Novel strains of Culex flavivirus and Hubei chryso-like virus 1 from the Anopheles mosquito in western Kenya.

Surveillance of mosquito vectors is critical for early detection, prevention and control of vector borne diseases. In this study we used advanced molecular tools, such as DNA barcoding in combination with novel sequencing technologies to discover new and already known viruses in genetically identified mosquito species. Mosquitoes were captured using BG sentinel traps in Western Kenya during May and July 2019, and homogenized individually before pooled into groups of ten mosquitoes. The pools and individual samples were then used for molecular analysis and to infect cell cultures. Of a total of fifty-four (54) 10-pools, thirteen (13) showed cytopathic effect (CPE) on VeroB4 cells, eighteen (18) showed CPE on C6/36 cells. Eight (8) 10-pools out of the 31 CPE positive pools showed CPE on both VeroB4 and C6/36 cells. When using reverse transcriptase polymerase chain reaction (RT-PCR), Sanger sequencing and Twist Comprehensive Viral Research Panel (CVRP) (Twist Biosciences), all pools were found negative by RT-PCR when using genus specific primers targeting alphaviruses, orthobunyaviruses and virus specific primers towards o'nyong-nyong virus, chikungunya virus and Sindbis virus (previously reported to circulate in the region). Interestingly, five pools were RT-PCR positive for flavivirus. Two of the RT-PCR positive pools showed CPE on both VeroB4 and C6/36 cells, two pools showed CPE on C6/36 cells alone and one pool on VeroB4 cells only. Fifty individual mosquito homogenates from the five RT-PCR positive 10-pools were analyzed further for flavivirus RNA. Of these, 19 out of the 50 individual mosquito homogenates indicated the presence of flavivirus RNA. Barcoding of the flavivirus positive mosquitoes revealed the mosquito species as Aedes aegypti (1), Mansonia uniformis (6), Anopheles spp (3), Culex pipiens (5), Culex spp (1), Coquilletidia metallica (2) and Culex quinquefasciatus (1). Of the 19 flavivirus positive individual mosquitoes, five (5) virus positive homogenates were sequenced. Genome sequences of two viruses were completed. One was identified as the single-stranded RNA Culex flavivirus and the other as the double-stranded RNA Hubei chryso-like virus 1. Both viruses were found in the same Anopheles spp. homogenate extracted from a sample that showed CPE on both VeroB4 and C6/36 cells. The detection of both viruses in a single mosquito homogenate indicated coinfection. Phylogenetic analyses suggested that the Culex flavivirus sequence detected was closely related to a Culex flavivirus isolated from Uganda in 2008. All four Hubei chryso-like virus 1 segments clusters closely to Hubei chryso-like virus 1 strains isolated in Australia, China and USA. Two novel strains of insect-specific viruses in Anopheles mosquitoes were detected and characterized.

Evidence of orthohantavirus and leptospira infections in small mammals in an endemic area of Gampaha district in Sri Lanka.

BACKGROUND: Orthohantaviruses and leptospira are emerging zoonotic pathogens of high public health significance. The epidemiology of orthohantavirus infections and leptospirosis is similar and presents related clinical pictures in humans. However, a paucity of data on actual reservoir hosts for orthohantaviruses and leptospira exists. Therefore, this study aimed at determining the occurrence of orthohantaviruses and leptospira in small mammals captured in an endemic region of Sri Lanka. METHODS: Rodents and shrews were morphologically and/or genetically identified using morphological keys and DNA barcoding techniques targeting the cytochrome oxidase b subunit gene (Cytb). Lung tissues and sera were subsequently analyzed for the presence of orthohantavirus RNA using qRT-PCR. Sera of rats were tested for IgG antibodies against orthohantaviruses and leptospira. RESULTS: Forty-three (43) small mammals representing: Rattus (R.) rattus (black rat) or R. tanezumi (Asian rat), Suncus murinus (Asian house shrew), R. norvegicus (brown rat) and Mus musculus (house mouse) were investigated. No orthohantavirus RNA was detected from the lung tissue or serum samples of these animals. Elevated levels of IgG antibodies against Puumala orthohantavirus (PUUV) and/or Seoul orthohantavirus (SEOV) antigens were detected in sera of 28 (72%) out of the 39 rats analysed. Interestingly, 36 (92%) of the 39 rats also showed presence of anti leptospira-IgG antibodies in their serum, representing dual infection or dual exposure in 26/39 (66.7%) of examined rats. CONCLUSIONS: This project targets important public health questions concerning the occupational risk of orthohantavirus infections and/or leptospirosis in an endemic region of Sri Lanka. Most rats (72%) in our study displayed antibodies reacting to orthohantavirus NP antigens, related to PUUV and/or SEOV. No correlation between the orthohantavirus and leptospira IgG antibody levels were noticed. Finally, a combination of both morphological and DNA barcoding approaches revealed that several species of rats may play a role in the maintenance and transmission of orthohantavirus and leptospira in Sri Lanka.

Emerging Mosquito-Borne Viruses Linked to Aedes aegypti and Aedes albopictus: Global Status and Preventive Strategies.

Emerging mosquito-borne viruses continue to cause serious health problems and economic burden among billions of people living in and near the tropical belt of the world. The highly invasive mosquito species Aedes aegypti and Aedes albopictus have successively invaded and expanded their presence as key vectors of Chikungunya virus, dengue virus, yellow fever virus, and Zika virus, and that has consecutively led to frequent outbreaks of the corresponding viral diseases. Of note, these two mosquito species have gradually adapted to the changing weather and environmental conditions leading to a shift in the epidemiology of the viral diseases, and facilitated their establishment in new ecozones inhabited by immunologically naive human populations. Many abilities of Ae. aegypti and Ae. albopictus, as vectors of significant arbovirus pathogens, may affect the infection and transmission rates after a bloodmeal, and may influence the vector competence for either virus. We highlight that many collaborating risk factors, for example, the global transportation systems may result in sporadic and more local outbreaks caused by mosquito-borne viruses related to Ae. aegypti and/or Ae. albopictus. Those local outbreaks could in synergy grow and produce larger epidemics with pandemic characters. There is an urgent need for improved surveillance of vector populations, human cases, and reliable prediction models. In summary, we recommend new and innovative strategies for the prevention of these types of infections.

Seewis hantavirus in common shrew (Sorex araneus) in Sweden.

BACKGROUND: Rodent borne hantaviruses are emerging viruses infecting humans through inhalation. They cause hemorrhagic fever with renal syndrome and hemorrhagic cardiopulmonary syndrome. Recently, hantaviruses have been detected in other small mammals such as Soricomorpha (shrews, moles) and Chiroptera (bats), suggested as reservoirs for potential pandemic viruses and to play a role in the evolution of hantaviruses. It is important to study the global virome in different reservoirs, therefore our aim was to investigate whether shrews in Sweden carried any hantaviruses. Moreover, to accurately determine the host species, we developed a molecular method for identification of shrews. METHOD: Shrews (n = 198), caught during 1998 in Sweden, were screened with a pan-hantavirus PCR using primers from a conserved region of the large genome segment. In addition to morphological typing of shrews, we developed a molecular based typing method using sequencing of the mitochondrial cytochrome C oxidase I (COI) and cytochrome B (CytB) genes. PCR amplified hantavirus and shrew fragments were sequenced and phylogenetically analysed. RESULTS: Hantavirus RNA was detected in three shrews. Sequencing identified the virus as Seewis hantavirus (SWSV), most closely related to previous isolates from Finland and Russia. All three SWSV sequences were retrieved from common shrews (Sorex araneus) sampled in Västerbotten County, Sweden. The genetic assay for shrew identification was able to identify native Swedish shrew species, and the genetic typing of the Swedish common shrews revealed that they were most similar to common shrews from Russia. CONCLUSION: We detected SWSV RNA in Swedish common shrew samples and developed a genetic assay for shrew identification based on the COI and CytB genes. This was the first report of presence of hantavirus in Swedish shrews.

Globe-Trotting Aedes aegypti and Aedes albopictus: Risk Factors for Arbovirus Pandemics.

Introduction: Two species of Aedes (Ae.) mosquitoes (Ae. aegypti and Ae. albopictus) are primary vectors for emerging arboviruses that are a significant threat to public health and economic burden worldwide. Distribution of these vectors and the associated arboviruses, such as dengue virus, chikungunya virus, yellow fever virus, and Zika virus, was for a long time restricted by geographical, ecological, and biological factors. Presently, arbovirus emergence and dispersion are more rapid and geographically widespread, largely due to expansion of the range for these two mosquitoes that have exploited the global transportation network, land perturbation, and failure to contain the mosquito population coupled with enhanced vector competence. Ae. aegypti and Ae. albopictus may also sustain transmission between humans without having to depend on their natural reservoir forest cycles due to arthropod adaptation to urbanization. Currently, there is no single strategy that is adequate to control these vectors, especially when managing arbovirus outbreaks. Objective: This review aimed at presenting the characteristics and abilities of Ae. aegypti and Ae. albopictus, which can drive a global public health risk, and suggests strategies for prevention and control. Methods: This review presents the geographic range, reproduction and ecology, vector competence, genetic evolution, and biological and chemical control of these two mosquito species and how they have changed and developed over time combined with factors that may drive pandemics and mitigation measures. Conclusion: We suggest that more efforts should be geared toward the development of a concerted multidisciplinary approach.

Population Dynamics of Bank Voles Predicts Human Puumala Hantavirus Risk.

Predicting risk of zoonotic diseases, i.e., diseases shared by humans and animals, is often complicated by the population ecology of wildlife host(s). We here demonstrate how ecological knowledge of a disease system can be used for early prediction of human risk using Puumala hantavirus (PUUV) in bank voles (Myodes glareolus), which causes Nephropathia epidemica (NE) in humans, as a model system. Bank vole populations at northern latitudes exhibit multiannual fluctuations in density and spatial distribution, a phenomenon that has been studied extensively. Nevertheless, existing studies predict NE incidence only a few months before an outbreak. We used a time series on cyclic bank vole population density (1972-2013), their PUUV infection rates (1979-1986; 2003-2013), and NE incidence in Sweden (1990-2013). Depending on the relationship between vole density and infection prevalence (proportion of infected animals), either overall density of bank voles or the density of infected bank voles may be used to predict seasonal NE incidence. The density and spatial distribution of voles at density minima of a population cycle contribute to the early warning of NE risk later at its cyclic peak. When bank voles remain relatively widespread in the landscape during cyclic minima, PUUV can spread from a high baseline during a cycle, culminating in high prevalence in bank voles and potentially high NE risk during peak densities.

Experimental Infection and Transmission Competence of Sindbis Virus in Culex torrentium and Culex pipiens Mosquitoes from Northern Sweden.

INTRODUCTION: Sindbis virus (SINV) is a mosquito-borne Alphavirus known to infect birds and cause intermittent outbreaks among humans in Fenno-Scandia. In Sweden, the endemic area has mainly been in central Sweden. Recently, SINV infections have emerged to northern Sweden, but the vectorial efficiency for SINV of mosquito species in this northern region has not yet been ascertained. OBJECTIVE: Mosquito larvae were sampled from the Umeå region in northern Sweden and propagated in a laboratory to adult stage to investigate the infection, dissemination, and transmission efficiency of SINV in mosquitoes. MATERIALS AND METHODS: The mosquito species were identified by DNA barcoding of the cytochrome oxidase I gene. Culex torrentium was the most abundant (82.2%) followed by Culex pipiens (14.4%), Aedes annulipes (1.1%), Anopheles claviger (1.1%), Culiseta bergrothi (1.1%), or other unidentified species (1.1%). Mosquitoes were fed with SINV-infected blood and monitored for 29 days to determine the viral extrinsic incubation period. Infection and dissemination were determined by RT-qPCR screening of dissected body parts of individual mosquitoes. Viral transmission was determined from saliva collected from individual mosquitoes at 7, 14, and 29 days. SINV was detected by cell culture using BHK-21 cells, RT-qPCR, and sequencing. RESULTS: Cx. torrentium was the only mosquito species in our study that was able to transmit SINV. The overall transmission efficiency of SINV in Cx. torrentium was 6.8%. The rates of SINV infection, dissemination, and transmission in Cx. torrentium were 11%, 75%, and 83%, respectively. CONCLUSIONS: Cx. torrentium may be the key vector involved in SINV transmission in northern Sweden.

Mosquitoborne Sindbis Virus Infection and Long-Term Illness.

An unexpected human outbreak of the mosquitoborne Sindbis virus occurred in a previously nonendemic area of Sweden. At follow-up, 6-8 months after infection, 39% of patients had chronic arthralgia that affected their daily activities. Vectorborne infections may disseminate rapidly into new areas and cause acute and chronic disease.

Quantification and kinetics of viral RNA transcripts produced in Orthohantavirus infected cells.

BACKGROUND: Rodent borne viruses of the Orthohantavirus genus cause hemorrhagic fever with renal syndrome among people in Eurasia, and hantavirus cardiopulmonary syndrome in the Americas. At present, there are no specific treatments or efficient vaccines against these diseases. Improved understanding of viral transcription and replication may instigate targeted treatment of Orthohantavirus infections. For this purpose, we investigated the kinetics and levels of viral RNA transcription during an ongoing infection in-vitro. METHODS: Vero E6 cells were infected with Puumala Orthohantavirus (strain Kazan) before cells and supernatants were collected at different time points post infection for the detection of viral RNAs. A plasmid containing primer binding sites of the three Orthohantavirus segments small (S), medium (M) and large (L) was constructed and standard curves were generated to calculate the copy numbers of the individual transcripts in the collected samples. RESULTS: Our results indicated a rapid increase in the copy number of viral RNAs after 9 h post infection. At peak days, 2-6 days after infection, the S- and M-segment transcripts became thousand and hundred-fold more abundant than the copy number of the L-segment RNA, respectively. The presence of viral RNA in the cell culture media was detected at later time-points. CONCLUSIONS: We have developed a method to follow RNA transcription in-vitro after synchronous infection of Vero cells. The obtained results may contribute to the understanding of the viral replication, and may have implications in the development of antiviral drugs targeting transcription or replication of negative stranded RNA viruses.

Mosquito-borne Inkoo virus in northern Sweden - isolation and whole genome sequencing.

BACKGROUND: Inkoo virus (INKV) is a less known mosquito-borne virus belonging to Bunyaviridae, genus Orthobunyavirus, California serogroup. Studies indicate that INKV infection is mainly asymptomatic, but can cause mild encephalitis in humans. In northern Europe, the sero-prevalence against INKV is high, 41% in Sweden and 51% in Finland. Previously, INKV RNA has been detected in adult Aedes (Ae.) communis, Ae. hexodontus and Ae. punctor mosquitoes and Ae. communis larvae, but there are still gaps of knowledge regarding mosquito vectors and genetic diversity. Therefore, we aimed to determine the occurrence of INKV in its mosquito vector and characterize the isolates. METHODS: About 125,000 mosquitoes were collected during a mosquito-borne virus surveillance in northern Sweden during the summer period of 2015. Of these, 10,000 mosquitoes were processed for virus isolation and detection using cell culture and RT-PCR. Virus isolates were further characterized by whole genome sequencing. Genetic typing of mosquito species was conducted by cytochrome oxidase subunit I (COI) gene amplification and sequencing (genetic barcoding). RESULTS: Several Ae. communis mosquitoes were found positive for INKV RNA and two isolates were obtained. The first complete sequences of the small (S), medium (M), and large (L) segments of INKV in Sweden were obtained. Phylogenetic analysis showed that the INKV genome was most closely related to other INKV isolates from Sweden and Finland. Of the three INKV genome segments, the INKV M segment had the highest frequency of non-synonymous mutations. The overall G/C-content of INKV genes was low for the N/NSs genes (43.8-45.5%), polyprotein (Gn/Gc/NSm) gene (35.6%) and the RNA polymerase gene (33.8%) This may be due to the fact that INKV in most instances utilized A or T in the third codon position. CONCLUSIONS: INKV is frequently circulating in northern Sweden and Ae. communis is the key vector. The high mutation rate of the INKV M segment may have consequences on virulence.

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 Pearson's 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.

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

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.

Molecular Diagnosis of Hemorrhagic Fever with Renal Syndrome Caused by Puumala Virus.

Rodent-borne hantaviruses cause two severe acute diseases: hemorrhagic fever with renal syndrome (HFRS) in Eurasia, and hantavirus pulmonary syndrome (HPS; also called hantavirus cardiopulmonary syndrome [HCPS]) in the Americas. Puumala virus (PUUV) is the most common causative agent of HFRS in Europe. Current routine diagnostic methods are based on serological analyses and can yield inconclusive results. Hantavirus-infected patients are viremic during the early phase of disease; therefore, detection of viral RNA genomes can be a valuable complement to existing serological methods. However, the high genomic sequence diversity of PUUV has hampered the development of molecular diagnostics, and currently no real-time reverse transcription-quantitative (RT)-PCR assay is available for routine diagnosis of HFRS. Here, we present a novel PUUV RT-PCR assay. The assay was validated for routine diagnosis of HFRS on samples collected in Sweden during the winter season from 2013 to 2014. The assay allowed detection of PUUV RNA in 98.7% of confirmed clinical HFRS samples collected within 8 days after symptomatic onset. In summary, this study shows that real-time RT-PCR can be a reliable alternative to serological tests during the early phase of HFRS.

Global emergence of Alphaviruses that cause arthritis in humans.

Arthropod-borne viruses (arboviruses) may cause severe emerging and re-emerging infectious diseases, which pose a significant threat to human and animal health in the world today. These infectious diseases range from mild febrile illnesses, arthritis, and encephalitis to haemorrhagic fevers. It is postulated that certain environmental factors, vector competence, and host susceptibility have a major impact on the ecology of arboviral diseases. Presently, there is a great interest in the emergence of Alphaviruses because these viruses, including Chikungunya virus, O'nyong'nyong virus, Sindbis virus, Ross River virus, and Mayaro virus, have caused outbreaks in Africa, Asia, Australia, Europe, and America. Some of these viruses are more common in the tropics, whereas others are also found in temperate regions, but the actual factors driving Alphavirus emergence and re-emergence remain unresolved. Furthermore, little is known about the transmission dynamics, pathophysiology, genetic diversity, and evolution of circulating viral strains. In addition, the clinical presentation of Alphaviruses may be similar to other diseases such as dengue, malaria, and typhoid, hence leading to misdiagnosis. However, the typical presence of arthritis may distinguish between Alphaviruses and other differential diagnoses. The absence of validated diagnostic kits for Alphaviruses makes even routine surveillance less feasible. For that purpose, this review describes the occurrence, genetic diversity, clinical characteristics, and the mechanisms involving Alphaviruses causing arthritis in humans. This information may serve as a basis for better awareness and detection of Alphavirus-caused diseases during outbreaks and in establishing appropriate prevention and control measures.

Acetylcholinesterases from the Disease Vectors Aedes aegypti and Anopheles gambiae: Functional Characterization and Comparisons with Vertebrate Orthologues.

Mosquitoes of the Anopheles (An.) and Aedes (Ae.) genus are principal vectors of human diseases including malaria, dengue and yellow fever. Insecticide-based vector control is an established and important way of preventing transmission of such infections. Currently used insecticides can efficiently control mosquito populations, but there are growing concerns about emerging resistance, off-target toxicity and their ability to alter ecosystems. A potential target for the development of insecticides with reduced off-target toxicity is the cholinergic enzyme acetylcholinesterase (AChE). Herein, we report cloning, baculoviral expression and functional characterization of the wild-type AChE genes (ace-1) from An. gambiae and Ae. aegypti, including a naturally occurring insecticide-resistant (G119S) mutant of An. gambiae. Using enzymatic digestion and liquid chromatography-tandem mass spectrometry we found that the secreted proteins were post-translationally modified. The Michaelis-Menten constants and turnover numbers of the mosquito enzymes were lower than those of the orthologous AChEs from Mus musculus and Homo sapiens. We also found that the G119S substitution reduced the turnover rate of substrates and the potency of selected covalent inhibitors. Furthermore, non-covalent inhibitors were less sensitive to the G119S substitution and differentiate the mosquito enzymes from corresponding vertebrate enzymes. Our findings indicate that it may be possible to develop selective non-covalent inhibitors that effectively target both the wild-type and insecticide resistant mutants of mosquito AChE.

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.

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.

Development and evaluation of a broad reacting SYBR-green based quantitative real-time PCR for the detection of different hantaviruses.

BACKGROUND: Hantaviruses are endemic in most parts of the world and cause hundreds of thousand human cases of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) annually throughout Eurasia and the Americas. They are zoonotic viruses, most commonly transmitted to humans by aerosolized rodent excreta. New hantaviruses are frequently discovered in previously unknown reservoir species and geographic areas. Consequently, there is a need to improve hantavirus diagnostics. OBJECTIVES: This paper describes the design and evaluation of a rapid and robust quantitative real-time PCR (QRT-PCR) assay able to detect a wide range of hantaviruses. STUDY DESIGN: Primers with the potential to detect different hantaviruses were designed from conserved regions of different hantavirus L segments, as identified from multiple sequence alignments. RESULTS: By using SYBR-green-based QRT-PCR 100-1000 target molecules of in vitro produced RNA and less than 100 copies of hantavirus RNA from different hantavirus clades and regions of the world were detected. When using the assay on clinical samples from patients with acute HFRS, Puumala hantavirus (PUUV) RNA was confirmed in all previously positive samples. Notably, the broad reacting L-segment QRT-PCR also detected viral RNA in HFRS patient samples, previously negative by a QRT-PCR targeting the S segment of PUUV. CONCLUSIONS: This novel assay provides a powerful tool for diagnosis of hantaviruses from different clades and regions and may also be useful in surveys with the purpose of finding new hantaviruses in rodent or insectivore species.

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 ß-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 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.