SV40 lymphomagenesis in Syrian golden hamsters

Simian virus 40 (SV40) isolates differ in oncogenic potential in Syrian golden hamsters following intraperitoneal inoculation. Here we describe the effect of intravenous exposure on tumor induction by SV40. Strains SVCPC (simple regulatory region) and VA45-54(2E) (complex regulatory region) were highly oncogenic following intravenous inoculation, producing a spectrum of tumor types. Three lymphoma cell lines were established; all expressed SV40 T-antigen, were immortalized for growth in culture, and were tumorigenic following transplantation in vivo. New monoclonal antibodies directed against hamster lymphocyte surface antigens are described. The cell lines expressed MHC class II and macrophage markers and were highly phagocytic, indicating a histiocytic origin. Many hamsters that remained tumor-free developed SV40 T-antigen antibodies, suggesting that viral replication occurred. This study shows that route of exposure influences the pathogenesis of SV40-mediated carcinogenesis, that SV40 strain VA45-54(2E) is lymphomagenic in hamsters, that hamster lymphoid cells of histiocytic origin can be transformed in vivo and established in culture, and that reagents to hamster leukocyte differentiation molecules are now available.     

Detection of polyomavirus SV40 in tonsils from immunocompetent children

BACKGROUND: BK virus (BKV), JC virus (JCV) and simian virus 40 (SV40) are nonenveloped DNA viruses, members of the family Polyomaviridae. BK and JC viruses establish persistent infections in humans, and evidence suggests that SV40 can infect humans, as well. Whether persistence occurs in the lymphoid system is unknown. METHODS: Paraffin-embedded tonsils from 220 immunocompetent children (mean age 9.3 years) were examined by polymerase chain reaction (PCR) to detect viral DNA of BKV, JCV, SV40, and Epstein-Barr virus (EBV). RESULTS: Polyomavirus-specific DNA sequences were detected in 8.3% (29/351) of specimens collected from 220 children. Twenty-one (9.5%) children had polyomavirus DNA present in at least one tonsil, with sequences identified as SV40 (n=20) and BKV (n=1). Polyomavirus JCV was not detected. Among patients positive for SV40, 8 of 14 (57%) contained viral DNA in both available tonsils. EBV DNA was detected in 99 (28.2%) samples from 67 (30.5%) patients. Eleven samples (3.1%) from 8 (3.6%) children were positive for both polyomavirus and EBV. SV40-positive children were significantly older than the SV40-negative subjects (P<0.001). T-antigen expression was detected in an SV40 DNA-positive tonsil by immunohistochemistry. CONCLUSIONS: These results suggest that SV40 can infect tonsils, that lymphoid tissue may represent a site for polyomavirus persistence, and that immunohistochemistry is not a useful detection assay when there are very few virus-infected cells in a tissue.     

Differential ability of two simian virus 40 strains to induce malignancies in weanling hamsters

Different strains of simian virus 40 (SV40) exist and are associated with some human malignancies, but it is not known if SV40 strains differ in biological potential in vivo. In two long-term experiments, Syrian golden hamsters 21 days of age were inoculated by the intraperitoneal route with two different strains of SV40 (10(7) plaque-forming units/animal) and were followed for 8 or 12 months. In vivo responses to strain VA45-54, isolated originally from monkey kidney cells, and to strain SVCPC, recovered from human cancers, were compared. Control animals of the same age were inoculated intraperitoneally with cell culture media. Malignancies developed only in animals infected with SV40 and not in controls. The rate of tumor development was more frequent among animals infected with strain SVCPC than with VA45-54, both in experiments held for 8 months (11/22, 50% vs. 4/20, 20%) and for 12 months (7/15, 47% vs. 3/13, 23%). Histologically, the tumors resembled mesotheliomas, osteosarcoma, and poorly differentiated sarcomas. Metastases to lung and lymph nodes occurred with both viral strains. T-antigen expression was detected in most tumor cells by immunohistochemistry. Anti-T-antigen antibodies were produced by almost all tumor-bearing animals and by about two-thirds of those that did not develop tumors after virus inoculation. SV40 viral neutralizing antibodies were detected in all tumor-bearing animals and in 92% and 38% of those inoculated with SVCPC and VA45-54, respectively, that failed to develop tumors. Antibody titers were usually higher in animals with tumors than in those without. Control animals did not develop viral antibodies. Infectious virus was recovered from 2 of 15 tumors tested. This study showed that there are biological differences between these two SV40 strains that influence the outcome of infections in normal hosts, including the development of malignancies and neutralizing antibody, and proved the principle that SV40 strains from different clades can vary in biological properties in vivo.     

Variable frequency of polyomavirus SV40 and herpesvirus EBV in lymphomas from two different urban population groups in Houston, TX

BackgroundStudies have reported differing frequencies of detection of polyomavirus simian virus 40 (SV40) in association with human lymphomas.ObjectiveWe addressed the hypothesis that SV40 positivity in lymphomas can vary among sampled populations.Study designArchival paraffin-embedded lymphoma specimens (n = 171) from patients at two urban hospitals in Houston, TX, USA, were analyzed following a cross-sectional study design. Extracted DNAs were characterized by quantitative polymerase chain reaction for the cellular RNase P gene and for SV40 and herpesvirus Epstein–Barr virus (EBV) sequences.ResultsPatient characteristics of the two study populations differed significantly whereas the classification of tumor types studied did not. SV40 DNA was detected more frequently in lymphomas from the public hospital population (10/44, 23%) than in lymphomas from the veterans’ hospital (VAMC) (4/127, 3%; P < 0.0001). EBV detection in lymphomas also differed between the two groups (17/44, 39% vs. 23/127, 18%; P = 0.01). SV40 positivity was associated with a younger age category of VAMC lymphoma patients (P = 0.02). Expression of T-antigen was detected by immunohistochemistry in half of lymphomas that contained SV40 DNA. Variation was observed in the quality and quantity of DNA recovered from paraffin-embedded specimens, but there was no difference in recoveries of DNA from samples from the two hospitals.ConclusionsThis study demonstrated that, in a direct comparison, the prevalence of SV40 DNA in lymphomas can differ significantly between groups with different demographic distributions.     

Effects of Mutations within the SV40 Large T Antigen ATPase/p53 Binding Domain on Viral Replication and Transformation

The simian virus 40 (SV40) large T antigen is a 708 amino-acid protein possessing multiple biochemical activities that play distinct roles in productive infection or virus-induced cell transformation. The carboxy-terminal portion of T antigen includes a domain that carries the nucleotide binding and ATPase activities of the protein, as well as sequences required for T antigen to associate with the cellular tumor suppressor p53. Consequently this domain functions both in viral DNA replication and cellular transformation. We have generated a collection of SV40 mutants with amino-acid deletions, insertions or substitutions in specific domains of the protein. Here we report the properties of nine mutants with single or multiple substitutions between amino acids 402 and 430, a region thought to be important for both the p53 binding and ATPase functions. The mutants were examined for the ability to produce infectious progeny virions, replicate viral DNA in vivo, perform in trans complementation tests, and transform established cell lines. Two of the mutants exhibited a wild-type phenotype in all these tests. The remaining seven mutants were defective for plaque formation and viral DNA replication, but in each case these defects could be complemented by a wild-type T antigen supplied in trans. One of these replication-defective mutants efficiently transformed the REF52 and C3H10T1/2 cell lines as assessed by the dense-focus assay. The remaining six mutants were defective for transforming REF52 cells and transformed the C3H10T1/2 line with a reduced efficiency. The ability of mutant T antigen to transform REF52 cells correlated with their ability to induce increased levels of p53. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of single and repeated pregnancy on frequency of origin of primary tumors induced by SV40 virus in syrian hamsters

The effect of single and repeated pregnancy on the frequency of origin of primary tumors induced by SV₄₀ virus was investigated in Syrian hamsters. Females developed tumors after 1 to 5 pregnancies significantly less frequently during the latent period of SV₄₀ carcinogenesis than females not becoming pregnant in the same experiment. However, these differences are evidently not attributable to immunization of the gravid females by embryonic antigens, for the frequency and times of origin of primary tumors in males were the same as in previously gravid females.Laboratory of Immunology of Tumors, Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 11, pp. 595–597, November, 1977.     

HBV感染孕妇体内病毒前C区G1896A变异与母婴垂直传播的关系

目的 分析孕妇体内乙型肝炎病毒(HBV)前C区G1896A变异对母婴垂直传播的影响.方法 收集40例本院妇产科HBeAg(-)/HBsAg( )的孕妇血标本,同时收集这些孕妇分娩时新生儿脐带血40例.荧光定量PCR(FQ-PCR)检测HBV孕妇血清和新生儿脐带血中HBV-DNA栽量,PCR-ELISA法检测孕妇血清中HBV-DNA前C区G1896A变异.分析HBV前C区G1896A变异及孕妇血清HBV-DNA载量对母婴垂直传播率的影响.结果 40例孕妇血清共检测到25例HBV前C区G1896A变异(62.5%);变异组母婴垂直传播发生率为44.0%(11/25),未变异组母婴垂直传播发生率为40.0%(6/15),二组比较无统计学差异(x2=0.0614,P＞0.05).孕妇血中HBV-DNA高载量组(≥1×105 copies/ml)母婴垂直传播发生率为62.5%(10/16),低栽量组(＜1 × 105 copies/ml)母婴垂直传播发生率为29.2%(7/24),二组相比有显著性差异(x2=4.3649,P＜0.05).结论 HBV前C区G1896A变异未增加母婴HBV垂直传播率,孕妇血清中HBV-DNA栽量升高是母婴HBV垂直传播危险因素.     

重组人白介素2对小鼠弓形虫垂直传播的影响

目的　研究rhuIL - 2对弓形虫垂直传播的影响。方法　用不同剂量rhuIL - 2处理感染弓形虫孕鼠 ,于妊娠第12天取孕鼠子宫 ,观察记录平均活胎率的变化 ,并将胚胎及胎盘固定 ,做免疫组化染色 ,观察胎盘胎鼠感染情况 ,计算孕鼠的垂直传播率。结果　高剂量组与感染组相比活胎率明显升高 (P <0 0 5 ) ;两处理组的垂直传播率均较感染组明显下降 (P<0 0 5 ) ;免疫组化结果显示 ,胎盘滋养层细胞、绒毛间隙、蜕膜细胞均有大量弓形虫抗原表达 ;部分孕鼠的胎盘有弓形虫抗原表达 ,但其胚胎组织弓形虫抗原的表达却呈阴性。结论　rhuIL - 2一定程度上可以降低弓形虫的垂直传播率 ,对感染孕鼠及胚胎起到一定保护作用。胎盘作为局部免疫屏障具有重要的研究价值。     

First detection of the OXA-40 carbapenemase in P. aeruginosa isolates, located on a plasmid also found in A. baumannii

The aim of this work was to carry out the molecular investigation of the OXA-40 carbapenemase detected in two isolates of Pseudomonas aeruginosa resistant to imipenem. The sequence showed 100% of homology with the gene previously described in Acinetobacter baumannii. Hybridization experiments located the gene on a plasmid also found in the OXA-40 control strain of A. baumannii.     

A novel disease-causing CD40L mutation reduces expression of CD40 ligand,but preserves CD40 binding capacity

Mutations in CD40 ligand (CD40L) that permit residual CD40L expression typically impair binding of CD40. We report a male patient who presented with recurrent bacterial respiratory tract infections, normal IgM, decreased IgG, absent IgA levels, and CD40L expression at ~ 50% of the level observed in the normal control. He subsequently developed autoimmunity, inflammatory bowel disease, severe opportunistic infections suggestive of a combined immunodeficiency, and a cervical spine schwannoma. Whole exome sequencing of the patient's genomic DNA revealed a novel missense mutation (p.H47Y) in CD40L. Although this mutation was predicted to be benign in silico, flow cytometry at 13 years of age demonstrated markedly decreased CD40L expression (~ 32% of normal control) that retained the capacity to bind soluble CD40-Ig, suggesting that the mutation impairs CD40L surface expression without affecting its affinity for CD40. This case highlights the variability in the clinical evolution and phenotype of CD40L deficiency.     

A novel colony-print immunoassay reveals differential patterns of distribution and horizontal transmission of four unrelated mycoviruses in Rosellinia necatrix

A colony-print immunoassay (CPIA) using an anti-dsRNA antibody was developed to visualize the distribution of four unrelated mycoviruses with dsRNA genomes, a partitivirus (RnPV1), mycoreovirus (RnMyRV3), megabirnavirus (RnMBV1), and an unidentified virus (RnQV1), in mycelia of the white root rot fungus, Rosellinia necatrix. CPIA revealed different distribution patterns within single colonies for each virus. Both RnPV1 and RnMBV1 were distributed throughout single colonies, RnMyRV3 was absent from some colony sectors, and RnQV1 exhibited varied accumulation levels between sectors. RnMyRV3 and RnQV1 were transmitted to the recipient virus-free colonies of virus-infected and virus-free colony pairs more slowly than were RnPV1 or RnMBV1. The presence of RnMyRV3 in recipient colonies restricted horizontal transmission of RnPV1 and RnMBV1. These results imply that one or more mechanisms are present in host-virus and virus-virus interactions that restrict the spread of viruses within and between colonies.     

Molecular evolution of West Nile virus in a northern temperate region: Connecticut, USA 1999-2008

West Nile virus (WNV) has become firmly established in northeastern US, reemerging every summer since its introduction into North America in 1999. To determine whether WNV overwinters locally or is reseeded annually, we examined the patterns of viral lineage persistence and replacement in Connecticut over 10 consecutive transmission seasons by phylogenetic analysis. In addition, we compared the full protein coding sequence among WNV isolates to search for evidence of convergent and adaptive evolution. Viruses sampled from Connecticut segregated into a number of well-supported subclades by year of isolation with few clades persisting ≥ 2 years. Similar viral strains were dispersed in different locations across the state and divergent strains appeared within a single location during a single transmission season, implying widespread movement and rapid colonization of virus. Numerous amino acid substitutions arose in the population but only one change, V → A at position 159 of the envelope protein, became permanently fixed. Several instances of parallel evolution were identified in independent lineages, including one amino acid change in the NS4A protein that appears to be positively selected. Our results suggest that annual reemergence of WNV is driven by both reintroduction and local-overwintering of virus. Despite ongoing evolution of WNV, most amino acid variants occurred at low frequencies and were transient in the virus population.     

The number of Foxp3-positive regulatory T cells is increased in Helicobacter pylori gastritis and gastric cancer

Helicobacter pylori (H. pylori) colonization induces vigorous innate and specific immune responses; however, the infection is not removed, a state of chronic active gastritis persists for life if untreated. Recent studies have shown that CD4⁺ CD25⁺ Foxp3-positive regulatory T cells (Tregs) suppress the immune response to H. pylori. Persistent H. pylori-associated gastritis is closely associated with gastric carcinogenesis. We investigated the number of Tregs in the context of H. pylori colonization in chronic gastritis, examined the relationship between it and histopathological findings and compared it with that of gastric dysplasia and adenocarcinoma. This study was based on the analysis of gastric biopsy specimens from 126 cases of H. pylori-associated gastritis, 16 cases of H. pylori-negative gastritis, 17 cases of gastric dysplasia, and 25 cases of gastric adenocarcinoma. The number of Tregs was elevated in H. pylori-associated gastritis, where it was positively correlated with the grade of chronic inflammation and the number of lymphoid follicles. It was significantly elevated in adenocarcinomas compared to chronic gastritis and gastric dysplasia. In summary, the number of Tregs is increased in H. pylori-associated gastritis and gastric cancer.     

The role of hypermethylation of the hMLH1 promoter region in HNPCC versus MSI+ sporadic colorectal cancers

INTRODUCTION—Hypermethylation of the promoter region of the hMLH1 gene is associated with absent expression of MLH1 protein in sporadic colorectal cancers with microsatellite instability (MSI+), and it has been proposed that methylation may be a mechanism of inactivation in Knudson's hypothesis. The incidence of hypermethylation of the hMLH1 promoter in hereditary non-polyposis colorectal cancer (HNPCC) versus MSI+ sporadic colorectal cancer was investigated and compared.
METHODS—DNA was available from 10 HNPCC colorectal cancers (median age 58 years, range 39-67) with germline mutations in hMLH1 and 10 MSI+ sporadic colorectal cancers (mean age 79 years, range 41-85). MSI was determined by amplification of BAT26 and TGF-β RII. The methylation status of the hMLH1 promoter was studied by the polymerase chain reaction (PCR) based HpaII restriction enzyme assay technique. Evidence of allelic loss at hMLH1 was searched for in the HNPCC colorectal cancers.
RESULTS—All cases were confirmed to be MSI+. The promoter region of hMLH1 was hypermethylated in seven of 10 MSI+ sporadic cancers versus 0 of 10 HNPCC cancers (p<0.002). Evidence of loss of heterozygosity at hMLH1 was observed in eight of the 10 HNPCC colorectal cancers.
CONCLUSION—While mutations and allelic loss are responsible for the MSI+ phenotype in HNPCC cancers, the majority of MSI+ sporadic cancers are hypermethylated in the promoter region of hMLH1. These data further support our argument that tumours from HNPCC patients, which almost always acquire a raised mutation rate, mostly follow a different pathway from MSI+ sporadic tumours.


Keywords: hMLH1 promoter region; HNPCC; hypermethylation; colorectal cancer     

The protruding domain of the coat protein of Melon necrotic spot virus is involved in compatibility with and transmission by the fungal vector Olpidium bornovanus

The Chi and W strains of Melon necrotic spot virus (MNSV) are efficiently transmitted by isolates Y1 and NW1, respectively, of the fungal vector Olpidium bornovanus. Analysis of chimeric viruses constructed by switching the coat protein (CP) gene between the two strains unveiled the involvement of the CP in the attachment of MNSV to zoospores of a compatible isolate of O. bornovanus and in the fungal transmission of the virus. Furthermore, analysis of the chimeric virus based on the Chi strain with the protruding domain of the CP from strain W suggested the involvement of the domain in compatibility with zoospore. Comparison of the three-dimensional structures between the CP of the two MNSV strains showed that many of the differences in these amino acid residues are present on the surface of the virus particles, suggesting that these affects the recognition of fungal vectors by the virus.     

A conserved secondary structure in the hypervariable region at the 5' end of Bamboo mosaic virus satellite RNA is functionally interchangeable

Satellite RNA (satRNA) associated with Bamboo mosaic virus (BaMV) is dependent on BaMV for replication and encapsidation. Molecular analyses of total RNA extracted from bamboo species collected worldwide revealed that 26 out of 61 BaMV isolates harbored satBaMV. Among them, two phylogenetically distinguishable groups, A and B, with a genetic diversity of 6.9 +/- 0.7% were identified. Greatest sequence diversity occurred in the 5' untranslated region (UTR) that contained one hypervariable region with variations of up to 20.7%. Concurrent covariations in the 5' hypervariable sequences support the existence of a conserved apical hairpin stem-loop structure, which was earlier mapped by enzymatic probings and functional analyses [Annamalai, P., Hsu, Y.H., Liu, Y.P., Tsai, C.H., Lin, N.S., 2003. Structural and mutational analyses of cis-acting sequences in the 5'-untranslated region of satellite RNA of bamboo mosaic potexvirus. Virology 311 (1), 229-239]. Furthermore, chimeric satBaMVs generated by interchanging the hypervariable region between groups A and B demonstrated the replication competence of satBaMV isolates in Nicotiana benthamiana protoplasts co-inoculated with BaMV RNA. The results suggest that an evolutionarily conserved secondary structure exists in the hypervariable region of 5' UTR of satBaMV.