Proteolytic digestion of reovirus in the intestinal lumens of neonatal mice.

Two approaches were used to demonstrate proteolysis of reovirus in the intestine of the neonatal mouse. The first approach utilized peroral inoculation of radiolabeled virus into neonatal mice; the intestinal washings were harvested at 0 to 30 min postinoculation. The virus recovered from the intestinal washings was electrophoresed in polyacrylamide to determine whether proteolytic digestion of viral proteins had occurred. Complete loss of sigma 3 and generation of the mu 1c cleavage product delta demonstrated that digestion occurred within 10 to 30 min after the inoculation, resulting in the rapid generation of intermediate subviral particles (ISVPs). The products formed resembled those seen when the virus is digested in vitro with chymotrypsin. The second approach took advantage of the fact that ISVPs grow in cells treated with NH4Cl, whereas intact virus does not grow under these conditions (L. J. Sturzenbecker, M. Nibert, D. Furlong, and B. N. Fields, J. Virol. 61:2351-2361, 1987). Thus, assaying virus for its ability to grow in NH4Cl-treated cells represents a means of ascertaining whether the samples contain ISVPs. Using this approach, we demonstrated that up to 8 h postinoculation ISVPs predominate in the intestinal tissue and in the intestinal lumen. Between 8 and 15 h postinoculation, there is a loss in the proportion of ISVPs in the tissue so that by 15 h postinoculation ISVPs are no longer detectable in intestinal tissue washed of lumen contents and virus. In contrast, the lumen of the intestine contains some ISVPs at all times postinoculation. Thus, after peroral inoculation, the mammalian reoviruses are converted to proteolytically cleaved virus, suggesting that proteolysis plays an important role in initiation of infection in the gastrointestinal tract.

Growth and survival of reovirus in intestinal tissue: role of the L2 and S1 genes.

Reovirus serotype 1 Lang can be recovered in high titer from the intestines of neonatal mice up to day 8 after peroral inoculation. By contrast, reovirus serotype 3 Dearing cannot be recovered from intestinal tissue past day 4 after peroral inoculation. This difference between the two reoviruses was mapped by using reassortants generated from nonmutagenized laboratory stocks. When the L2 and S1 genes of reovirus serotype 3 Dearing were present in reassortants, the reassortants behaved like serotype 3 Dearing in exhibiting a decreased capacity to be recovered from intestinal tissue. Likewise, viruses which contained the L2 and S2 genes from serotype 1 Lang exhibited an enhanced capacity to grow and survive, which is characteristic of serotype 1 Lang. Thus, the capacity of reovirus to survive in intestinal tissue was determined by the L2 and S1 genes.

Intra- and inter-serogroup genetic relatedness of orbiviruses. II. Blot hybridization and reassortment in vitro of epizootic haemorrhagic disease serogroup, bluetongue type 10 and Pata viruses.

Viruses which belong to the epizootic haemorrhagic disease (EHD), bluetongue and Eubenangee serogroups of orbiviruses exhibit low level cross-reactions in some serological tests. Although Pata virus cross-reacts at low levels with members of the EHD and bluetongue serogroups, it was assigned originally to the Eubenangee serogroup. RNA-RNA blot hybridization data, however, suggest that Pata virus is not a member of the Eubenangee serogroup. In this study, the genetic relatedness of the EHD serogroup viruses, bluetongue virus type 10 (BTV-10) and Pata virus was assessed by RNA-RNA blot hybridization and by gene reassortment experiments in vitro. The five members of the EHD serogroup examined were highly related by reciprocal RNA RNA blot hybridization. Genes 1, 3, 4 and 6 to 9 were highly conserved with three unique types of gene 2, four variant types of gene 5 and two variant types of gene 10. Geographical boundaries could not be correlated with sequence relatedness because viruses isolated from the same locality were distant relatives when compared with another virus isolated on a different continent. The significance of the unique and variant genes is discussed. The EHD isolates, BTV-10 and Pata virus exhibited distinct profiles on agarose and polyacrylamide gel electrophoresis, and they are related distantly as shown by weak hybridization signals in blot hybridization. Gene 2 was a unique gene among the EHD isolates, BTV-10 and Pata virus. One BTV-10 gene hybridized more strongly to gene 9 of the EHD viruses than the other BTV-10 genes, and its role in encoding the cross-reactive antigen is discussed. Intra-serogroup gene reassortment in vitro was demonstrated in the six crosses among EHD serogroup members. In contrast, gene reassortment was not observed in inter-serogroup crosses between EHD 1 and BTV-10, BTV-10 and Pata virus, and between EHD 1 and Pata virus. Correlation of blot hybridization and gene reassortment indicated that viruses must share high sequence conservation in the majority of their genes before genetic interaction is likely. The usefulness of blot hybridization as an indicator of the likelihood of gene reassortment is discussed. These hybridization and gene reassortment data indicated that Pata virus is not a member of the bluetongue or EHD serogroups and that it should be assigned to the ungrouped set of orbiviruses. The hybridization and gene reassortment data suggest that members of the EHD serogroup, BTV-10 and Pata virus represent three distinct gene pools, and the role of reassortment in the generation of genetic diversity is discussed.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetic relatedness of Colorado tick fever virus isolates by RNA-RNA blot hybridization.

The sequence relatedness of ten isolates of the Colorado tick fever (CTF) serogroup of orbiviruses was examined by RNA-RNA blot hybridization. The 12 dsRNA genome segments of each of the isolates were electrophoresed in a 10% polyacrylamide gel, the segments were transferred electrophoretically to membranes and hybridized to radiolabelled genomic RNA from CTF Florio mouse-adapted strain (CTF FMA) or CTF SS-18. All genome segments of the ten CTF viruses exhibited cross-hybridization signals with either CTF FMA or CTF SS-18, under conditions in which greater than or equal to 74% sequence homology was required to form stable hybrids. Although the dsRNA polyacrylamide gel profiles were unique for each isolate examined, the CTF genes did not exhibit sequence divergence as has been seen among other Orbivirus serogroups. These hybridization analyses suggest that the CTF gene pool is relatively homogeneous which may be a reflection of the lack of multiple serotypes of CTF strains in neutralization tests. Nevertheless, the hybridization signals of segments 4 and 6 were lighter than those of the other genes, indicating that these two genes exhibited the highest degree of sequence variability among these isolates. These data are compared with hybridization data on other Orbivirus serogroups.

Genetic relatedness of Palyam serogroup viruses by RNA-RNA blot hybridization.

Cognate genes of members of the Palyam serogroup of orbiviruses have been identified previously, and their relatedness to the prototype virus was determined by blot hybridization of the genome segments of members of the serogroup using Palyam genomic RNA and isolated Palyam RNA segments as probes. In this study, the genetic relatedness of nine Palyam serogroup isolates was determined by reciprocal blot hybridizations of genomic RNA from each virus to the segments of all members of the group. The number and identity of highly related genes varied between isolates. For example. CSIRO Village and Palyam were related in genes 2 and 6, while Bunyip Creek and Vellore were related in genes 2 and 6. However, CSIRO Village and Bunyip Creek were highly related to D'Aguilar in all genes except 2 and 6, suggesting that there may have been genetic reassortment of Palyam serogroup dsRNA segments. Genes 2 and 6 were correlated consistently with serotype specificity. Genes 5, 7 and 9 were highly related among all members of the group. The Indian strains, Palyam and Vellore, were highly related in genes 1, 3 and 8, and they exhibited weak homology to genes 1, 3 and 8 of the Australian and African strains. However, one Indian isolate, Kasba, was more closely related to strains from Africa and Australia than it was to other Indian strains. There was little evidence which indicated that geography was predictive of the genetic relationships of the strains. Thus, immunological pressure may be the most important factor affecting the Palyam serogroup gene pool.

Biological and antigenic characterization of Netivot virus, an unusual new Orbivirus recovered from mosquitoes in Israel.

The antigenic and biological characteristics of a new Orbivirus, designated Netivot virus, are described. This agent was originally recovered in cultures of the C6/36 clone of Aedes albopictus cells from a pool of Culex pipiens captured in Israel. Netivot virus is not pathogenic for newborn mice, nor did it initially produce detectable cytopathic effect (CPE) in Vero cells. It is closely related antigenically to Umatilla and Llano Seco viruses; these 3 agents appear to constitute a new serogroup within the genus Orbivirus. Netivot virus is also more distantly related to a number of other orbiviruses in the blue-tongue, epizootic hemorrhagic disease of deer, and Eubenangee serogroups. Netivot virus replicated to high titer and produced CPE in a variety of mosquito cell cultures, but it did not grow in 2 sand fly cell lines. Inoculation of Ae. aegypti and Ae. albopictus with Netivot virus resulted in almost 100% mortality in both species within 15 days after infection. The recovery of this and a number of other yet unidentified viral agents from field-collected mosquitoes in cultures of C6/36 cells, but not in the conventional vertebrate assay systems, suggests the existence in nature of many yet unrecognized mosquito-associated viruses. It also demonstrates the value of using new isolation methods in arbovirus studies.

Sequence relatedness of Palyam virus genes to cognates of the Palyam serogroup viruses by RNA-RNA blot hybridization.

Cognate genes of nine members of the Palyam serogroup of orbiviruses have been identified and their relatedness to the prototype, Palyam virus, has been determined. Viral dsRNA segments were electrophoresed through 10% polyacrylamide gels, transferred to membranes, and hybridized to labeled RNA from Palyam virus under hybridization conditions using 52 degrees, 50% formamide, 5 X SSC. Cognate genes of each virus isolate were identified by hybridizing their genomes to [5'-32P]pCp-labeled, isolated segments from Palyam virus. Single segments from Palyam hybridized to no more than one segment in the other isolates. Nine of the 10 genes exhibited nucleic acid sequence homology between Palyam and seven of the other eight isolates. Gene 2 of Palyam hybridized only with gene 2 of CSIRO Village, and it was correlated with serotype specificity. Since CSIRO Village is the only member of the serogroup which cross-reacts with Palyam in neutralization tests, gene 2 may encode the neutralization antigen. Variation in the intensity of the hybridization signals of the remaining nine genes within a given virus indicated that the number and identity of conserved genes differed between members of the group. Genes 5, 7, and 9 were the most conserved genes for all members of the serogroup, while the levels of relatedness of Palyam genes 1, 3, 4, 8, and 10 to their cognates in the other isolates varied under these hybridization conditions.

Assessment of sequence relatedness of double-stranded RNA genes by RNA-RNA blot hybridization.

Three well-characterized reovirus serotypes were used to investigate the usefulness of RNA-RNA blot hybridization as a means to assess the genetic relatedness of double-stranded RNA (dsRNA) viruses. [5'-32P]pCp-labeled genomic dsRNAs from reovirus 1, 2 and 3 were used as probes in hybridization experiments in which segments of the three serotypes were separated in 10% polyacrylamide gels and transferred electrophoretically to membranes. Nine of the 10 reovirus genes cross-hybridized between the serotypes. The S1 gene was serotype specific. The L2 gene of reovirus 2 showed a lower level of cross-hybridization with types 1 and 3 when compared to the hybridization signal observed for L2 when types 1 and 3 were hybridized to each other. The data were consistent with previous studies on the relatedness of the three virus serotypes. Since RNA-RNA blot hybridization allows the number and identity of conserved genes to be determined, this approach may prove useful for assessing the genetic relatedness among other viruses in the family Reoviridae.