Superinfection of Simian Virus 40-Transformed Permissive Cells with Simian Virus 40

Evidence that the resistance of simian virus (SV40)-transformed permissive cells to superinfection with SV40 is due to lack of virus uptake is presented. When virus uptake is enhanced, the events of infection proceed as in normal permissive cells, resulting in production of infectious virus.     

Interaction of Simian Virus 40 chromatin with Simian Virus 40 T-antigen.

We have studied the binding of the tumor antigen (T-antigen) of simian virus 40 to simian virus 40 chromatin (minichromosomes). The minichromosomes isolated from infected cells by a modification of standard techniques were relatively free of contaminating RNA and cellular DNA and had a ratio (by weight) of protein to DNA of approximately 1; their DNA was 50 to 60% digestible to an acid-soluble form by staphylococcal nuclease. Cleavage of this chromatin with restriction endonucleases indicated that the nuclease-resistant regions were randomly distributed in the population of minichromosomes, but were not randomly distributed within minichromosomes. Only 20 to 35% of these minichromosomes adsorbed nonspecifically to nitrocellulose filters, permitting binding studies between simian virus 40 T-antigen and chromatin to be performed. Approximately two to three times as much T-antigen was required to bind chromatin as to bind an equivalent amount of free DNA. When T-antigen was present in excess, both chromatin and free DNA were quantitatively retained on the filters. On the other hand, when DNA or chromatin was present in excess, only one-third as much chromatin as DNA was retained. We suggest that T-antigen-chromatin complexes may be formed by the cooperative binding of T-antigen to chromatin, whereas T-antigen-DNA complexes may be formed by simple bimolecular interactions.     

Association of Simian Virus 40 T Antigen with Simian Virus 40 Nucleoprotein Complexes

Viral nucleoprotein complexes were extracted from the nuclei of simian virus 40 (SV40)-infected TC7 cells by low-salt treatment in the absence of detergent, followed by sedimentation on neutral sucrose gradients. Two forms of SV40 nucleoprotein complexes, those containing SV40 replicative intermediate DNA and those containing SV40 (I) DNA, were separated from one another and were found to have sedimentation values of 125 and 93S, respectively. [(35)S]methioninelabeled proteins in the nucleoprotein complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to VP1, VP3, and histones, a protein with a molecular weight of 100,000 (100K) is present in the nucleoprotein complexes containing SV40 (I) DNA. The 100K protein was confirmed as SV40 100K T antigen, both by immunoprecipitation with SV40 anti-T serum and by tryptic peptide mapping. The 100K T antigen is predominantly associated with the SV40 (I) DNA-containing complexes. The 17K T antigen, however, is not associated with the SV40 (I) DNA-containing nucleoprotein complexes. The functional significance of the SV40 100K T antigen in the SV40 (I) DNA-containing nucleoprotein complexes was examined by immunoprecipitation of complexes from tsA58-infected TC7 cells. The 100K T antigen is present in nucleoprotein complexes extracted from cells grown at the permissive temperature but is clearly absent from complexes extracted from cells grown at the permissive temperature and shifted up to the nonpermissive temperature for 1 h before extraction, suggesting that the association of the 100K T antigen with the SV40 nucleoprotein complexes is involved in the initiation of SV40 DNA synthesis.     

Identification of the Simian Virus 40 Which Replicates When Simian Virus 40-Transformed Human Cells Are Fused with Simian Virus 40-Transformed Mouse Cells or Superinfected with Simian Virus 40 Deoxyribonucleic Acid

Simian virus 40 (SV40) was rescued from heterokaryons of transformed mouse and transformed human cells. To determine whether the rescued SV40 was progeny of the SV40 genome resident in the transformed mouse cells, the transformed human cells, or both, rescue experiments were performed with mouse lines transformed by plaque morphology mutants of SV40. The transformed mouse lines that were used yielded fuzzy, small-clear, or large-clear plaques after fusion with CV-1 (African green monkey kidney) cells. The transformed human lines that were used did not release SV40 spontaneously or after fusion with CV-1 cells. From each mouse-human fusion mixture, only the SV40 resident in the transformed mouse cells was recovered. Fusion mixtures of CV-1 and transformed mouse cells yielded much more SV40 than those from transformed human and transformed mouse cells. The rate of SV40 formation was also greater from monkey-mouse than from human-mouse heterokaryons. Deoxyribonucleic acid (DNA) from SV40 strains which form fuzzy, largeclear, or small-clear plaques on CV-1 cells was also used to infect monkey (CV-1 and Vero), normal human, and transformed human cell lines. The rate of virion formation and the final SV40 yields were much higher from monkey than from normal or transformed human cells. Only virus with the plaque type of the infecting DNA was found in extracts from the infected cells. Two uncloned sublines of transformed human cells [W18 Va2(P363) and WI38 Va13A] released SV40 spontaneously. Virus yields were not appreciably enhanced by fusion with CV-1 cells. However, clonal lines of W18 Va2(P363) did not release SV40 spontaneously or after fusion with CV-1 cells. In contrast, several clonal lines of WI38 Va13A cells did continue to shed SV40 spontaneously.     

Zoonotic Potential of Simian Arteriviruses

Wild nonhuman primates are immediate sources and long-term reservoirs of human pathogens. However, ethical and technical challenges have hampered the identification of novel blood-borne pathogens in these animals. We recently examined RNA viruses in plasma from wild African monkeys and discovered several novel, highly divergent viruses belonging to the family Arteriviridae. Close relatives of these viruses, including simian hemorrhagic fever virus, have caused sporadic outbreaks of viral hemorrhagic fever in captive macaque monkeys since the 1960s. However, arterivirus infection in wild nonhuman primates had not been described prior to 2011. The arteriviruses recently identified in wild monkeys have high sequence and host species diversity, maintain high viremia, and are prevalent in affected populations. Taken together, these features suggest that the simian arteriviruses may be “preemergent” zoonotic pathogens. If not, this would imply that biological characteristics of RNA viruses thought to facilitate zoonotic transmission may not, by themselves, be sufficient for such transmission to occur.     

Simian homologues of Epstein-Barr virus

Gamma-herpesviruses closely related to the Epstein-Barr virus (EBV) are known to naturally infect Old World non-human primates and are classified in the same lymphocryptovirus (LCV) genera. LCV infecting humans and Old World primates share similar biology, and recent studies have demonstrated that these viruses share a similar repertoire of viral genes. Surprisingly, the latent infection genes associated with cell growth transformation demonstrate the most striking sequence divergence, but the functional mechanisms for these genes are generally well conserved. The recent discovery of LCVs naturally infecting New World primates has rewritten the old paradigm of LCV host range restriction to humans and Old World non-human primates, so that these viruses are more widespread than previously believed. However, the New World LCV genome has significant and interesting differences from EBV and other Old World LCVs despite similar biological properties. Thus, the simian homologues of EBV can provide an important animal model for studying LCV pathogenesis, and the similarities and differences that have evolved among these related viruses can provide a unique perspective towards a better understanding of EBV.     

Simian AIDS: an historical perspective

The author has had the unique opportunity to participate, over the last 35 years, in the retrovirus research field that proceeded and followed the discovery of human, simian and feline AIDS. The onset of human AIDS was certainly unanticipated, but in retrospect, the warning signs had been present for at least a decade in captive macaques. I will briefly summarize the key scientific knowledge and 'mindset' leading up to these events and will outline the major contributions and unanswered questions arising from the simian model of AIDS.     

猴腺病毒研究进展

猴腺病毒（simian adenovirus,SAdV）是猴类呼吸道和消化道的常在病毒之一,可引起肺炎、咽炎、肠胃炎、结膜炎等以粪口途径传播为主,主要感染猕猴、非洲绿猴、黑猩猩和狒狒等[1]属腺病毒科,哺乳动物腺病毒属。有学者认为SAdV感染有严格的种属特异性,也有报道认为其种间交叉传播在非人灵长类中可能普遍发生,是动物传染病似的感染。SAdV是否感染人类尚未见报道,但已从猴的分泌物中发现了可能和人腺病毒（humanadenovirus,HAdV）发生重组的新型腺病毒。SAdV与HAdV亲缘关系很近,感染引起的临床症状也相似,是人腺病毒研究的理想模型。目前已经制定了研发猴腺病毒模型的目标,用于研究感染机制,促进腺病毒基因治疗和疫苗开发进程[3]。但猴腺病毒自然感染流行情况的研究依然很少。本文将就SAdV的发现与分类、生物学特性、感染特点及检测方法做一综述,为研究者提供一些有用参考。     

Properties of Simian Virus 40 Rescued from Cell Lines Transformed by Ultraviolet-Irradiated Simian Virus 40

Simian virus 40 (SV40) strains have been rescued from various clonal lines of mouse kidney cells that had been transformed by ultraviolet (UV)-irradiated SV40. To learn whether some of the rescued SV40 strains were mutants, monkey kidney (CV-1) cells were infected with the rescued virus strains at 37 C and at 41 C. The SV40 strains studied included strains rescued from transformed cell lines classified as "good," "average," "poor," and "rare" yielders on the basis of total virus yield, frequency of induction, and incidence of successful rescue trials. Four small plaque mutants isolated from "poor" yielder lines and fuzzy and small plaque strains isolated from an "average" and a "good" yielder line, respectively, were among the SV40 strains tested. Virus strains rescued from all classes of transformed cells were capable of inducing the transplantation antigen, and they induced the intranuclear SV40-T-antigen, thymidine kinase, deoxyribonucleic acid (DNA) polymerase, and cellular DNA synthesis at 37 C and at 41 C. With the exception of four small plaque strains rescued from "poor" yielders, the rescued SV40 strains replicated their DNA and formed infectious virus with kinetics similar to parental SV40 at either 37 or 41 C. The four exceptional strains did replicate at 37 C, but replication was very poor at 41 C. Thus, only a few of the rescued virus strains exhibited defective SV40 functions in CV-1 cells. All of the virus strains rescued from the "rare" yielder lines were similar to parental SV40. Several hypotheses consistent with the properties of the rescued virus strains are discussed, which may account for the significant variations in virus yield and frequency of induction of the transformed cell lines.     

Carrier Cultures of Simian Foamy Virus

The production of cultures of HEp-2 and BHK-21 cells persistently infected with a type 1 simian foamy virus is described. After infection, HEp-2 cells showed no structural changes, whereas BHK-21 cells lost their normal spindle shape and showed mitochondrial damage, and some cells contained many lysosomes. Thin sections also showed that a few BHK-21 cells contained virus particles in low concentration, and infectious virus could be isolated from both the cells and the supernatant fluid. No virus was seen in thin sections of HEp-2 cells, although infectious virus in low titer could be recovered intermittently from lysed cells. Both carrier cultures were immune to challenge with homologous virus and antigen could be detected in over 90% of the cells even after growth for 9 weeks in the presence of virus-neutralizing serum. The distribution of antigen in carrier cultures of both cell types is described and compared with that seen in cytocidal infections.     

Structural Polypeptides of Simian Virus 40

To determine the number and molecular weights of the structural polypeptides of simian virus 40, we have analyzed purified virus by electrophoresis on 14% polyacrylamide gels containing sodium dodecyl sulfate. Full virus purified by several different methods showed six distinct bands with molecular weights of approximately 43,000 (VP1, containing 70% of virion protein), 32,000 (VP2, 9%), 23,000 (VP3, 10%), 14,000 (VP4, 6%), 12,500 (VP5, 4%), and 11,000 (VP6, 3%) both by analysis of radioactively labeled virions and by visualization of the polypeptide bands after staining. “Empty” virions contain decreased amounts of VP4, 5, and 6. The approximate molecular ratios of the polypeptides were 6.0, 1.0, 1.5, 1.5, 1.1, and 1.0. When virus degraded in an alkaline buffer was analyzed by velocity centrifugation in sucrose gradients, the two larger polypeptides (VP1 and VP2) remained at the top of the gradient, whereas the three smallest polypeptides (VP4, 5, and 6) sedimented as a complex with the viral deoxyribonucleic acid. VP3 was found in association with either VP1 and 2 or VP4, 5, and 6, depending on the conditions of degradation. Presumably, VP1 and VP2, comprising about 80% of the protein, form the capsid of the virus. VP4, 5, and 6 may form a nucleoprotein in the virion, and VP3 may serve as an intermediate structural component.     

Airborne Stability of Simian Virus 40

The influence of relative humidity on the airborne survival of simian virus 40 (SV40) was studied by allowing virus aerosols to age in rotating drums at 21 or 32 C and at a relative humidity (RH) value ranging from 22 to 88%. Airborne SV40 virus was stable at every RH tested at 21 C, but aerosols maintained at 32 C were inactivated within 60 min at mid-range RH values. The unusual stability at 21 C over a broad RH range indicates that potentially biohazardous situations may occur under laboratory conditions if this virus becomes accidentally airborne.     

Simian Varicella in Old World Monkeys

Simian varicella virus (SVV) causes a natural erythematous disease in Old World monkeys and is responsible for simian varicella epizootics that occur sporadically in facilities housing nonhuman primates. This review summarizes the biology of SVV and simian varicella as a veterinary disease of nonhuman primates. SVV is closely related to varicella–zoster virus, the causative agent of human varicella and herpes zoster. Clinical signs of simian varicella include fever, vesicular skin rash, and hepatitis. Simian varicella may range from a mild infection to a severe and life-threatening disease, and epizootics may have high morbidity and mortality rates. SVV establishes a lifelong latent infection in neural ganglia of animals in which the primary disease resolves, and the virus may reactivate later in life to cause a secondary disease corresponding to herpes zoster. Prompt diagnosis is important for control and prevention of epizootics. Antiviral treatment for simian varicella may be effective if administered early in the course of infection.Abbreviations: FEAU, 1-(2′-deoxy-2′-flouro-β-D-arabinofuranosyl)-5-iodouracil, IE, immediate early, ORF, open reading frame, PBL, peripheral blood lymphocyte, SVV, simian varicella virus, VZV, varicella–zoster virusSimian varicella is a natural erythematous disease of Old World primates (Superfamily Cercopithecoidea, Subfamily Cercopithecinae), involving particularly patas (Erythrocebus patas), African green or vervet (Chlorocebus aethiops), and various species of macaque (Macaca spp.) monkeys. Epizootics of simian varicella occur sporadically in facilities housing nonhuman primates. These outbreaks are sometimes associated with high morbidity and mortality and the loss of valuable research animals. Simian varicella virus (SVV; Cercopithecine herpesvirus 9), a primate herpesvirus, is the etiologic agent of the disease. SVV is antigenically and genetically related to varicella–zoster virus (VZV; Human herpesvirus 3), the cause of human varicella (chickenpox) and herpes zoster (shingles). The clinical similarities between simian and human varicella and the relatedness of SVV and VZV, indicate that SVV infection of nonhuman primates is a useful model for study of varicella pathogenesis and development of antiviral therapies. A previous comprehensive review emphasized simian varicella as an experimental model for VZV infections.²² This review focuses on simian varicella as a veterinary disease of nonhuman primates. Simian varicella outbreaks and their epidemiology are considered, and the etiologic agent, clinical manifestations, pathogenesis, diagnosis, treatment, and control of the disease are discussed.     

Susceptibility of Human Cell Strains to Transformation by Simian Virus 40 and Simian Virus 40 Deoxyribonucleic Acid

Marked differences were found in the susceptibility of human fibroblasts to transformation by simian virus 40 (SV40). Highly susceptible cell strains were derived from patients with diseases associated with chromosomal abnormalities and a high incidence of tumors. In the present study, SV40 transformation-susceptible cell strains were not found to have a generalized increase in viral sensitivity. The differences in transformation frequency among cell strains with whole virus are eliminated by the use of isolated SV40 deoxyribonucleic acid, suggesting that the relative resistance of most cell strains to transformation by whole virus is due to a block at an early step in infection.     

Thymidine Kinase from Normal, Simian Virus 40-Transformed and Simian Virus 40-Lytically Infected Cells

Simian virus 40 (SV40) infection of human diploid cells failed to cause an enhanced production of thymidine kinase during the first 10 days after infection. Thymidine kinase activities from extracts of SV40-transformed cultures (human or simian) were considerably higher than the activity levels in extracts from the normal cells of origin. In addition, whereas the kinase activities obtained for human diploid cultures decreased as the cell sheet became confluent, the kinase activities for SV40-transformed human cells remained high after confluence was reached. Antisera obtained from hamsters bearing SV40 or adeno-7-SV40 hybrid virus tumors selectively inhibited enzyme from transformed sources (human or simian). Also, the antisera selectively inhibited enzyme extracted from SV40-lytically infected monkey cells. Sera from normal animals or from hamsters bearing polyoma tumors failed to inhibit enzymes from normal, SV40-transformed, or SV40-lytically infected cells. The Michaelis constant of partially purified enzyme from SV40-transformed cells was two to five times as high as that obtained for partially purified enzyme from human diploid cell cultures.