Lymphotropism of Merkel Cell Polyomavirus Infection, Nova Scotia, Canada

Lymphoid cells may be a site for virus persistence

Pokusy o ovlivneni imunizacnich vlastnosti viru vakcinie a jeho rekombinant.

Available from STL, Prague, CZ / NTK - National Technical LibrarySIGLECZCzech Republi

Viral MicroRNA Effects on Pathogenesis of Polyomavirus SV40 Infections in Syrian Golden Hamsters

Shaojie Zhang, Vojtech Sroller, Preeti Zanwar, Steven J. Halvorson, Nadim J. Ajami, Corey W. Hecksel, Jody L. Swain, Connie Wong, Janet S. Butel, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, United States of AmericaChun Jung Chen, Christopher S. Sullivan, Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, United States of AmericaJody L. Swain, Center for Comparative Medicine, Baylor College of Medicine, Houston, Texas, United States of AmericaEffects of polyomavirus SV40 microRNA on pathogenesis of viral infections in vivo are not known. Syrian golden hamsters are the small animal model for studies of SV40. We report here effects of SV40 microRNA and influence of the structure of the regulatory region on dynamics of SV40 DNA levels in vivo. Outbred young adult hamsters were inoculated by the intracardiac route with 1×107 plaque-forming units of four different variants of SV40. Infected animals were sacrificed from 3 to 270 days postinfection and viral DNA loads in different tissues determined by quantitative real-time polymerase chain reaction assays. All SV40 strains displayed frequent establishment of persistent infections and slow viral clearance. SV40 had a broad tissue tropism, with infected tissues including liver, kidney, spleen, lung, and brain. Liver and kidney contained higher viral DNA loads than other tissues; kidneys were the preferred site for long-term persistent infection although detectable virus was also retained in livers. Expression of SV40 microRNA was demonstrated in wild-type SV40-infected tissues. MicroRNA-negative mutant viruses consistently produced higher viral DNA loads than wild-type SV40 in both liver and kidney. Viruses with complex regulatory regions displayed modestly higher viral DNA loads in the kidney than those with simple regulatory regions. Early viral transcripts were detected at higher levels than late transcripts in liver and kidney. Infectious virus was detected infrequently. There was limited evidence of increased clearance of microRNA-deficient viruses. Wild-type and microRNA-negative mutants of SV40 showed similar rates of transformation of mouse cells in vitro and tumor induction in weanling hamsters in vivo. This report identified broad tissue tropism for SV40 in vivo in hamsters and provides the first evidence of expression and function of SV40 microRNA in vivo. Viral microRNA dampened viral DNA levels in tissues infected by SV40 strains with simple or complex regulatory regions.This work was supported in part by research grants R01 CA134524 (JSB) and R01 AI077746 (CSS) from the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Molecular BiosciencesEmail: [email protected]

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

Frequent persistence of SV40 in hamster tissues following intracardiac inoculation.

<p>ND, not done; p.i., postinoculation; SV40, simian virus 40.</p>a<p>A tissue was considered positive if viral DNA copies were detected by real-time quantitative polymerase chain reaction (described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003912#s4" target="_blank">Materials and Methods</a>).</p

Preferential persistence of SV40 in kidney compared to liver and spleen.

<p>SV40, simian virus 40.</p>a<p>Values represent the ratio of the average observed (nonadjusted) results from day 45 and day 3 for each virus in the indicated tissues. The numbers of tissues from different animals tested per virus at each time point (usually n = 4) are listed (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003912#ppat-1003912-t001" target="_blank">Table 1</a>). Note the higher level of persistence of each virus in the kidney as compared to liver and spleen.</p

Viral loads in different hamster tissues during early stages of infection by SV40.

<p>p.i., postinoculation; SV40, simian virus 40; Und, undetectable.</p>a<p>Numbers represent the average of observed (nonadjusted) results from the tissues analyzed at each time point. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003912#ppat-1003912-t001" target="_blank">Table 1</a> for the number of tissues from different animals tested per virus at each time point (usually n = 4). Two or three separate fragments were assayed and the values averaged from each tissue from each animal (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003912#s4" target="_blank">Materials and Methods</a>).</p>b<p>The inoculum for each virus was 10⁷ plaque-forming units (PFU) per animal by the intracardiac route. The number of SV40 genome DNA copies per PFU varied for each viral stock: 776-WT, 1700; 776-SM1, 130; SVCPC-WT, 1800; SVCPC-SM2, 590. This resulted in the following ratios of viral genome copies inoculated: 776-WT:776-SM1, 13; SVCPC-WT:SVCPC-SM2, 3.</p>c<p>Note that higher viral DNA copy numbers were more often detected in liver and kidney samples from animals exposed to microRNA-negative viruses than in those infected with wild-type viruses, even though fewer viral genome copies were inoculated with the mutants than with the wild-type viruses. This pattern held for most specimen collections through day 45 p.i.</p

Lack of effect of SV40 microRNA on tumor development in Syrian golden hamsters following intraperitoneal inoculation.

<p>SV40, simian virus 40.</p>a<p><i>p</i> values for comparisons of % tumors and of time to tumors were determined by the Z test and the proportional hazards regression test, respectively.</p

Detection of simian virus 40 (SV40) early and late mRNAs in hamster tumors induced by wild-type viruses and microRNA-negative mutants following intraperitoneal inoculation.

<p>SV40 transcripts in hamster tumors induced by wild-type and microRNA-negative viral strains were reverse transcribed, quantitated by real-time quantitative polymerase chain reaction and expressed as the average number of SV40 mRNA copies per 10⁶ copies of 18S ribosomal RNA. The numbers of tumors analyzed for each viral system were the following: SVCPC-776 = 5, SVCPC-776-SM1 = 5, SVCPC = 3, and SVCPC-SM2 = 4. The error bars represent the standard deviation.</p