The monkey pathogenicity of the Columbia Sk virus and the mouse-adapted lansing strain of poliomyelitis virus,and the influence of monkey passage on the characteristics of the virus

Summary The intracerebral inoculation of cynomolgus monkeys with Columbia SK virus (mouse brain suspension) produced flaccid paralysis after an incubation period of 3 to 5 days. In the spinal cord leucocytic infiltrations, acute necrosis and neuronophagia of anterior horn cells were found. Similar lesions, but far less extensive were found in the medulla and the brain stem, whereas leucocytic perivascular infiltrations were present in the motor area of the cerebral cortex. Small accumulations of leucocytes were observed in the heart muscle and in the epicardium. The Lansing strain of poliomyelitis virus produced essentially similar lesions, though less extensive, and the incubation period was 14 days. In the heart muscle and the epicardium of a Lansing-infected monkey small mononuclear infiltrations were found. The mouse infectivity titer of the ColSK virus decreased rapidly after monkey passage, and there was a simultaneous decrease of the hemagglutination titer with sheep red cells. No close antigenic relationship between the Y-SK and the ColSK virus was demonstrated by the hemagglutination inhibition reaction with sera from monkeys immunized against Y-SK and ColSK virus. The question is discussed, whether or not the ColSK group of viruses, which should be considered to be of animal origin, has to be classed into the family of poliomyelitis viruses as a fourth immunological type.     

The effect of serial passage on Sabin oral poliomyelitis vaccine virus in secondary monkey kidney versus Vero cells as measured by the monkey neurovirulence test

The three Sabin strains of poliomyelitis seed virus were serially passaged in either secondary monkey kidney or Vero cell cultures and the tenth passage of each virus harvest compared to non-passaged Sabin reference virus of the same type using the monkey neurovirulence test. All three types were further attenuated by passage in Vero cells, whereas only type 2 became further attenuated after passage in secondary monkey kidney cells. After passage in Vero cells, type 3 poliomyelitis virus became more heat stable, as measured by its replicative capacity at 40°C.     

A naturally occurring highly attenuated strain of type 1 poliomyelitis virus

Summary Twenty strains of poliomyelitis virus recovered from cases of abortive, non-paralytic, poliomyelitis have been tested for neurotropism. Only one strain possessed a low intraspinal activity inCynomolgus monkeys. Six single particle cultures were derived from this naturally occurring highly attenuated strain of type 1 poliomyelitis virus. It has been shown that the original virus consisted of a mixture of virus particles with varying degree of neurotropism. The antigenic and immunizing capacity of one highly attenuated plaque progeny has been tested in guinea pigs and monkeys. Aided with a grant from the National Health Research Council T.N.O.     

Haematogenous spread of poliomyelitis virus

Summary In rhesus monkeys inoculated intramuscularly with the Leon strain of poliomyelitis virus, the agent could be recovered from the blood during the first five days of the incubation period. It was apparently associated with the blood cells. It was also demonstrable in the inoculated muscle during the first 7 days. Moreover, virus was present in the sciatic nerves and in the regional lymphnodes. The significance of these findings in relation to the mechanism of infection, and in relation to the possibility of active or passive immunization is discussed. Aided by a grant from the Dr Simon Baruch Foundation and from the National Health Research Council T.N.O.     

A mixed epidemic of poliomyelitis and Bornholm's disease (pleurodynia)

Summary During a mixed epidemic of poliomyelitis and Bornholm's disease in the summer of 1951, evidence was obtained of the involvement of at least 6 different immunological types of Coxsackie virus, among which the Albany A₂ type dominated. Poliomyelitis virus was isolated from the stools of 6 out of 20 patients suffering from paralytic poliomyelitis; Coxsackie virus from 1, and both poliomyelitis and Coxsackie virus from 2 out of these 20 patients. During the whole year, Coxsackie virus was recovered from the stools of patients suffering from paralytic poliomyelitis, aseptic meningitis, pleurodynia and summer grippe in approximately equal percentages (11 to 14%), but during the epidemic months from July to October, 25% of the patients with poliomyelitis, and 16% of the patients with pleurodynia gave positive results for Coxsackie virus. The sparing or the enhancing effect of Coxsackie virus infection on the development of paralysis in patients with dual infections is discussed. Aided by a grant from the National Health Research Council T.N.O.     

A single-blind, placebo-controlled comparison of the reactivity and antigenicity of trivalent oral poliomyelitis vaccine prepared in monkey kidney or human diploid cell substrates

Vaccination with a single dose of trivalent oral poliomyelitis vaccine elicited fourfold or greater antibody responses to one or more poliomyelitis virus types in 59% of volunteers (16/27) receiving vaccine prepared from virus grown in monkey kidney cells and in 69% of volunteers (16/23) receiving vaccine prepared from virus grown in MRC5 human diploid cells. Type for type the antibody titres and percentages of volunteers responding to the two vaccines were broadly equivalent. The clinical reactivities of both vaccines were similar to that of a placebo in terms of the overall incidence, duration and severity of reactions. The nature of the reactions observed did, however, vary, in that headaches were more frequently reported by recipients of vaccine prepared from virus grown in monkey kidney cells. This difference was not, however, statistically significant.     

Coronavirus isolates SK and SD from multiple sclerosis patients are serologically related to murine coronaviruses A59 and JHM and human coronavirus OC43, but not to human coronavirus 229E.

Two coronaviruses (SK and SD), isolated from fresh autopsy brain tissue from two multiple sclerosis patients, were compared with known human and murine coronaviruses. In plaque neutralization assays, antisera prepared against multiple sclerosis isolates SK and SD demonstrated significant cross-reactivity to each other and to murine coronavirus A59, weak cross-reactivity to murine coronavirus JHM, but no cross-reactivity to the human coronavirus 229E. Antiserum to SK or SD failed to inhibit hemagglutination of chicken erythrocytes by the human coronavirus OC43. However, OC43 antiserum neutralized both SD and SK. Specific coronavirus polypeptides were identified and compared by immunoprecipitation and polyacrylamide gel electrophoresis. Infected and mock-infected 17Cl-1 cells were pretreated with actinomycin D and labeled with [35S]methionine. Polypeptides in Nonidet P-40 cytoplasmic extracts were immunoprecipitated with homologous and heterologous antisera. Identical polypeptides were precipitated from A59-, SD-, or SK-infected cell extracts by SD, SK, OC43, or A59 antisera. The polypeptides of human virus 229E were antigenically distinct, with the exception of weak recognition of a polypeptide of 50,000 molecular weight. We conclude that the two multiple sclerosis virus isolates SK and SD are closely related serologically to the murine coronavirus A59 and the human coronavirus OC43.     

The molecular epidemiology of poliomyelitis: the characteristics of the strains isolated from patients in Moscow in 1973-1986

Eleven virus strains isolated from poliomyelitis patients in Moscow in 1973-1986 were analyzed by the method of oligonucleotide mapping of RNA. The genome of the isolates showed considerable similarity to the genomes of Sabin's vaccine strains and mainly to the vaccine strain of antigenic type 2. The conclusion was made that the sporadic cases of poliomyelitis registered in this region were etiologically linked with the vaccine strains of poliomyelitis virus. Only in one case the disease appeared in the recipient of the vaccine, in all other cases the patients were infected through contacts.     

A novel isoform of SK2 assembles with other SK subunits in mouse brain

The SK2 subtype of small conductance Ca2+-activated K+ channels is widely distributed throughout the central nervous system and modulates neuronal excitability by contributing to the afterhyperpolarization that follows an action potential. Western blots of brain membrane proteins prepared from wild type and SK2-null mice reveal two isoforms of SK2, a 49-kDa band corresponding to the previously reported SK2 protein (SK2-S) and a novel 78-kDa form. Complementary DNA clones from brain and Western blots probed with an antibody specific for the longer form, SK2-L, identified the larger molecular weight isoform as an N-terminally extended SK2 protein. The N-terminal extension of SK2-L is cysteine-rich and mediates disulfide bond formation between SK2-L subunits or with heterologous proteins. Immunohistochemistry revealed that in brain SK2-L and SK2-S are expressed in similar but not identical patterns. Heterologous expression of SK2-L results in functional homomeric channels with Ca2+ sensitivity similar to that of SK2-S, consistent with their shared core and intracellular C-terminal domains. In contrast to the diffuse, uniform surface distribution of SK2-S, SK2-L channels cluster into sharply defined, distinct puncta suggesting that the extended cysteine-rich N-terminal domain mediates this process. Immunoprecipitations from transfected cells and mouse brain demonstrate that SK2-L co-assembles with the other SK subunits. Taken together, the results show that the SK2 gene encodes two subunit proteins and suggest that native SK2-L subunits may preferentially partition into heteromeric channel complexes with other SK subunits.     

Failure to detect interfering agents in febrile respiratory illnesses

Throat swab specimens from 57 military recruits with febrile respiratory illness in whom no virologic or serologic evidence of infection with respiratory viruses orMycoplasma pneumoniae was found were inoculated and passaged into fresh cultures of monkey kidney cells and continuous lines of human, simian and rabbit cells. Cultures of second passage were challenged with poliovirus type 2 or vaccinia virus and subsequently tested for interference by reading of cytopathic effect and titration of challenge virus. No interference was shown.     

Passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein E(rns)

Infection of cells with Classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoprotein E(rns) and E2 with the cell surface. In this report we studied the role of the cell surface glycoaminoglycans (GAGs), chondroitin sulfates A, B, and C (CS-A, -B, and -C), and heparan sulfate (HS) in the initial binding of CSFV strain Brescia to cells. Removal of HS from the surface of swine kidney cells (SK6) by heparinase I treatment almost completely abolished infection of these cells with virus that was extensively passaged in swine kidney cells before it was cloned (clone C1.1.1). Infection with C1.1.1 was inhibited completely by heparin (a GAG chemically related to HS but sulfated to a higher extent) and by dextran sulfate (an artificial highly sulfated polysaccharide), whereas HS and CS-A, -B, and -C were unable to inhibit infection. Bound C1.1.1 virus particles were released from the cell surface by treatment with heparin. Furthermore, C1.1.1 virus particles and CSFV E(rns) purified from insect cells bound to immobilized heparin, whereas purified CSFV E2 did not. These results indicate that initial binding of this virus clone is accomplished by the interaction of E(rns) with cell surface HS. In contrast, infection of SK6 cells with virus clones isolated from the blood of an infected pig and minimally passaged in SK6 cells was not affected by heparinase I treatment of cells and the addition of heparin to the medium. However, after one additional round of amplification in SK6 cells, infection with these virus clones was affected by heparinase I treatment and heparin. Sequence analysis of the E(rns) genes of these virus clones before and after amplification in SK6 cells showed that passage in SK6 cells resulted in a change of an Ser residue to an Arg residue in the C terminus of E(rns) (amino acid 476 in the polyprotein of CSFV). Replacement of the E(rns) gene of an infectious DNA copy of C1.1.1 with the E(rns) genes of these virus variants proved that acquisition of this Arg was sufficient to alter an HS-independent virus to a virus that uses HS as an E(rns) receptor.     

Preferential replication of a class of host-substituted defective simian virus 40 variants at low temperature

The host-substituted variant termed CVP8/1/P2 (EcoRI res) was first isolated several years ago after serial passage of simian virus 40 strain 777 on BSC-1 cells at 37 degrees C. When BSC-1 are coinfected with wild-type simian virus 40 strain 777 and variant CVP8/1/P2 (EcoRI res), the variant rapidly becomes the dominant species produced, often representing as much as 80% of the total DNA I synthesized after infection. We present evidence that the replicative advantage of the variant was increased when the infection was carried out at 33 rather than 37 degrees C. Also described are nine new and independent serial passage experiments carried out at 33 degrees C with several purified wild-type virus stocks, including strain 776, and both BSC-1 and primary African green monkey kidney cells. In each series variants related to CVPs/1/P2 (EcoRI res) were detected in the progeny viral genomes after four serial passages. Hybridization data suggest that at least some of these variant DNA I molecules contain simian virus 40 DNA sequences, monkey alpha-component DNA sequences (highly repetitive), and the infrequently reiterated monkey DNA sequences found in CVP8/1/P2 (EcoRI res), all covalently linked as in CPV8/1/P2 (EcoRI res). It appears that this type of variant emerges with some frequency during infection and is then preferentially replicated at 33 degrees C, thereby becoming readily detectable in passaged stocks. A variety of control experiments indicated that the repeated emergence of similar, if not identical, variants is unlikely to be the result of inadvertent cross-contamination or the presence of detectable amounts of the variant in the plaque-purified viral stocks.     

Complementary action of two independent dominant genes in Columbia soybean for resistance to Soybean mosaic virus

A stem-tip necrosis disease was observed in the soybean [Glycine max (L.) Merr.] cultivar Columbia and its derivative OX686 when infected with a necrosis-causing strain of Soybean mosaic virus (SMV) in Canada. A dominant gene named Rsv3 was found in OX686 for the necrotic reaction. In the present research we have found that Columbia is resistant to all known SMV strains G1-G7, except G4. Genetic studies were conducted to investigate the inheritance of resistance in Columbia and interactions of resistance gene(s) with SMV strains. Columbia was crossed with a susceptible cultivar, Lee 68, and with resistant lines PI96983, Ogden, and LR1, each possessing a resistance gene at the Rsv1 locus. F(1) individuals, F(2) populations, and F(2:3) lines from these crosses were inoculated with G7 or G1 in the greenhouse. Our inheritance data confirmed the presence of two independent dominant genes for SMV resistance in Columbia. Results from allelism tests further demonstrate that the two genes (referred to as R3 and R4 in this article) in Columbia were independent of the Rsv1 locus. R3 appears to be the same gene previously reported as Rsv3 in OX686, which was derived from Columbia. The R3 gene confers resistance to G7, but necrosis to G1. The other gene, R4, conditions resistance to G1 and G7 at the early seedling stage and then a delayed mild mosaic reaction (late susceptible) 3 weeks later. Plants carrying both the R3 and R4 genes were completely resistant to both G1 and G7, indicating that the two genes interact in a complementary fashion. Plants heterozygous for R3 or R4 exhibited systemic necrosis or late susceptibility, suggesting that the resistance is allele dosage dependent. The R4 gene appeared epistatic to R3 since it masked expression of necrosis associated with the response of R3. The complementary interaction of two resistance genes, as exhibited in Columbia, can be useful in development of soybean cultivars with multiple and durable resistance to SMV.     

Recurring defective variants of simian virus 40 containing monkey DNA segments.

Four independently and newly isolated defective variants of simian virus 40 have been characterized. All four are very similar, if not identical, to two previously and independently isolated variants (Wakamiya et al., J. Biol. Chem. 254:3584-3591, 1979; J. Papamatheakis, E. Kuff, E. Winocour, and M. F. Singer, J. Biol. Chem. 255:8919-8927, 1980). The documented similarities include restriction endonuclease maps and the presence of the same monkey DNA segments covalently linked to simian virus 40 DNA sequences. Each of the newly described variants was first detected upon serial passaging of wild-type simian virus 40 at a high multiplicity of infection at 33 degrees C as recently described (M. F. Singer and R. E. Thayer, J. Virol. 35:141-149, 1980). A variety of experiments support the idea that the various isolates were independent and do not reflect inadvertent cross-contamination. Two of the new isolates arose during passage of wild-type strain 777 virus in BSC-1 cells, one during passage of strain 776 in BSC-1 cells, and one during passage of strain 776 in primary African green monkey kidney cells. The two variants obtained after passage of strain 776 were shown to contain a particular recognition site for restriction endonuclease MboII within their simian virus 40 DNA segments, as do the two previous isolates. This site is not present in wild-type strain 776 DNA but is shown here to be present in wild-type strain 777 DNA. The surprising recurrence of closely related variants and particularly the unexpected presence of the endo R.MboII site in variants derived from passaging strain 776 suggest that these variants may arise by mechanisms other than recombination between the initial infecting viral genome and the host DNA.     

State of collective immunity to poliomyelitis in Moscow donors

Immunity induced by immunization with oral poliomyelitis vaccine has long been considered to last for life, similarly to immunity developing after infection with wild poliomyelitis virus. Vaccine virus cannot circulate among the immune population for a long time. The vaccination of children against poliomyelitis, carried out in the course of many years, has made it possible to suggest that a considerable number of immune persons were present among the adult population. The examination of 1,030 Moscow donors has revealed that antibodies to poliomyelitis virus of types 1, 2 and 3 were detected in 47.3%, 45.5% and 76.4% of the examinees respectively, the values of the average geometric titers being low. It is known that passages of poliomyelitis vaccine virus through nonimmune persons may result in emergence of revertant viruses with increased neurovirulence. The nonimmune adult population, especially the mothers of vaccinated and revaccinated children, may serve as favorable environment for the circulation of vaccine viruses and the appearance of revertant viruses.     

Adaptation of hepatitis A virus strain (JaM-55) originally pathogenic for monkeys to a cell culture

The results of adaptation of hepatitis A viral strain JaM-55 to the culture of embryo kidney cells FRhk-4 from macaque Rhesus are presented. The viral strain was isolated from a M. fascicularis suffering from spontaneous hepatitis. Before inoculating the cell culture the virus was passaged twice in the M. arctoides capable of reproducing hepatitis. FRhk-4 cell line inoculation by the monkey liver extract, containing the strain HAV-YaM-55, resulted in isolation of single viral particles of hepatitis A in the preparations obtained at the first 3 passages by the 28-31 day of cultivation. Beginning from the fourth passage the abrupt increase in the number of viral particles and hepatitis A antigen was registered. There were no traces of cytopathogenic effect at any level of viral passages in the inoculated cell culture. The adapted virus contains hepatitis A viral RNA identified by spot hybridization with the cloned cDNA of hepatitis A virus.     

猴泡沫病毒（SFV）RT-nestedPCR检测方法的建立及初步应用

目的建立实验猴群及相关生物制品猴泡沫病毒（SFV）的PCR检测方法。方法选择SFV-1、SFV-3、SFVCPZ前病毒序列的pol基因同源性较高的区域设计嵌套引物对SFV-1毒种进行RT-nestedPCR扩增并克隆测序,以确定其准确性,通过验证方法的特异性和敏感性,初步应用该方法对恒河猴外周血淋巴细胞（PBLs）,常用猴肾传代细胞及猴源性生物制品进行检测。结果经RT-nestedPCR扩增出的片断与SFV-1 cDNA序列同源性达到99%,对10只恒河猴的检测结果为5只阳性,5只阴性,对常用猴肾传代细胞及脊髓灰质炎疫苗的检测结果均为阴性。结论所建立的SFV RT-nestedPCR检测方法能准确的检测出恒河猴SFV的感染情况,对控制实验猴群的质量具有重要意义。该方法可用于检测猴源性生物制品中SFV的污染情况,为保证生物制品应用的安全性提供一定依据。     

Age-dependent poliomyelitis of mice: expression of endogenous retrovirus correlates with cytocidal replication of lactate dehydrogenase-elevating virus in motor neurons.

The widespread presence of endogenous retroviruses in the genomes of animals and humans has suggested that these viruses may be involved in both normal and abnormal developmental processes. Previous studies have indicated the involvement of endogenous ecotropic murine leukemia virus (MuLV) in the development of age-dependent poliomyelitis caused by infection of old C58 or AKR mice by lactate dehydrogenase-elevating virus (LDV). The only genetic components which segregate with susceptibility to LDV-induced paralytic disease are multiple proviral copies of ecotropic MuLV and the permissive allele, at the Fv-1 locus, for N-tropic, ecotropic virus replication (Fv-1n/n). Using in situ hybridization and Northern (RNA) blot hybridization, we have correlated the expression of the endogenous MuLV, both temporally and spatially, with LDV infection of anterior horn motor neurons and the development of paralysis. Our data indicate that treatment of 6- to 7-month-old C58/M mice with cyclophosphamide, which renders these mice susceptible to LDV-induced paralytic disease, results in transient increases in ecotropic MuLV RNA levels in motor neurons throughout the spinal cord. Peripheral inoculation of C58/M mice with LDV, at the time of elevated MuLV RNA levels, results in a rapid spread of LDV to some spinal cord motor neurons. LDV infections then spread slowly but progressively throughout the spinal cord, involving an increasing number of motor neurons. LDV replication is cytocidal and results in neuron destruction and paralysis of the infected animals 2 to 3 weeks postinfection. The slow replication of LDV in the spinal cord contrasts sharply with the rapid replication of LDV in macrophages, the normal host cells for LDV, during the acute phase of infection. The data indicate that the interaction between the endogenous MuLV with the generally nonpathogenic murine togavirus LDV occurs at the level of the motor neuron. We discuss potential mechanisms for the novel dual-virus etiology of age-dependent poliomyelitis of mice.     

Isolation of an intertypic poliovirus capsid recombinant from a child with vaccine-associated paralytic poliomyelitis

The isolation of a capsid intertypic poliovirus recombinant from a child with vaccine-associated paralytic poliomyelitis is described. Virus 31043 had a Sabin-derived type 3-type 2-type 1 recombinant genome with a 5'-end crossover point within the capsid coding region. The result was a poliovirus chimera containing the entire coding sequence for antigenic site 3a derived from the Sabin type 2 strain. The recombinant virus showed altered antigenic properties but did not acquire type 2 antigenic characteristics. The significance of the presence in nature of such poliovirus chimeras and the consequences for the current efforts to detect potentially dangerous vaccine-derived poliovirus strains are discussed in the context of the global polio eradication initiative.