John F. Obijeski

Multiple Genetic Changes Can Occur in the Oral Poliovaccines upon Replication in Humans

Poliovirus isolates of serotypes 2 and 3 from patients whose paralytic poliomyelitis cases were classified as oral vaccine-associated were analysed by oligonucleotide mapping of the virus genomes and by polyacrylamide gel electrophoresis of the virus proteins. Oligonucleotide maps of all isolates were similar to the maps of the corresponding oral vaccine strain. No two isolates gave identical maps. Most maps differed from that of the vaccine strain by at least one oligonucleotide spot. Maps of some isolates revealed numerous differences, indicating that multiple (greater than 100) genetic changes had occurred in the vaccine virus genomes during replication in one or two individuals. In contrast, maps of some neural tissue isolates showed minimal differences from the reference vaccine maps, raising the possibility that neurovirulence may be restored by a small number of genetic changes. For many isolates, changes were also detected in the mobilities of processing rates of the virus proteins.

Comparative electrophoretic analysis of the virus proteins of four rhabdoviruses

Summary The structural proteins of vesicular stomatitis virus (VSV) Indiana, VSV New Jersey, Chandipura virus and Piry virus were compared by using two different SDS-polyacrylamide gel electrophoresis systems. The G and M proteins of the Indiana serotype had different electrophoretic mobilities from the corresponding proteins of VSV New Jersey, Chandipura virus and Piry virus in a continuous-SDS (CONT-SDS) gel system. The three major proteins of VSV Indiana G, N and M, were distinctly different from their counterpart proteins in the other three viruses when a high-resolution discontinuous-SDS (DISC-SDS) system was used. Chandipura and Piry virus glycoproteins migrated differently when co-electrophoresed in the CONT-SDS system, whereas both their N and M proteins were different in the DISC-SDS method. During infection both Chandipura and Piry viruses produced defective T particles which contained the same four structural proteins as virus particles.

Orthopoxvirus DNA: strain differentiation by electrophoresis of restriction endonuclease fragmented virion DNA.

Abstract Procedures were developed for purifying intact intracytoplasmic poxvirus particles from infected cells and for isolating DNA from virions by equilibrium centrifugation in sodium diatrizoate density gradients. The buoyant density of twelve closely related orthopoxviruses purified in these gradients was determined to be 1.25 g/ml, and that of the isolated virion DNAs was 1.1 g/ml. Virion DNA from each of the 12 selected prototype and wild-type viruses was cleaved with three separate site-specific restriction endonucleases, Hin d III, Sal I, and Bam HI, and the fragments (molecular weights 0.5 × 10 6 to 20 × 10 6 ) were separated by agarose gel electrophoresis. Characteristic DNA fragment migration patterns observed in the gels permitted classification of the viruses. By comparing profiles of Hin d III cleaved DNAs we were able to group the viruses into 4 species: cowpox, vaccinia, monkeypox (2 isolates), and variola (8 isolates). Viruses from variola major infection could be differentiated from viruses from variola minor infection. Isolates within species (strains) were also differentiated, mainly by comparing the gel electrophoresis profiles of Sal I digested DNA from the viruses.

Bunyaviridae: Recent Biochemical Developments

Introduction. The scope of this review is limited to the basic biochemical constituency of the bunyaviruses and the structure-function relationships of their proteins and nucleic acids. The complex antigenic relationships among bunyaviruses have been reviewed elsewhere (Casals, 1971; Murphy, Harrison & Whitfield, 1973; Murphy, 1975; Porterfield et al. 1973/4, 1975/6), and extensive ecological and epidemiological information can be found in several other timely reviews (Proceedings of the Symposium on Arboviruses of the California Complex and the Bunyamwera Groups, 1969; Henderson & Coleman, 1970; Sudia et al. 1971; Parkin, Hammon & Sather, 1972; Berge, 1975). The family Bunyaviridae is a newly defined taxonomic group of arboviruses which contain lipid envelopes and segmented RNA genomes. Previously, about 90 viruses had been assigned to 11 serogroups, and these were brought together on the basis of distant serological cross-reactions to form the Bunyamwera serological supergroup.

The structural proteins of rabies virus and evidence for their synthesis from separate monocistronic RNA species.

Purified preparations of the CVS strain of rabies virus, which were labelled during the infectious growth cycle with different isotopes or labelled in vitro by iodination or acetylation, contained five major proteins, L, G, N, M1, and M2, when examined by polyacrylamide gel electrophoresis (PAGE). The major surface glycoprotein, G, could be separated into two components, G1, G2, in some PAGE systems; they were present in about equimolar amounts and had apparent mol. wt. of 70.5 X 10(3) and 65 X 10(3), respectively. The virus nucleocapsid (p = 1.28 g/ml) could be isolated after detergent treatment of purified virus. It contained the virus RNA, the major nucleocapsid protein, N (mol. wt. 58.5 X 10(3)), and small amounts of a large protein, L (mol. wt. 170 X 10(3)). Two membrane proteins, M1 (mol. wt. 39.5 X 10(3)) and M2 (mol. wt. 25 X 10(3)), were also observed. Chromatography of dissoliated rabies virus in agarose columns with guanidine hydrochloride did not resolve any additional virus structural proteins. Two-dimensional peptide may analysis of iodinated structural proteins indicated that they were unique gene products and not derived from a precursor polypeptide by cleavage. The peptide maps of the two glycoproteins, G1 and G2, appeared identical. The approximate number of proteins molecules per virion has been determined. Rabies virus-directed protein synthesis in BHK21 cultures was detected as early as 6 h p.i. and all the proteins were present 12h p.i. Additional non-structural virus-specific proteins were not observed. The NaCl hypertonic shock procedure, which differentially inhibits polypeptide chain initiation in different classes of mRNAs, was used to ihibit the synthesis of the G and M1 proteins relative to the others selectively. All the rabies virus proteins were synthesized simultaneously following release from hypertonic treatment, suggesting that there are independent polypeptide chain initiation sites for the synthesis of each of the rabies proteins and that each protein is derived via translation of monocistronic mRNA species.

Applications of Oligonucleotide Fingerprinting to the Identification of Viruses

Publisher Summary This chapter focuses on applications of oligonucleotide fingerprinting to the identification of viruses. Fingerprinting is a technique by which oligonucleotides, produced by cleavage of RNA molecules with specific ribonucleases, are separated in two dimensions. It is a definitive method of identifying RNA viruses according to their genotypes. It is not subject to the problems of antigenic drift or antigenic convergence that complicate serological identification. Furthermore, it provides a semiquantitative means of following the evolution of viral genomes in nature. Because all regions of the genome are represented by the large diagnostic oligonucleotides, a survey of the total genomic changes can be monitored. Fingerprinting has two limitations as a diagnostic tool. First, although highly definitive, fingerprinting is not as rapid or inexpensive as serological techniques and cannot be as easily scaled up for routine identification of a large number of samples. Second, the evolutionary range of fingerprinting is short and relationships may not be evident for isolates of rapidly evolving viruses obtained over long intervals. However, these limitations are not large, compared to the full benefits offered to the virologist by the fingerprinting method.

Rabies virus-induced RNA synthesis in BHK21 cells.

Rabies virus polysomes contained two sizes of messenger RNAs, one of which had a sedimentation value of 30S and another which sedimented at 12 to 16S. RNA extracted from infected cultures contained virion-size RNA, 42S, as well as 30S and 12 to 16S RNA species. Hybridization studies indicated that the 30S and 12 to 16S RNAs had nucleotide sequences which were complementary to virion RNA. RNA. RNA isolated from virus polysomes contained adenylate-rich sequences which were heterogeneous in size and were determined to be about 100 to 250 nucleotides in length on the basis of their migration rates in polyacrylamide gels. Acid-urea agarose gel electrophoresis established that the 30S RNA material was composed of a single RNA species (mol. wt. greater than or equal to 1.65 X 10(6)), whereas the 12 to 16S material could be resolved into at least four distinct species whose mol. wt. ranged from 0.28 to 0.87 X 10(6). When labelled rabies-infected cell RNAs, which were purified by oligo(dT)-cellulose chromatography, were annealed to excess unlabelled virus RNA, digested with ribonuclease T2 and the RNA duplex molecules analysed by polyacrylamide gel electrophoresis, five duplexes could be separated. The mol. wt. of these duplexes were estimated to be 3.2, 1.4, 0.96, 0.55 and 0.39 X 10(6), when compared to the known mol. wt. of vesicular stomatitis virus (VSV) RNA duplexes. This study suggests that the replicative processes of rabies virus are very similar to VSV and that rabies virus proteins are probably translated from smaller than virion-size RNAs.

Studies on the poxvirus cotia

The poxvirus Cotia was studied by electron microscopy and by serological and biochemical analyses. Thin-sectioned preparations of infected Vero cells indicated that Cotia virus morphogenesis was similar to other mammalian poxviruses; unique filamentous structures and inclusion matrices were apparent in the cytoplasm. Complement fixation tests that included purified Cotia virions showed a reciprocal cross-reaction with rabbit myxoma virus and no cross-reaction with vaccinia virus. Serological results coupled with gradient polyacrylamide gel electropherograms of the structural proteins of purified Cotia, vaccinia, myxoma and fibroma viruses suggested that Cotia virus was similar to the latter two viruses. Agarose gel electropherograms of cleavage fragments of each of these virus DNAs digested with three separate restriction endonucleases showed that each of these viruses had a unique DNA gel profile.

Biochemical Characterization of Infantile Gastroenteritis Virus (IGV)

Enzymic and biophysical studies with purified infantile gastroenteritis virus (IGV) nucleic acid indicated that the virion contained a double-stranded RNA genome of approx. 14 x 10(6) daltons which could be separated by gel electrophoresis into eight bands of RNA which were comprised of 15 RNA species. Two major virus proteins, VP2 (mol. wt. = 135,000) and VP8 (mol. wt. = 40,000), which composed about 85% of the total virion protein, were detected in IGV particles by polyacrylamide gel electrophoresis. Eight additional minor proteins were also resolved.

High Resolution Polyacrylamide Gradient Gel Electrophoresis

A method was developed for high resolution electrophoresis of proteins in linear gradient (3 to 30%) polyacrylamide gel rods in a neutral phosphate buffer containing 0.1% sodium dodecyl sulfate. Well-defined protein zones were observed and improved resolution was attained especially for low molecular weight proteins in preparations containing a variety of polypeptides, e.g. viruses that are often separated by continuous gel methods. Electropherograms of continuous (8%) and gradient (3 to 30%) gels were made of purified vesicular stomatitis virus, variola virus, Rickettsia rickettsii, and alpha and beta chains of hemoglobin in order to demonstrate the resolution of the gradient system.