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  • 1.
    Lwande, Olivia Wesula
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Luande, Verah Nafula
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Pereira de Freitas, Amanda
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Tajedin, Leila
    Department of Microsystems, University of South-Eastern Norway, Vestfold, Norway.
    Ahlm, Clas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Näslund, Jonas
    Swedish Defence Research Agency, CBRN, Defence and Security, Umeå, Sweden.
    Evander, Magnus
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Bucht, Göran
    Umeå University, Faculty of Science and Technology, European CBRNE Center.
    Mismatch amplification mutation assays of Chikungunya virus and O'Nyong-Nyong virus: a simple and reliable method for surveillance and identification of emerging alphaviruses2022In: Frontiers in Virology, ISSN 2673-818X, Vol. 2, article id 769354Article in journal (Refereed)
    Abstract [en]

    Background: The mosquito-borne alphaviruses chikungunya virus (CHIKV) and o'nyong-nyong virus (ONNV) are closely related Alphaviruses that belong to the Semliki forest virus serocomplex. The two viruses are associated with large outbreaks with significant morbidity. However, they are transmitted by different mosquito vectors and accordingly need different prevention strategies. The viruses are difficult to distinguish clinically and there is a lack of sensitive and specific assays that can discriminate between CHIKV and ONNV. Therefore, there is a need for new methods that may be able to determine the true burden of the diseases caused by these viruses, especially in resource-poor settings.

    Method: To distinguish between CHIKV and ONNV, we designed and optimized two genetic methods, melt analysis of mismatch amplification mutation assay (Melt-MAMA) and agarose gel-based mismatch amplification mutation assay (Agarose-MAMA). The identification was based on single nucleotide polymorphisms using two competing forward primers and a common reverse primer that targeted selected sites in the envelope genes (E1 and E2). A specific shift in the melting point and mobility on agarose gels was obtained by tailing one of the two competing primers with a G/C-rich stretch of nucleotides.

    Results: The melting point analyses by real-time polymerase chain reaction (qPCR Melt-MAMA) or gel-shift assay (Agarose-MAMA assay) for CHIKV and ONNV were found to be reproducible and the sensitivity of the two assays was estimated at under 100 template copies/reaction. Furthermore, no cross-reactivity with related viruses of the same serocomplex such as Mayaro virus, Ross River virus or Semliki forest virus was detected, or with other viruses such as Sindbis virus (Alphavirus), West Nile virus, dengue virus (Flavivirus), Inkoo virus and Tahyna virus (Orthobunyavirus). The results from the two assays were comparable when the obtained amplicons were analyzed by Melt-MAMA or by Agarose-MAMA.

    Conclusion: Herein we present reliable and robust methods that can discriminate between CHIKV and ONNV. These methods can be used in well-equipped laboratories and basic clinical settings (e.g., in developing countries), as well as in field situations. The approach may also be applicable in the distinction of other closely-related mosquito-borne viruses that belong to the same serogroup.

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  • 2.
    Lwande, Olivia Wesula
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.
    Mohamed, Nahla
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.
    Bucht, Göran
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.
    Ahlm, Clas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Olsson, Gert
    Evander, Magnus
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.
    Seewis hantavirus in common shrew (Sorex araneus) in Sweden2020In: Virology Journal, E-ISSN 1743-422X, Vol. 17, no 1, article id 198Article in journal (Refereed)
    Abstract [en]

    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.

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  • 3.
    Lwande, Olivia Wesula
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Näslund, Jonas
    Swedish Defence Research Agency, CBRN, Defence and Security, Umeå, Sweden.
    Sjödin, Andreas
    Swedish Defence Research Agency, CBRN, Defence and Security, Umeå, Sweden.
    Lantto, Rebecca
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Luande, Verah Nafula
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine.
    Bucht, Göran
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Ahlm, Clas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Agwanda, Bernard
    Mammalogy Section, National Museums of Kenya, Nairobi, Kenya.
    Obanda, Vincent
    Department of Research Permitting and Compliance Wildlife Research and Training Institute, Naivasha, Kenya.
    Evander, Magnus
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Novel strains of Culex flavivirus and Hubei chryso-like virus 1 from the Anopheles mosquito in western Kenya2024In: Virus Research, ISSN 0168-1702, E-ISSN 1872-7492, Vol. 339, article id 199266Article in journal (Refereed)
    Abstract [en]

    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.

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  • 4.
    Lwande, Olivia Wesula
    et al.
    Umeå University, Arctic Research Centre at Umeå University. Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Obanda, Vincent
    Lindstrom, Anders
    Ahlm, Clas
    Umeå University, Arctic Research Centre at Umeå University. Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Evander, Magnus
    Umeå University, Arctic Research Centre at Umeå University. Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Näslund, Jonas
    Umeå University, Faculty of Science and Technology, European CBRNE Center.
    Bucht, Göran
    Umeå University, Faculty of Science and Technology, European CBRNE Center.
    Globe-Trotting Aedes aegypti and Aedes albopictus: Risk Factors for Arbovirus Pandemics2020In: Vector Borne and Zoonotic Diseases, ISSN 1530-3667, E-ISSN 1557-7759, Vol. 20, no 2, p. 71-81Article in journal (Refereed)
    Abstract [en]

    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.

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  • 5. Näslund, Jonas
    et al.
    Ahlm, Clas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Arctic Research Centre at Umeå University.
    Islam, Md. Koushikul
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Evander, Magnus
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Arctic Research Centre at Umeå University.
    Bucht, Göran
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Lwande, Olivia Wesula
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Arctic Research Centre at Umeå University.
    Emerging Mosquito-Borne Viruses Linked to Aedes aegypti and Aedes albopictus: Global Status and Preventive Strategies2021In: Vector Borne and Zoonotic Diseases, ISSN 1530-3667, E-ISSN 1557-7759, Vol. 21, no 10, p. 731-746Article, review/survey (Refereed)
    Abstract [en]

    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.

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  • 6.
    Rusanganwa, Vincent
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. College of Medicine and Health Sciences, University of Rwanda, Kigali, Rwanda; Teaching Coordination and Quality Assurance Department, Ministry of Health, Kigali, Rwanda.
    Lwande, Olivia W.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Bainda, Brenda
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Chiyo, Patrick
    Department of Biology, Duke University, Durham, North Carolina, USA.
    Seruyange, Eric
    College of Medicine and Health Sciences, University of Rwanda, Kigali, Rwanda;e Internal Medicine Department, Rwanda Military Hospital, Kigali, Rwanda.
    Bucht, Göran
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Evander, Magnus
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Arbovirus surveillance in febrile patients attending selected health facilities in Rwanda2024In: Infection Ecology & Epidemiology, ISSN 2000-8686, E-ISSN 2000-8686, Vol. 14, no 1, article id 2289872Article in journal (Refereed)
    Abstract [en]

    Arthropod-borne (arbo) viruses cause emerging diseases that affect the livelihoods of people around the world. They are linked to disease outbreaks resulting in high morbidity, mortality, and economic loss. In sub-Saharan Africa, numerous arbovirus outbreaks have been documented, but the circulation and magnitude of illness caused by these viruses during inter-epidemic periods remains unknown in many regions. In Rwanda, there is limited knowledge on the presence and distribution of arboviruses. This study aimed at determining the occurrence and distribution of selected arboviruses, i.e., chikungunya virus (CHIKV), o’nyong-nyong virus (ONNV), dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), Rift Valley fever virus (RVFV) and Crimean-Congo haemorrhagic fever virus (CCHFV), among febrile patients visiting health centres in Rwanda. A total of 2294 dry blood spots (DBS) were collected on filter papers during August 2019 – December 2020. Reverse-transcription polymerase chain reaction (RT-PCR) was performed on samples in pools of ten, using both quantitative (DENV, ZIKV, RVFV) and conventional PCR (CHIKV, ONNV, WNV, CCHFV) with virus specific primers, followed by sequencing. Demographic data and clinical manifestations of illness were analysed. ONNV infection was detected in 12 of 230 pools (5.2%) and ZIKV in three pools (1.3%). The other arboviruses were not detected. All ONNV cases were found in the Rwaniro health centre, while ZIKV infection was found among patients visiting the Kirinda and Zaza health centres. There was temporal variability in ONNV infections with most cases being recorded during the long dry season, while ZIKV infection occurred during both dry and wet seasons. Patients with ONNV were older and more were females. In conclusion, ONNV and ZIKV infection were detected in acute patients and can explain some of the feverish diseases in Rwanda.

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  • 7.
    Sunil-Chandra, N. P.
    et al.
    Department of Medical Microbiology, Faculty of Medicine, University of Kelaniya, Ragama, Sri Lanka; Sri Lanka Institute of Biotechnology, Homagama, Sri Lanka.
    Fahlman, Åsa
    Swedish Biodiversity Centre, Department of Rural and Urban Development, Faculty of Natural Resources and Agricultural Sciences, Swedish University of Agricultural Sciences, P.O. Box 7016, SE-750 07, Uppsala, Sweden.
    Waidyarathna, Shantha
    Department of Medical Microbiology, Faculty of Medicine, University of Kelaniya, Ragama, Sri Lanka.
    Näslund, Jonas
    Swedish Defence Research Agency, CBRN Defence and Security, Umeå, Sweden.
    Jayasundara, M. V. M. L.
    Department of Medical Microbiology, Faculty of Medicine, University of Kelaniya, Ragama, Sri Lanka.
    Lwande, Olivia Wesula
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.
    Bucht, Göran
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Swedish Defence Research Agency, CBRN Defence and Security, Umeå, Sweden.
    Evidence of orthohantavirus and leptospira infections in small mammals in an endemic area of Gampaha district in Sri Lanka2022In: One health outlook, ISSN 2524-4655, Vol. 4, no 1, article id 17Article in journal (Refereed)
    Abstract [en]

    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.

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  • 8.
    Wesula Lwande, Olivia
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Bucht, Göran
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Ahlm, Clas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Evander, Magnus
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Inkoo virus: a common but unrecognized mosquito-borne virus in northern Europe2017In: Infectious Diseases HubArticle, review/survey (Other academic)
    Abstract [en]

    Inkoo virus (INKV) is a common but less explored mosquito-borne virus belonging to family Bunyaviridae, genus Orthobunyavirus of the California serogroup. The virus was originally isolated in Finland in 1964 and has since then been detected in other northern European countries including Norway, Russia and Sweden. The virus has been associated with fever and encephalitis, in addition, patients considered positive for INKV antibodies have been linked with neurologic disease.

  • 9.
    Wigren Byström, Julia
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Näslund, Jonas
    Umeå University, Faculty of Science and Technology, European CBRNE Center.
    Trulsson, Fredrik
    Umeå University, Faculty of Science and Technology, European CBRNE Center. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Evander, Magnus
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Wesula Lwande, Olivia
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Ahlm, Clas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Bucht, Göran
    Umeå University, Faculty of Science and Technology, European CBRNE Center.
    Quantification and kinetics of viral RNA transcripts produced in Orthohantavirus infected cells2018In: Virology Journal, E-ISSN 1743-422X, Vol. 15, article id 18Article in journal (Refereed)
    Abstract [en]

    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.

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