Comparison of polypeptides of two strains of murine hepatitis virus.

Viral polypeptides labeled with [³⁵S]methionine in mouse L fibroblasts infected with either of two strains (JHM or MHV3) of mouse hepatitis virus (MHV) were analyzed by polyacrylamide gel electrophoresis (PAGE). Four major polypeptides of apparent molecular weights, 180,000 (p′180′), 56,000 (p′56′), 24,000 (p′24′), and 22,000 (p′22′) were detected after a 15-min pulse of methionine. During a 2-hr chase performed late in infection (5.5 hr PI) a polypeptide of molecular weight 50,000 (p′50) was observed which appears to be derived from p′56′ by proteolytic processing. Both p′56′ and p′50′ were enriched in the amino acid arginine. Of the five major viral polypeptides only p′180′ could be labeled by growing infected cells in the presence of [¹⁴C]glucosamine; moreover, it was the only polypeptide whose mobility of PAGE was altered after labeling infected cells with [³⁵S]methionine in the presence of glycosylation-inhibiting drugs. Despite the extreme similarity in polypeptide patterns specified by JHM and MHV3, the apparent molecular weights of each of the five major JHM polypeptides were consistently lower (by about 500–1000) daltons than the corresponding ones of MHV3.     

Antibodies for autonomous parvoviruses of lower animals detected in human serum

Summary Isolates of ADV replicate to rather high quantities in lungs from neonatally infected mink kits. The virus was analysed for polypeptide composition, and for the first time high molecular weight polypeptides have been observed inin vivo produced ADVs. These polypeptides are analogous to those ofin vitro produced ADVs. The molecular weights of the structural polypeptides of the low virulence Pullman ADV and the highly virulent DK and Utah I isolates of ADV were found to be 88kD and 78kD andin vivo produced ADV-G polypeptides were found to be 85kD and 75kD, the same molecular weights as those described forin vitro produced ADV-G. Presence of the ADV coded, non-structural polypeptide with the molecular weight of 71kD (p71) was also demonstrated in the lung tissue from mink kits.With 4 Figures     

The biological relationship of mouse hepatitis virus (MHV) strains and interferon:In vitro induction and sensitivities

Summary Five prototype strains of mouse hepatitis virus (MHV) -1,-3, -S,- A59 and -JHM were analyzed for their ability to induce interferon (IFN) in seven cell lines of rodent origin. Induction of IFN by all of the prototype MHV strains was infrequent and unpredictable, while IFN was produced consistently by five cell lines treated with known inducers. Priming and/or aging of cells did not enhance IFN induction by the MHV strains except in the case of MHV-A59 which consistently induced moderate levels of IFN on L-cells which were both primed and aged. Kinetic studies of MHV-A59-induced IFN on primed and aged L-cells demonstrated that detectable levels of IFN were not produced until 24 hours post-inoculation (p.i.). Peak levels were attained at 30 hours p.i. with no additional IFN produced through 48 hours p.i. MHV-induced IFN was similar in composition and properties to Newcastle disease virus-induced IFN.The sensitivities of the five MHV strains to eight concentrations of preformed L-cell IFN were also assessed. All strains except MHV-S fit a linear model with MHV-3, MHV-A59 and MHV-JHM having similar slopes. At most concentrations MHV-3 was less sensitive than MHV-1, -A59 or -JHM to IFN. The response curve for MHV-S was non-linear. This strain was more sensitive to the antiviral effects of the pre-formed IFN except at the highest concentrations of IFN used.With 1 Figure     

Polypeptides associated with inclusion bodies from leaves of turnip infected with cauliflower mosaic virus

Inclusion bodies, the major intracellular site of accumulation of cauliflower mosaic virus (CaMV), have been partially purified by centrifugation onto a saturated sucrose cushion, followed by detergent treatment to lyse chloroplasts, then centrifugation on a saturated sucrose cushion again. The resulting preparation was enriched for inclusion bodies as judged by light and electron microscopic observation and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the polypeptides in the preparation. Two major polypeptides of 61,000 and 43,000 apparent molecular weights were found in inclusion body preparations from leaves infected separately with each of three different isolates of CaMV. Polypeptides of apparent molecular weights 56,000, 50,000, 39,000, 37,000, and 34,000 were also detected in preparations from infected leaves, but not in comparable preparations from healthy leaves. The mobilities of all but the 61,000 and 34,000 molecular weight polypeptides were slightly different from the CM4-184 isolate, as compared with Cabbage B Davis or NY8153. The 61,000 molecular weight polypeptide did not correspond in mobility to any of the virion polypeptides. The relative intensities of polypeptide bands common to inclusion bodies and isolated virions were different in these two preparations, suggesting that considerable degradation took place during virion isolation.     

RNA and polypeptide homology among murine coronaviruses

Using a ³²P complementary DNA (cDNA) prepared against the A59 nucleocapsid protein messenger RNA, we have investigated the extent of homology between A59 and four other strains of murine hepatitis virus (MHV). Analysis by hybridization kinetics of the annealing between A59 [³²P]cDNA and infected cell RNA from the other four MHV strains demonstrated 70–80% homology. By gel transfer analysis, the A59 [³²P]cDNA was able to detect subgenomic-size virus-specific RNAs in cells infected with all of the five MHV strains. A similar pattern of seven viral RNAs was detected in cells infected with A59, MHV-1, MHV-3, and JHM. In contrast, cells infected with MHV-S contained seven virus-specific RNAs, of which only the two smallest species comigrated with RNAs from the other four strains. The results suggest that as previously shown with A59 (S. Cheley, R. Anderson, M. J. Cupples, E. C. M. Lee Chan, and V. L. Morris (1981)Virology, 112, 596–604), all MHV strains tested encode a nested set of subgenomic RNAs. Analysis of [³⁵S]methionine-labeled viral proteins by SDS-polyacrylamide gel electrophoresis revealed that each strain of MHV specified four major viral polypeptides with apparent molecular weights very similar to those previously reported for the E2, N, El, and PEI polypeptides of A59. The strong degree of interstrain homology among the five MHV strains investigated was confirmed by comparative chymotryptic peptide mapping of the viral N proteins. A majority of the chymotryptic peptides from each of the [³⁵Sknethionine-labeled N proteins was found to coelute by high-performance liquid chromotography. Moreover, this technique of peptide mapping indicated a particularly strong relatedness between MHV-1 and MHV-S and among MHV-3, JHM, and A59.     

Analysis of genomic homology of murine gammaherpesvirus (MHV)-72 to MHV-68 and impact of MHV-72 on the survival and tumorigenesis in the MHV-72-infected CB17 scid/scid and CB17+/+ mice

Murine gammaherpesvirus (MHV)-68-infected mice are well-known as models for Epstein-Barr virus (EBV)-related lymphoproliferative diseases. MHV-72 may be a relative of MHV-68, but any genetic comparison between the two (except for the M7 gene) has never been reported. The genetic compositions of MHV-72 and MHV-68 were compared and the pathology of MHV-72 infection studied in CB-17 severe combined immunodeficiency (scid/scid; SCID) and CB17 wild-type (CB17+/+) mice. The MHV-72 DNA sequence was almost identical to MHV-68 except for approximately 7000 bp corresponding to the MHV-68 M1-M3 genes. Twenty-seven of 30 MHV-72-infected SCID mice (90%) died from generalized infection with intranuclear viral inclusions for approximately 1 month, while MHV-72-infected CB17+/+ mice recovered from acute infection. Long observation and pathological study of 68 MHV-72-infected mice for up to 24 months revealed that the survival rate (29.4%) and survival time (21.3 months) of MHV-72-infected CB17+/+ mice were significantly lower (P = 0.0127) and shorter (P = 0.0065) than those of the controls (61.1% and 22.9 months), respectively. The malignancy development rate (60.3%) of the infected CB17+/+ mice was also significantly higher (P = 0.004) than those of the controls (22.2%). However, no MHV-72 DNA was detected in the tumors of infected mice. MHV-72 may have some tumor-promoting effects but the tumorigenesis in infected CB17+/+ mice is different from EBV-associated tumors.     

Comparative polypeptide analysis of human, murine and strigis herpesviruses with murine cytomegalovirus by polyacrylamide gel electrophoresis

The polypeptide composition of five purified murine herpesvirus (MHV) strains grown in a stable line of rabbit embryo fibroblasts (REF) was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and compared with herpes simplex virus type 1 (HSV-1). About 24 structural polypeptides of molecular mass ranging from 275,000 to 25,000 were identified in MHV and HSV-1. The polypeptide profiles of MHV and HSV-1, showed a close similarity. The polypeptides of MHV were further compared with those of HSV-1, HSV-2, herpes virus strigis (HVS) and murine cytomegalovirus (MCMV). Differences were found between herpesviruses of different origin and MCMV. SDS-PAGE analysis of the six strains of MCMV labelled with 14C-amino acid hydrolysate also revealed differences in electrophor eticprofiles of MHV and MCMV proteins, what was confirmed by densitometric scanning of HSV-1, MHV and MCMV.     

Pathogenetical characterization of isolate MHV-60 of mouse herpesvirus strain 68

Infection of mice with mouse herpesvirus strain 68 (MHV-68) is an excellent small animal model of gammaherpesvirus pathogenesis in a natural host. We carried out comparative studies on MHV-60, another isolate of MHV-68. The acute infection of BALB/c mice inoculated intranasally (i.n.) with MHV-60 as well as its impact on tumor development were investigated. During the acute phase of infection the lungs were the main tissues infected. Our results show that MHV-60 has similar pathological features like other 4 isolates so far examined, namely MHV-72, MHV-78, MHV-Sumava inclusive of MHV-68. Nevertheless, MHV-60 differed from other isolates in following features: (i) the acute phase of infection was established very soon and lasted 10 days post infection (p.i.) in contrast to 14-28 days p.i. in the abovementioned isolates with a peak on days 3-5 p.i. The virus could also be recovered from the spleen, thymus and kidneys but not in other investigated organs. A lymphoproliferative response was associated with splenomegaly. At this time an increase in the number of leukocytes and appearance of atypical leukocytes in peripheral blood were observed. (ii) the infection was localized in the lungs and spleen, while in other isolates it was detected in a much broader scale of organs, and (iii) the acute phase of infection was accompanied by a massive splenomegaly, which was characteristic for the chronic phase of infection. Despite the fact that after clearance of the acute infection the virus was hardly detected, the tumor formation was later observed in 22% of infected mice as compared to 5% in control non-infected mice.     

Effect of pertussigen on inflammation caused by Freund adjuvant.

A gene library of Chlamydia trachomatis (serovar L1) DNA has been prepared in the phage vector lambda 1059. From this bank, 20 recombinant phage-expressing components which reacted with serum from a patient with a C. trachomatis (L1) infection were chosen. Selective expression and radiolabeling of phage polypeptides in irradiated Escherichia coli demonstrated that one of these clones encoded a polypeptide doublet with an apparent molecular weight similar to that of the C. trachomatis (L1) major outer membrane protein. Both species of this cloned doublet (40 and 41 kilodaltons) could be immunoprecipitated by serum from a patient with a C. trachomatis (L1) infection but not by normal human serum. Components of this apparent molecular weight were not precipitated from irradiated E. coli infected with vector phage lambda 1059 by either of these sera. Comparison of the Staphylococcus aureus-V8 protease peptide maps of these two cloned polypeptides and chlamydial major outer membrane protein extracted from elementary bodies showed all three polypeptides to produce peptide fragments of 15.5, 13.8, and 11.5 kilodaltons. Due to the identical apparent molecular weights of the fragments produced from the 40- and 41-kilodalton cloned polypeptides, these were concluded to be different conformational forms of the same molecular species. These cloned components were indistinguishable from C. trachomatis (L1) major outer membrane protein.     

Precursor-product relationship between nonglycosylated polypeptides of A and B particles of mouse mammary tumor virus.

Polypeptides and antigens of various preparations of intracytoplasmic A particles were analyzed in detail in comparison with those of B particles of mouse mammary tumor virus (MTV). SDS-polyacrylamide-gel electrophoresis (PAGE) revealed that B particles consisted of three major nonglycosylated polypeptides (B-p25, B-P15, and B-P7; numerals indicate calculated molecular weights in 10³ daltons) and six glycopeptides. All A-particle polypeptides were nonglycosylated. The particles purified by a conventional method contained seven major bands in the 70,000- to 37,000-molecular weight region, but their relative amounts varied from preparation to preparation. In most preparations, one other major band, A-p7 (an A-particle polypeptide with a molecular weight of 7000), was also observed. Thus, the polypeptide composition of A particles was quite different from that of B particles, having in common only one major band, p7, and three other bands of variable amounts, p43, p37, and p13. Those A particles that had been incubated at 37° for 20 hr showed a systematic change in PAGE pattern; major bands disappeared except for A-p43, A-p37, and A-p7, while a strong new band, A-p25, appeared. Consequently, incubated A particles were now similar to B particles in their PAGE pattern except for the absence of p15 in the-A particles. In spite of such a profound change in components, no ultrastructural alteration was observed in the A particles after incubation. Polypeptide conversion induced by incubation was completely inhibited by diisopropylfluorophosphonate (DFP). A particles purified in the presence of phenylmethanesulfonyl fluoride (PMSF) consisted of a single major polypeptide, A-p70. Incubation of these A particles resulted in generation of A-p15 in addition to A-p25 and A-p7 at the sacrifice of A-p70, although the rate of polypeptide conversion was much retarded. Antigenic analysis of individual polypeptides eluted from polyacrylamide gels shows that (i) B-p25, B-p15, and B-p7 carried distinct antigens; (ii) B-p25 and B-p7 were antigenically identical with A-p25 and A-p7, respectively; (iii) A-p70 carried all three of these different antigenicities of B particles; and (iv) other major polypeptides of A particles also carried these antigens in ways characteristic to each antigen. This indicates that three major internal components of B particles are generated from a common precursor, A-p70, through enzymatic cleavage and, hence, that A particles are the real pronucleocapsids of B particles. Present observations are discussed in connection with the precursor-product relationships proposed in other RNA tumor virus systems.     

Molecular size variations in an immunoprotective protein complex among isolates of Anaplasma marginale.

A major surface protein complex from the Florida isolate of Anaplasma marginale has been previously shown to induce protection in immunized cattle and has been proposed as the basis of a subunit vaccine against anaplasmosis. This complex in the Florida isolate is composed of two noncovalently associated polypeptides with molecular masses of 105 and 100 kilodaltons (kDa). The analogous protein complex from four geographically different isolates of A. marginale was immunoprecipitated and compared with the protein complex of the Florida isolate. The polypeptides of the complex varied in apparent molecular mass among the isolates. By using antibodies recognizing epitopes on each polypeptide of the Florida isolate, the antigenic identity of the polypeptides in the analogous complexes was determined. The polypeptides recognized by the neutralizing monoclonal antibody 22B1, which recognizes a 105-kDa polypeptide in the Florida isolate, ranged from 70 to 100 kDa in the other isolates. Those polypeptides recognized by rabbit antiserum R911, which recognizes a 100-kDa polypeptide in the Florida isolate, ranged from 97 to 100 kDa. The surface-exposed peptides in the complexes were compared by limited enzymatic digestion to assess structural homology among isolates. Despite the marked variations in molecular weight, there were conserved peptides between the 22B1-reactive polypeptides and between the R911-reactive peptides. Determination of the role of the conserved peptides in inducing immunity will be critical in the application of these polypeptides as the basis of a subunit vaccine for bovine anaplasmosis.     

A comparison of the polypeptides of four measles virus strains

The polypeptides of measles virions from four different sources have been analyzed and compared by polyacrylamide gel electrophoresis in both phosphate buffer and discontinuous Tris buffer systems. A similar pattern of six polypeptides was found with the four different virus strains. Only one polypeptide (G) was found to contain detectable amounts of carbohydrate label; this was the largest virion polypeptide with an estimated molecular weight of 80,000. Slight differences in electrophoretic mobilities of the nucleocapsid protein subunits (NP) were found among the different strains; with estimated molecular weights in the 60,000 to 62,000 range. Another polypeptide with a molecular weight of ～ 70,000 has been found to be associated with the nucleocapsids isolated from infected cells. The three remaining polypeptides identified have estimated molecular weights of 55,000, 42,000, and 37,000, and the smallest of these has been designated M, by analogy to other paramyxoviruses. The differences between the present results and previous findings with measles virus, and the possible relationship of measles virus polypeptides to those of other paramyxoviruses, have been discussed.     

Equine infectious anemia virus, a putative lentivirus, contains polypeptides analogous to prototype-C oncornaviruses

The polypeptide composition of purified radioactive leucine or glucosamine-labeled equine infectious anemia virus (EIAV) was investigated using guanidine hydrochloride gel filtration (GHCI-GF) and high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and compared to Friend murine leukemia virus (FLV), a prototype-C oncornavirus. The apparent molecular weights and relative abundance of each EIAV polypeptide were calculated. The results of these studies indicate that EIAV contains four major nonglycosylated proteins (p26, p15, p11, and p9) and two glycoproteins (gp90 and gp45), which together account for greater than 95% of the total virion protein. Four minor polypeptides of unknown significance were also detected reproducibly. EIAV gp90 appears to be the more heavily glycosylated polypeptide, while the gp45 component aggregates in 6 M GHCI, evidently reflecting a hydrophobic character. No disulfide linkages were detected between the EIAV glycoproteins. These observations demonstrate for the first time that EIAV contains polypeptides analogous to prototype-C oncornaviruses, such as FLV. However, the demonstrated serological unrelatedness between EIAV and FLV was reflected in biochemical differences in protein apparent molecular weights and by the resistance of EIAV nonglycosylated proteins to dissociation in GHCI, a property shared by visna virus.     

Structural polypeptides of the murine coronavirus DVIM

Summary The structural polypeptides of the murine coronavirus DVIM (diarrhoea virus of infant mice) have been analysed in comparison with other strains MHV-2, MHV-3, MHV-4 (JHM) and MHV-S by SDS-PAGE. In the presence of 2-mercaptoethanol, three major glycopolypeptides, gp180, gp69, gp25 (as a group of similar species) and one major non-glycosylated polypeptide p58 were detected. The gp69 is a DVIM specific glycopolypeptide, in which the glycosidic moieties are linked to the core polypeptide through N-glycosidic bonds, and hence may be correlated with the short projections of the viral envelope. Further gp140, which appears in the absence of reducing agents, is apparently a dimer of gp69 held together by disulfide linkages. The gp25 family, on the other hand, consists of four polypeptides, two of which are not metabolically inhibited by tunicamycin suggesting that they are O-linked glycopolypeptides. DVIM seems to be serologically closely related to the MHV-S strain as shown by neutralization.With 3 Figures     

Identification of the structural proteins of turkey enteric coronavirus

Summary Coronaviruses in the intestinal contents of turkey poults from Quebec flocks with outbreaks of enteritis were detected by electron microscopy. Five isolates were serially propagated in embryonic turkey eggs by inoculation of clarified intestinal contents into the amniotic cavity. Viral particles contained in the intestinal contents of infected embryos were purified by differential and isopycnic centrifugation in sucrose gradients. Intact virions present in fractions of densities 1.18 to 1.20 g/ml were precipitated with trichloroacetic acid and the protein content was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At least seven polypeptides with molecular weights ranging from 27,000 to 180,000 were regularly detected after electrophoresis of SDS-solubilized virions. Homologous rabbit antisera to turkey coronaviruses and pooled sera from convalescent turkeys immunochemically stained five polypeptides of apparent molecular weights of 95,000, 72–75,000, 66,000, 52,000 and 27,000. A further polypeptide with a molecular weight of 140,000 appeared in the absence of 2-mercaptoethanol and probably represents a dimer of the 66,000 polypeptide. One Quebec isolate differed from the Minnesota strain by two of its polypeptides, but no antigenic variations could be demonstrated amongst the various isolates either by immuno-electron microscopy, hemagglutination inhibition, or enzyme immuno assays on nitrocellulose.This study was partly supported by the Medical Research Council of Canada and the Quebec Federation of Poultry Producers.     

Sequence analysis of the regions flanking terminal repeats of the genome of umava isolate of murine gammaherpesvirus

The umava isolate of murine gammaherpesvirus (MHV-umava) slightly differs from Murine gammaherpesvirus 68 (MHV-68) and two other isolates of murine gammaherpesvirus (MHV), MHV-76 and MHV-72 in some biological properties. To identify the region(s) in the MHV-umava genome responsible for this phenomenon, we compared the sequences flanking terminal repeats (TRs) of the MHV-umava genome with those of MHV-68, MHV-76 and MHV-72. Restriction and sequence analyses revealed in MHV-umava as compared to MHV-68 approximately 9.3 kbp deletion at the left end of the genome and approximately 1.5 kbp deletion at the right end of the genome. While the approximately 9.3 kbp deletion was similar to that in MHV-76, the approximately 1.5 kbp deletion was unique for MHV-umava.     

New strain of mouse hepatitis virus as the cause of lethal enteritis in infant mice.

A new strain of mouse hepatitis virus (MHV) was isolated from pooled gut suspensions from an epizootic of lethal enteritis in newborn mice. Negative-contrast electron microscopy showed an abundance of coronavirus particles in the intestinal contents and intestinal epithelium of moribund mice. We found no other virus in the epizootic. Dams seroconverted to MHV polyvalent antigen and to the agent isolated, but did not develop antibodies to other known mouse pathogens. Virus propagated in NCTC-1469 tissue culture produced enteric disease in suckling mice but not fatal diarrhea; the dams of these mice also developed antibodies to MHV and to the isolates. By complement fixation, single radial hemolysis, and quantal neutralization tests, we found the isolates antigenically most closely related to MHV-S, unilaterally related to MHV-JHM, and more distantly related to MHV-1, MHV-3, MHV-A59, and human coronavirus OC-43. We also studied cross-reactions among the murine and human coronaviruses in detail. Tissues of infected newborn mice were examined by light microscopy, thin-section electron microscopy, and frozen-section indirect immunofluorescence, revealing that viral antigen, virus particles, and pathological changes were limited to the intestinal tract. We have designated our isolates as MHV-S/CDC.     

A murine gammaherpesvirus

In 1976, within a project on isolation of herpesviruses from small rodents in former Czechoslovakia, the mouse herpesvirus strain 68 (MHV-68) was isolated (Blaskovic et al., 1980). This virus was accepted by The International Committee on Taxonomy of Viruses (ICTV) as a new, so far unassigned species (member) of the Gammaherpesvirinae subfamily of the Herpesviridae family (Murphy et al., 1995). Besides MHV-68, four more isolates (MHV-60, MHV-72, MHV-76, and MHV-78) similar to MHV-68 were obtained in that field experiment in Slovakia. Later, three more isolates (MHV-Sumava from Bohemia and MHV-4556 and MHV-5682 from Slovakia) were obtained in other field experiments. All these isolates are in some properties similar but in others different from each other. Nevertheless, as their comparative genome sequence analysis is not yet available, we propose at present to regard all the abovementioned isolates as different isolates of the same virus and strain, i.e. MHV-68. It is not excluded that a more detailed characterization of these isolates in the future will lead to proposals of designating some of these isolates as new strains of the virus of concern. This review summarizes the up to date knowledge of various biological and physico-chemical properties of MHV-68. At least three isolates, MHV-68, MHV-72 and MHV-Sumava seem to be involved in malignant neoplasm development in mice. It should be stressed that the pathogenesis of the induced lymphoproliferative disease in mice is similar to that caused by Epstein-Barr virus (EBV) in humans.