Search

Vero cells have been used as a convenient laboratory substrate for the isolation of mumps virus but may not be very sensitive and may select for particular adapted variants from clinical specimens. Continuous cell lines were evaluated for their ability to support the replication of mumps virus. Criteria included the production of infectious virus, detection of intracellular mumps proteins by immunofluorescence and electron microscopy and detection of specific nucleic acid by RT-PCR. Of the cells tested, CaCo-2, PLC/PRF/5, and Vero cells produced infectious virus, with Vero and CaCo-2 being the most permissive. The other substrates tested included cells of murine, canine and human origin showed signs of intracellular proteins and RNA but the amounts produced were much lower, and no infectious virus was detected in some cases. The virus use was a low passage of a Vero derived wild type strain, and it will ultimately be necessary to continue the studies with an unpassaged clinical specimen to identify a cell line able to isolate mumps virus at high efficiency and in unmodified form.

Comparative evaluation of TaqMan RT-polymerase chain reaction (PCR) methodology developed during this study with the conventional RT-PCR-nested PCR methodology developed earlier, using measles virus RNA templates derived from synthetic and natural sources against a number of primer sets belonging to various regions of the genome, revealed the existence of similar assay thresholds for both methods. An exception to this finding was, however, noted using primer sets of the N and M genes regions with RNA templates extracted from the wild type measles virus strain where the nested PCR method proved to be 10- to 100-fold more sensitive than the end points established with the N gene specific TaqMan RT-PCR method with synthetic RNA templates. These differences were not evident when the same primer sets were evaluated with RNA templates extracted from a brain sample of SSPE patient. These findings indicate that the genetic make up of measles virus strain in any given clinical specimen, in relation to the amplifying primers/probe sequences, can have impact on the overall sensitivity and specificity of the methodology applied. Both methods are equally suitable for the molecular detection of measles virus sequences in clinical specimens, although the TaqMan RT-PCR method may be preferred due to its advantages of contamination control, automation, and real-time product quantitation., (Copyright 2004 Wiley-Liss, Inc.)

Of the 55 point mutations which distinguish the type 1 poliovirus vaccine strain (Sabin 1) from its neurovirulent progenitor (P1/Mahoney), two have been strongly implicated by previous studies as determinants of the attenuation phenotype. A change of an A to a G at position 480, located within the 5' noncoding region, has been suggested to be the major attenuating mutation, analogous to the mutations at positions 481 and 472 in poliovirus types 2 and 3, respectively. In addition, the change of a U to a C at position 6203, resulting in an amino acid change in the polymerase protein 3D, has also been implicated as a determinant of attenuation, albeit to a lesser extent. To assess the contributions of these mutations to attenuation and temperature sensitivity, reciprocal changes were generated at these positions in infectious cDNA clones of Sabin 1 and P1/Mahoney. Assays in tissue culture and primates indicated that the two mutations make some contribution to the temperature sensitivity of the Sabin 1 strain but that neither is a strong determinant of attenuation.

This paper describes an efficient method of RNA isolation, enabling RNA templates to be obtained for reverse transcription within 20 min. The procedure avoids the use of hazardous organic chemicals and ethanol precipitation, and does not require ultracentrifugation. Results are presented for extraction of RNA from virus-infected tissue culture fluid, infected cell sheets, vaccine bulk material and lyophilized commercial vaccines.

Part of the 5' noncoding regions of all three Sabin vaccine strains of poliovirus contains determinants of attenuation that are shown here to influence the ability of these strains to grow at elevated temperatures in BGM cells. The predicted RNA secondary structure of this region (nt 464-542 in P3/Sabin) suggests that both phenotypes are due to perturbation of base-paired stems. Ts phenotypes of site-directed mutants with defined changes in this region correlated well with predicted secondary structure stabilities. Reversal of base-pair orientation had little effect whereas stem disruption led to marked increases in temperature sensitivity. Phenotypic revertants of such viruses displayed mutations on either side of the stem. Mutations destabilizing stems led to intermediate phenotypes. These results provided evidence for the biological significance of the predicted RNA secondary structure.

Enterovirus type 70 (EV70) is the causative agent of acute haemorrhagic conjunctivitis and may also give rise to a rare neurological complication closely resembling poliomyelitis. The complete nucleotide sequence of the genome of EV70 has been determined from cDNA cloned in Escherichia coli. The genome consists of a 5' non-coding region of 726 nucleotides (nt), a long open reading frame of 6582 nt and a 3' non-coding region of 82 nt prior to the poly(A) tract. Comparison of the nucleotide sequence and the predicted amino acid sequence of the polyprotein with those published for other enteroviruses reveals sufficiently high similarity to predict antigenic regions and polyprotein cleavage sites. The P1 region of EV70 is as similar to those of the entero- as to those of the rhinoviruses, whereas the P2 and P3 regions are more closely related to the coxsackie B and swine vesicular disease viruses than other entero- or rhinoviruses.

Previous studies have shown that the genome of Sabin type 3 poliovaccine strain (P3/Leon 12a1b) possesses a diminished translation efficiency as compared to genomes of closely related neurovirulent strains, the neurovirulent progenitor (P3/Leon/37), or a revertant (P3/119/70) of the vaccine (Y.V. Svitkin, S.V. Maslova, and V.I. Agol, 1985, Virology 147, 243-252). Here we attempted to evaluate the contribution of each mutation in the genome of the vaccine to this translation deficiency. Recombinants between P3/Leon 12a1b and P3/Leon/37 or P3/119/70 were constructed in vitro and their RNAs were translated in a cell-free system derived from Krebs-2 cells. The results show that of 10 nucleotide differences between the genomes of P3/Leon 12a1b and P3/Leon/37 9 have minor or no effect on translation and that the only mutation of significance is C472----U which is known to reduce the neurovirulence of the virus. Reversion from uridine to cytosine at position 472 in type 3 poliovaccine upon replication in the human gut resulted in an increase of both translation efficiency of polio RNAs and neurovirulence of corresponding strains. The data provide evidence for a common nucleotide sequence regulatory element for protein synthesis of the virus and its neurovirulence. In vitro translation assays may therefore prove to be useful for detection of attenuating mutations in the 5' noncoding region of poliovirus genome. The apparent involvement of the translation mechanism in the expression of neurovirulent or attenuated phenotype of poliovirus is briefly discussed.

T1 RNase oligonucleotide maps of RNA prepared from type 3 poliovirus were shown to be highly characteristic of the strain from which they were prepared. Strains isolated from paralytic cases of poliomyelitis temporally associated with live poliovirus vaccination were shown to be very closely related to the Sabin vaccine strain. Comparison of strains isolated in 1962, and strains isolated between 1973 and 1975 indicated that strains derived from the Sabin vaccine strain have displaced other circulating strains of type 3 poliovirus in the U.K. as a result of the use of live polio vaccine.

Considerable genetic and antigenic heterogeneity was detected among a collection of 17 poliovirus type 3 strains isolated between 1939 and 1958 in studies using monoclonal antibodies and by T1 oligonucleotide mapping. Heterogeneity was detected even amongst a collection of nine viruses designated Saukett and assumed to originate from the same prototype virus. The monoclonal antibodies were found to differ in their strain specificities for poliovirus type 3 strains in virus neutralization or single-radial-immunodiffusion tests. Relationships between strains detected in this way were in general consistent with those detected by oligonucleotide mapping. One of the monoclonal antibodies (NIBp 56) was able to distinguish between certain Saukett virus strains which differed by as little as a single specific oligonucleotide. The heterogeneity detected amongst Saukett viruses is of potential practical importance since these strains are used widely in the manufacture of inactivated poliovirus vaccine.

A study of the biochemistry of type 3 poliovirus strains which involves the examination of the virus-coded polypeptides in infected cells and the preparation of oligonucleotide maps is reported. The polypeptide patterns were shown to be a relatively stable property of virus strains and distinguished Sabin vaccine strains from wild strains of poliovirus type 3. This approach may be of value in deciding the origin (vaccine or nonvaccine) of field isolates of poliovirus. Oligonucleotide maps were found to be sensitive indicators of differences among strains and appear to form a basis for determining genetic relationships among strains. The nucleotide maps of two viruses isolated from human cases of paralytic poliomyelitis temporally associated with the administration of attenuated vaccine suggested a vaccine origin for the strain. In one case the nucleotide map was indistinguishable from that of the vaccine strain.

In vivo intertypic RNA recombination has previously been observed in excreted type 3 poliovirus isolates from a normal asymptomatic primary vaccinee. This study examines isolates from additional primary vaccinees to determine whether intertypic recombination is a general occurrence in excreted polioviruses. T1 RNase oligonucleotide finger-printing and limited dideoxy primer extension RNA sequencing demonstrated no evidence of intertypic recombination among type 1 or type 2 excreted strains. However, vaccinees excreting type 3 strains for long periods of time eventually produced recombinant strains involving either type 1 or type 2 poliovirus. Moreover, a characteristic time course of appearance of excreted type 3 intertypic recombinant polioviruses was established. Type 3/type 1 and type 3/type 2 recombinant strains appeared at Days 10-11 with a single crossover site in the gene for nonstructural protein 2C. Type 3/type 2/type 3 complex recombinant strains with an additional crossover site in the polymerase gene replaced type 3/type 2 strains at approximately Day 28. A significant portion of the genome of the type 3 Sabin vaccine strain is thus replaced during long-term excretion by vaccinees, and the appearance of some genomic arrangements coincided with a base deletion at the 3' terminus.

The genome of the prototype stain of coxsackievirus B4 (J.V.B. Benschoten) has been cloned in Escherichia coli and its complete nucleotide sequence determined. Excluding the poly(A) tract, the RNA genome is 7395 nucleotides in length and appears to encode a single polyprotein of 2183 amino acids. The predicted amino acid sequence of the polyprotein shows close homology (88%) to that of the previously sequenced coxsackievirus B3 and to certain regions of the polyproteins of the polioviruses and human rhinovirus 14. This allows identification of putative polyprotein cleavage signals, antigenic domains and other structural features likely to be important to the biological integrity of the virus.

The optimum conditions for the coupling of fowl plague virus (FPV) transcription to an in vitro reticulocyte translation system have been established and shown to be close to those required for maximum RNA synthesis by purified FPV virions. Products have been characterized by the peptides they yield on limited proteolysis in SDS and it has been shown that virus nucleoprotein (NP) and matrix (M) protein are made. The smallest virus coded polypeptide, the non-structural protein (NS), is made in only small amounts in the coupled system although it is a major virus coded product of infected cells early in infection.

A secondary structure model for the 5' non-coding RNA of poliovirus has been derived by comparing computer-generated folding patterns of equivalent sequences from a number of related enteroviruses and rhinoviruses and identifying compensating mutations that suggest conservation of a common secondary structure. Although certain elements are similar, the new model differs considerably from a previously published minimal energy structure and is consistent with the observed sensitivity of in vitro RNA transcripts of infectious poliovirus cDNA to RNases and modifying chemicals. The sequence of a neurovirulent revertant of an attenuated mutant provides additional evidence for an interaction between a region known to be important for neurovirulence, sequence 471-483, and nucleotides 528 to 538.

We have determined the complete nucleotide sequence of human rhinovirus 1B and made comparisons with other rhinoviruses. Extensive homology was found with serotypes 2 and 89 but the similarity to serotype 14 was considerably less. Rhinovirus-specific characteristics have been noted, in particular the length of the 5' non-coding region and the pattern of codon usage, and these may be sufficient to define the rhinoviruses as a distinct genus rather than being considered as members of the enteroviruses as has been suggested previously.