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interactions between the ibv nucleocapsid protein and rna sequences specific for the 3' end of the genome.the infectious bronchitis virus (ibv) nucleocapsid protein was expressed as a fusion protein in bacteria. the coding sequence differed from the native protein only in the addition of six histidine residues at the amino terminus which were used for enrichment with a nickel affinity column. in gel shift assays, the mobility of labelled g rna was decreased with increasing concentrations of the fusion protein. competitive gel shift assays with labelled g rna indicated that the protein interacted wit ...19958830535
california encephalitis in alabama.arthropod-borne virus (arbovirus) infections in humans are primarily central nervous system infections, but other clinical manifestations include febrile illness and fever with hemorrhagic diathesis. in the genus bunyavirus there are several viruses that cause disease in humans, especially in north america; these include lacrosse, jamestown canyon, trivittatus, and snowshoe hare viruses. the disease seen mainly in children is california encephalitis (usually of the lacrosse subtype); this infect ...19968865793
the extracellular domain of la crosse virus g1 forms oligomers and undergoes ph-dependent conformational changes.the la crosse virus g1 glycoprotein plays a critical role in virus binding to susceptible cells and in the subsequent fusion of viral and cellular membranes. a soluble form of the g1 glycoprotein (sg1) prepared in a recombinant baculovirus system mimics the cell-binding pattern of la crosse virus and inhibits la crosse virus infection (a. pekosz et al., virology 214, 339-348, 1995), presumably by competing for a cellular receptor, a finding that implies that sg1 can perform some functions absent ...19968918555
[arbovirus circulation in the republic of guinea].in 1978-1991 the ussr-guinea virological and microbiological laboratory functioned in kindia, the republic of guinea. arbovirus activity in this country was studied by a number of virologists and other specialists. their personal contribution and achievements in this collaboration are reflected in the present paper. about 74,000 mosquitoes, 100,000 ixodidae ticks, 1,500 wild birds, 2,700 bats, 106 monkeys, and 308 other mammals, 927 blood samples collected from febrile patients were examined in ...19968926934
molecular determinants of golgi retention in the punta toro virus g1 protein.the g1 glycoprotein of punta toro virus, a member of the bunyavirus family, accumulates in the golgi complex when it is expressed from cloned cdna. we previously reported that the information necessary for golgi retention of the g1 protein is located within the transmembrane domain and a portion of the cytoplasmic domain adjacent to the transmembrane domain (matsuoka, y., chen, s.-y., and compans, r. w. (1994) j. biol. chem. 269, 22565-22573). to determine the features of the amino acid sequence ...19968951050
rescue of a segmented negative-strand rna virus entirely from cloned complementary dnas.we provide the first report, to our knowledge, of a helper-independent system for rescuing a segmented, negative-strand rna genome virus entirely from cloned cdnas. plasmids were constructed containing full-length cdna copies of the three bunyamwera bunyavirus rna genome segments flanked by bacteriophage t7 promoter and hepatitis delta virus ribozyme sequences. when cells expressing both bacteriophage t7 rna polymerase and recombinant bunyamwera bunyavirus proteins were transfected with these pl ...19968986823
immunocytochemical analysis of uukuniemi virus budding compartments: role of the intermediate compartment and the golgi stack in virus maturation.previous studies have suggested that uukuniemi virus, a bunyavirus, matures at the membranes of the golgi complex. in this study we have employed immunocytochemical techniques to analyze in detail the budding compartment(s) of the virus. electron microscopy of infected bhk-21 cells showed that virus particles are found in the cisternae throughout the golgi stack. within the cisternae, the virus particles were located preferentially in the dilated rims. this would suggest that virus budding may b ...19978995638
[outbreak of oropouche virus fever in serra pelada, municipality of curionópolis, pará, 1994].in the final of november 1994, an outbreak of a febrile disease was observed in the serra pelada gold mine (5 degrees 35's: 49 degrees 30'w) in the southeast region of pará state. twenty samples were collected and sent to the laboratory of arbovirus of instituto evandro chagas. the tests showed that the disease was caused by oropouche virus (bunyaviridae, bunyavirus, simbu serological group). between 8-22 december 296 serum samples were taken (54 from febrile patients, 16 paired samples and 242 ...19969011877
completion of the impatiens necrotic spot virus genome sequence and genetic comparison of the l proteins within the family bunyaviridae.the nucleotide sequence of the large (l) genome segment of impatiens necrotic spot virus (insv) has been determined, and herewith the complete nucleotide sequence of the whole, tripartite genome of this important tospovirus has been elucidated. the l rna is 8776 nucleotides long and of negative polarity, containing one large orf on the viral complementary strand. comparison of the deduced amino acid sequence of the insv l rna primary translation product (330.3 kda) with those of the l rnas of ot ...19979049402
ovine fetal malformations induced by in utero inoculation with main drain, san angelo, and lacrosse viruses.the teratogenic potential of three bunyaviruses, two california serogroup bunyaviruses, lacrosse virus and san angelo virus, and a bunyamwera serogroup member, main drain virus, in sheep was studied following in utero inoculation of ewes in early gestation. although main drain virus appeared to be most teratogenic, all three viruses induced a range of lesions including arthrogryposis, hydrocephalus, fetal death, axial skeletal deviations, anasarca, and oligohydramnios. the teratogenic effects of ...19979080876
antigenic diversity of akabane virus detected by monoclonal antibodies.the antigenic properties of 21 japanese field isolates and two australian strains of akabane (aka) virus (simbu serogroup, bunyavirus) isolated from 1959 to 1990 were compared by enzyme-linked immunosorbent assay (elisa), plaque-reduction neutralization (prnt) and hemagglutination inhibition (hi) tests using monoclonal antibodies (mabs) to the obe-1 strain of aka virus. sixteen mabs were established by fusing p3x63ag8u1 mouse myeloma cells and spleen cells from balb/c mice immunized with the obe ...19979085550
evidence that fatal human infections with la crosse virus may be associated with a narrow range of genotypes.la crosse (lac) virus belongs to the california (cal) serogroup of the genus bunyavirus, family bunyaviridae. it is considered one of the most important mosquito-borne pathogens in north america, especially in the upper mid-west, where it is associated with encephalitis during the time of year when mosquitoes are active. infections occur most frequently in children and young adults and, while most cases are resolved after a period of intense illness, a small fraction (< 1%) are fatal. at present ...19979175252
[a circulation study of california serogroup and batai viruses (fam. bunyaviridae, genus bunyavirus) on the territory of saratov province].examining 337 sera from saratov healthy residents in the neutralization test with tyaginya and inco viruses has revealed 56 positive results (16.6%), of which 19 (5.6%) reacted only with tyaginya virus, 13 (3.9%) did only with inco virus, and 24 (7.1%) simultaneously with these two viruses. batai virus antibodies were not detected in the population. among 80 bovine serum samples collected in the saratov district of the region, type-specific antibodies to tyaginya virus were found in 10 (12.5%) a ...19979182198
lack of serologic evidence for an association between cache valley virus infection and anencephaly and other neural tube defects in texas.we tested the hypothesis that cache valley virus (cvv), an endemic north american bunyavirus, may be involved in the pathogenesis of human neural tube defects. this investigation followed a 1990 and 1991 south texas outbreak of neural tube defects with a high prevalence of anencephaly and the demonstration in 1987 that in utero infection by cvv was the cause of outbreaks of central nervous system and musculoskeletal defects in north american ruminants. sera from 74 women who gave birth to infant ...19979204303
rapid identification of australian bunyavirus isolates belonging to the simbu serogroup using indirect elisa formats.the bunyavirus genus, belonging to the bunyaviridae family, is comprised of a large group of antigenically and geographically disparate arthropod-borne viruses of medical and veterinary significance. in australia, viruses belonging to the simbu serogroup of the bunyavirus genus, akabane, tinaroo, peaton, aino, douglas, thimiri and facey's paddock have been isolated. in this communication we describe two indirect elisas, referred to as the simbu serogroup elisa (sg-elisa), and the simbu typing el ...19979220398
venezuelan equine encephalitis and oropouche virus infections among peruvian army troops in the amazon region of peru.an outbreak of a febrile illness characterized by headache, ocular pain, myalgia, and arthralgia occurred during june 1994 among peruvian army troops in northern peru. on june 14-16, 1994, clinical data and blood samples were obtained from eight soldiers with a febrile illness, and from 26 others who had a history of febrile illness during the past three months. a follow-up blood sample was obtained 107 days later from four of the febrile and seven of the afebrile soldiers. serum samples were te ...19979230800
cladistic analysis of tospovirus using molecular characters.the genus tospovirus was thought to be composed only of the tomato spotted wilt virus (tswv), but now at least four tospovirus species have been proposed based on serological and molecular data. a classification of tospoviruses has been proposed taking into account global similarities of the n gene and n protein sequences of 7 isolates of tospovirus. because phylogenetic analyses based on global similarities can lead to classifications which do not mirror the genealogy of the group, we have empl ...19979242593
immunohistochemical and in situ localization of crimean-congo hemorrhagic fever (cchf) virus in human tissues and implications for cchf pathogenesis.crimean-congo hemorrhagic fever (cchf) is a potentially fatal disease that occurs in parts of africa, asia, and eastern europe, and that is caused by a recently emerged bunyavirus. rapid laboratory diagnosis of cchf infection is essential and is currently performed by virus isolation and serology. histopathologic studies have been limited to a small number of cases, and little is known about the cellular tropism of cchf virus and the pathogenesis of this disease.19979278612
antigenic and genetic comparisons of japanese and australian simbu serogroup viruses: evidence for the recovery of natural virus reassortants.the antigenicity and rna genome structures of five simbu serogroup bunyaviruses isolated in japan and australia were analyzed using monoclonal antibodies (mabs) raised to akabane (aka) virus and oligonucleotide fingerprinting. the virion surface glycoprotein (g1) and the nucleocapsid (n) protein of heterologous viruses showed no reactivity to the mabs, while the aka-derived anti-g1 mab (2f1) reacted with peaton virus and all three aka anti-n mabs reacted with tinaroo (tin) virus at almost the sa ...19979282785
[the mx protein that confers the resistance to influenza virus].the murine mx1 protein was identified as an interferon-inducible host factor that confers the resistance to influenza virus. mx proteins homologous to the murine mx1 are subsequently found in a wide variety of species including human, rat, chicken, fish and so on. mx proteins have the typical gtp-binding motif, and in fact, are shown to be gtp-binding proteins and gtpases. nuclear mx proteins selectively inhibit the primary transcription of orthomyxoviruses. in contrast, the cytoplasmic mx prote ...19979360386
sequence determination and phylogenetic analysis of the akabane bunyavirus s rna genome segment.the nucleotide sequence of the small (s) rna segment of akabane (aka) bunyavirus was determined. the segment is 858 nucleotides long and contains two overlapping open reading frames (orfs), which encode the nucleocapsid (n) and nonstructural (nss) proteins, consistent with other bunyaviruses. comparisons with the aino virus s rna sequence indicated that there is 73.5% identity in nucleotide sequence. however, the sequence identity of the 5' non-coding region of the genomic rna between these two ...19979367371
[distribution of viruses from the californian encephalitis serogroup (bunyaviridae, bunyavirus) in the northern expanses of russia].the study was carried out in 1983-1991 and covered a territory of about 10 x 10(6) km2 in various physico-geographic areas (east fennoscandia, northern russian plain, west siberia, central siberia, north-eastern siberia, and northern pacific region) in the arctic, subarctic, northern-central-southern taiga, forest-steppe, and steppe in northern russia. a total of 251 strains were isolated from 1391,900 mosquitoes, identified as the california group snowshoe hare (83), inkoo (44), and tahyna (2) ...19979424850
toscana virus infections of the central nervous system in children: a report of 14 cases.to evaluate the pathogenicity of a recently discovered arthropod-transmitted bunyavirus (toscana virus) on the cns in children and to provide information on the epidemiologic and clinical aspects of toscana virus infection.19989470016
emerging infections in australia.over the last 10 years, novel infectious agents including equine morbillivirus, lyssavirus, barmah forest virus, rickettsia honei and two as-yet-unnamed bunyaviruses have been identified as causes of human disease in australia. previously described agents, such as japanese b encephalitis virus, dengue virus, ross river virus, orientia tsutsugamushi, rickettsia australis, burkholderia pseudomallei, mycobacterium ulcerans and trichinella pseudospiralis, have increased their geographical distributi ...19979494666
rapid detection of antigenic diversity of akabane virus isolates by dot immunobinding assay using neutralizing monoclonal antibodies.akabane (aka) virus is an arthropod-borne virus belonging to the simbu group of the genus bunyavirus. neutralizing monoclonal antibodies (mabs) against aka virus were prepared, and the neutralizing epitopes of the virus were defined by competitive binding assay. five distinct antigenic domains were identified and were designated a, b, c, d, and e. domains a and c consisted of two epitopes each. it was demonstrated that seven neutralizing epitopes exist on the g1 glycoprotein of aka virus. dot im ...19989521142
antibodies to alphavirus, flavivirus, and bunyavirus arboviruses in house sparrows (passer domesticus) and tree sparrows (p. montanus) in poland.sparrows from central poland were examined by a hemagglutination-inhibition test (titer > or = 20) for the presence of antibodies to arboviruses, between 1995 and 1996. in house sparrows (passer domesticus) (n = 179), antibodies to sindbis, west nile, tick-borne encephalitis, tahyna, and calovo viruses were detected at seroprevalences of 1.1%, 2.8%, 1.1%, 2.8%, and 1.1%, respectively. in tree sparrows (p. montanus) (n = 33), antibodies to the sindbis, west nile, and tahyna viruses were detected ...19989533098
[circulation of viruses of the california serocomplex (bunyaviridae, bunyavirus) in the central and southern parts of the russian plain].seventy strains of california group viruses were isolated in the central and southern territories of the russian plain situated in the southern taiga, mixed forest, broad-leaved forest, forest-steppe, steppe, and semiarid zones. sixty-three of these were isolated from 873,300 mosquitoes, 4 from patients, and 3 from rodents. 57.1% of strains were isolated from aedes communis and the associated species, 28.5% from aedes excrucians and associated species, and rarely from other mosquito species. the ...19989559528
[diseases associated with viruses of the california encephalitis serogroup, in russia].studies of 1986-1995 revealed diseases etiologically connected with california serogroup viruses (bunyaviridae, bunyavirus) all over the country. highly endemic zones are the tundra, taiga, and leafy forest. the disease occurs mainly in summer, the patients are mostly young: under 30 years of age. analysis of 183 cases confirmed by laboratory findings enabled us to distinguish the following forms: influenza-like (70.9%) with the predominant involvement of the bronchopulmonary system (bronchitis ...19989559529
pseudotype formation with la crosse virus glycoproteins.pseudotype formation is a powerful tool for analysing mechanisms of virus neutralization and entry, since it allows for analysis of glycoprotein properties without the necessity for preparing recombinant genomes. using recombinant vaccinia viruses, we prepared pseudotypes of la crosse virus with recombinant glycoproteins cloned from the monoclonal antibody (mab)-resistant variant v31. the resulting pseudotypes became partially resistant to mab 807-31. furthermore, when the v31 glycoproteins were ...19989568959
transmission of tick-borne bunyaviruses by cofeeding ixodid ticks.palma and bhanja bunyaviruses replicated in dermacentor marginatus, d. reticulatus, rhipicephalus sanguineus, r. appendiculatus and ixodes ricinus ticks after parenteral inoculation and appropriate incubation and feeding. palma virus was transmitted to d. marginatus and d. reticulatus males, d. reticulatus and r. sanguineus females, and r. appendiculatus nymphs while cofeeding with infected ticks on laboratory mice. bhanja virus was transmitted to d. marginatus males and r. appendiculatus nymphs ...19979607090
mosquito feeding-induced enhancement of cache valley virus (bunyaviridae) infection in mice.cache-valley (cv) virus, an arthropod-borne bunyavirus, recently has emerged as a significant veterinary pathogen causing infertility and congenital malformations in north american ruminants. to investigate the role of vector feeding on cv infection, adult mice were injected subcutaneously with cv, cv and vector thorax extract (a source of vector saliva), or cv into the site of intense, noninfected-mosquito feeding. mice did not become infected after injection of cv or cv and vector saliva, nor ...19989615544
mx proteins: mediators of innate resistance to rna viruses.mx proteins are interferon-induced members of the dynamin superfamily of large guanosine triphosphatases. these proteins have attracted attention because some display antiviral activity against pathogenic rna viruses, for example against members of the orthomyxovirus (influenzavirus) family or the bunyavirus family. transfected cells and transgenic mice expressing mx proteins are highly resistant to mx-sensitive viruses, demonstrating that mx proteins are powerful antiviral agents. in humans, sy ...19989638812
vectors vs. humans in australia--who is on top down under? an update on vector-borne disease and research on vectors in australia.australia has a diversity of vectors and vector-borne human diseases. mosquito-borne arboviruses are of greatest concern, but there are issues with other vector and pathogen systems. mosquitoes were responsible for more than 35,000 cases of ross river virus during 1991-1997. barmah forest virus is increasing nationwide, and unidentified bunyaviruses suspected of causing illness have been isolated. cases of murray valley encephalitis have occurred in 14 of the past 20 years in northern australia. ...19989673928
mxa gtpase: oligomerization and gtp-dependent interaction with viral rnp target structures.mxa protein is an interferon-induced gtpase of human cells that inhibits the multiplication of several rna viruses, including influenza viruses and bunyaviruses. studies on mxa transgenic mice have shown that mxa is a powerful antiviral agent in vivo. it has been suggested that this cellular protein also protects humans from viral disease, but the mechanism(s) by which mxa exerts its antiviral action is still poorly understood. using an in vitro cosedimentation assay, we now demonstrate that mxa ...19989735310
zoonoses and haemorrhagic fever.virus zoonoses causing haemorrhagic fever have been recognized in three major families: arenaviridae, bunyaviridae and filoviridae. all are negative-stranded rna viruses, with genomes in two segments, three segments, or non-segmented, respectively. acquisition of haemorrhagic fever in man generally requires close contact with a vertebrate vector species, usually rodents, for the arenaviruses and bunyaviruses. in the case of filoviruses, the vector is currently unknown, but these viruses may infe ...19989737374
role of anopheles quadrimaculatus and coquillettidia perturbans (diptera: culicidae) in the transmission cycle of cache valley virus (bunyaviridae: bunyavirus) in the midwest, usa.midwestern populations of coquillettidia perturbans (walker) and anopheles quadrimaculatus (say) were tested for their ability to transmit cache valley virus (cv), a recognized human and animal pathogen. field-collected mosquitoes were fed artificial blood meals containing 5.2-6.2 log10 pfu/ml of cv. after 9-23 d at 28 degrees c, 75-93% of blood-fed cq. perturbans had disseminated infections and 6-62% transmitted the virus to suckling mice. however, when infected with a lower virus titer (3.3 lo ...19989775588
[preparation and use of monoclonal antibodies to the inkoo virus (bunyaviridae, bunyavirus) for differentiating between california serogroup viruses].five types of monoclonal antibodies (mab) to inkoo virus were obtained. the direction of mabs to virus proteins was determined and reactions of mabs with other california group viruses studied by the fluorescent antibody method.19989791883
a retrospective serological survey of humans in india for wanowrie virus.wanowrie virus (wan) is an ungrouped arbovirus having morphogenetic characters in common with bunyavirus. it was isolated for the first time from ticks, hyalomma marginatum isacci (6700 strain) and subsequently from a human case (68923 strain). in the absence of any reports on the prevalence of this virus amongst humans in india, 600 human sera were tested in the complement fixation (cf) test against both the strains of wan virus. one hundred human sera were randomly selected from six different ...19989914674
[identification of california serogroup viruses (bunyaviridae, bunyavirus) using monoclonal antibodies to inkoo virus].five types of monoclonal antibodies to inkoo virus were used to detect antigenic relationships between 12 inkoo-like strains and the prototype inkoo virus strain. eight strains were found antigenically very close or identical to inkoo virus, and the rest 4 are probably original variants (viruses) of california serogroup.199910190233
seroepidemiology of california and bunyamwera serogroup (bunyaviridae) virus infections in native populations of alaska.this study investigated the geographic distribution and prevalence of antibodies to california and bunyamwera serogroup viruses in native populations of alaska, and demographic and ecologic risk factors associated with exposure. sera (n = 1,635) from 18 communities were screened using an elisa. all age groups were tested for antibodies to jamestown canyon (jc), inkoo (ink), snowshoe hare (ssh), and northway (nor) viruses; persons > or = 45 years old (n = 90) from six communities were additionall ...199910344657
mosquito-borne viruses in western europe: a review.several mosquito-borne arboviruses belonging to the genera alphavirus, flavivirus, and bunyavirus have been reported to occur in mosquitoes and to infect humans and other vertebrates in western europe. these zoonotic viruses circulate in nature either in an aedes-mammal, anopheles-mammal, or culex-bird transmission cycle. infected humans normally do not contribute to the virus circulation. west nile virus (flavivirus) caused an outbreak of fever, malaise, pain in eyes and muscles, and headache a ...199910436876
west nile fever in czechland. 199910458974
potential for evolution of california serogroup bunyaviruses by genome reassortment in aedes albopictus.aedes albopictus was introduced into the united states in used tires in 1985. its successful colonization of the upper midwest has potential to alter the current epidemiology of bunyaviruses that circulate in the region. it is permissive for the replication of several arboviruses, including la crosse (lacv) and jamestown canyon (jcv) bunyaviruses. in this study, we demonstrate the ability of lacv and jcv to coinfect ae. albopictus mosquitoes and to form all six possible reassortant genotypes. al ...199910466972
sequence comparisons of medium rna segment among 15 california serogroup viruses.the complete nucleotide sequences have been determined for the m segment of 12 california (cal) serogroup bunyaviruses. a method is described here of long reverse transcription-polymerase chain reaction (rt-pcr) that yields the full-length medium (m) rna genomic segment. a phylogenetic tree was constructed by comparison of the open reading frames (orfs) in the m rna segment of 15 cal serogroup viruses. three distinct branches were identified and they are represented by the california encephaliti ...199910475083
murine leukemia virus pseudotypes of la crosse and hantaan bunyaviruses: a system for analysis of cell tropism.we have developed a system for the preparation of la crosse virus (lac) and hantaan virus (htn) pseudotypes using a murine leukemia virus vector. after concentration, the pseudotypes were present in quantities sufficient to analyze cell tropism and neutralization. cells resistant to lac could not be infected with the mlv (lac) pseudotypes, and the pseudotypes were sensitive to neutralizing monoclonal antibodies prepared against lac glycoproteins, as well as to inhibition by a soluble form of the ...199910500280
bunyavirus superinfection and segment reassortment in transovarially infected mosquitoes.rapid evolution of bunyaviruses may occur by rna segment reassortment between closely related viruses. reassortment between viruses occurs in dually infected mosquitoes when two different viruses are simultaneously ingested or when the second virus is ingested within 2 days of the first virus. by 3 days after oral infection, interference to superinfection occurs, thus limiting the potential for evolution. aedes triseriatus mosquitoes can also be transovarially infected (ti+) with lacrosse (lac) ...199910567649
genetic engineering of influenza and other negative-strand rna viruses containing segmented genomes. 199910582103
detection and differentiation of aino and akabane simbu serogroup bunyaviruses by nested polymerase chain reaction.reverse transcription-polymerase chain reaction (rt-pcr) and nested pcr were developed to detect and differentiate aino (aino) and akabane (aka) virus s rna. two pairs of aino- and aka-specific primers for nested pcr were synthesized and examined for their capacity to amplify pcr products using 7 simbu serogroup viruses isolated in japan and australia. rt- and nested pcr using aka-specific primers amplified cdna from tinaroo virus rna as well as homologous rna. nested pcr products were different ...199910603165
nucleotide sequences and phylogeny of the nucleocapsid gene of oropouche virus.the nucleotide sequence of the s rna segment of the oropouche (oro) virus prototype strain trvl 9760 was determined and found to be 754 nucleotides in length. in the virion-complementary orientation, the rna contained two overlapping open reading frames of 693 and 273 nucleotides that were predicted to encode proteins of 231 and 91 amino acids, respectively. subsequently, the nucleotide sequences of the nucleocapsid genes of 27 additional oro virus strains, representing a 42 year interval and a ...200010675412
preferential infection of neuronal and astroglia cells by akabane virus in primary cultures of fetal bovine brain.akabane virus is a member of the genus bunyavirus; it is pathogenic for ruminants and transmitted by arthropod vectors. infection of adult cattle and sheep causes a transient viremia without obvious clinical signs, while infection of pregnant animals often causes fetal abnormalities including hydranencephaly, poliomyelitis and arthrogryposis. infectious virus or viral antigens is present in the brain, spinal cord and skeletal muscle of infected fetuses. to understand the interaction between akab ...200010781726
detection of anti-arboviral immunoglobulin g by using a monoclonal antibody-based capture enzyme-linked immunosorbent assay.monoclonal antibody (mab)-based capture enzyme-linked immunosorbent assays (elisas) for the detection of anti-arboviral immunoglobulin g (igg elisas) were developed for a comprehensive array of medically important arboviruses from the alphavirus, flavivirus, and bunyavirus genera. tests were optimized and standardized so that maximum homology could be maintained among working parameters for the different viral agents, enabling a wide range of viruses to be easily tested for at one time. mabs wer ...200010790108
identification and localization of conserved antigenic epitopes on the g2 proteins of california serogroup bunyaviruses.california (cal) serogroup bunyaviruses are significant agents of arboviral encephalitis in humans. they are maintained and transmitted in nature by mosquitoes to preferred vertebrate amplifying hosts. the g2 envelope glycoprotein of la crosse virus (lac) was proposed by ludwig et al. to be a determinant for virus attachment to mosquito midgut cells. monoclonal antibodies to g2 neutralize the infectivity of pronase-treated virus for mosquito cells. we determined the location of antigenic sites o ...200010893000
rna binding properties of bunyamwera virus nucleocapsid protein and selective binding to an element in the 5' terminus of the negative-sense s segment.the genome of bunyamwera virus (bun) (family bunyaviridae, genus bunyavirus) comprises three negative-sense rna segments which act as transcriptional templates for the viral polymerase only when encapsidated by the nucleocapsid protein (n). previous studies have suggested that the encapsidation signal may reside within the 5' terminus of each segment. the bun n protein was expressed as a 6-histidine-tagged fusion protein in escherichia coli and purified by metal chelate chromatography. an rna pr ...200011024122
tomato spotted wilt virus glycoproteins exhibit trafficking and localization signals that are functional in mammalian cells.the glycoprotein precursor (g1/g2) gene of tomato spotted wilt virus (tswv) was expressed in bhk cells using the semliki forest virus expression system. the results reveal that in this cell system, the precursor is efficiently cleaved and the resulting g1 and g2 glycoproteins are transported from the endoplasmic reticulum (er) to the golgi complex, where they are retained, a process that could be blocked by tunicamycin. expression of g2 alone resulted in transport to and retention in the golgi c ...200111134314
inhibition of interferon-mediated antiviral responses by influenza a viruses and other negative-strand rna viruses. 200111162793
nucleotide sequence and deduced amino acid sequence of the medium rna segment of oropouche, a simbu serogroup virus: comparison with the middle rna of bunyamwera and california serogroup viruses.the bunyavirus genus of the family bunyaviridae contains 18 serogroups. to date nucleotide sequence data has been obtained for three serogroups, bunyamwera, california and simbu, based on analysis of the small (s) rna segment. in comparison, there is only nucleotide sequence data for the large and medium (m) rna segments for members of the bunyamwera and california serogroups. in this paper we report the nucleotide sequence of the m rna of oropouche (oro) virus, a member of the simbu serogroup. ...200111172919
west nile virus investigations in south moravia, czechland.seven virus isolates were obtained from 11,334 mosquitoes after the 1997 morava river flooding in south moravia (czech republic): 6 strains of tahyna bunyavirus, california antigenic group (5 from aedes vexans, 1 from ae. cinereus), and 1 strain of west nile flavivirus (wnv) from culex pipiens. in 1999, one isolate of tahyna virus from ae. vexans and one isolate of wnv from cx. pipiens were recovered from a total of 14,354 mosquitoes examined in the same area, whereas no virus was detected there ...200011192289
constitutive expression of interferon-induced human mxa protein in transgenic tobacco plants does not confer resistance to a variety of rna viruses.mxa is a key component in the interferon-induced antiviral defense in humans. after viral infections, mxa is rapidly induced and accumulates in the cytoplasm. the multiplication of many rna viruses, including all bunyaviruses tested so far, is inhibited by mxa. these findings prompted us to express mxa in plants in an attempt to create resistance to tospoviruses. here, we report the generation of transgenic tobacco plants that constitutively express mxa under the control of the 35 s cauliflower ...200011206971
bunyamwera bunyavirus nonstructural protein nss is a nonessential gene product that contributes to viral pathogenesis.bunyamwera virus (family bunyaviridae, genus bunyavirus) contains a tripartite negative-sense rna genome. the smallest rna segment, s, encodes the nucleocapsid protein n and a nonstructural protein, nss, in overlapping reading frames. we have generated a mutant virus lacking nss, called bundelnss, by reverse genetics. compared with the wild-type (wt) virus, bundelnss exhibited a smaller plaque size and generated titers of virus approximately 1 log lower. in mammalian cells, the mutant expressed ...200111209062
[rift valley fever]. 200011221696
the bunyamwera virus nonstructural protein nss inhibits viral rna synthesis in a minireplicon system.the small (s) genomic segment of bunyamwera virus (family bunyaviridae, genus bunyavirus) encodes the nucleocapsid protein, n, and a nonstructural protein, nss, in overlapping reading frames. in order to elucidate the function of nss, we established a plasmid-based minireplicon system using mammalian cells that express large amounts of t7 rna polymerase. expression of n, the viral polymerase protein (l), and a minireplicon containing a reporter gene was sufficient to reconstitute functional viru ...200111222097
identification of simbu, california and bunyamwera serogroup bunyaviruses by nested rt-pcr.we describe a reverse transcription-polymerase chain reaction (rt-pcr) with primers that anneal to the 5' and 3' ends and amplify the bunyavirus s rna segments. the rt-pcr was done on the fluids of c6/36 cells infected with each of 21 bunyaviruses. the bunyaviruses studied, with the exception of catu virus, produced amplicons having 700 to 1300 base pairs and probably contained the whole s rna segment sequence. a nested pcr performed with these amplicons distinguished california and most bunyamw ...200111280054
use of the squirrel monkey saimiri sciureus to optimize serological tests for diagnosis of bunyavirus infection in humans.several arthropod-borne viruses of the large bunyaviridae virus family have been isolated in south america. there are few data about the incidence of these viruses in man, except for the oropuche virus. since haemagglutination inhibition tests are difficult to perform, only enzyme-linked immunosorbent assays (elisas) are used. nevertheless, positive controls are necessary for elisa, and infected humans are rare. squirrel monkeys (saimiri sciureus) were therefore infected experimentally to assess ...200111311344
comparative analysis of g1 glycoprotein-coding sequences of cache valley virus (bunyaviridae: bunyavirus) isolates.the complete 4463 nucleotide sequence for the medium segment viral rna of cache valley virus has been cloned and sequenced in four isolates; in addition, the g1 glycoprotein extracellular coding domains are completed for nine additional isolates, including two subtypes, ft. sherman (86msp18) and tlacotalpan (61d240) viruses. the 13 represent isolations spanning over 45 years and a large geographic area, including the u.s., mexico, canada, and panama. glycosylation sites in g1 are generally conse ...200111324749
detection of arboviral rna directly from mosquito homogenates by reverse-transcription-polymerase chain reaction.many arthropod-borne viruses (arboviruses) are important human pathogens medically. the development of an effective technique to detect the viruses by using nucleic acid amplification, such as polymerase chain reaction (pcr), improves not only clinical diagnosis but also virologic surveillance of mosquito vectors in the field. in this study, the development of an improved and simplified assay is described for detection of mosquitoes infected with eastern equine encephalitis (eee) virus, cache va ...200111337046
jatobal virus is a reassortant containing the small rna of oropouche virus.jatobal (jat) virus was isolated in 1985 from a carnivore (nasua nasua) in tucuruí, pará state, brazil and was classified as a distinct member of the simbu serogroup of the bunyavirus genus, family bunyaviridae on the basis of neutralization tests. on the basis of nucleotide sequencing, we have found that the small (s) rna of jat virus is very similar (>95% identity) to that of oropouche (oro) virus, in particular, the peruvian genotype of oro virus. in comparison, limited nucleotide sequencing ...200111451484
arbovirus surveillance from 1990 to 1995 in the barkedji area (ferlo) of senegal, a possible natural focus of rift valley fever virus.surveillance for mosquito-borne viruses was conducted in barkedji area from 1990 to 1995, following an outbreak of rift valley fever (rvf) virus in southern mauritania. mosquitoes, sand flies, and midges were collected from human bait and trapped by solid-state u.s. army battery-powered cdc miniature light traps baited with dry ice or animals (sheep or chickens) at four ponds. overall, 237,091 male and female mosquitoes representing 52 species in eight genera, 214,967 phlebotomine sand flies, an ...200111476327
phylogeny of the simbu serogroup of the genus bunyavirus.the simbu serogroup of the genus bunyavirus, family bunyaviridae contains 25 viruses. previous serological studies provided important information regarding some but not all of the relationships among simbu serogroup viruses. this report describes the nucleotide sequence determination of the nucleocapsid (n) gene of the small genomic segment of 14 simbu serogroup viruses and partial nucleotide sequence determination of the g2 glycoprotein-coding region (encoded by the medium rna segment) of 19 vi ...200111514727
[anti-arboviral properties of different substances of chemical and plant origin].the paper deals with the results of search for inhibitors of two arboviral infections, including alfavirus of venezuelan equine encephalomyelitis (family togaviridae) and bunyavirus tahyna (family bunyaviridae), interferon inducers among 99 substances of organic synthesis and 92 compounds of plant origin. antiarboviral activity of some of these chemicals were investigated as well. reproduction of the studied viruses in cell culture was inhibited by 8 derivatives of triazole, triazine and uracile ...200111558241
interferon-induced rat mx proteins confer resistance to rift valley fever virus and other arthropod-borne viruses.mx proteins belong to the interferon (ifn)-induced antiviral defense. the rat genome contains three mx genes, ratmx1, ratmx2, and ratmx3. the mx gene products differ in their subcellular localization and antiviral specificity. the nuclear ratmx1 protein confers resistance to influenza a virus, and the cytoplasmic ratmx2 is active against vesicular stomatitis virus (vsv), whereas the cytoplasmic ratmx3 protein is antivirally inactive. to investigate the antiviral potential of the rat mx proteins ...200111576460
hantavirus infections and their prevention.hantaviruses are rodent-borne bunyaviruses which cause haemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome in humans. this review covers the host interactions of the viruses, including the rodent reservoirs, the clinical outcome of human infections as well as the pathogenesis and laboratory diagnosis of infections. the current stage in prophylaxis and therapy of hantaviral diseases is described and different approaches in vaccine development are discussed.200111709294
first isolation of la crosse virus from naturally infected aedes albopictus.la crosse (lac) virus, a california serogroup bunyavirus, is the leading cause of pediatric arboviral encephalitis in the united states and an emerging disease in tennessee, west virginia, and north carolina. human cases of lac encephalitis in tennessee and north carolina have increased above endemic levels during 1997 to 1999 and may represent an expansion of a new southeastern endemic focus. this report describes the isolation of lac virus from the exotic mosquito aedes albopictus. the discove ...200111747692
diagnosis of oropouche virus infection by rt-nested-pcr.using the rt-pcr with primers that anneal to the 5' and the 3' extremities of the genome segments of bunyaviruses and internal primers that anneal to the s segment of simbu serogroup viruses in a nested pcr it was possible to amplify the oropouche virus (oro) genome from the sera of three patients. these results show that this rt-nested-pcr is a useful tool for rapid diagnosis of oropouche fever infections.200211748670
a reassortant bunyavirus isolated from acute hemorrhagic fever cases in kenya and somalia.in late 1997 and early 1998, a large outbreak of hemorrhagic fever occurred in east africa. clinical samples were collected in kenya and southern somalia, and 27 of 115 (23%) hemorrhagic fever patients tested showed evidence of acute infection with rift valley fever (rvf) virus as determined by igm detection, virus isolation, detection of virus rna by reverse transcription-polymerase chain reaction (rt-pcr), or immunohistochemistry. however, two patients (one from kenya and the other from somali ...200111878887
antivirally active mxa protein sequesters la crosse virus nucleocapsid protein into perinuclear complexes.bunyaviruses replicate in the cytoplasm of infected cells. new viral particles are formed by budding of nucleocapsids into the golgi apparatus. we have previously shown that the ifn-induced human mxa protein inhibits bunyavirus replication by an unknown mechanism. here we demonstrate that mxa binds to the nucleocapsid protein of la crosse virus (lacv) and colocalizes with the viral protein in cytoplasmic complexes. electron microscopy revealed that these complexes accumulated in the perinuclear ...200211880649
serological and genetic characterization of newly isolated peaton virus in japan. brief report.the viruses were isolated from the blood of sentinel cattle and culicoides biting midges in the kyushu district, southwestern japan, in 1999 and identified by neutralization tests as peaton (pea) viruses. before this study, pea virus had been isolated in australia only. the nucleotide identity of the nucleocapsid (n) protein encoded by the s segment ranged from 91.1 to 91.6% between the australian and japanese strains. a phylogenetic analysis of the n protein sequence revealed that the pea virus ...200211890531
an outbreak of rift valley fever in northeastern kenya, 1997-98.in december 1997, 170 hemorrhagic fever-associated deaths were reported in garissa district, kenya. laboratory testing identified evidence of acute rift valley fever virus (rvfv). of the 171 persons enrolled in a cross-sectional study, 31(18%) were anti-rvfv immunoglobulin (ig) m positive. an age-adjusted igm antibody prevalence of 14% was estimated for the district. we estimate approximately 27,500 infections occurred in garissa district, making this the largest recorded outbreak of rvfv in eas ...200211897064
ultrastructural, antigenic and physicochemical characterization of the mojuí dos campos (bunyavirus) isolated from bat in the brazilian amazon region.the mojuí dos campos virus (mdcv) was isolated from the blood of an unidentified bat (chiroptera) captured in mojuí dos campos, santarém, state of pará, brazil, in 1975 and considerated to be antigenically different from other 102 arboviruses belonging to several antigenic groups isolated in the amazon region or another region by complement fixation tests. the objective of this work was to develop a morphologic, an antigenic and physicochemical characterization of this virus. mdcv produces cytop ...200212048556
bunyamwera bunyavirus nonstructural protein nss counteracts the induction of alpha/beta interferon.production of alpha/beta interferons (ifn-alpha/beta) in response to viral infection is one of the main defense mechanisms of the innate immune system. many viruses therefore encode factors that subvert the ifn system to enhance their virulence. bunyamwera virus (bun) is the prototype of the bunyaviridae family. by using reverse genetics, we previously produced a recombinant virus lacking the nonstructural protein nss (bundelnss) and showed that nss is a nonessential gene product that contribute ...200212133999
characterization and identification of oya virus, a simbu serogroup virus of the genus bunyavirus, isolated from a pig suspected of nipah virus infection.a virus, named oya virus, was isolated in vero cell cultures from the lungs of a pig suspected of nipah virus infection. the virus was revealed as a spherical enveloped rna virus with a diameter of 79 nm. for identification of oya virus, rt-pcr was performed. a common primer set for s-rna of the simbu serogroup of the genus bunyavirus was able to amplify a cdna from oya virus rna. the sequence data of the product revealed that the partial gene of oya virus s-rna segment had 65-70% homology with ...200212181680
[bunyavirus--virus and host relationship: the coevolution between hantavirus and rodent]. 200212227181
interferon-induced mx proteins: dynamin-like gtpases with antiviral activity.mx proteins are interferon-induced gtpases that belong to the dynamin superfamily of large gtpases. similarities include a high molecular weight, a propensity to self-assemble, a relatively low affinity for gtp, and a high intrinsic rate of gtp hydrolysis. a unique property of mx gtpases is their antiviral activity against a wide range of rna viruses, including bunya- and orthomyxoviruses. the human mxa gtpase accumulates in the cytoplasm of interferon-treated cells, partly associating with the ...200212230469
[microbial warfare and bioterrorism].infectious diseases have been used as warfares since ancient times. since the 1920s military organizations have studied bacteria of anthrax, plague, tularemia, botulism, brucelloses, glander, q-fever, and smallpox virus, filo-, arena-, bunyaviruses causing hemorrhagic fever or alphaviruses eliciting encephalitis. these can be dispersed by aerosol. salmonellae, shigellae, vibrio cholerae, distinguished escherichia coli strains are suitable to contaminate food, water, pharmaceutical products. fana ...200212244657
turlock-like bunyavirus associated with encephalomyelitis and myocarditis in an ostrich chick.in the fall of 1995, a 20-day-old female ostrich chick, 1 of a group of 20, was presented live with clinical signs of 2 days duration characterized by unsteady gait, circling to the left, and walking backward. another bird with similar clinical signs had died and another had recovered. the bird was euthanized and examined at necropsy. twenty-five milliliters of serous fluid was in the abdominal cavity and there was increased pericardial fluid. histopathology of the brain revealed mild to moderat ...200212296386
antigenic drift, antigenic shift and interferon antagonists: how bunyaviruses counteract the immune system.members of the bunyaviridae family are amongst the most widespread viruses in the world. they can be found on every inhabited continent at virtually every latitude, and are able to infect a wide range of arthropods, plants and mammals including humans. more than 300 named viruses are contained within the family bunyaviridae (virus taxonomy: seventh report of the international committee on taxonomy of viruses (2000) 599), and several members cause significant disease in humans or domestic animals ...200212297332
effect of triethylamine on the recovery of selected south american alphaviruses, flaviviruses, and bunyaviruses from mosquito (diptera: culicidae) pools.we evaluated the effect of triethylamine (tea) on the recovery of infectious virus from pools of mosquitoes for two south american alphaviruses (eastern equine encephalomyelitis and venezuelan equine encephalomyelitis subtypes iiic and id), one flavivirus (ilheus) and two bunyaviruses (mirim [guama group] and itaqui [group c]). mosquitoes were inoculated intrathoracically with virus, held for 7-10 d at 26 degrees c, and handled under one of four regimens before testing for the presence of virus ...200212349865
genetic organisation of iris yellow spot virus m rna: indications for functional homology between the g(c) glycoproteins of tospoviruses and animal-infecting bunyaviruses.the complete nucleotide sequence (4838 nucleotides) of iris yellow spot virus (iysv) m rna indicates, typical for tospoviruses, the presence of two genes in ambisense arrangement. the vrna orf codes for the potential cell-to-cell movement (nsm) protein (34.8 kda) and the vcrna orf for the viral glycoprotein (g1/g2) precursor (128.6 kda). multiple sequence alignment of the nsm and g1/g2 precursor proteins of iysv with those of other tospoviruses, showed highest homologies to peanut bud necrosis v ...200212491100
imported viral haemorrhagic fever with a potential for person-to-person transmission: review and recommendations for initial management of a suspected case in belgium.viral haemorrhagic fevers are caused by a wide range of viruses. there are 4 types of viruses well known to spread from person to person and able to cause nosocomial outbreaks with a high case fatality rate: an arenavirus (lassa fever and more exceptionally the junin and machupo virus), a bunyavirus (crimean-congo haemorrhagic fever) and the filoviridae (ebola and marburg viruses). so far there have been only a limited number of imported cases of viral haemorrhagic fever in industrialized countr ...200212534129
analysis of oropouche virus l protein amino acid sequence showed the presence of an additional conserved region that could harbour an important role for the polymerase activity.we described here the complete nucleotide sequence of the l rna segment of oropouche virus (genus orthobunyavirus, family bunyaviridae). we found the l rna segment is 6846 nucleotides long and encodes a putative rna polymerase of 2250 amino acids. phylogenetic analysis showed that oro virus cluster to the orthobunyavirus genus confirming the serological classification. it also showed that bunyamwera and california viruses, from the orthobunyavirus genus, are more closely related to each other th ...200312536293
[specific markers for the detection of circulation of tahyna, inko and batai viruses (bunyaviridae, bunyavirus) in humans, mosquitoes, ticks and cattle of the ul'ianovsk region].comprehensive examinations of the population, sanguivorous arthropoda and domestic animals were for the first time held in the territory of the ulyanovsk region. an active circulation of certain arboviruses was detected and a predominantly ecological relation of california encephalitis serogroup viruses with aedes mosquitoes, on the one hand, and of batai viruses with anopheles mosquitoes, on the other hand, was established. the most reliable detection of antibodies to california encephalitis vi ...200312608063
effects of a point mutation in the 3' end of the s genome segment of naturally occurring and engineered bunyamwera viruses.the genome of bunyamwera virus (bun) consists of three segments of single-stranded rna of negative polarity. the smallest segment, s, encodes the n protein and a nonstructural protein called nss. we recently described a mutant virus (bundelnss) that does not express nss but overexpresses n and grows to lower titres than wild-type (wt) bun. here we report a bundelnss variant that expresses lower levels of n protein and grows to higher titres. sequencing of the 3' and 5' termini of the bundelnss s ...200312655079
bunyavirus infections in north carolina white-tailed deer (odocoileus virginianus).we analyzed serum from white-tailed deer (odocoileus virginianus) collected in southeastern north carolina in 1991 for neutralizing antibodies to six mosquito-borne bunyaviruses (lacrosse, jamestown canyon, keystone,cache valley, potosi, and tensaw), including several of public health importance. evidence was found for all six to be locally transmitted, although greatest seroprevalence was found for potosi, jamestown canyon, and cache valley viruses.200112678047
activation of pkr by bunyamwera virus is independent of the viral interferon antagonist nss.double-stranded rna (dsrna) is a by-product of viral rna polymerase activity, and its recognition is one mechanism by which the innate immune system is activated. cellular responses to dsrna include induction of alpha/beta interferon (ifn) synthesis and activation of the enzyme pkr, which exerts its antiviral effect by phosphorylating the eukaryotic initiation factor eif-2 alpha, thereby inhibiting translation. we have recently identified the nonstructural protein nss of bunyamwera virus (bunv), ...200312692253
reverse genetics for crimean-congo hemorrhagic fever virus.the widespread geographical distribution of crimean-congo hemorrhagic fever (cchf) virus (more than 30 countries) and its ability to produce severe human disease with high mortality rates (up to 60%) make cchf a major public health concern worldwide. we describe here the successful establishment of a reverse genetics technology for cchf virus, a member of the genus nairovirus, family bunyaviridae: the rna polymerase i (pol i) system was used to generate artificial viral rna genome segments (mini ...200312719591
sequence analysis of the medium rna segment of three simbu serogroup viruses, akabane, aino, and peaton viruses.the sequence analysis was carried out for the medium (m) rna segment of the akabane virus (akav), aino virus (ainv), and peaton virus (peav) of the simbu serogroup of the genus orthobunyavirus of the family bunyaviridae. the complementary sequences of the m rna segments of akav, ainv, and peav contain a single large open reading frame (orf), like other orthobunyaviruses. the orfs potentially encode 1401 amino acids (aa), 1404 aa, and 1400 aa polypeptides, respectively. the identity of the m segm ...200312727343
natural infections of pigs with akabane virus.akabane (aka) virus is considered a pathogen of herbivores in nature. however, we found that pig populations in fields were infected in taiwan. an isolate (nt-14) of aka virus was obtained from pigs. the nt-14 virus was able to infect pigs by the oronasal route. subsequently, low levels of infectious virus particles were excreted into the oronasal discharge during the stage of viremia but they were not sufficient to infect new porcine hosts via contact transmission. the prevalence of serum neutr ...200312742710
isolation of a new flavivirus related to cell fusing agent virus (cfav) from field-collected flood-water aedes mosquitoes sampled from a dambo in central kenya.cell fusing agent virus (cfav) is an rna insect virus that was isolated from a line of aedes aegypti mosquito cells and has been assigned to the family flaviviridae, genus flavivirus. we report here the first isolation of a cfa-like virus from field-collected mosquitoes. mosquito larvae and pupae were sampled from flooded dambos in central province, kenya during the short rain season of 1999. specimens were reared to adults, identified and pooled by species and were tested for the presence of vi ...200312756616
isolations of bwamba virus from south central uganda and north eastern tanzania.bwamba virus (genus bunyavirus, family bunyaviridae) is widely distributed in africa. it causes many unidentified fevers because of its benign nature.200212789111
bunyamwera virus nonstructural protein nss counteracts interferon regulatory factor 3-mediated induction of early cell death.the genome of bunyamwera virus (bun; family bunyaviridae, genus orthobunyavirus) consists of three segments of negative-sense rna. the smallest segment, s, encodes two proteins, the nonstructural protein nss, which is nonessential for viral replication and transcription, and the nucleocapsid protein n. although a precise role in the replication cycle has yet to be attributed to nss, it has been shown that nss inhibits the induction of alpha/beta interferon, suggesting that it plays a part in cou ...200312829839
segment-specific terminal sequences of bunyamwera bunyavirus regulate genome replication.bunyamwera virus (bunv) is the prototype of both the orthobunyavirus genus and the bunyaviridae family of segmented negative sense rna viruses. the tripartite bunv genome consists of small (s), medium (m), and large (l) segments that are transcribed to give a single mrna and replicated to generate an antigenome that is the template for synthesis of further genomic rna strands. we modified an existing cdna-derived rna synthesis system to allow identification of bunv rna replication and transcript ...200312842622
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