Attenuation Of Neurovirulence Research Articles

Molecular basis of attenuation of neurovirulence of wild-type Japanese encephalitis virus strain SA14.

To identify the molecular determinants for attenuation of wild-type Japanese encephalitis (JE) virus strain SA14, the RNA genome of wild-type strain SA14 and its attenuated vaccine virus SA14-2-8 were reverse transcribed, amplified by PCR and sequenced. Comparison of the nucleotide sequence of SA14-2-8 vaccine virus with virulent parent SA14 virus and with two other attenuated vaccine viruses derived from SA14 virus (SA14-14-2/PHK and SA14-14-2/PDK) revealed only seven amino acids in the virulent parent SA14 had been substituted in all three attenuated vaccines. Four were in the envelope (E) protein (E-138, E-176, E-315 and E-439), one in non-structural protein 2B (NS2B-63), one in NS3 (NS3-105), and one in NS4B (NS4B-106). The substitutions at E-315 and E-439 arose due to correction of the SA14/CDC sequence published previously by Nitayaphan et al. (Virology 177, 541-552, 1990). The mutations in NS2B and NS3 are in functional domains of the trypsin-like serine protease. Attenuation of SA14 virus may therefore, in part, be due to alterations in viral protease activity, which could affect replication of the virus.

Identification of a sequence in the unique 5' open reading frame of the gene encoding glycosylated Gag which influences the incubation period of neurodegenerative disease induced by a murine retrovirus.

Neonatal inoculation of the wild-mouse ecotropic retrovirus CasBrE (clone 15-1) causes a noninflammatory spongiform neurodegenerative disease with an incubation period of > or = 6 months. Introduction of sequences from Friend murine leukemia virus (clone FB29) into the genome of CasBrE results in a marked shortening of the incubation period. The FB29 sequences which influence the incubation period were previously localized to the 5' leader sequence of the viral genome (M. Czub, F. J. McAtee, and J. L. Portis, J. Virol. 66:3298-3305, 1992). In the current study, we constructed a series of chimeric viruses consisting of the genome of CasBrE containing various segments of the leader sequence from FB29. A 41-nucleotide element (positions 481 through 521) near the 3' end of the leader was found to have a strong influence on the incubation period. This element influenced the kinetics of virus replication and/or spread in nonneuronal tissues, a property which was shown previously to determine the extent of central nervous system infection (M. Czub, F. J. McAtee, and J. L. Portis, J. Virol. 66:3298-3305, 1992). Curiously, this sequence had no demonstrable effect on virus replication in vitro in a fibroblastic cell line from Mus dunni. This segment encodes 14 of the unique 88-amino-acid N terminus of pr75gag, the precursor of a glycosylated form of the gag polyprotein which is expressed at the cell surface. Previous in vitro studies of mutants of Moloney murine leukemia virus lacking expression of glycosylated Gag failed to reveal a function for this protein in virus replication. We mutated the Kozak consensus sequence around the initiation codon for this protein in the chimeric virus CasFrKP, a virus which induces neurologic disease with a short (18- to 23-day) incubation period. M. dunni cells infected with the mutants lacked detectable cell surface Gag, but, compared with CasFrKP, no effect on replication kinetics in vitro was observed. In contrast, there was a marked slowing of the replication kinetics in vivo and a dramatic attenuation of neurovirulence. These studies indicate that glycosylated Gag has an important function in virus replication and/or spread in the mouse and further suggest that the sequence of its N terminus is a critical, though likely indirect, determinant of neurovirulence.

Albert B. Sabin and the Development of Oral Poliovaccine

Abstract. There are many reasons for the modern interest in viral vaccines, but there is no doubt that the key role played by viral vaccines in public health is the major factor since other prophylactic or therapeutic anti-vital products simply do not exist. Viral vaccines have a long history that has been marked by successful events and by tragic accidents. Live viral vaccines are an extraordinary category of biologicals since, despite their reputed efficacy, they were developed by empirical experiments and patient epidemiological observation. From this point of view oral polio vaccine should be considered a 'miracle' since it became a major tool for public health in the 20th century, before we were able to understand the molecular basis of polio virus neurovirulence attenuation. The first evidence that polio virus can be attenuated was provided in the early 1940s by Max Theiler, but it was Hilary Koprowsky who demonstrated further in 1952, that a rodent adapted strain was safe and able to immunise a limited number of volunteers. Koprowsky studies were confirmed later during a mass field trial in Africa. However it is undeniable that the patient and systematic work of Albert B. Sabin was primordial in developing live oral attenuated poliovaccine. The excellence of Sabin's testing of poliovirus neurovirulence in the accurate studies that he developed, enabled him to select, after the cloning of viral populations by plaque assay, the best attenuated variants. It is interesting to remember that the real selective factor that allowed the isolation of attenuated variants was ignored by Sabin and was put forward by Lwoff in the Pasteur Institute, when he described the role of temperature in the selection of cold attenuated mutants. Historically, the first to perform a successful mass vaccination with Sabin oral live poliovaccine were Russian scientists. Oral live poliovaccine was in some cases the origin of paralytic accidents and Sabin strains were involved occasionally in such events. Other attenuated poliovirus strains used in clinical trials as oral vaccine, such as Cox-Lederle type 1 and Usol-D bac type 3, generated in some instances clusters of vaccinees that developed paralysis. An important achievement in the consistency of the Sabin vaccine was the transfer by Albert Sabin to the WHO of the seed material and the responsibility for surveying the quality control and licensing procedure of oral poliovaccine.

The 5′-Untranslated Region of Picornaviral Genomes

Publisher SummaryPicornaviruses are small naked icosahedral viruses with a single-stranded RNA genome of positive polarity. According to current taxonomy, the family includes four genera: Enterouirus (polioviruses, coxsackieviruses, echoviruses, and other enteroviruses), Rhinovirus, Curdiouirus [encephalomyocarditis virus (EMCV), mengovirus, Theiler's murine encephalomyelitis virus (TMEV)], and Aphthouirus [foot-and-mouth disease viruses (FMDV)]. There are also some, as yet, unclassified picornaviruses [e.g., hepatitis A virus (HAW] that should certainly be assessed as a separate genus. Studies on the molecular biology of picornaviruses might be divided into two periods: those before and after the first sequencing of the poliovirus genome. The 5'-untranslated region (5-UTR) of the viral genome was one of the unexpected problems. This segment proved to be immensely long: about 750 nucleotides or ∼10% of the genome length. There were also other unusual features (e.g., multiple AUG triplets preceding the single open reading frame (ORF) that encodes the viral polyprotein). This chapter shows that the picornaviral 5-UTRs are not only involved in such essential events as the synthesis of viral proteins and RNAs that could be expected to some extent, although some of the underlying mechanisms appeared to be quite a surprise, but also may determine diverse biological phenotypes from the plaque size or thermosensitivity of reproduction to attenuation of neurovirulence. Furthermore, a close inspection of the 5-UTR structure unravels certain hidden facets of the evolution of the picornaviral genome. Finally, the conclusions drawn from the experiments with the picornaviral5-UTRs provide important clues for understanding the functional capabilities of the eukaryotic ribosomes.

Attenuating mutations in glycoproteins E1 and E2 of Sindbis virus produce a highly attenuated strain when combined in vitro

Alterations in either the E1 or the E2 glycoprotein of Sindbis virus can affect pathogenesis in animals. Previously, we identified two distinct E1 glycoprotein gene sequences which differed in their effect on pathogenesis. One had an attenuation phenotype following subcutaneous inoculation of neonatal mice (E1 Ala-72, Gly-75, and Ser-237), while the other was virulent (E1 Val-72, Asp-75, and Ala-237). In this study, we examined the basis for this difference in pathogenesis by using a full-length cDNA clone of Sindbis virus from which infectious RNA could be transcribed in vitro. The relative contribution of each E1 residue to the pathogenesis phenotype was determined by using site-directed mutagenesis to alter each codon individually and in combination. Residues 75 and 237, in combination, appeared to be the major E1 determinants affecting pathogenesis. In addition, the effect of directly combining independently attenuating E1 and E2 mutations in the same virus was examined. The attenuating E1 sequences characterized in this study were coupled to a previously characterized attenuating mutation at E2 residue 114. The resulting recombinant virus, constructed in vitro, exhibited an increased attenuation of neurovirulence as compared with recombinant viruses containing either of the attenuating elements alone.

Altered substrate specificity of herpes simplex virus thymidine kinase confers acyclovir-resistance.

Acyclovir (9-[2-hydroxyethoxymethyl]guanine or ACV) is a nucleoside analogue with considerable potential for the treatment of herpes simplex virus (HSV) infections in man. Two virus-coded enzymes are important in the mechanism of action of this drug: thymidine kinase (TK) which initiates its activation by converting it to the monophosphate and DNA polymerase whose action is inhibited by ACV triphosphate. Changes in either gene may confer resistance, but all reported mutations in the TK gene have resulted in failure of the resistant virus to induce appreciable levels of the enzyme. Such TK- mutants arise readily in tissue culture systems where the enzyme is non-essential for virus replication, but in animals they show considerably reduced pathogenicity and neurovirulence. We now describe the isolation of a resistant mutant which induces a TK of altered substrate specificity and we show that this virus retains pathogenicity for mice with only a slight attenuation of neurovirulence.