the synaptosome-associated protein of 25 kDa (SNAP-25) are the most promising targets for the NSm protein of the virus during an infection.

Detection and Isolation of Sindbis Virus from Mosquitoes Captured During an Outbreak in Sweden, 2013

Mosquito-borne alphaviruses have the potential to cause large outbreaks throughout the world. Here we investigated the causative agent of an unexpected Sindbis virus (SINV) outbreak during August-September, 2013, in a previously nonendemic region of Sweden. Mosquitoes were collected using carbon dioxide-baited CDC traps at locations close to human cases. The mosquitoes were initially screened as large pools by SINV-specific quantitative RT-PCR, and the SINV-positive mosquitoes were species determined by single-nucleotide polymorphism (SNP) analysis, followed by sequencing the barcoding region of the cytochrome oxidase I gene. The proportion of the collected mosquitoes was determined by a metabarcoding strategy. By using novel strategies for PCR screening and genetic typing, a new SINV strain, Lövånger, was isolated from a pool of 1600 mosquitoes composed of Culex, Culiseta, and Aedes mosquitoes as determined by metabarcoding. The SINV-positive mosquito Culiseta morsitans was identified by SNP analysis and sequencing. After whole-genome sequencing and phylogenetic analysis, the SINV Lövånger isolate was shown to be most closely similar to recent Finnish SINV isolates. In conclusion, within a few weeks, we were able to detect and isolate a novel SINV strain and identify the mosquito vector during a sudden SINV outbreak.

Detection of Sindbis and Inkoo Virus RNA in Genetically Typed Mosquito Larvae Sampled in Northern Sweden

Abstract Introduction: Mosquito-borne viruses have a widespread distribution across the globe and are known to pose serious threats to human and animal health. The maintenance and dissemination of these viruses in nature are driven through horizontal and vertical transmission. In the temperate climate of northern Sweden, there is a dearth of knowledge on whether mosquito-borne viruses that occur are transmitted transovarially. To gain a better understanding of mosquito-borne virus circulation and maintenance, mosquito larvae were sampled in northern Sweden during the first and second year after a large outbreak of Ockelbo disease in 2013 caused by Sindbis virus (SINV). Materials and Methods: A total of 3123 larvae were sampled during the summers of 2014 and 2015 at multiple sites in northern Sweden. The larvae were homogenized and screened for viruses using RT-PCR and sequencing. Species identification of selected larvae was performed by genetic barcoding targeting the cytochrome C oxidase subunit I gene. Results and Discussion: SINV RNA was detected in mosquito larvae of three different species, Ochlerotatus (Oc.) communis, Oc. punctor, and Oc. diantaeus. Inkoo virus (INKV) RNA was detected in Oc. communis larvae. This finding suggested that these mosquitoes could support transovarial transmission of SINV and INKV. Detection of virus in mosquito larva may serve as an early warning for emerging arboviral diseases and add information to epidemiological investigations before, during, and after outbreaks. Furthermore, our results demonstrated the relevance of genetic barcoding as an attractive and effective method for mosquito larva typing. However, further mosquito transmission studies are needed to ascertain the possible role of different mosquito species and developmental stages in the transmission cycle of arboviruses.