Lasting biological effects of early environmental influences. IV. Notes on the physicochemical and immunological characteristics of an enterovirus that depresses the growth of mice.

Physicochemical and immunological techniques have been used in an attempt to characterize a filterable agent, separated from the intestines of mice raised under ordinary conditions of husbandry, which produces a lasting depression of weight in specific pathogen-free (SPF) mice when administered to them orally shortly after birth. Although this agent has not yet been identified, it will be tentatively designated here as enterovirus. The mouse enterovirus can be readily sedimented by ultracentrifugation and by precipitation at pH 4.3; it does not pass through cellophane membranes. Its infective power is completely destroyed by ultraviolet radiation, but is resistant to heating at 56 degrees C, exposure to ether, treatment with trypsin, ribonuclease, and deoxyribonuclease. Dialysis and treatment with ether and nucleases greatly increase the infective activity of the intestinal filtrates containing the enterovirus, a finding which suggests that these procedures eliminate or destroy some inhibitory substance(s). The mouse enterovirus causes hemagglutination of mouse red blood cells. When injected into rabbits, it elicits in them an immune response that renders their serum capable of neutralizing its weight-depressing activity. As measured by inhibition of hemagglutination or complement fixation, the sera of infected mice do not exhibit any significant activity against usual mouse viruses. Centrifugation of the mouse enterovirus in 50%-20% sucrose gradient gave almost complete recovery of the infectivity and of hemagglutinating activity in the same fraction. In contrast, the protein content of the material was distributed through the various fractions. Consequently, this procedure resulted in a marked increase of specific activity.

Comparison of structural and nonstructural proteins of virulent and less virulent Theiler's virus isolates using two-dimensional gel electrophoresis.

The Theiler's murine encephalomyelitis viruses (TMEV) are important neurotropic picornaviruses because they persist in the central nervous system (CNS) and produce an inflammatory demyelinating disease in the mouse, their natural host. Insight into the pathogenesis of this disease may come from studying the genetic and biochemical compositions of these viruses; therefore, in this report, the structural and nonstructural proteins specified by both highly and less virulent TMEV were examined. Using two-dimensional gel electrophoresis, structural and nonstructural proteins, originating from each of the three regions of the picornavirus genome (Kitamura et al., 1981; Rueckert and Wimmer, 1984), from nine TMEV isolates were compared on the basis of isoelectric points (pI). Proteins of two virulent TMEV (GDVII and FA viruses) had almost indistinguishable pI values, whereas two of the three major capsid proteins of the less virulent TMEV varied considerably. For example, the structural proteins VP1 and VP3 from seven less virulent viruses ranged from pI 6.3 to 6.9 and 6.5 to 8.3, respectively. On the other hand, the pI values of VP2 and nonstructural proteins from the less virulent TMEV varied relatively little. In general, structural proteins of each TMEV group had pI ranges unique to their respective biological group, while most nonstructural proteins were similar for all TMEV. The virus-specified proteins of Vilyuisk virus, which is serologically related to the TMEV and a possible cause of encephalomyelitis in man, had pI values similar to the less virulent TMEV. Finally, VP3 not only showed the greatest variation in pI among the less virulent TMEV, but it also was preferentially radioiodinated in intact virus from each of the two biological groups using the lactoperoxidase technique.

Spot hybridization for the detection of adenoviruses and enteroviruses.

Nucleic acid spot hybridization was used for detection of adenovirus DNA directly in clinical specimens and enterovirus RNA in infected cells. The test gave results comparable to those obtained with antigen detection assays for adenoviruses. Spot hybridization could also be used for detection of enterovirus growth in cell cultures after a single passage from clinical specimens.

[Physicochemical properties of Teschen disease virus RNA]. / Nekotorye fiziko-khimicheskie svoistva RNK virusa bolenzi Teshnena.

The specific infectivity of virion RNA of teschen disease virus in a sensitive PP cell culture was 4-5 lg TCD50/ml per 1 microgram RNA. When virion RNA was inoculated into cell cultures insusceptible to the native virus, the virus replicated to a titre of 2.0-3.5 lg TCD50/ml. The molecular weight of virion RNA determined by two independent methods was 2.7 x 10(6) daltons. Tm calculated from the curve of virion RNA melting temperature was 57 degrees C. The double-stranded replicative form of RNA recovered from virus-infected PP cells was shown to have sucrose gradient sedimentation coefficient of 20 S. The specific infectivity was 2-3 lg TCD50/ml per 1 microgram of RNA.

[Physicochemical properties of the Teschen disease virus]. / Fiziko-khimicheskie svoistva virusa bolezni Teshena.

The physical stability of Teschen disease virus (TDV) was tested. It was found that 8 M urea at 37 degrees C and 0.5% Tween-20 for 1-2 hours destroyed TDV with formation of morphologically distinct subunits. The morphology of TDV virions and its subunits formed under the effect of urea and Tween-20 was studied. Sedimentation constant (120 S) and the polypeptide composition of TDV were determined. In TDV, major polypeptides (VP1 less than or equal to 4) and minor polypeptides (VP1m and VP2m) were found their molecular weights being 82,000, 67,000, 35,000, 30,000, 77,000, and 46,000 daltons respectively.

Structural proteins of hand, foot and mouth disease viruses.

Coxsackievirus A16 (CA16) and enterovirus 71 (E71), which cause hand, foot and mouth disease, were compared with respect to their polypeptide composition by PAGE. Only three proteins were resolved for CA16, whereas the four characteristic structural proteins of E71, (VP1, VP2, VP3 and VP4) were separated. The distribution of labeled proteins suggested that the molecular weights of VP2 and VP3 of CA16 were very similar. The smallest protein, VP4, of CA16 and E71 had the same molecular weight, but the other proteins of the two serotypes were different in their molecular weights.

Using different sorbents for the concentration of enteroviruses.

Comparative investigations were carried out to evaluate the efficiency of concentration (EC) of enteroviruses (poliovirus type 1 and simian rotavirus SA-11) using macroporous glass (brands MPG-1000 VGKh, USSR, and SO1, Czechoslovakia) and membrane filters (MF): (nitrocellulose PNTs 0.45, USSR, Millipore HAWP 0.45, USA, Synpor 0.45, Czechoslovakia as well as polycapromide MF Pall 0.2, FRG, and FMPA 0.55, USSR). To assess the sorption properties of filters, poliovirus preparations and rabbit serum gamma-globulin were labelled with radioactive isotopes. Nitrocellulose filters (Millipore and PNTs) proved to be superior in providing for 64-90% virus sorption and 20-24% protein sorption. Elution experiments using solutions of different chemical nature--protein solutions (triptosophosphate broth and beef extract), amino acid mixture (glycine + arginine), chaotropic salt (sodium trichloroacetate mixed with lysine)--showed that protein solutions better eluted rotavirus SA-11 from nitrocellulose filters and macroporous glass (MPG). The utilization of nitrocellulose filters and MPG as sorbents enables a 10-40-fold concentration of enteroviruses depending on the chosen eluent. Comparisons of EC values for rotavirus SA-11 obtained using porous glass MPS-1000 VGKh and SO1 indicated that MPS-1000 VGKh was superior both with respect to recovery efficiency (R) and concentration factor (CF).

Oligonucleotide fingerprint analysis of coxsackievirus A10 isolated in Japan.

Eight coxsackievirus A10 strains isolated in 1978 and in 1981 and 1982 from patients with hand, foot-and-mouth disease and with herpangina at a dispensary in Matsue city were compared by RNA fingerprinting techniques. The oligonucleotide maps of the four 1978 isolates were related to each other by 85 to 93% with respect to their large T1 oligonucleotides. In contrast, the oligonucleotide maps of the four 1981 and 1982 isolates were very different from each other. Co-electrophoresis experiments revealed that the 1981 and 1982 strains shared only 17 to 34% of their large oligonucleotides. In addition, some large oligonucleotides were found in most of the fingerprint maps of isolates from 1978 to 1982, suggesting that there are regions in the genome of coxsackievirus A10 which are not subject to mutational changes.

Purification of Theiler's murine encephalomyelitis virus and analysis of the structural virion polypeptides: correlation of the polypeptide profile with virulence.

Theiler's murine encephalomyelitis viruses (TMEV) are separable into two groups based on their biological behavior: those highly virulent isolates which are unable to cause persistent infection and the less virulent isolates which regularly produce persistent central nervous system infection in mice. Two highly virulent and five less virulent TMEV were found to have the same buoyant density (1.34 g/ml) on isopycnic centrifugation and virion structure by electron microscopy. Negatively stained virus particles purified in Cs(2)SO(4) gradients appeared to have icosahedral symmetry and measured 28 nm in diameter. Mature virions were found to possess three major structural polypeptides, VP1, VP2 and VP3, in the range of 25,000 to 35,000 daltons, and a smaller fourth major polypeptide, VP4, of 6,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The precursor of VP2 and VP4, VP0, which is a minor polypeptide of mature picornavirus particles, was also identified. However, a slight but consistent difference in several of the capsid polypeptides between the highly virulent and less virulent TMEV was found. VP1 was slightly larger (34,000 versus 33,500 daltons) and VP2 was slightly smaller (31,000 versus 32,000 daltons) for the highly virulent strains compared to the same polypeptide species in the less virulent viruses. VP0 was also slightly smaller (35,500 versus 36,000 daltons) for the highly virulent isolates compared to their less virulent counterparts. Finally, trypsin which was used initially in our purification procedure resulted in preferential cleavage of a 2,000-molecular-weight fragment or fragments from VP1 of only the less virulent isolates.

Use of cRNA probes for the detection of enteroviruses by molecular hybridization.

Subgenomic fragments of cDNA from poliovirus type 1 were inserted downstream from the SP6 or the T7 promoter in a Gemini riboprobe vector and their in vitro synthesized RNA transcripts were used as radiolabeled probes for the detection of enteroviral RNAs by molecular hybridization. The cRNA transcripts appeared to be more sensitive probes than the corresponding cDNAs. In vitro transcripts of the 5' noncoding region (5' nc riboprobe) were able to detect all of 14 reference enterovirus strains tested, as well as human rhinovirus 2, by dot blot hybridization with infected cell lysates. The same riboprobe also detected the enteroviral RNAs present in 16 of 18 samples of successive passages of stools in tissue culture and in some cases even in crude stool extracts. A riboprobe from the VP 1 region detected specifically poliovirus types 1, 2, and 3 in lysates of infected cells and in 50% of the infected stool specimens tested. These probes could be of particular interest for the epidemic survey of poliovirus infections.

Relation of serological- and CPE- classification of porcine enteroviruses to the classification by immunoperoxidase (IP) staining, and observation of CPE by IP staining method.

Characteristics of CPE produced by porcine enteroviruses (PEV) were examined by Immunoperoxidase (IP) staining method. Viral antigens were detected earlier than appearance of CPE. Distinctive characteristics of the three CPE types were clearly showed by the method. In cross reactions by IP staining, the titers of the staining were high (1:6,400 to 1:25,600), even though neutralizing titers of PEV with CPE II or III were low (1:200 to 1:400). The very close relationship was detected between PEV with the same CPE type, but the very low relationships were detected between PEV with the different CPE type. The relationships in CPE I group were various. The results by IP staining were more relative to CPE type than the serotype. Thus, IP staining is one method to classify PEV clearly.

A high density component in several vertebrate enteroviruses.

In addition to the major infective component, which bands at a density of 1:34 g/ml in caesium chloride ("light component"), a component with a density of 1:44 g/ml ("heavy component") has been found in harvests of poliovirus (type I), Coxsackie B5 virus, a bovine enterovirus (VG-5-27) and swine vesicular disease virus (SVDV). With SVDV about 98% of the infectivity equilibrated at 1 . 34 g/ml but approx. 2% was present as a peak at 1 . 44 g/ml. The morphology of the two forms was similar but the heavy component had a smaller diameter (28 nm) than the light component (30 nm). No inter-conversion of the two forms was observed on re-cycling in fresh caesium chloride gradients and the two components had the same proportions of RNA and protein and the same polypeptide composition. Each component gave a similar proportion of the light and heavy forms on replication, but the light component had a specific infectivity about fourfold higher than that of the heavy component and was also much more efficient in eliciting the formation of neutralizing antibodies in guinea pigs. Although these results suggest that the two particles are alternative stable configurations of the virus, iodination failed to reveal any differences in the extent or pattern of labelling of the polypeptides in the two forms.

Determination of the molecular weight of bovine enterovirus RNA by nuclease digestion.

The mol. wt. of a [32P]-labelled bovine enterovirus RNA has been determined by digesting with pancreatic RNase and separating the resulting oligonucleotides using a two-stage fractionation method on DEAE-Sephadex in 7 M-urea at pH 7-6 and pH 3-0. We have estimated the number of nucleotides as 8612 plus or minus 55. This corresponds to a mol. wt. of 2-93 plus or minus 0-02 X 10-6 which is in aggrement with estimates obtained by sedimentation and gel electrophoresis techniques.

The surface nature of proteins of a bovine enterovirus, before and after neutralization.

The surface nature of the proteins of a bovine enterovirus have been determined by using 125I and pyridoxal phosphate-sodium borohydride labelling techniques. As found previously, 125I labels only VP1 in intact capsid particles, whereas reaction with pyridoxal phosphate followed by reduction with tritiated sodium borohydride labels VP1, VP2 and VP3. Only VP4 is found to have no surface tyrosine, histidine or lysine available for reaction. After neutralization with homologous antisera, however, VP4 becomes exposed and is then available for labelling with 125I. This must reflect a substantial conformational change in the virus particle after neutralization.