Genetic analysis of simian virus 40 from brains and kidneys of macaque monkeys.

Simian virus 40 (SV40) was isolated from the brains of three rhesus monkeys and the kidneys of two other rhesus monkeys with simian immunodeficiency virus-induced immunodeficiency. A striking feature of these five cases was the tissue specificity of the SV40 replication. SV40 was also isolated from the kidney of a Taiwanese rock macaque with immunodeficiency probably caused by type D retrovirus infection. Multiple full-length clones were derived from all six fresh SV40 isolates, and two separate regions of their genomes were sequenced: the origin (ori)-enhancer region and the coding region for the carboxy terminus of T antigen (T-ag). None of the 23 clones analyzed had two 72-bp enhancer elements as are present in the commonly used laboratory strain 776 of SV40; 22 of these 23 clones were identical in their ori-enhancer sequences, and these had only a single 72-bp enhancer element. We found no evidence for differences in ori-enhancer sequences associated with tissue-specific SV40 replication. The T-ag coding sequence that was analyzed was identical in all clones from kidney. However, significant variation was observed in the carboxy-terminal region of T-ag in SV40 isolated from brain tissues. This sequence variation was located in a region previously reported to be responsible for SV40 host range in cultured cell lines. Thus, SV40 appears to be an opportunistic pathogen in the setting of simian immunodeficiency virus-induced immunodeficiency, similarly to JC virus in human immunodeficiency virus-infected humans, the enhancer sequence organization generally attributed to SV40 is not representative of natural SV40 isolates, and sequence variation near the carboxy terminus of T-ag may play a role in tissue-specific replication of SV40.     

Natural Isolates of Simian Virus 40 from Immunocompromised Monkeys Display Extensive Genetic Heterogeneity: New Implications for Polyomavirus Disease

Simian virus 40 (SV40) DNAs in brain tissue and peripheral blood mononuclear cells (PBMCs) of eight simian immunodeficiency virus-infected rhesus monkeys with SV40 brain disease were analyzed. We report the detection, cloning, and identification of five new SV40 strains following a quadruple testing-verification strategy. SV40 genomes with archetypal regulatory regions (containing a duplication within the G/C-rich regulatory region segment and a single 72-bp enhancer element) were recovered from seven animal brains, two tissues of which also contained viral genomes with nonarchetypal regulatory regions (containing a duplication within the G/C-rich regulatory region segment as well as a variable duplication within the enhancer region). In contrast, PBMC DNAs from five of six animals had viral genomes with both regulatory region types. It appeared, based on T-antigen variable-region sequences, that nonarchetypal virus variants arose de novo within each animal. The eighth animal exclusively yielded a new type of SV40 strain (SV40-K661), containing a protoarchetypal regulatory region (lacking a duplication within the G/C-rich segment of the regulatory region and containing one 72-bp element in the enhancer region), from both brain tissue and PBMCs. The presence of SV40 in PBMCs suggests that hematogenous spread of viral infection may occur. An archetypal version of a virus similar to SV40 reference strain 776 (a kidney isolate) was recovered from one brain, substantiating the idea that SV40 is neurotropic as well as kidney-tropic. Indirect evidence suggests that maternal-infant transmission of SV40 may have occurred in one animal. These findings provide new insights for human polyomavirus disease.     

Specific cleavage and physical mapping of simian-virus-40 DNA by the restriction endonuclease of Arthrobacter luteus.

Simian virus 40 (SV40) DNA (strain 776) is cleaved by the restriction endonuclease from Arthrobacter luteus into 32 specific fragments including 20 large pieces designated Alu-A through T as well as 12 minor products named Alu m1 through m8. These were mapped on the SV40 genome by double digestion experiments. Alu fragments were treated with Hind enzymes and vice versa. Similar reciprocal digestions were also carried out with Hae III enzyme. In this way a detailed cleavage map of the SV40 genome could be constructed.     

Phylogenetic analysis of polyomavirus simian virus 40 from monkeys and humans reveals genetic variation

A phylogenetic analysis of 14 complete simian virus 40 (SV40) genomes was conducted in order to determine strain relatedness and the extent of genetic variation. This analysis included infectious isolates recovered between 1960 and 1999 from primary cultures of monkey kidney cells, from contaminated poliovaccines and an adenovirus seed stock, from human malignancies, and from transformed human cells. Maximum-parsimony and distance methods revealed distinct SV40 clades. However, no clear patterns of association between genotype and viral source were apparent. One clade (clade A) is derived from strain 776, the reference strain of SV40. Clade B contains isolates from poliovaccines (strains 777 and Baylor), from monkeys (strains N128, Rh911, and K661), and from human tumors (strains SVCPC and SVMEN). Thus, adaptation is not essential for SV40 survival in humans. The C terminus of the T-antigen (T-ag-C) gene contains the highest proportion of variable sites in the SV40 genome. An analysis based on just the T-ag-C region was highly congruent with the whole-genome analysis; hence, sequencing of just this one region is useful in strain identification. Analysis of an additional 16 strains for which only the T-ag-C gene was sequenced indicated that further SV40 genetic diversity is likely, resulting in a provisional clade (clade C) that currently contains strains associated with human tumors and human strain PML-1. Four other polymorphic regions in the genome were also identified. If these regions were analyzed in conjunction with the T-ag-C region, most of the phylogenetic signal could be captured without complete genome sequencing. This report represents the first whole-genome approach to establishing phylogenetic relatedness among different strains of SV40. It will be important in the future to develop a more complete catalog of SV40 variation in its natural monkey host, to determine if SV40 strains from different clades vary in biological or pathogenic properties, and to identify which SV40 strains are transmissible among humans.     

恒河猴外周血及肾组织SV40病毒基因分析

SV40即猿猴病毒40 (simian virus 40),是DNA肿瘤病毒的原型代表,其基因结构为共价闭合环状双股DNA分子,标准参考株SV40-776含5243个核甘酸。不同分离株bp数略有差异。SV40病毒为强DNA肿瘤病毒,具有使啮齿类动物及人源多种组织培养细胞永生化和转化能力。SV40病毒早期基因编码两个早期非结构蛋白即小T抗原(ST-ag、)和大T抗原(LT-ag),与病毒诱导的肿瘤发生有关。近年来研究表明,从猴体组织新分离的SV40株与实验室参考株SV40-776及SV40-B株相比较,具有明显的遗传异质性,并且SV40遗传变     

Variable defectiveness for lytic growth of the dl 54/59 mutants of simian virus 40.

When viral growth in TC-7 cells is compared with that in the simian virus 40 (SV40) transformed CV-1 line C6 some mutants of SV40 deleted between 0.54 and 0.59 on the standard map (dl 54/59 mutants) give relative bursts similar to those of wild-type strain 776, whereas others grow markedly poorer in the untransformed cell. In general, viruses which are defective by this criterion have been found to produce neither a fragmentary small-t protein nor a mature small-t mRNA.     

Nucleotide sequence of the Hind-C fragment of simian virus 40 DNA. Comparison of the 5'-untranslated region of wild-type virus and of some deletion Mutants.

We report here the nucleotide sequence of the wild-type simian virus 40 (strain 776) restriction fragment Hind-C-P1 DNA and of the homologous region of various mutant DNAs which lack part of this fragment. During this work, we detected between EcoRII fragments N and G an additional, 17-base-pair EcoRII fragment, fragment P, which had previously been overlooked. Also, an additional dTpdG dinucleotide at residues L 339--340 was observed by sequence analysis of the DNA minus (E) strand; the presence of this dinucleotide was masked on sequencing patterns of the plus strand due to the persistence (during gel electrophoresis) of some secondary structures in the strand's 5'-terminal region. These nucleotide additions raise the total length of SV40 DNA to 5243 base pairs. The longest tandemly repeated segment in SV40 DNA now extends over 72 base pairs. SV40 deletion mutants dl 893 and dl 894 and SV40 strains Rh 911 and 1801 all lack an identical 72-base-pair-long DNA segment in the Hind-C region. This deletion corresponds precisely to one of the two aforementioned large tandemly repeated sequences. Mutant dl 895 lacks 66 base pairs, 63 of which are part of the former repetition. All these mutants, except dl 895, very probably were generated by an intramolecular, homologous recombination event. The 40-base-pair deletion in mutant dl 1811 includes the major capping site of SV40 late RNA. dl 1812 lacks only three base pairs, which are part of the overlapping HhaI and HpaII restriction sites at position 0.725--0.726.     

Acquisition of Sequences Homologous to Host DNA by Closed Circular Simian Virus 40 DNA II. Further Studies on the Serial Passage of Virus Clones

Three plaque isolates of SV40 strain 777 and 1 plaque isolate of strain 776 were grown to high-titer stocks and serially passaged, undiluted, in monkey BS-C-1 cells. In each case, the serial passaging procedure resulted in the accumulation of closed-circular SV40 DNA molecules containing covalently linked sequences homologous to reiterated host cell DNA (called substituted virus DNA). The relative yields, at a given passage level, of SV40 DNA with measurable homology to host DNA varied in different sets of serial passages, including passages of the same virus clone. More reproducible yields of substituted viral DNA progeny were obtained when the serial passaging procedure was initiated from earlier passages rather than from the original plaque-purified stock. Fractionation of closed-circular SV40 DNA molecules on alkaline sucrose gadients indicated that the majority of substituted virus DNA molecules are not plaque producers and are slightly smaller in size than plaque-forming DNA molecules which display no detectable homology to host DNA. Evidence that substituted SV40 DNA molecules replicate during serial undiluted passage was obtained from experiments which demonstrated (i) the presence of host sequences in replicative forms of the viral DNA and (ii) the incorporation of (3)H-thymidine into host sequences isolated from the mature substituted virus DNA molecule.     

野生及笼养猕猴SV40T抗原基因检测方法的建立

目的建立猴外周血单核细胞SV40DNA的PCR检测方法,对猕猴SV40T抗原基因进行检测。方法使用PCR方法对分别来自野外（云南宁蒗71只、景东60只）及笼养猕猴（64只）的SV40大T抗原基因进行检测,同时对扩增出的阳性结果进行测序。结果在195份样本中,有4只来自野生猴样本扩增出SV40大T抗原基因（3．1％,4／131）,1只来自笼养猴样本扩增出SV40大T抗原基因（1．6％,1／64）。测序结果显示：猴血样本扩增片段序列与GenBank中的SV40大T抗原C-末端的基因序列片段有15个核苷酸不同（3．3％差异）,与SV40．776标准株的序列基本一致,但SV40—776在nt3020处有一缺口。结论云南野生及笼养猕猴猴群均能检出SV40T抗原基因。因此建立SV40病毒的DNA检测技术,对用于科研及疫苗生产的实验猕猴的病毒学质量控制具有重要意义。     

Comparison of two viable variants of simian virus 40.

The DNAs of two viable strains of simian virus 40, 776 and 777, have been compared by using restriction endonucleases. Differences between the two strains were detected at five separate points on the simian virus 40 genome. One of these differences, in the region of DNA coding for the major viral coat protein, was confirmed by tryptic peptide analysis of coat proteins from the two strains. Some physiological differences between the two strains were examined and can, in general, be explained by differences observed between the DNAs of the two strains. In addition, defective variants derived from strain 777 interfere more efficiently with the replication of strain 777 than with the replication of strain 776.     

Excision of integrated simian virus 40 DNA involving homologous recombination between viral DNA sequences

We have investigated the structure of simian virus 40 (SV40) DNA integrated into the genome of transformed mouse mKS-A cells. We have identified at least six independent integration units containing intact or truncated SV40 DNA sequences. One integration unit was isolated from a genomic mKS-A cell library and investigated by restriction enzyme analysis and partial nucleotide sequencing. This integration unit contains one apparently intact SV40 genome flanked on both sides by truncated versions of the SV40 genome. One of the flanking elements contains a large deletion in the SV40 "late" region and an abbreviated SV40 "early" region. This element was efficiently excised and mobilized after fusion of mKS-A to COS cells. The excision products invariably included the entire SV40 early region even though they were derived from an integrated element lacking this part of the SV40 genome. An analysis of this discrepancy led to the conclusion that the early region sequences were acquired by homologous recombination and, furthermore, that homologous excisional recombination was clearly preferred over non-homologous recombination.     

The early region of SV40 DNA may have more than one gene.

The nucleotide sequence of 70 base pairs (bp) around 0.545 map units (Alu I C and B junction) of the genome from the single Eco RI cleavage site within SV40 DNA is presented. The mRNA transcribed from the early strand template from this stretch contains two copies of the nonsense triplet UAA in each of the three reading frames. Thus at least 25% of the early region of SV40 DNA does not code for the SV40 "A" protein, and the viral contribution to events in the lytic cycle and transformation may be more complex than is generally appreciated.     

Hairpin structures are the primary amplification products: a novel mechanism for generation of inverted repeats during gene amplification.

Early events of DNA amplification which occur during perturbed replication were studied by using simian virus 40 (SV40)-transformed Chinese hamster cells (CO60) as a model system. The amplification is observed shortly after carcinogen treatment, and the amplified sequences contain molecules organized as inverted repeats (IRs). SV40 amplification in vitro was studied by using extracts from carcinogen-treated CO60 cells. In the amplified DNA the SV40 origin region was rereplicated, while more distal sequences were not replicated even once. Using several experimental procedures such as sucrose gradients, "snap-back" assay, and two-dimensional gel electrophoresis, we show that the overreplicated DNA contains IRs which are synthesized de novo as hairpins or stem-loop structures which were detached from the template molecules. The fully replicated SV40 molecules synthesized by the HeLa extracts do not contain such IRs. We propose "U-turn replication" as a novel mechanism for gene amplification, accounting for the generation of extrachromosomal inverted duplications as a result of perturbed replication and template switching of the DNA polymerases.     

Influence of the viral regulatory region on tumor induction by simian virus 40 in hamsters

Most of the simian virus 40 (SV40) genome is conserved among isolates, but the noncoding regulatory region and the genomic region encoding the large T-antigen C terminus (T-ag-C) may exhibit considerable variation. We demonstrate here that SV40 isolates differ in their oncogenic potentials in Syrian golden hamsters. Experimental animals were inoculated intraperitoneally with 10⁷ PFU of parental or recombinant SV40 viruses and were observed for 12 months to identify genetic determinants of oncogenicity. The viral regulatory region was found to exert a statistically significant influence on tumor incidence, whereas the T-ag-C played a minor role. Viruses with a single enhancer (1E) were more oncogenic than those with a two-enhancer (2E) structure. Rearrangements in the 1E viral regulatory region were detected in 4 of 60 (6.7%) tumors. Viral loads in tumors varied, with a median of 5.4 SV40 genome copies per cell. Infectious SV40 was rescued from 15 of 37 (40%) cell lines established from tumors. Most hamsters with tumors and many without tumors produced antibodies to T antigen. All viruses displayed similar transforming frequencies in vitro, suggesting that differences in oncogenic potential in vivo were due to host responses to viral infection. This study shows that SV40 strains differ in their biological properties, suggests that SV40 replicates to some level in hamsters, and indicates that the outcome of an SV40 infection may depend on the viral strain present.     

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.     

SV40病毒的培养、增殖及鉴定

目的：建立SV40病毒在Vero细胞上培养的方法,观察其生长过程,获得SV40病毒,并建立相应的SV40病毒检测方法,为SV40灭活疫苗的制备奠定良好的基础。方法：在Vero细胞上培养和增殖SV40-776株病毒。收获病毒后,应用PCR、免疫荧光以及克隆特异性片段进行测序比较来鉴定SV40病毒。结果：SV40在Vero细胞中增殖很快,并且使细胞出现明显的病变。小规模分离到了SV40病毒颗粒,获得了病毒DNA。不同的鉴定方法均显示出良好的特异性。结论：探讨了SV40病毒病变的基本过程,建立了病毒的培养、增殖和鉴定的方法。     

Complete genomic sequence analysis of a new SV40 isolate

The genome of a new SV40 strain(SV-IMB) isolated from a rhesus monkey was completely sequenced and compared with other isolates. The results showed that the whole genome contains 5246bp, and the aver age identity of SV-IMB was 98.1% as compared to other SV40 isolates. Its regulatory region is composed of a complete enhancer and a defective e enhancer. Amino acid changes occurred to some extent in both the large T antigen (T-Ag) and VP1 region. The findings demonstrate that the SV-IMB is a new SV40 isolate.     

Complete Genomic Sequence Analysis of a New SV40 Isolate

The genome of a new SV40 strain (SV-IMB) isolated from a rhesus monkey was completely sequenced and compared with other isolates. The results showed that the whole genome contains 5246bp, and the average identity of SV-IMB was 98.1% as compared to other SV40 isolates. Its regulatory region is composed of a complete enhancer and a defective enhancer. Amino acid changes occurred to some extent in both the large T antigen (T-Ag) and VP1 region. The findings demonstrate that the SV-IMB is a new SV40 isolate.