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Heartland bandavirus (HRTV), which is an emerging tick-borne virus first identified in Missouri in 2009, causes fever, fatigue, decreased appetite, headache, nausea, diarrhea, and muscle or joint pain in humans. HRTV is genetically close to Dabie bandavirus, which is the causative agent of severe fever with thrombocytopenia syndrome (SFTS) in humans and is known as SFTS virus (SFTSV). The generation of infectious HRTV entirely from cloned cDNAs has not yet been reported. The absence of a reverse genetics system for HRTV has delayed efforts to understand its pathogenesis and to generate vaccines and antiviral drugs. Here, we developed a reverse genetics system for HRTV, which employs an RNA polymerase I-mediated expression system. A recombinant nonstructural protein (NSs)-knockout HRTV (rHRTV-NSsKO) was generated. We found that NSs interrupted signaling associated with innate immunity in HRTV-infected cells. The rHRTV-NSsKO was highly attenuated, indicated by the apparent absence of symptoms in a mouse model of HRTV infection. Moreover, mice immunized with rHRTV-NSsKO survived a lethal dose of HRTV. These findings suggest that NSs is a virulence factor of HRTV and that rHRTV-NSsKO could be a vaccine candidate for HRTV. IMPORTANCE Heartland bandavirus (HRTV) is a tick-borne virus identified in the United States in 2009. HRTV causes fever, fatigue, decreased appetite, headache, nausea, diarrhea, and muscle or joint pain in humans. FDA-approved vaccines and antiviral drugs are unavailable. The lack of a reverse genetics system hampers efforts to develop such antiviral therapeutics. Here, we developed a reverse genetics system for HRTV that led to the generation of a recombinant nonstructural protein (NSs)-knockout HRTV (rHRTV-NSsKO). We found that NSs interrupted signaling associated with innate immunity in HRTV-infected cells. Furthermore, rHRTV-NSsKO was highly attenuated and immunogenic in a mouse model. These findings suggest that NSs is a virulence factor of HRTV and that rHRTV-NSsKO could be a vaccine candidate for HRTV.

Negative-strand (-) RNA viruses (NSVs) comprise a large and diverse group of viruses that are generally divided in those with non-segmented and those with segmented genomes. Whereas most NSVs infect animals and humans, the smaller group of the plant-infecting counterparts is expanding, with many causing devastating diseases worldwide, affecting a large number of major bulk and high-value food crops. In 2018, the taxonomy of segmented NSVs faced a major reorganization with the establishment of the order Bunyavirales . This article overviews the major plant viruses that are part of the order, i.e., orthospoviruses ( Tospoviridae ), tenuiviruses ( Phenuiviridae ), and emaraviruses ( Fimoviridae ), and provides updates on the more recent ongoing research. Features shared with the animal-infecting counterparts are mentioned, however, special attention is given to their adaptation to plant hosts and vector transmission, including intra/intercellular trafficking and viral counter defense to antiviral RNAi.

A possible etiological link between the onset of endemic pemphigus in Tunisia and bites of Phlebotomus papatasi, the vector of zoonotic cutaneous leishmaniasis, has been previously suggested. We hypothesized that the immunodominant P. papatasi salivary protein PpSP32 binds to desmogleins 1 and 3 (Dsg1 and Dsg3), triggering loss of tolerance to these pemphigus target autoantigens. Here, we show using far-Western blot that the recombinant PpSP32 protein (rPpSP32) binds to epidermal proteins with a MW of approximately 170 kDa. Coimmunoprecipitation revealed the interaction of rPpSP32 with either Dsg1 or Dsg3. A specific interaction between PpSP32 and Dsg1 and Dsg3 was further demonstrated by ELISA assays. Finally, mice immunized with rPpSP32 twice per week exhibited significantly increased levels of anti-Dsg1 and -Dsg3 antibodies from day 75 to 120. Such antibodies were specific for Dsg1 and Dsg3 and were not the result of cross-reactivity to PpSP32. In this study, we demonstrated for the first time to our knowledge a specific binding between PpSP32 and Dsg1 and Dsg3, which might underlie the triggering of anti-Dsg antibodies in patients exposed to sand fly bites. We also confirmed the development of specific anti-Dsg1 and -Dsg3 antibodies in vivo after PpSP32 immunization in mice. Collectively, our results provide evidence that environmental factors, such as the exposure to P. papatasi bites, can trigger the development of autoimmune antibodies.

Rift Valley Fever (RVF) is an emerging zoonotic arbovirus with a complex cycle of transmission that makes difficult the prediction of its expansion. Recent outbreaks outside Africa have led to rediscover the human disease but it remains poorly known. The wide spectrum of acute and delayed manifestations with potential unfavorable outcome much complicate the management of suspected cases and prediction of morbidity and mortality during an outbreak. We reviewed literature data on bio-clinical characteristics and treatments of RVF human illness. We identified gaps in the field and provided a practical algorithm to assist clinicians in the cases assessment, determination of setting of care and prolonged follow-up.

Viral haemorrhagic fever can be caused by one of a diverse group of viruses that come from four different families of RNA viruses. Disease severity can vary from mild self-limiting febrile illness to severe disease characterized by high fever, high-level viraemia, increased vascular permeability that can progress to shock, multi-organ failure and death. Despite the urgent need, effective treatments and preventative vaccines are currently lacking for the majority of these viruses. A number of factors preclude the effective study of these diseases in humans including the high virulence of the agents involved, the sporadic nature of outbreaks of these viruses, which are typically in geographically isolated areas with underserviced diagnostic capabilities, and the requirements for high level bio-containment. As a result, animal models that accurately mimic human disease are essential for advancing our understanding of the pathogenesis of viral haemorrhagic fevers. Moreover, animal models for viral haemorrhagic fevers are necessary to test vaccines and therapeutic intervention strategies. Here, we present an overview of the animal models that have been established for each of the haemorrhagic fever viruses and identify which aspects of human disease are modelled. Furthermore, we discuss how experimental design considerations, such as choice of species and virus strain as well as route and dose of inoculation, have an influence on animal model development. We also bring attention to some of the pitfalls that need to be avoided when extrapolating results from animal models., (Copyright © 2016. Published by Elsevier Ltd.)

Infection caused by the severe fever and thrombocytopenia syndrome virus (SFTSV) causes a hemorrhagic illness with a mortality between 20% and 40%. Initially recognized in 2009 in China, cases have additionally been documented in Japan and Korea although retrospective studies have documented seroprevalence since 1996. Although case rates have increased due to increased awareness and more widely available diagnostics, SFTSV infection remains rare with the highest rates documented in Korea for Jeju Province (3.5 cases per 100,000 population) and the Inje-gun region (66.2 cases per 100,000). Because of the very low incidence of infection, a placebo-controlled study with 1:1 randomization to evaluate an SFTSV vaccine would require a sample size that is 25% greater than the region of study. We discuss alternatives to licensure. Vaccine effectiveness may be assessed through a registry, comparing rates of infection over time between vaccine recipients versus regional populations. Modeled data can be updated based on actual case rates and population changes over the years of follow-up. Using one model, statistically significant differences are seen after 10 years in Inje-gun and 15 years of follow-up in Jeju. This approach may be applicable to other uncommon infectious diseases for which a standard study design is difficult.

The International Committee on Taxonomy of Viruses (ICTV) has implemented numerous changes to the taxonomic classification of bunyaviruses over the years. Whereas most changes have been justified and necessary because of the need to accommodate newly discovered and unclassified viruses, other changes are a cause of concern, especially the decision to demote scores of formerly recognized species to essentially strains of newly designated species. This practice was first described in the seventh taxonomy report of the ICTV and has continued in all subsequent reports. In some instances, viruses that share less than 75% nucleotide sequence identity across their genomes, produce vastly different clinical presentations, possess distinct vector and host associations, have different biosafety recommendations, and occur in nonoverlapping geographic regions are classified as strains of the same species. Complicating the matter is the fact that virus strains have been completely eliminated from ICTV reports; thus, critically important information on virus identities and their associated biological and epidemiological features cannot be readily related to the ICTV classification. Here, we summarize the current status of bunyavirus taxonomy and discuss the adverse consequences associated with the reclassification and resulting omission of numerous viruses of public health importance from ICTV reports. As members of the American Committee on Arthropod-borne Viruses, we encourage the ICTV Bunyavirus Study Group to reconsider their stance on bunyavirus taxonomy, to revise the criteria currently used for species demarcation, and to list additional strains of public and veterinary importance.

The evolutionary origins of arboviruses are unknown because their typical dual host tropism is paraphyletic within viral families. Here we studied one of the most diversified and medically relevant RNA virus families, the Bunyaviridae, in which four of five established genera are transmitted by arthropods. We define two cardinally novel bunyavirus groups based on live isolation of 26 viral strains from mosquitoes (Jonchet virus [JONV], eight strains; Ferak virus [FERV], 18 strains). Both viruses were incapable of replicating at vertebrate-typical temperatures but replicated efficiently in insect cells. Replication involved formation of virion-sense RNA (vRNA) and mRNA, including cap-snatching activity. SDS/PAGE, mass spectrometry, and Edman degradation identified translation products corresponding to virion-associated RNA-dependent RNA polymerase protein (RdRp), glycoprotein precursor protein, glycoproteins Gn and Gc, as well as putative nonstructural proteins NSs and NSm. Distinct virion morphologies suggested ancient evolutionary divergence, with bunyavirus-typical morphology for FERV (spheres of 60-120 nm) as opposed to an unusual bimorphology for JONV (tubular virions of 60 × 600 nm and spheres of 80 nm). Both viruses were genetically equidistant from all other bunyaviruses, showing <15% amino acid identity in the RdRp palm domain. Both had different and unique conserved genome termini, as in separate bunyavirus genera. JONV and FERV define two novel sister taxons to the superclade of orthobunyaviruses, tospoviruses, and hantaviruses. Phylogenetic ancestral state reconstruction with probabilistic hypothesis testing suggested ancestral associations with arthropods at deep nodes throughout the bunyavirus tree. Our findings suggest an arthropod origin of bunyaviruses.

The family Bunyaviridae of segmented, negative-stranded RNA viruses includes over 350 members that infect a bewildering variety of animals and plants. Many of these bunyaviruses are the causative agents of serious disease in their respective hosts, and are classified as emerging viruses because of their increased incidence in new populations and geographical locations throughout the world. Emerging bunyaviruses, such as Crimean-Congo hemorrhagic fever virus, tomato spotted wilt virus and Rift Valley fever virus, are currently attracting great interest due to migration of their arthropod vectors, a situation possibly linked to climate change. These and other examples of continued emergence suggest that bunyaviruses will probably continue to pose a sustained global threat to agricultural productivity, animal welfare and human health. The threat of emergence is particularly acute in light of the lack of effective preventative or therapeutic treatments for any of these viruses, making their study an important priority. This review presents recent advances in the understanding of the bunyavirus life cycle, including aspects of their molecular, cellular and structural biology. Whilst special emphasis is placed upon the emerging bunyaviruses, we also describe the extensive body of work involving model bunyaviruses, which have been the subject of major contributions to our overall understanding of this important group of viruses.

Reverse-genetic systems are often used to study different aspects of the viral life cycle. To date, three rescue systems have been developed for the family Bunyaviridae. These systems use T7 RNA polymerase, which is generally used in rescue systems for Mononegavirales. In the present study, we describe a rescue system for Akabane virus (family Bunyaviridae) that uses cDNAs and RNA polymerase I instead of T7 RNA polymerase. The utility of this system was demonstrated by the generation of a mutant with a deletion of the non-structural protein (NSs) on the S RNA segment. These results offer a new option for bunyavirus rescue.

The Bunyaviridae are a large group of viruses that infect a diversity of arthropod vectors and animal hosts. They have a worldwide distribution and can be the cause of human illness ranging from mild asymptomatic infection to hemorrhagic fever and fatal encephalitis. The growth of the human population, the expansion of agricultural and economic development, climatic changes, and the speed and frequency of global transportation all favor the emergence of bunyaviruses and other arthropod borne viruses. International monitoring of the Bunyaviridae and a greater understanding of their ecology and biology are needed to prepare for future outbreaks.

To determine the teratogenic potential of Aino virus (AINOV) in cattle, pregnant cows and fetal cattle were infected with a fresh isolate of AINOV. Five pregnant cows were inoculated intravenously with the virus at 122 to 162 days of gestation and allowed to give birth. All of the cows developed neutralizing antibodies to the virus, indicating that the cows had been infected with the virus; however, no clinical abnormalities were seen in their six newborn calves, and no specific antibodies to the virus were detected in the precolostral serum of calves. Five fetuses with fetal ages ranging from 132 to 156 days were inoculated in utero with the virus. One weak newborn and four stillborn calves were delivered at gestation days 256 to 263, i.e., less than the standard gestation term; they had congenital abnormalities including arthrogryposis, hydranencephaly and cerebellar hypoplasia. Antibodies specific to AINOV were detected in their precolostral serum. These results demonstrate that AINOV is a potential etiological agent of congenital malformation of cattle.

A virus isolated from dead Chaerephon plicata bats collected near Kampot, Cambodia, was identified as a member of the family Bunyaviridae by electron microscopy. The only bunyavirus previously isolated from Chaerephon species bats in South-East Asia is Kaeng Khoi (KK) virus (genus Orthobunyavirus), detected in Thailand over 30 years earlier and implicated as a public health problem. Using RT-PCR, nucleotide sequences from the M RNA segment of several virus isolates from the Cambodian C. plicata bats were found to be almost identical and to differ from those of the prototype KK virus by only 2.6-3.2 %, despite the temporal and geographic separation of the viruses. These results identify the Cambodian bat viruses as KK virus, extend the known virus geographic range and document the first KK virus isolation in 30 years. These genetic data, together with earlier serologic data, show that KK viruses represent a distinct group within the genus Orthobunyavirus.

Adult golden hamsters inoculated subcutaneously with either of two sandfly fever group viruses, Punta Toro and Gabek Forest (Phlebovirus, Bunyaviridae), developed a fulminating fatal illness characterized by hepatic and splenic necrosis and interstitial pneumonitis. Most animals died within three days after infection; this was accompanied by high levels of viremia. Necropsy and histopathologic examination of the infected animals revealed pathologic changes involving multiple organs that resembled those described in Rift Valley fever. These two hamster-phlebovirus systems may serve as alternative animal models for Rift Valley fever and should be useful in studying the pathogenesis of severe phlebovirus infection and for testing potential therapeutic agents.

A viral genetic system was used to map the determinants of the ability of Tomato spotted wilt virus (TSWV) to overcome the R gene (Sw-5) in tomato and the resistance conferred by the nucleocapsid gene of TSWV (N gene) in tobacco. A complete set of reassortant genotypes was generated from TSWV isolates A and D. TSWV-A was able to overcome the Sw-5 gene in tomato and the TSWV N gene in tobacco, whereas TSWV-D was repressed by both forms of resistance. The ability to overcome both forms of resistance was associated with the M RNA segment of TSWV-A (M(A)). Overcoming the Sw-5 gene was linked solely to the presence of M(A), and the ability of M(A) to overcome the TSWV N gene was modified by the L RNA and the S RNA of TSWV-A, which is consistent with previous reports that suggest that the nucleocapsid gene is not the primary determinant for overcoming the nucleocapsid-mediated resistance. Sequence analysis of the M RNA segment of TSWV-A, -D, and the type isolate BR-01 revealed multiple differences in the coding and noncoding regions, which prevented identification of the resistance-breaking nucleotide sequences.

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. All reassortant viruses infect Ae. albopictus orally and can be transmitted to suckling mice. All reassortants are neurovirulent in mice. However, reassortant viruses carrying the LACV M segment in the foreign genetic background of JCV are more neuroinvasive than JCV, or any other reassortant genotype. In addition, these reassortants can replicate in gerbils and infect Ae. triseriatus, characteristics of LACV, but not JCV. Because Ae. albopictus is spreading into new geographic areas and feeds on a variety of mammals, including humans, it has the potential to transmit new, emerging bunyaviruses in nature.

A neuroattenuated variant bunyavirus, designated RFC/25B.5 (B.5), was selected by serial passage of a reassortant clone (RFC virus) of a California serogroup virus in BHK-21 cells, followed by plaque purification of that passaged stock. Based on its virulence index (ratio of PFU/50% lethal dose), clone B5 was over 40,000-fold less virulent than its unpassaged RFC parent after intracerebral (i.c.) inoculation into adult mice. Clone B.5 also exhibited markedly reduced neuroinvasiveness after subcutaneous injection into neonatal mice, although it retained its ability to replicate and kill suckling mice after i.c. injection. A murine neuroblastoma line (NA cells) can be used as an in vitro surrogate for the adult mouse brain, since clone B.5 replicated to at least 1,000-fold-lower titers in NA cells than did several neurovirulent California serogroup viruses. Clone B.5 replicated in BHK-21 cells at 37 degrees C to titers similar to those achieved by other California serogroup viruses but was temperature sensitive (ts) since its replication was markedly restricted at 38.9 degrees C. Ten ts revertant clones of B.5 virus were selected at 38.9 degrees C, and all of them lost their ts phenotype and regained the ability to replicate to high titer in NA cells and to kill adult mice after i.c. injection. Clone B.5 is the first described California serogroup virus which is truly attenuated after i.c. inoculation, and its availability will permit genetic analysis of bunyavirus neurovirulence.

Susceptibility to lethal infection with the KT281/75 strain of the tick-borne nairovirus, Dugbe virus, was similar in an outbred strain and several inbred strains of mice. For the outbred strain, both neural and extraneural routes of virus inoculation resulted in lethal infection, but susceptibility decreased with age and only intracerebral inoculation produced a lethal infection in adults. In newborn mice, subcutaneous (s.c.) inoculation of virus (analogous to a tick-bite) produced a disseminated infection, titres being highest in the upper respiratory tract (URT), spleen and liver at 5 days post-inoculation (p.i.), the heart at 7 days p.i. and brain by 8 days p.i. In neonates inoculated intranasally (i.n.), by contrast, virus spread rapidly from the URT to the brain by 2 days p.i., in the absence of a detectable viraemia. Virus was undetectable in the blood of s.c. and i.n. inoculated adults; in the former, virus replication was limited to the site of inoculation, and in the latter virus grew in the respiratory tract and again spread to the brain. Immunosuppression of i.n. inoculated adult mice with cyclophosphamide produced some mortality indicating that host defences are important in protecting the adult, especially as newborn and adult lung tissue were equally able to support the growth of Dugbe virus in culture. The similarity between the pattern of Dugbe virus infection in the mouse and that of other, more pathogenic nairoviruses suggests that, although haemorrhagic disease was not observed, this may be a useful model for studying the genetic basis of nairovirus virulence and for testing vaccines and anti-viral drugs.

The respiratory infectivity of a strain of Rift Valley fever virus isolated in Egypt (strain ZH-501) was compared with that of one isolate from Uganda (Entebbe strain) and two isolates from South Africa (strains SA-51 and SA-75). Studies were performed with ICR mice which were infected by exposure to infectious aerosols composed of particles with a mass median diameter of 0.96 micrometer. The respiratory median lethal doses for ZH-501, Entebbe, SA-51, and SA-75 were 2.2, 1.9, 2.6, and 1.9 log10 plaque-forming units, respectively. Although these values are statistically different, the biological implications of such differences seem unimportant. In an additional study of pathogenesis, a single group of mice was infected with 3.1 log10 plaque-forming units of ZH-501, and tissues were assayed sequentially through 96 h postinfection. Between 6 and 30 h, demonstration of an increasing virus concentration only in the lungs indicated that initial replication occurred there; however, determination of histopathological changes did not reveal evidence of pneumonia. Virus was isolated from the liver by 48 h, and the ultimate outcome of infection was a fulminating and fatal hepatic necrosis.

Two experiments were conducted with five gallinaceous and one passerine bird species to determine their responses to Turlock (TUR) virus inoculations. Inoculation of TUR strain 847-32 into bobwhites, chukars, ring-necked pheasants, chickens, and Japanese quail did not product detectable viremias. The first four species, however, did respond with neutralizing antibody. Inoculation of chickens with strain 69V-1095 resulted in a viremia which lasted 5 days and had a peak mean titer of 2.0 log 10 PFU per 0.2 ml of blood on the third day after infection. The observation that viremic birds exhibited no noticeable virus-associated morbidity or mortality suggested that TUR virus does not have a detrimental effect on free-ranging populations of the avian hosts studied during this investigation.

Homologous and heterologous haemagglutination-inhibition (HAI), complement-fixation (CF), immunodiffusion (ID) and mouse neutralization tests were performed with the Lunyo (LUN) and a Zimbabwean strain of Rift Valley fever (RVF) virus, the prototype and a South African strain of Arumowot (AMT) virus and prototype strains of Gordil (GOR), Saint-Floris (SAF) and Gabek Forest (GF) viruses, using immune mouse ascitic fluids prepared against these viruses. Reactions of identity occurred in all tests between LUN and the Zimbabwean strains of RVF and between the two strains of AMT virus. Otherwise, cross-reactions occurred between all the phleboviruses in HAI tests, while reactions in CF, ID and neutralization tests were monospecific for virus serotypes, except that weak cross-reaction occurred between GOR and SAF viruses in CF and ID tests. Four sheep infected subcutaneously with the Zimbabwean strain of RVF virus developed transient fever, viraemia, leucopaenia, relative thrombocytopaenia, haemoconcentration and raised serum enzyme levels, which indicated that the sheep had developed necrotic hepatitis. Disseminated focal necrotic hepatitis was confirmed in a sheep killed for examination on day 4 post-infection. The other three sheep recovered uneventfully after only mild depression and anorexia. Groups of three sheep infected with SAF, GOR, AMT and GF viruses had no demonstrable viraemia or other sign of infection or illness, except that the sheep infected with AMT developed mild fever lasting less than 24 h. Antibody responses were monitored at intervals over a period of 24 weeks in all sheep by homologous and heterologous HAI, CF and cell culture neutralization (CPENT) tests. Homologous antibody responses were marked in the RVF-infected sheep and their sera cross-reacted strongly in HAI tests with antigens of the other viruses. The sera of the RVF-infected sheep cross-reacted less markedly in CF and CPENT tests. Homologous antibody responses were poor in all the sheep infected with phleboviruses other than RVF, and the cross-reactivity of their sera for RVF antigen or virus was negligible. All sheep were challenged with RVF virus 48 weeks after their initial infection. The sheep which had originally been infected with RVF virus were immune and developed neither fever nor viraemia. All other sheep developed fever, viraemia and antibodies to RVF virus. It was concluded that the African phleboviruses, other than RVF, are unlikely to cause disease in livestock or to induce antibodies which could cause confusion in the diagnosis of RVF.

We have studied the transport of the Uukuniemi virus membrane glycoproteins in baby hamster kidney and chick embryo cells by using a temperature-sensitive mutant (ts12). Uukuniemi virus assembles in the Golgi complex, where both glycoproteins G1 and G2 and nucleocapsid protein N accumulate (E. Kuismanen, B. Bång, M. Hurme, and R. F. Pettersson, J. Virol. 51:137-146, 1984). At the restrictive temperature (39 degrees C), the glycoproteins of ts12 were transported to the Golgi complex as in wild-type, virus-infected cells, whereas the nucleocapsid protein failed to accumulate there. Pulse-chase labeling followed by immunoprecipitation and treatment with endo-beta-N-acetylglucosaminidase H showed that G1 synthesized at 39 degrees C in ts12-infected cells had an altered mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting a lack of terminal glycosylation. The typical Uukuniemi virus-induced vacuolization and expansion of the Golgi complex could be seen also in ts12-infected cells at 39 degrees C, although no virus particles were formed. This suggests that the morphological changes were induced by the Uukuniemi virus glycoproteins. In wild-type virus- or ts12-infected cells, G1 and G2 could not be chased out from the Golgi complex even after 6 h of treatment with cycloheximide. The glycoproteins were thus retained in the Golgi even under conditions when no virus maturation took place and when nucleocapsids did not accumulate in the Golgi region. Accordingly, the glycoproteins of Uukuniemi virus were found to have properties resembling those of Golgi-specific proteins. This virus model system may be useful in studying the synthesis and transport of membrane proteins that are transported to and retained in the Golgi.

In an examination of the molecular basis of oral transmission of bunyaviruses by mosquitoes., La Crosse (LAC), snowshoe hare (SSH), and LAC-SSH reassortant viruses were compared in their ability to be transmitted to laboratory mice by the natural mosquito vector of LAC virus, Aedes triseriatus. Both LAC virus and the reassortment viruses containing the middle-sized (M) segment from the LAC parent were efficiently transmitted. In contrast, SSH virus and reassortment viruses containing the M RNA from the SSH parent were inefficiently transmitted. Thus, the M RNA segment, which codes for the virion glycoproteins, may be a major determinant of oral transmission of bunyaviruses by mosquitoes.

To identify which RNA segments of the California serogroup bunyaviruses determine virulence, we prepared reassortant viruses by coinfecting BHK-21 cells with two wild-type parents, La Crosse/original and Tahyna/181-57 viruses, which differed about 30,000-fold in virulence. The progeny clones were screened by polyacrylamide gel electrophoresis to ascertain the phenotype of the M and S RNA segments, and RNA-RNA hybridization was used to determine the genotype of selected clones. Two or three clones of each of the six possible reassortant genotypes were characterized quantitatively for neuroinvasiveness by determining the PFU/50% lethal dose (LD50) ratio after subcutaneous injection into suckling mice. The reassortants fell into two groups. (i) Six of seven reassortants with a La Crosse M RNA segment were as virulent as the parent La Crosse virus (about 1 PFU/LD50); the one exception was strikingly different (about 1,000 PFU/LD50) and probably represents a spontaneous mutant. (ii) The seven reassortants with a Tahyna M RNA segment were about 10-fold more virulent than the parent Tahyna virus (median 1,600 PFU/LD50 for reassortants and 16,000 PFU/LD50 for Tahyna virus). A comparative pathogenesis study in suckling mice of one reassortant virus and the parent Tahyna virus confirmed the greater neuroinvasiveness of the reassortant virus. From these data it was concluded that the M RNA segment was the major determinant of virulence, but that the other two gene segments could modulate the virulence of a nonneuroinvasive California serogroup virus.

Two young female rabbits (Oryctolagus cuniculus var. edulis) were inoculated subcutaneously with Bhanja virus (BHA) in a dose of 85 intracerebral LD50 for suckling mice (SMicLD50) and 8500 SMicLD50 (rabbit A and B, resp.). In rabbit A no clinical symptoms, nor viremia were observed, only seroconversion was revealed on day 7 p.i. On the other hand, in the rabbit B hypothermy on days 7--11 p.i., a mild adynamia on day 11 p.i. and a slight paresis on one hind leg on days 23--30 p.i. were observed; traces of BHA virus in the blood were detected on day 9 p.i. and seroconversion on day 7 p.i. The virus was not isolated from the interior organs (brain, liver, spleen, kidneys, lungs and heart) of the two rabbits dissected 38 days p.i.

La Crosse virus, a member of the California serogroup of the family Bunyaviridae, causes encephalitis in humans and laboratory rodents. A variant virus (V22) selected with a monoclonal antibody against the large (G1) glycoprotein showed diminished neuroinvasiveness after peripheral inoculation. This variant has an alteration in its fusion function, requiring a lower pH for the activation of fusion and demonstrating reduced efficiency of cell-to-cell fusion of BHK-21 cultures. V22 was studied in detail following the infection by intraperitoneal or intracerebral routes in suckling, weanling, or adult CD-1 mice. It exhibited a marked reduction in its ability to replicate in striated muscle and to produce viremia; however, after intracerebral injection V22 virus replicated almost as rapidly in brain as its parent, La Crosse virus. V22 virus thus represents an example of reduced neuroinvasiveness associated with an alteration at a specific epitope of the G1 glycoprotein. This same epitope also influences the fusion activity of the glycoprotein.

The wild-type ZH501 strain of Rift Valley fever (RVF) virus and two small-plaque strains (T1 and T46) derived from it were characterized by plaque size, pathogenicity for hamsters and ability to replicate in Vero cells. Additionally, a mutagenized, attenuated, large-plaque, vaccine-candidate strain of RVF virus (ZH548-M12) was also studied. Infections with either the ZH501 or T46 strain were uniformly fatal to hamsters. In contrast, nearly all hamsters infected with either the T1 or ZH548-M12 strains survived and were immune to challenge with 10(5) LD50 of the ZH501 strain. Both of these attenuated strains failed to replicate in Vero cells maintained at 41 degrees C, whereas the more virulent strains (ZH501 and T46) replicated at this temperature. The low virulence and ability to induce protection against lethal RVF virus challenge that is associated with the T1 and ZH548-M12 strains make them potential vaccine candidates.

Comparative analysis of the structural and possible non-structural proteins of seven Simbu serogroup bunyaviruses isolated in Australia revealed them all to be similar in size to those of Bunyamwera virus, the prototype of the Bunyavirus genus. The molecular weights of the structural proteins for these bunyaviruses (Akabane, Aino, Tinaroo, Douglas, Peaton, Facey's Paddock and Thimiri viruses) were 193K to 205K (L), 103K to 125K (G1), 33K to 37K (G2) and 25K to 26K (N). Analysis of the virion RNA of three viruses (Akabane, Douglas and Facey's Paddock) showed them all to be similar to Bunyamwera virus RNA, apparent Mr values being 2.6 X 10(6) (L), 1.4 X 10(6) to 1.9 X 10(6) (M) and 0.24 X 10(6) to 0.42 X 10(6) (S). Host cell protein synthesis was switched off late during infection, revealing four structural proteins L, G1, G2 and N. Comparative analysis of these protein profiles in infected Vero cells showed each virus, although similar, to be unique and easily identified; this method of comparison was efficient and rapid compared to the difficulty in obtaining adequate amounts of purified virus for analysis. Additionally, for all viruses except Douglas, two to four possible non-structural proteins were identified, with an Mr range from 12K to 30K. The viruses Akabane and Tinaroo, which have previously been shown to cross-react by plaque inhibition virus neutralization tests, were readily distinguished in migration of the G1 glycoprotein and by analysis of plaque reduction virus neutralization data using linear regression analysis of the dose-response curves. Using these same analyses, the differences between Aino and Douglas viruses, also related by plaque inhibition, were even greater. Application of the biochemical analysis of virus-specified proteins and some serological comparisons identified a mixed pool of different viruses in two unknown isolates grouped as Simbu serogroup viruses, and further identified a potential teratogenic strain in one of the two pools.

The use of embryonated chicken eggs as a model for assessing the teratogenic potential of animal viruses was investigated with 12 members of the Bunyaviridae family. Infection of 4-day-old embryonated chicken eggs via the yolk sac with 10 of the viruses resulted in deaths or congenital deformities that were similar to those observed in Akabane virus infections of fetal ruminants and included arthrogryposis, scoliosis, mandible defects, and retarded development. Statistical analysis showed that the viruses fell into three main groupings, namely, those that caused both death and deformities (Akabane, Aino, Tinaroo, and Belmont viruses), those that mainly caused death (Peaton, Thimiri, and Facey's Paddock viruses), and those that required very high doses to cause either death or deformities (Douglas and CSIR0296 viruses). In addition, two viruses (Kowanyama and Mapputta viruses) caused neither death nor deformities. A difference in the pathogenic potential between two Akabane isolates (B8935 and CSIR016) in the embryonated chicken egg model was found to correlate with differences previously observed in experimentally infected sheep; Akabane CSIR016 was the more pathogenic. It is concluded that the embryonated chicken egg model should also be of value in assessing the teratogenic potential of other Bunyaviridae and attenuated vaccine viruses, although it does not assess the ability of the virus to cross the placenta.