Sindbis Virus Strains of Divergent Origin Isolated from Humans and Mosquitoes During a Recent Outbreak in Finland.

Sindbis virus (SINV) is a mosquito-borne avian hosted virus that is widely distributed in Europe, Africa, Asia, and Oceania. Disease in humans is documented mainly from Northern Europe and South Africa and associated with genotype I. In 2018 under extremely warm climatic conditions, a small outbreak of 71 diagnosed SINV infections was recorded in Finland. We screened 52 mosquito pools (570 mosquitoes) and 223 human sera for SINV with real-time RT-PCR and the positive samples with virus isolation. One SINV strain was isolated from a pool (n = 13) of genus Ochlerotatus mosquitoes and three strains from patient serum samples. Complete genome analysis suggested all the isolates to be divergent from one another and related to previous Finnish, Swedish, and German strains. The study provides evidence of SINV strain transfer within Europe across regions with different epidemiological characteristics. Whether these are influenced by different mosquito genera involved in the transmission remains to be studied.

Sindbis virus polyarthritis outbreak signalled by virus prevalence in the mosquito vectors.

Polyarthritis and rash caused by Sindbis virus (SINV), was first recognised in northern Europe about 50 years ago and is known as Ockelbo disease in Sweden and Pogosta disease in Finland. This mosquito-borne virus occurs mainly in tropical and sub-tropical countries, and in northern Europe it is suggested to cause regularly reoccurring outbreaks. Here a seven-year cycle of SINV outbreaks has been referred to in scientific papers, although the hypothesis is based solely on reported human cases. In the search for a more objective outbreak signal, we evaluated mosquito abundance and SINV prevalence in vector mosquitoes from an endemic area in central Sweden. Vector mosquitoes collected in the River Dalälven floodplains during the years before, during, and after the hypothesised 2002 outbreak year were assayed for virus on cell culture. Obtained isolates were partially sequenced, and the nucleotide sequences analysed using Bayesian maximum clade credibility and median joining network analysis. Only one SINV strain was recovered in 2001, and 4 strains in 2003, while 15 strains were recovered in 2002 with significantly increased infection rates in both the enzootic and the bridge-vectors. In 2002, the Maximum Likelihood Estimated infection rates were 10.0/1000 in the enzootic vectors Culex torrentium/pipiens, and 0.62/1000 in the bridge-vector Aedes cinereus, compared to 4.9/1000 and 0.0/1000 in 2001 and 0.0/1000 and 0.32/1000 in 2003 Sequence analysis showed that all isolates belonged to the SINV genotype I (SINV-I). The genetic analysis revealed local maintenance of four SINV-I clades in the River Dalälven floodplains over the years. Our findings suggest that increased SINV-I prevalence in vector mosquitoes constitutes the most valuable outbreak marker for further scrutinising the hypothesized seven-year cycle of SINV-I outbreaks and the mechanisms behind.

Evolutionary analyses of Sindbis virus strains isolated from mosquitoes in Kenya.

Sindbis virus (SINV) is a mosquito borne virus maintained in nature in a mosquito-bird cycle, with human outbreaks known to occur in Northern Europe and parts of Africa. We analyzed five SINV strains isolated in Kenya from five different mosquito species and geographic locations between 2007 and 2013. Phylogenetic relationships and evolutionary inferences were performed using maximum likelihood and Bayesian phylogenetic inference approaches. Selection analyses were carried out based on the virus envelope glycoproteins (E1, E2) and non-structural protein (nsP4) genes. Phylogenetic analysis revealed that all the Kenyan SINV isolates belonged to genotype 1 with selection analyses suggesting that SINV E1, E2 and nsP4 protein encoding genes were predominantly evolving under negative selection.

Operational mosquito and vector-borne diseases surveillance at Incirlik Air Base, Turkey.

Arboviruses on Incirlik Air Base, Turkey, pose a threat to military personnel and civilians, but might also be relevant for understanding the threats in neighboring conflict zones such as Syria. We reviewed 6 years of mosquito and arbovirus surveillance at Incirlik Air Base. Over 6,000 mosquitoes were identified as Aedes caspius, Anopheles claviger, Culex mimeticus, Cx. perexiguus, Cx. pipiens, Cx. sinaiticus, and Culiseta longiareolata. Almost all of the mosquitoes (more than 90%) were Cx. perexiguus or Cx. pipiens. Both West Nile virus and Sindbis virus were detected in 6 mosquito pools among collections made in 2013, 2014, and 2015.

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.

Concentration of Sindbis virus with optimized gradient insulator-based dielectrophoresis.

Biotechnology, separation science, and clinical research are impacted by microfluidic devices. Separation and manipulation of bioparticles such as DNA, protein and viruses are performed on these platforms. Microfluidic systems provide many attractive features, including small sample size, rapid detection, high sensitivity and short processing time. Dielectrophoresis (DEP) and electrophoresis are especially well suited to microscale bioparticle control and have been demonstrated in many formats. In this work, an optimized gradient insulator-based DEP device was utilized for concentration of Sindbis virus, an animal virus with a diameter of 68 nm. Within only a few seconds, the concentration of Sindbis virus can be increased by two to six times in the channel under easily accessible voltages as low as about 70 V. Compared with traditional diagnostic methods used in virology, DEP-based microfluidics can enable faster isolation, detection and concentration of viruses in a single step within a short time.

Enveloped virus flocculation and removal in osmolyte solutions.

Our ability to reduce infectious disease burden throughout the world has been greatly improved by the creation of vaccines. However, worldwide immunization rates are low. The two most likely reasons are the lack of sufficient distribution in underdeveloped countries and the high cost of vaccine products. The high costs are due to the difficulties of manufacturing individual vaccine products with specialized purification trains. In this study, we propose to use virus flocculation in osmolytes, followed by microfiltration, as an alternative vaccine purification operation. In our previous work, we demonstrated that osmolytes preferentially flocculate a non-enveloped virus, porcine parvovirus (PPV). In this work we show that osmolytes flocculate the enveloped virus, Sindbis virus heat resistant strain (SVHR), and demonstrate a >80% removal with a 0.2 µm microfilter membrane while leaving proteins in solution. The best osmolytes were tested for their ability to flocculate SVHR at different concentrations, pH and ionic strengths. Our best removal was 98% of SVHR in 0.3M mannitol at a pH of 5. We propose that osmolytes are able to flocculate hydrophobic non-enveloped and enveloped virus particles by the reduction of the hydration layer around the particles, which stimulates virus aggregation. Now that we have demonstrated that protecting osmolytes flocculate viruses, this method has the potential to be a future platform purification process for vaccines.

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 VENEZUELAN EQUINE ENCEPHALOMYELITIS VIRUS RNA IN BIOLOGICAL SAMPLES BY REVERSE-TRANSCRIPTION POLYMERASE CHAIN REACTION].

AIM: Detection-and identification of Venezuelan equine encephalomyelitis (VEE) virus RNA in biological samples by reverse-transcription polymerase chain reaction (RT-PCR) and RT-PCR in real time (rRT-PCR). MATERIALS AND METHODS: VEE, Sindbis, West Nile, Japanese and tick-borne encephalitis viruses were studied. Cell culture of chicken fibroblasts, outbred mice and rats, Javanese macaques were used in the experiments. Biological activity determination of the running culture of causative agents used in the experiments was carried out by negative colony method in monolayer cell culture under agar coating. and using intra-cerebral infection of mice. Reagent kits developed in the 48th Central Research Institute and Institute of Analytical Instrument Engineering were used during execution of experiments of VEE virus RNA detection by RT-PCR and rRT-PCR. RESULTS: VEE virus was detected in biological samples by various methods. Data from RT-PCR and rRT-PCR are in accordance with the results of virus detection in samples using sensitive animals. CONCLUSION: Use of molecular-diagnostics methods for detection in biological samples of a causative agent of a dangerous infectious disease is important for procuring biological safety of Russian Federation.

Culex gelidus: an emerging mosquito vector with potential to transmit multiple virus infections.

Culex gelidus Theobald has emerged as a major vector of Japanese encephalitis virus (JEV) in India, Southeast Asian countries and Australia. The species has expanded its geographic distribution from the Indian subcontinent to Japan, China, other Southeast Asian countries, Island nations in Australasian region and Australia. In recent years, a sudden increase in its population especially in the urban and sub-urban areas has been observed in several countries, thus, becoming a dominant mosquito species. The virus has been repeatedly isolated from from different geographical locations making it one of the most important vectors of JEV. Apart from JEV, other viruses of public health importance, viz. Getah, Ross River (RRV), Sindbis and Tembusu have been isolated from the mosquito. Experimental studies have shown that the mosquito Cx. qelidus is highly competent to transmit West Nile, Kunjin and Murray valley encephalitis viruses with infection and transmission rates of >80 and >50%, respectively for each virus. The species is also found competent to transmit RRV, but at a lower rate. Experimental studies have shown that the species is susceptible to chikungunya, Chandipura and Chittoor (Batai) viruses. Development of resistance to DDT and malathion has also been detected in the species recently. The invasive nature, ability to breed both in fresh and dirty waters, development of resistance to insecticides, high anthropophily and its potential to transmit important human viruses pose an increased threat of viral encephalitis in India and Oriental region especially in the light of explosive increase in its population.

Isolation of sindbis virus from a hooded crow in Germany.

Sindbis virus (SINV) is an arbovirus that causes clinical symptoms, including arthritis, rash, and fever during acute human infections. In Europe, SINV outbreaks are largely restricted to northern Europe. Intrigued by the isolation of SINV from mosquitoes in southwestern Germany in 2009, we initiated a passive arbovirus-monitoring program in birds and analyzed a total of 685 samples. By this approach, we were able to detect a SINV in a Hooded Crow in Germany for the first time. It was possible to isolate SINV virus in cell cultures and even to visualize virus particles by electron microscopy. After the determination of the complete SINV genome sequence, the phylogenetic analysis revealed its close relationship to SINV genotype I sequences previously obtained from mosquitoes in Germany and Scandinavia. This first report on the isolation of viable SINV indicates the potential involvement of crows in an enzootic circulation of SINV in Germany and Central Europe.

Seroprevalence of Sindbis virus and associated risk factors in northern Sweden.

Mosquito-borne Sindbis virus (SINV) cause disease characterized by rash, fever and arthritis which often leads to long-lasting arthralgia. To determine the seroprevalence of SINV and associated risk factors in northern Sweden, a randomly selected population aged between 25 and 74 years were invited to join the MONICA study. Serum from 1611 samples were analysed for specific IgG antibodies. Overall, 2·9% had IgG against SINV. More men (3·7%) than women (2·0%) were SINV seropositive (P = 0·047) and it was more common in subjects with a lower educational level (P = 0·013) and living in small, rural communities (P < 0·001). Seropositivity was associated with higher waist circumference (P = 0·1), elevated diastolic blood pressure (P = 0·037), and history of a previous stroke (P = 0·011). In a multiple logistic regression analysis, adjusting for known risk factors for stroke, seropositivity for SINV was an independent predictor of having had a stroke (odds ratio 4·3, 95% confidence interval 1·4-13·0, P = 0·011).

[Complete genome characterization of the Kyzylagach virus (KYZV) (Togaviridae, Alphavirus, Sindbis serogroup) isolated from mosquitoes Culex modestus Ficalbi, 1889 (Culicinae) collected in a colony of herons (Ardeidae Leach, 1820) in Azerbaijan].

Complete genome sequencing of the Kyzylagach virus (KYZV) LEIV-65A strain isolated from the Culex modestus Ficalbi, 1889 (Culicinae), which was collected in the colony of the Ardeidae Leach, 1820 birds on the coast of Caspian sea, Kyzyl-Agach bay, in the southern part of Azerbaijan, was carried out. KYZV has high homology (about 99%) with the Chinese XJ-160 strain of the Sindbis virus (SINV) isolated from Anopheles sp. in Xinjiang Uyghur autonomic region (north-eastern China). Homologies of KYZV and XJ-160 with European SINV strains are 82% and 93% for the nucleotide and amino acid sequences, respectively (GenBank ID: KF981618). The difference between the nucleotide sequences of KYZV and Australian SINV/SW6562 strain is 19%; amino acid sequences, 12%. Since XJ-160 strain is extremely similar to KYZV, the first could be considered as the KYZV strain. The geography of the KYZV and XJ-160 isolation points and their genetic distance from the European viruses allow the KYZV to be suggested as a SINV (genotype IV) topotypic variant typical of Transcaucasia and Central Asia.

Phenotypic and molecular characteristics of plaque-purified MX10 virus, an Oriental-Australian genotype of Sindbis virus from Yunnan, China.

A previous investigation showed that MX10 virus, recently isolated in China, belongs to the Oriental-Australian (O/A) genotype of Sindbis virus (SINV) (Wang Jinglin, 2011, ATMH). Similar to the MRE16 isolate, the prototype O/A genotype of SINV, two derivate viruses with obviously different plaque morphologies were derived from MX10 virus, which were accordingly denoted as MX10-LP and MX10-SP. MX10-LP virus exhibited higher neurovirulence in neonatal mice than MX10-SP virus. Analysis of the complete genome revealed seven nucleotide differences between MX10-LP and MX10-SP. Compared with MRE16 virus, MRE16SP virus has a deletion of 30 aa in the E2 gene (200-229), which has been shown to be the molecular basis for the different plaque morphology. However, the MX10-SP virus did not have the 30-amino-acid deletion in the E2 gene. These results demonstrate that the molecular basis for the different plaque morphology of MX10 virus, the first strain of the O/A genotype of SINV isolated from China, is different from that of the prototype MRE16 virus.

Climatic, ecological and socioeconomic factors as predictors of Sindbis virus infections in Finland.

Mosquito-borne Sindbis virus (SINV) causes rash-arthritis syndrome in Finland. Major outbreaks with approximately 7-year cycles have caused substantial burden of illness. Forest dwelling grouse are suspected to be amplifying hosts, with the infection transmitted to humans by mosquito bites. SINV infection surveillance data for 1984­2010 were used to create a negative binomial hurdle model, with seasonality, long-term cycles, climatic, ecological and socioeconomic variables. Climatic factors during early summer and amount of snow in April described the occurrence and incidence of SINV infections. Regulated water shore and hatch-year black grouse density described the occurrence, while population working in agriculture, agricultural land(negative) and income (negative) described the incidence of the disease. The prediction for 2009 was 85 cases (95% prediction interval 2-1187), while the actual occurrence was 106. We identified novel and known risk factors. The prevention of SINV infections in regulated water areas by infected mosquito populations should be targeted.

Generation of recombinant alphaviral vectors.

The alphaviruses Semliki Forest virus and Sindbis virus have been used frequently as expression vectors in vitro and in vivo. Usually, these systems consist of replication-deficient vectors that require a helper vector for packaging of recombinant particles. Replication-proficient vectors have also been engineered. Alphaviral vectors can be used as nucleic-acid-based vectors (DNA and RNA) or infectious particles. High-titer viral production is achieved in <2 d. The broad host range of alphaviruses facilitates studies in mammalian and nonmammalian cell lines, primary cells in culture, and in vivo. The strong preference for expression in neuronal cells has made alphaviruses particularly useful in neurobiological studies. Unfortunately, their strong cytotoxic effect on host cells, relatively short-term transient expression patterns, and the reasonably high cost of viral production remain drawbacks. However, novel mutant alphaviruses have showed reduced cytotoxicity and prolonged expression. Membrane proteins (which are generally difficult to express at high levels in recombinant systems) have generated high yields and facilitate applications in structural biology. Alphaviruses have also been applied in vaccine development and gene therapy. This protocol describes the production of recombinant alphaviral vectors. The SFV- and SIN-based expression systems apply two vectors for recombinant particle production. In addition to the RNA-based vectors described here, DNA vectors with cytomegalovirus or other RNA polymerase type II promoters can be used for direct plasmid DNA transfections. Cotransfection of SFV-based pSCA expression and pSCA helper vectors generates recombinant viral particles.

Purification and concentration of alphavirus.

The alphaviruses Semliki Forest virus and Sindbis virus have been used frequently as expression vectors in vitro and in vivo. Usually, these systems consist of replication-deficient vectors that require a helper vector for packaging of recombinant particles. Replication-proficient vectors have also been engineered. Alphaviral vectors can be used as nucleic-acid-based vectors (DNA and RNA) or infectious particles. High-titer viral production is achieved in <2 d. The broad host range of alphaviruses facilitates studies in mammalian and nonmammalian cell lines, primary cells in culture, and in vivo. The strong preference for expression in neuronal cells has made alphaviruses particularly useful in neurobiological studies. Unfortunately, their strong cytotoxic effect on host cells, relatively short-term transient expression patterns, and the reasonably high cost of viral production remain drawbacks. However, novel mutant alphaviruses have showed reduced cytotoxicity and prolonged expression. Membrane proteins (which are generally difficult to express at high levels in recombinant systems) have generated high yields and facilitate applications in structural biology. Alphaviruses have also been applied in vaccine development and gene therapy. Generally, purification or concentration of alphaviruses is not necessary. However, for instance, the medium derived from baby hamster kidney cells is toxic to primary neurons in culture. Including a purification step substantially improves the survival of the transduced neurons. Viral concentration and purification may also be advantageous for in vivo studies in animal models and are mandatory for clinical applications. This protocol describes three methods for purification and concentration of alphavirus.

Determination of alphaviral titers.

The alphaviruses Semliki Forest virus and Sindbis virus have been used frequently as expression vectors in vitro and in vivo. Usually, these systems consist of replication-deficient vectors that require a helper vector for packaging of recombinant particles. Replication-proficient vectors have also been engineered. Alphaviral vectors can be used as nucleic-acid-based vectors (DNA and RNA) or infectious particles. The broad host range of alphaviruses facilitates studies in mammalian and nonmammalian cell lines, primary cells in culture, and in vivo. The strong preference for expression in neuronal cells has made alphaviruses particularly useful in neurobiological studies. Unfortunately, their strong cytotoxic effect on host cells, relatively short-term transient expression patterns, and the reasonably high cost of viral production remain drawbacks. However, novel mutant alphaviruses have showed reduced cytotoxicity and prolonged expression. Alphaviruses have also been applied in vaccine development and gene therapy. Before use in vitro or in vivo, it is essential to determine the titer of the generated alphaviral particles. Because defective alphaviruses do not produce plaques, their titers cannot be determined by conventional methods. However, viral titers can be determined readily in cases where the recombinant viruses express reporter genes such as green fluorescent protein or ß-galactosidase, as well as indirectly by immunofluorescence methods. The potency of viral stocks can also be evaluated by light microscopic analysis. Alphavirus-infected cells show a dramatic decrease in growth and can be easily distinguished from noninfected control cells through their rounded morphology.

[Recombinant interferon-alpha suppression of Karelian fever virus replication in human blood cells].

The active replication of Karelian fever virus (KFV) in human blood vessels and the protective activity of the Russian agent reaferon were first shown. KFL was highly susceptible to interferon (IFN)-alpha. In control (uninfected) cells, reaferon caused low gene expressions of the IFN-dependent enzymes dsRNA-dependent protein kinase and 2'5'-oligoadenylate synthetase, by exerting a little effect on the activity of its family genes. KFV suppressed the reaferon-induced gene expression of IFN-dependent enzymes, but IFN-alpha gene transcription was increased in the reaferon-treated infected cells.

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.