Characterization of Batai virus isolated from a domestic Muscovy duck (Cairina moschate).

Batai virus (BATV) belongs to the genus Orthobunyavirus of the family Bunyaviridae. It has been isolated from mosquitos, pigs, cattle, and humans throughout Africa, Asia, and Europe, and causes clinical signs in domestic animals and humans. Here, we report the isolation of BATV from a domestic duck flock. Genome sequence analysis revealed clustering of this isolate in the Africa-Asia lineage. The virus replicated in mosquitos and vertebrate host cells, showing different phenotypic characteristics, and showed the potential to infect mice. This is the first report of BATV in domestic birds and indicates the wide circulation of BATV in China.

Isolation and complete nucleotide sequence of a Batai virus strain in Inner Mongolia, China.

BACKGROUND: Batai virus (BATV) is a member of the Orthobunyavirus genus of the family Bunyaviridae, and a vector-borne pathogen. Genomic variations of BATV exist in different regions of the world, due to genetic reassortment. Whole-genome sequencing of any isolate is necessary for a phylogenetic analysis. In 1998, a BATV strain was isolated from an Anopheles philippines mosquito in Yunnan Province, China. This strain has not been found to infect any other host. We investigated BATV infection in cattle in Inner Mongolia, China and performed deep sequencing of the genome of the BATV isolate. FINDINGS: Ninety-five blood samples were collected from cattle in Inner Mongolia, China in 2012. The BATV infection rate was 2.1%. Previously, BATV strain NM/12 was isolated from two cattle in Inner Mongolia, China, and the whole genomic sequence of the strain has been available. We determined the complete genomic nucleotide sequences of the small (S), medium (M), and large (L) genome segments using bovine blood obtained in 2012, and the nucleotide homologies of these segments with those from GenBank were 88.7%-97%, 84%-95.4%, and 72.6%-95.8%, respectively. The deduced amino acid identities were 87.2-99.7%, 64.2-96.8%, and 81.1-98.6%. Phylogenetic analyses based on full-length genomic sequences indicated that the M and L segments, and a portion of the S segment, of NM/12 are most closely related to the BATV strains isolated in Asia. The S and M segments of NM/12 were independent of phylogenetic lineages. The L segment was the most closely related to Chittoor/IG-20217 (isolated in India), and distantly related to isolated strains in Italy. Nucleotide substitution rates in the nucleotide sequences that code for the nucleocapsid, envelope glycoprotein, and polymerase protein of NM/12 strain were 2.56%, 4.69%, and 4.21%, respectively, relative to the original strain of MM2222. CONCLUSION: A novel BATV NM/12 strain from bovine serum collected in Inner Mongolia was isolated from cattle in China for the first time. Our findings elucidate the evolutionary status of the BATV NM/12 strain among different orthobunyavirus strains and may provide some clues to prevent the emergence of BATV infection in cattle and humans.

Orthobunyavirus ultrastructure and the curious tripodal glycoprotein spike.

The genus Orthobunyavirus within the family Bunyaviridae constitutes an expanding group of emerging viruses, which threaten human and animal health. Despite the medical importance, little is known about orthobunyavirus structure, a prerequisite for understanding virus assembly and entry. Here, using electron cryo-tomography, we report the ultrastructure of Bunyamwera virus, the prototypic member of this genus. Whilst Bunyamwera virions are pleomorphic in shape, they display a locally ordered lattice of glycoprotein spikes. Each spike protrudes 18 nm from the viral membrane and becomes disordered upon introduction to an acidic environment. Using sub-tomogram averaging, we derived a three-dimensional model of the trimeric pre-fusion glycoprotein spike to 3-nm resolution. The glycoprotein spike consists mainly of the putative class-II fusion glycoprotein and exhibits a unique tripod-like arrangement. Protein-protein contacts between neighbouring spikes occur at membrane-proximal regions and intra-spike contacts at membrane-distal regions. This trimeric assembly deviates from previously observed fusion glycoprotein arrangements, suggesting a greater than anticipated repertoire of viral fusion glycoprotein oligomerization. Our study provides evidence of a pH-dependent conformational change that occurs during orthobunyaviral entry into host cells and a blueprint for the structure of this group of emerging pathogens.

Role of the cytoplasmic tail domains of Bunyamwera orthobunyavirus glycoproteins Gn and Gc in virus assembly and morphogenesis.

The M RNA genome segment of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, encodes a precursor polyprotein that is proteolytically cleaved to yield two structural proteins, Gn and Gc, and a nonstructural protein called NSm. Gn and Gc are type I integral transmembrane glycoproteins. The Gn protein contains a predicted cytoplasmic tail (CT) of 78 residues, and Gc has a shorter CT of 25 residues. Little is known about the role of the Gn and Gc CT domains in the virus replication cycle. We generated a series of mutant glycoprotein precursor constructs containing either deletions or alanine substitutions in the CT domains of Gn and Gc. We examined the effects of these mutations on glycoprotein maturation, cell surface expression, and low pH-induced syncytium formation. In addition, the effects of these mutations were also assessed using a reverse genetics-based virus assembly assay and a virus rescue system. Our results show that the CT domains of both Gn and Gc play crucial roles in BUNV-mediated membrane fusion, virus assembly, and morphogenesis.

Studying cellular architecture in three dimensions with improved resolution: Ta replicas revisited.

Metal replicas have been used for surface analysis of biological structures with a variety of spatial resolutions. Platinum (Pt) has been the metal of choice because it provides very stable replicas and images of high contrast. Some other metals, such as tantalum (Ta) have been reported to provide better resolution on isolated macromolecular complexes and cellular structures. Our goal is to study the gain in detail with Ta and to evaluate if it provides enough detail and resolution to assist in the study of complex volumes of intact cellular structures obtained by methods that reach molecular resolution. To this purpose Pt and Ta replicas of cellular structures and viruses have been studied by transmission electron microscopy (TEM). Replicas of Ta show new details on the surface of two types of isolated viral particles such as 100 nm bunyaviruses and large, > 300 nm, vaccinia virus (VV). Inside cells, the structural pieces that build VV immature particles are visualized only in Ta replicas. Looking for smaller intracellular complexes, new details are also seen in nuclear pores from Ta replicas. Additional masses, most likely representing the cargo during transport, are distinguished in some of the pores. Visualization of proteins in plasma membranes strongly suggests that detail and resolution of Ta replicas are similar to those estimated for 3D maps currently obtained by electron tomography of viruses and cells.

Key Golgi factors for structural and functional maturation of bunyamwera virus.

Several complex enveloped viruses assemble in the membranes of the secretory pathway, such as the Golgi apparatus. Among them, bunyaviruses form immature viral particles that change their structure in a trans-Golgi-dependent manner. To identify key Golgi factors for viral structural maturation, we have purified and characterized the three viral forms assembled in infected cells, two intracellular intermediates and the extracellular mature virion. The first viral form is a pleomorphic structure with fully endo-beta-N-acetylglucosaminidase H (Endo-H)-sensitive, nonsialylated glycoproteins. The second viral intermediate is a structure with hexagonal and pentagonal contours and partially Endo-H-resistant glycoproteins. Sialic acid is incorporated into the small glycoprotein of this second viral form. Growing the virus in glycosylation-deficient cells confirmed that acquisition of Endo-H resistance but not sialylation is critical for the trans-Golgi-dependent structural maturation and release of mature viruses. Conformational changes in viral glycoproteins triggered by changes in sugar composition would then induce the assembly of a compact viral particle of angular contours. These structures would be competent for the second maturation step, taking place during exit from cells, that originates fully infectious virions.

Polymorphism and structural maturation of bunyamwera virus in Golgi and post-Golgi compartments.

The Golgi apparatus is the assembly site for a number of complex enveloped viruses. Using high-preservation methods for electron microscopy, we have detected two previously unknown maturation steps in the morphogenesis of Bunyamwera virus in BHK-21 cells. The first maturation takes place inside the Golgi stack, where annular immature particles transform into dense, compact structures. Megalomicin, a drug that disrupts the trans side of the Golgi complex, reversibly blocks transformation, showing that a functional trans-Golgi is needed for maturation. The second structural change seems to take place during the egress of viral particles from cells, when a coat of round-shaped spikes becomes evident. A fourth viral assembly was detected in infected cells: rigid tubular structures assemble in the Golgi region early in infection and frequently connect with mitochondria. In Vero cells, the virus induces an early and spectacular fragmentation of intracellular membranes while productive infection progresses. Assembly occurs in fragmented Golgi stacks and generates tubular structures, as well as the three spherical viral forms. These results, together with our previous studies with nonrelated viruses, show that the Golgi complex contains key factors for the structural transformation of a number of enveloped viruses that assemble intracellularly.

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 Somalia) whose illness met the hemorrhagic fever case definition yielded virus isolates that were not RVF. Electron microscopy suggested these two virus isolates were members of the family Bunyaviridae. RT-PCR primers were designed to detect bunyavirus RNA in these samples. Regions of the S and L segments of the two isolates were successfully amplified, and their nucleotide sequences exhibited nearly complete identity with Bunyamwera virus, a mosquito-borne virus not previously associated with severe human disease. Unexpectedly, the virus M segment appeared to be reassorted, as the sequences detected exhibited 32-33% nucleotide and 28% amino acid differences relative to the corresponding M segment sequence of Bunyamwera virus. The association of this reassortant bunyavirus, proposed name Garissa virus, with severe disease is supported by the detection of the virus RNA in acute-phase sera taken from 12 additional hemorrhagic fever cases in the region.

The effect of cicloxolone sodium on the replication in cultured cells of adenovirus type 5, reovirus type 3, poliovirus type 1, two bunyaviruses and Semliki Forest virus.

The effect of cicloxolone sodium (CCX) on the replication of typical representatives of different virus families [adenovirus type 5 (Ad-5), reovirus type 3 (Reo-3), Bunyamwera and Germiston viruses, poliovirus type 1 (Polio-1) and Semliki Forest virus (SFV)] in tissue culture was investigated. The Golgi apparatus inhibitor monensin (Mon) and CCX were shown to have analogous effects on some aspects of virus replication. Although the Mon-like effect of CCX played no role in the antiviral activity against Ad-5, Reo-3 or Polio-1, it could entirely account for the antiviral activity against the Bunyamwera and Germiston viruses, for which inhibition of glycoprotein processing was responsible for the antiviral activity. In the case of SFV, the Mon-like activity of CCX caused cytoplasmic assembly of fully infectious SFV within vacuoles and thus impaired virus release without altering total infectious virus yield. Fewer Ad-5 and Reo-3 progeny were produced in the presence of the drug. CCX had a dose-dependent biphasic effect on the particle:p.f.u. ratio of the Reo-3 yield. At low CCX concentration (less than 50 microM) the virus yield contained poor quality, non-infectious virus, but at higher CCX concentration (greater than or equal to 100 microM) low quality virus could no longer be successfully assembled. We conclude that the antiviral effect can be manifested in three ways: (i) by a reduction in the virus particle yield produced; (ii) by a loss of quality (relative infectivity); (iii) by a virucidal effect of the drug. We have previously defined three CCX sensitivity classes. Mechanisms (i), (ii) and (iii) operate against viruses belonging to class CCXs-1 [herpes simplex virus (HSV) type 1, HSV-2 and vesicular stomatitis virus], but essentially only (i) and (ii) affect Reo-3 (CCXs-2), whereas (i) and possibly (iii) affect Ad-5 (CCXs-2). In the case of SFV (CCXs-3) none of these mechanisms operate, but relocation of assembled virus is found.

[The circulation of viruses of the California encephalitis and Bunyamwera groups (Bunyaviridae, Bunyavirus) on the northeastern Russian plain]. / Tsirkuliatsiia virusov kompleksov Kaliforniiskogo éntsefalita i Bun'iamvera (Bunyaviridae, Bunyavirus) na severo-vostoke Russkoi ravniny.

In the Komi ASSR, 102.2 thousand mosquitoes, 207 small mammals, 1487 blood serum specimens from people, 793 cow blood sera, 140 blood serum specimens from reindeer were collected in June-August, 1988. Twenty seven virus strains isolated from mosquitoes were classified into Bunyaviridae family according to the data of electron microscopic studies. Identification of the isolates by CFT and IFA showed 18 of the strains to belong to California encephalitis complex and 9 to Bunyamwera complex. Serological studies by neutralization test demonstrates a high frequency of contact of the human population and domestic animals with viruses of the California complex practically in the entire study area: an average of 45% among human subjects, 48% among cattle and 33% among reindeer. Such high values of the immune portions indicate the activity of the discovered natural foci of this complex. The results for Batai virus from the Bunyamwera complex do not indicate its high activity: approximately 2% of positive findings in human subjects and about 4% in the cattle. The results permit a prognosis of California encephalitis and Bunyamwera complex viruses spread in the northern part of Western Siberia.

Isolations of Cache Valley virus in Texas, 1981.

Two strains of the same virus (isolates AR 168 and 7856), were isolated in 1981 from an apparently healthy cow and a sick sheep in TX, U.S.A. These isolates were shown to be members of the Bunyamwera serogroup (family Bunyaviridae, genus Bunyavirus) by complement-fixation tests. Serum dilution-plaque reduction neutralization test results indicated that the isolates are closely related to Cache Valley virus. The virus isolates were characterized by sensitivity to lipid solvent, size (50-100 nm by filtration and 70 nm by electron microscopy), heat (56 degrees C) and pH 3 lability, cytopathic effects or plaques in cultures of Vero, LLC-MK2, embryonic bovine testicle and PS cells, and pathogenicity for suckling and weaned mice by the intracranial but not the intraperitoneal route. Gnotobiotic and conventional sheep and goats were experimentally infected by inoculation with one of the isolates given either intravenously or intraperitoneally. Elevation of body temperature, depression, tremors, muscle spasms, disorientation, feeding anomalies, convulsions, or other signs of central nervous system disturbances were observed.

Characterization of Leanyer virus: resemblance to Bunyavirus.

The properties of Leanyer virus, isolated in Northern Australia in 1974, were compared with those of Bunyamwera virus. Leanyer virus replicated in BHK-21 and Vero cells. In sucrose gradients it had a density of 1.17 g/cm3 and sedimented with the same s value as Bunyamwera virus. The diameter of negatively stained virions was approximately 110 nm. Three species of RNA sedimenting at 30S (L), 26S (M) and 14S (S) and four virion proteins (L, G1, G2, N) were detected in preparations of purified virions. The results were consistent with the classification of Leanyer virus as a member of the family Bunyaviridae, possibly within the Bunyavirus genus.

The Mapputta group of arboviruses: ultrastructural and molecular studies which place the group in the bunyavirus genus of the family Bunyaviridae.

We have characterized members of the Mapputta group of 'bunyavirus-like' viruses in terms of morphology, structure, ultrastructural development and virus-directed RNA and protein synthesis. Our primary study has been with Maprik virus (MPK) as a representative of the group. The MPK virion is uniformly spherical (congruent to 90 nm diameter) and possesses a membrane envelope. Virus maturation is by budding into small vesicles in the perinuclear region. During infection of BHK cells which is cytopathic, maximum viral RNA synthesis is congruent to 20% of RNA synthesis in uninfected cells without actinomycin D. In Aedes albopictus cells, where high initial titres are followed by a persistent infection, maximum viral RNA synthesis is only 1.5% of levels in control cells. MPK virions contain three RNA species. MPK generates four virus-specific RNAs (L, M, S1 and S2) and three nucleocapsid species in infected BHK cells. Six virus-specific polypeptides are present in infected cells; the four largest correspond to L (a probable transcriptase component), G1 and G2 (envelope proteins) and N (the nucleocapsid protein), respectively. These results and complementary studies with Trubanaman and Gan Gan viruses indicate that the members of the Mapputta serogroup can be placed in the bunyavirus genus of the family Bunyaviridae.

Comparisons of Belmont virus, a possible bunyavirus unique to Australia, with bunyamwera virus.

Belmont virus is an arbovirus isolated from mosquitoes and has a preference for marsupial hosts. The diameter of virions by negative staining (122 nm before fixation and 91 nm after fixation) was greater than that of Bunyamwera virus (94 nm and 79 nm respectively). However, the particles of both viruses appeared morphologically identical and sedimented at the same rate in sucrose density gradients. Belmont virus had a tripartite segmented RNA genome (28S, 24S and 11S) similar to Bunyamwera virus RNA (33S, 26S and 16S). The mol. wt. of these RNA species of Belmont virus measured by gel electrophoresis was 3.2 x 10(6), 2.4 x 10(6) and 0.3 x 10(6) compared to 2.9 x 10(6), 1.8 x 10(6) and 0.3 x 10(6) for the L, M and S species of Bunyamwera virus RNA. Both viruses comprised four structural proteins of the same relative proportions and corresponding mol. wt. For Bunyamwera virus, these were 145 x 10(3) (L), 104 x 10(3) (G1), 32 x 10(3) (G2) and 22 x 10(3) (N). The equivalent proteins of Belmont virus had mol. wt. of 147 x 10(3) (P147), 107 x 10(3) (G107), 28 x 10(3) (P28) and 25 x 10(3) (P25). Under conditions in which the envelope glycoproteins G1 and G2 of Bunyamwera virus were labelled in glucosamine, only G107 of Belmont virus was labelled. However, both G107 and P28 of Belmont virus were solubilized by non-ionic detergent and were then separable from the nucleocapsid containing all the RNA and P25. Chymotrypsin treatment of Belmont virus digested only G107, leaving a residue of P25 and P28, and of visible spikes. Similarly, G2 and the spikes of Bunyamwera virus resisted digestion with chymotrypsin. It was concluded that P28 is an envelope protein, equivalent to G2. Belmont virus thus appears to be a typical member of the Bunyaviridae but is unique in that it lacks carbohydrate in the small envelope protein (P28).

Electron microscopy of Akabane virus.

Electron microscopy of negatively stained purified virus and of thin sections of infected cells and tissues showed Akabane virus being similar in morphology and morphogenesis to members of the family Bunyaviridae.

The structure of togaviruses and bunyaviruses.

This article reviews the properties of arthropod-borne and nonarbo togaviruses and members of the Bunyaviridae family. Averaged information is given on the morphology and substructure of the virion, its physical properties, its chemical composition and the function of its constituents. Special attention is given to distinctive traits which might be useful for virus classification.