Human DNA Viruses Research Articles

Phylogenetic studies of frequently diagnostically sampled herpesviruses – Possibilities for clinical applications?

With the modern sequencing technologies and the capability to sequence large viral genomes virtually overnight, full-genome sequencing of large DNA viruses and correlating any sequence variation to specific clinical manifestations is now eminently feasible – but is it worth doing, i.e. can such large-scale viral genomic analyses be clinically useful? A few clinical conditions, such as organ and bone marrow transplant recipients, require the regular diagnostic sampling and testing for certain viruses over an extended period of time. Such frequent sampling leads to the routine archiving of multiple samples from the same patient over an extended time period and it is the purpose of this mini-review to explore whether such routinely collected samples can be utilized for viral sequencing studies to produce clinically useful outcomes. Specific examples of this include the monitoring of Epstein-Barr virus (EBV) and cytomegalovirus (CMV) levels in transplant recipients, as rising levels of these viruses in these patients can cause serious and sometimes fatal disease during the post-transplant period when their immune system is gradually recovering from the transplant process. Other herpesviruses, such as herpes simplex virus (HSV) and varicella zoster virus (VZV), may also reactivate and cause disease in post-transplant and other types of immunocompromised patients, though the specific complications may differ between patients. Although the natural mutation rate for these human herpes DNA viruses are very low, all of these viruses are capable of developing specific drug resistance within a few weeks of therapy, demonstrating that these viruses do have the ability to adapt rapidly to their local environment if the need arises. Specific problems with analyzing these large DNA virus genomes include the selection of the appropriate gene target (unless a full-genome analysis is the aim), and how to deal with overlapping and inverted reading frames, which are discussed in this mini-review.

Identification of a Novel Inhibitor That Selectively Targets NF-κB Activity in KSHV-Infected Lymphoma Cells

Abstract 160Kaposi’s sarcoma herpesvirus (KSHV/HHV-8), a member of the γ-herpesvirus family of human DNA viruses, is the etiologic agent of several malignancies in immune-compromised individuals, such as Kaposi’s sarcoma (KS) and primary effusion lymphoma (PEL). Activated NF-κB is a critical mechanism by which lymphoma cells infected by KSHV are protected from apoptotic stress. vFLIP is the viral protein largely responsible for the constitutive NF-κB activity, and is essential for PEL cell survival. Using a subclone of the BC3 PEL cell line that stably expresses firefly luciferase under the control of an NF-κB promoter (BC3/NFκB-luc), we performed high throughput screening (HTS) of compound libraries to identify inhibitors of vFLIP/NF-κB in PEL cells.Assay optimization was done using the NCI Training set, following which screening of the NIH diversity set at 5μM picked up 60 hits (with >50% inhibition) from a total of ∼2000 compounds. To further test these compounds, we introduced a renilla luciferase controlled by a constitutive promoter (retroviral LTR) to obtain a double reporter cell line (BC3-REN#3). Three compounds showed preferential inhibition of firefly luciferase in the double-reporter cell line, and/or preferential inhibition of the BC3/NFκB-luc cell line when compared to a control non-lymphoid cell line with constitutive luciferase expression (U251-pGL3). Analogs of these three compounds were further tested in BC3-REN#3 cells, and three analogs were identified with increased activity/specificity for NF-κB.One of the compounds identified so far, here designated NSC-E, has shown dose-dependent inhibition of NF-κB in PEL cells, as evaluated independently by reporter and electrophoretic mobility shift assays (EMSA). Nuclear translocation of the NF-κB active subunit p65, but not inactive p50 is inhibited by this compound, although it does not alter upstream events in the NF-κB pathway, such as IKKα/β phosphorylation or IκBα degradation. Studies are ongoing to elucidate the direct molecular target of NSC-E.Inhibition of NF-κB by NSC-E occurs only in PEL cell lines, and not in other lymphoma cell types. In addition, this compound preferentially inhibits viability of KSHV-infected lymphoma cells, at LC50s ranging from 18nM to ∼200nM in various PEL cell lines tested. In contrast, an effect on EBV-infected lymphoma cell viability is only apparent at much higher doses (LC50s in the 20–80μM range). Hence, ∼300X the KSHV-effective dose of NSC-E would be required to achieve inhibition in EBV-associated or virus-negative lymphomas, suggesting this inhibitor is very KSHV-specific.Finally, NSC-E was evaluated in a traceable reporter xenograft mouse model of KSHV-associated PEL using in vivo imaging, and found to be highly efficacious against both incipient and well-established tumors, with 100% tumor responses and no apparent toxicity. Thus, we have identified a novel, specific, and highly effective inhibitor of KSHV-associated lymphoma that demonstrates clear pre-clinical promise for therapeutic applications. Disclosures:No relevant conflicts of interest to declare.

Abstract 4829: A purine scaffold Hsp90 inhibitor has antitumor activity in KSHV-associated malignancies by suppressing vFLIP

Abstract Background: Kaposi's sarcoma herpesvirus (KSHV/ HHV-8), a member of the α-herpesvirus family of human DNA viruses, is the etiologic agent of several malignancies in immune-compromised individuals, such as Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). Activated NF-κB is a critical mechanism by which KSHV-infected PEL cells are protected from apoptosis. KSHV vFLIP has been identified as a viral oncogene that is responsible for NF-κB-dependent anti-apoptotic gene expression in PEL cells, and also prevents autophagy in KSHV-infected endothelial cells. In particular, the IKK signaling complex consisting of IKKα, IKKα, IKKα, vFLIP and Hsp90 was shown to be essential for survival in PEL cells. The chaperone protein Hsp90 binds client proteins involved in the regulation of cell survival and apoptosis signal transduction, including Akt, IKK complex, and KSHV vFLIP. A lack of Hsp90 causes protein misfolding, ubiquitination and degradation. The Hsp90 inhibitor geldanamycin was previously tested in PEL cells and shown to inhibit activity of the IKK complex in vitro. However, geldanamycin is of limited therapeutic potential due to its undesirable pharmacophysiology. We tested a new purine-scaffold Hsp90 inhibitor with high selectivity for tumor versus normal cell Hsp90, which is water-soluble with high oral bioavailability and excellent therapeutic window. Materials and methods: We evaluated the sensitivity of several KSHV infected and uninfected cell lines to treatment with this inhibitor, designated PU-H71. We performed viability and NF-κB reporter luciferase assays, and immunoblot and qRT-PCR analyses of cellular and viral proteins. Finally, we used a mouse PEL xenograft model and in vivo imaging to assess tumor responses to PU-H71. Results: We found all KSHV-positive PEL cell lines to be exquisitely sensitive when compared to uninfected lymphoma cells, with LC50s in the nanomolar range. PU-H71 was shown to induce PEL cell death by apoptosis and autophagy within 48 hours of treatment. PU-H71 also showed preferential toxicity in an in vitro model of KS. Pulldown demonstrated association of the compound with the active vFLIP-IKKα signalosome. Western blot analysis indicated that the IKK signaling complex was destabilized, and components vFLIP and IKKα were degraded upon PU-H71 treatment, resulting in inhibition of NF-κB signaling, as confirmed by reporter luciferase assay. qRT-PCR and reporter analysis indicated low levels of concomitant lytic reactivation. PU-H71 was further shown to inhibit progression of tumor spread and confer a significant survival advantage (p<0.02) in a mouse xenograft model of PEL, with persistence of IKKα destabilization ex vivo. Conclusions: Our findings demonstrate that Hsp90 inhibition with PU-H71 leads to reduced vFLIP levels in KSHV-infected cells, and to tumor responses. Thus, PU-H71 is a promising targeted approach for the treatment of KS and PEL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4829. doi:1538-7445.AM2012-4829

Sequence characteristics of T4-like bacteriophage IME08 genome termini revealed by high throughput sequencing

BackgroundT4 phage is a model species that has contributed broadly to our understanding of molecular biology. T4 DNA replication and packaging share various mechanisms with human double-stranded DNA viruses such as herpes virus. The literature indicates that T4-like phage genomes have permuted terminal sequences, and are generated by a DNA terminase in a sequence-independent manner;Methodsgenomic DNA of T4-like bacteriophage IME08 was subjected to high throughput sequencing, and the read sequences with extraordinarily high occurrences were analyzed;Resultswe demonstrate that both the 5' and 3' termini of the IME08 genome starts with base G or A. The presence of a consensus sequence TTGGA|G around the breakpoint of the high frequency read sequences suggests that the terminase cuts the branched pre-genome in a sequence-preferred manner. Our analysis also shows that terminal cleavage is asymmetric, with one end cut at a consensus sequence, and the other end generated randomly. The sequence-preferred cleavage may produce sticky-ends, but with each end being packaged with different efficiencies;Conclusionsthis study illustrates how high throughput sequencing can be used to probe replication and packaging mechanisms in bacteriophages and/or viruses.

Epigenetic mechanisms in virus-induced tumorigenesis

About 15–20% of human cancers worldwide have viral etiology. Emerging data clearly indicate that several human DNA and RNA viruses, such as human papillomavirus, Epstein–Barr virus, Kaposi’s sarcoma-associated herpesvirus, hepatitis B virus, hepatitis C virus, and human T-cell lymphotropic virus, contribute to cancer development. Human tumor-associated viruses have evolved multiple molecular mechanisms to disrupt specific cellular pathways to facilitate aberrant replication. Although oncogenic viruses belong to different families, their strategies in human cancer development show many similarities and involve viral-encoded oncoproteins targeting the key cellular proteins that regulate cell growth. Recent studies show that virus and host interactions also occur at the epigenetic level. In this review, we summarize the published information related to the interactions between viral proteins and epigenetic machinery which lead to alterations in the epigenetic landscape of the cell contributing to carcinogenesis.

Mapping the landscape of host-pathogen coevolution: HLA class I binding and its relationship with evolutionary conservation in human and viral proteins.

The high diversity of HLA binding preferences has been driven by the sequence diversity of short segments of relevant pathogenic proteins presented by HLA molecules to the immune system. To identify possible commonalities in HLA binding preferences, we quantify these using a novel measure termed "targeting efficiency," which captures the correlation between HLA-peptide binding affinities and the conservation of the targeted proteomic regions. Analysis of targeting efficiencies for 95 HLA class I alleles over thousands of human proteins and 52 human viruses indicates that HLA molecules preferentially target conserved regions in these proteomes, although the arboviral Flaviviridae are a notable exception where nonconserved regions are preferentially targeted by most alleles. HLA-A alleles and several HLA-B alleles that have maintained close sequence identity with chimpanzee homologues target conserved human proteins and DNA viruses such as Herpesviridae and Adenoviridae most efficiently, while all HLA-B alleles studied efficiently target RNA viruses. These patterns of host and pathogen specialization are both consistent with coevolutionary selection and functionally relevant in specific cases; for example, preferential HLA targeting of conserved proteomic regions is associated with improved outcomes in HIV infection and with protection against dengue hemorrhagic fever. Efficiency analysis provides a novel perspective on the coevolutionary relationship between HLA class I molecular diversity, self-derived peptides that shape T-cell immunity through ontogeny, and the broad range of viruses that subsequently engage with the adaptive immune response.

Herpes simplex virus‐1 induces expression of a novel MxA isoform that enhances viral replication

MxA is an antiviral protein induced by interferon (IFN)-α/β that is known to inhibit the replication of many RNA viruses. In these experiments, the 76-kDa MxA protein expressed in IFN-α-treated cells was shown to have antiviral activity against herpes simplex virus-1 (HSV-1), a human DNA virus. However, MxA was expressed as a 56-kDa protein in HSV-1-infected cells in the absence of IFN-α. This previously unrecognized MxA isoform was produced from an alternatively spliced MxA transcript that had a deletion of Exons 14-16 and a frame shift altering the C-terminus. The variant MxA (varMxA) isoform was associated with HSV-1 regulatory proteins and virions in nuclear replication compartments. varMxA expression enhanced HSV-1 infection as shown by a reduction in infectious virus titers from cells in which MxA had been inhibited by RNA interference and by an increase in HSV-1 titers when the 56-kDa varMxA was expressed constitutively. Thus, the human MxA gene encodes two MxA isoforms, which are expressed differentially depending on whether the stimulus is IFN-α or HSV-1. These findings show that alternative splicing of cellular mRNA can result in expression of a novel isoform of a host defense gene that supports instead of restricting viral infection.

Human papillomavirus is not associated with breast carcinoma

Breast cancer is the most common cancer in women, accounting for 23% of all female cancers around the world. Research on its etiology has focused primarily on genetic and environmental factors [1]. Infectious etiology in carcinogenesis has long been suggested by epidemiological and experimental studies. Current estimates suggest that approximately 15–20% of all cancers worldwide appear to be associated with viral infections, and several human DNA viruses are now accepted as causative agent of specific malignancies [2]. Human papillomavirus (HPV) is one of the viruses and is responsible for causing virtually all cases of cervical cancer in women [2]. A number of studies have reported HPV DNA detection in extragenital cancers, although the etiological involvement of HPV in those malignancies is still controversial [3]. The aim of the present study was to investigate a possible presence of HPV DNA in breast cancer tissue in a population of Turkish women and discuss the results in the light of literature. In this study, 84 patients with invasive breast carcinoma as a study group and 49 patients with invasive cervical carcinoma and CIN lesions as a control group were selected from our pathology department’s archives. All of the cases were screened by nested MY/GP polymerase chain reaction (PCR) for HPV DNA in paraffin-embedded archival tissue samples. In the study group, all tested samples (84 breast carcinoma) were negative for HPV DNA (Fig. 1). In the control group (21 with invasive cervical carcinoma, 12 with CIN III, 7 with CIN II, and 9 with CIN I) 34 out of 49 (69.4%) cervical tissues were positive for the HPV DNA. Di Lonardo et al. were first to report the relationship between HPV and breast cancer in 1992. They investigated the presence of HPV DNA on paraffin-embedded tissue sections using PCR and in situ hybridization techniques. By PCR, they detected HPV 16 DNA in breast carcinomas, with a frequency of 29.4% positive cases. However, they were unable to identify any HPV DNA sequences by in situ hybridization [4]. Since then, some other studies which looked for HPV DNA in breast carcinoma by PCR were published and the prevalence of HPV DNA in breast carcinomas was found to be varied between 4 and 86% [5–7]. To date, the mechanism by which the virus reaches the breast has not been clearly identified. However, there are two proposed mechanisms that HPVs are transmitted to the breast, mechanical and systemic pathways [5, 7]. de Villiers et al. detected the viruses in both the nipple and the areolar tissue in patients with breast carcinoma. Their study is consistent with the mechanical pathway involving transfer of the virus in a retrograde fashion via the nipple, areola, lactiferous ducts, and sinuses [6]. On the other hand, Widschwendter et al. found the same type of HPV DNA positivity in breast carcinomas, as well as in regional lymph node metastases of the patients with invasive cervical cancer, and they suggested oncogenic HPV DNA might be transported from an original site of infection to the breast tissue by the blood or lymphatic system in a systemic pathway [8]. However, some other studies from different countries were failed to demonstrate the presence of HPV DNA in D. Yavuzer (&) N. Karadayi Department of Pathology, Dr. Lutfi Kirdar Kartal Training and Research Hospital, Cevizli, 34865 Istanbul, Turkey e-mail: dilekyavuzer@yahoo.com

Telbivudine in the treatment of chronic hepatitis B

The treatment of chronic hepatitis B virus (HBV) infection has been revolutionized in the past decade by the increased availability of effective antiviral agents. Telbivudine is an L-nucleoside that is structurally related to lamivudine and has recently been approved for use in patients with chronic HBV infection. Telbivudine is highly selective for HBV DNA and inhibits viral DNA synthesis with no effect on human DNA or other viruses. This article reviews the pharmacology, pharmacokinetics, therapeutic efficacy and safety of telbivudine, and discusses its place in the current armamentarium against HBV. Relevant publications were identified from searches of Medline and PubMed between 2000 and 2008, using the search terms "hepatitis B/HBV," "telbivudine/LdT," "beta-L-thymidine," "pharmacokinetics," "safety," "adverse events," and "resistance." The reference lists of retrieved articles were searched for relevant studies. Phase 3 clinical studies demonstrate that telbivudine is superior to lamivudine over a 2-year period in hepatitis B e-antigen (HBeAg)-positive and HBeAg-negative patients. Telbivudine was associated with a statistically significantly greater reduction in HBV DNA, greater proportion of alanine aminotransferase normalization, and greater histological response than lamivudine. Furthermore, telbivudine use resulted in fewer cases of treatment failure and less virological resistance than lamivudine. However, after 2 years of therapy, telbivudine resistance was appreciable (25%) and considerably higher than that seen with other new antivirals such as tenofovir and entecavir. Overall, telbivudine was found to be safe, although grade 3 or 4 adverse events, including elevations in creatine kinase, were more commonly found in patients receiving telbivudine than lamivudine. Telbivudine is not active against lamivudine-resistant HBV. Telbivudine is a new antiviral agent joining the armamentarium against HBV. It is superior to lamivudine in terms of therapeutic response and resistance profile. However, concerns about resistance with long-term use, along with inferior cost-effective analyses, have relegated telbivudine to a second-line agent in the management of chronic HBV infection.

Primase-based whole genome amplification

In vitro DNA amplification methods, such as polymerase chain reaction (PCR), rely on synthetic oligonucleotide primers for initiation of the reaction. In vivo, primers are synthesized on-template by DNA primase. The bacteriophage T7 gene 4 protein (gp4) has both primase and helicase activities. In this study, we report the development of a primase-based Whole Genome Amplification (pWGA) method, which utilizes gp4 primase to synthesize primers, eliminating the requirement of adding synthetic primers. Typical yield of pWGA from 1 ng to 10 ng of human genomic DNA input is in the microgram range, reaching over a thousand-fold amplification after 1 h of incubation at 37°C. The amplification bias on human genomic DNA is 6.3-fold among 20 loci on different chromosomes. In addition to amplifying total genomic DNA, pWGA can also be used for detection and quantification of contaminant DNA in a sample when combined with a fluorescent reporter dye. When circular DNA is used as template in pWGA, 108-fold of amplification is observed from as low as 100 copies of input. The high efficiency of pWGA in amplifying circular DNA makes it a potential tool in diagnosis and genotyping of circular human DNA viruses such as human papillomavirus (HPV).

Epstein-Barr Virus Nuclear Antigen 3C Interacts with and Enhances the Stability of the c-Myc Oncoprotein

Epstein-Barr virus (EBV) was the first human DNA virus to be associated with cancer. Its oncogenic potential was further demonstrated by its ability to transform primary B lymphocytes in vitro. EBV nuclear antigen 3C (EBNA3C) is one of a small subset of latent antigens critical for the transformation of human primary B lymphocytes. Although EBNA3C has been shown to modulate several cellular functions, additional targets involved in cellular transformation remain to be explored. EBNA3C can recruit key components of the SCF(Skp2) ubiquitin ligase complex. In this report, we show that EBNA3C residues 130 to 190, previously shown to bind to the SCF(Skp2) complex, also can strongly associate with the c-Myc oncoprotein. Additionally, the interaction of EBNA3C with c-Myc was mapped to the region of c-Myc that includes the highly conserved Skp2 binding domain. Skp2 has been shown to regulate c-Myc stability and also has been shown to function as a coactivator of transcription for c-Myc target genes. We now show that the EBV latent oncoprotein EBNA3C can stabilize c-Myc and that the recruitment of both c-Myc and its cofactor Skp2 to c-Myc-dependent promoters can enhance c-Myc-dependent transcription. This same region of EBNA3C also recruits and modulates the activity of retinoblastoma and p27, both major regulators of the mammalian cell cycle. The inclusion of c-Myc in the group of cellular targets modulated by this domain further accentuates the importance of these critical residues of EBNA3C in bypassing the cell cycle checkpoints.

High prevalence of human Torque teno virus in streams crossing the city of Manaus, Brazilian Amazon

Torque teno virus (TTV) is a human DNA virus chronically infecting most healthy individuals worldwide and can be transmitted by faecal-oral route. The occurrence of TTV was evaluated in the streams crossing the city of Manaus (Brazilian Amazon) over a 1-year period, four times a year. Fifty-two water samples were collected from 13 different locations. Viruses were concentrated from two litres of water by adsorption to negative membrane filters followed by ultrafiltration. TTV DNA was detected by PCR assays designed to detect all five TTV genomic groups. By conventional PCR, 19/52 (37%) samples were positive. By real-time PCR, TTV DNA could be detected in 48/52 (92%) samples. Viral loads ranged from 1300 to 746 000 genome equivalent per 100 ml of river water. Eleven distinct nucleotide sequences were obtained. Our results show the wide distribution and diversity of TTV among Manaus urban micro basins. The data presented here may contribute to substantiate TTV as a sensitive indicator of human contamination.

Regulation of Telomerase and Telomeres: Human Tumor Viruses Take Control

Human tumor viruses are responsible for one-fifth of all cancers worldwide. These viruses have evolved multiple strategies to evade immune defenses and to persist in the host by establishing a latent infection. Proliferation is necessary for pretumor cells to accumulate genetic alterations and to acquire a transformed phenotype. However, each cell division is associated with a progressive shortening of the telomeres, which can suppress tumor development by initiating senescence and irreversible cell cycle arrest. Therefore, the ability of virus-infected cells to circumvent the senescence program is essential for the long-term survival and proliferation of infected cells and the likelihood of transformation. We review the multiple strategies used by human DNA and RNA tumor viruses to subvert telomerase functions during cellular transformation and carcinogenesis. Epstein-Barr virus, Kaposi sarcoma-associated herpesvirus, human papillomavirus, hepatitis B virus, hepatitis C virus, and human T-cell leukemia virus-1 each can increase transcription of the telomerase reverse transcriptase. Several viruses appear to mediate cis-activation or enhance epigenetic activation of telomerase transcription. Epstein-Barr virus and human papillomavirus have each developed posttranscriptional mechanisms to regulate the telomerase protein. Finally, some tumor virus proteins can also negatively regulate telomerase transcription or activity. It is likely that, as future studies further expose the strategies used by viruses to deregulate telomerase activity and control of telomere length, novel mechanisms will emerge and underscore the importance of increased telomerase activity in sustaining virus-infected cells and its potential in therapeutic targeting.

MicroRNAs and viral infections

RNA silencing plays an important role in development through the action of micro (mi) RNAs that fine tune the expression of a large portion of the genome. But, in plants and insects, it is also a very important player in innate immune responses, especially in antiviral defense. It is now well established that the RNA silencing machinery targets plant as well as insect viruses. As the genetic basis underlying this defense mechanism in these organisms starts being elucidated, much less is known about the possible antiviral role ofRNAsilencing in mammals. No small interferingRNAs (siRNAs) of viral origin can be detected in human cells infected withRNAviruses. On the contrary, viral smallRNAs can be found in cells infected by the DNA virus Epstein-Barr, but they rather resemble miRNAs. This primary observation has been extended to other members of the herpesvirus family as well as other DNA viruses such as the polyomavirus SV40. These findings indicate that, rather than being targeted by RNA silencing, human DNA viruses seem to have evolved their own miRNAs to modulate the expression of their own genes or of their host's.

Micro ARN et infections virales chez les mammifères

RNA silencing plays an important role in development through the action of micro (mi) RNAs that fine tune the expression of a large portion of the genome. But, in plants and insects, it is also a very important player in innate immune responses, especially in antiviral defense. It is now well established that the RNA silencing machinery targets plant as well as insect viruses. While the genetic basis underlying this defense mechanism in these organisms starts being elucidated, much less is known about the possible antiviral role of RNA silencing in mammals. In order to identify siRNAs coming from viruses in infected human cells, small RNAs from cells infected with RNA viruses, such as hepatitis C virus, yellow fever virus or HIV-1, were cloned and sequenced, but no virus-specific siRNAs could be detected. On the contrary, viral small RNAs were found in cells infected by the DNA virus Epstein-Barr. A closer look at these revealed that they were not siRNAs, but rather resembled miRNAs. This finding indicated that, rather than being targeted by RNA silencing, human DNA viruses seem to have evolved their own miRNAs to modulate the expression of host genes. This primary observation has been extended to other members of the herpesvirus family as well as other DNA viruses such as the polyomavirus SV40. Viral miRNAs have the potential to act both in cis to regulate expression of viral genes, or in trans on host genes. There are good indications for the cis mode of action, but the identification of cellular targets of these small viral regulators is only in its infancy.

Inhibition of Simian Virus 40 Large T Antigen Helicase Activity by Fluoroquinolones

Fluoroquinolones represent a potent group of antibiotics that inhibit bacterial DNA replication by targeting the essential bacterial enzymes gyrase and topoisomerase IV. Inhibition of gyrase activity by quinolones involves the interaction of these drugs with the helicase component of bacterial gyrase. DNA tumour viruses also encode helicases that are essential for their DNA replication in the host. In this study we have evaluated the effect of fluoroquinolones on viral DNA replication using the DNA tumour virus Simian virus 40 (SV40) as our model. Four different fluoroquinolones, namely, levofloxacin, trovafloxacin, ciprofloxacin and ofloxacin, were tested for their ability to inhibit viral DNA replication. We show here that all four quinolones tested were effective in the inhibition of SV40 plaque formation and DNA replication in CV1-P cells. In addition, we found that each of these quinolones was inhibitory to the helicase activity of SV40 large tumour antigen. Fluoroquinolones and their derivates may therefore be useful in the treatment and/or prevention of infection by SV40-homologous human DNA viruses that encode helicase activity for their survival.

Compositional Bias and Size of Genomes of Human DNA Viruses

Genomes of 144 human DNA viruses were analyzed in the aspect of their compositional asymmetry. DNA viruses were divided into two groups according to their genome sizes. The analysis revealed that the level of guanine and cytosine (GC content) in the coding sequences of small genome DNA viruses was significantly lower than that of large genome DNA viruses. Because small genome viruses replicate their genomes using cellular enzymes, while large genome viruses use their own enzymes for genome replication, the two groups of viruses may be under different mutational bias and/or selection pressure. In these viruses, GC content at the third codon position correlated with GC content at the first and second codon position. However, the relationship in small genome DNA viruses was weaker than that in large genome DNA viruses, suggesting that their genome composition may be more strongly influenced by codon usage preference or restriction on amino acid composition.

Molecular Detection of Transfusion Transmitted Virus Coinfection With Some Hepatotropic Viruses

A novel human DNA virus was isolated from the serum of a patient with posttransfusion hepatitis; it was named transfusion transmitted virus (TTV). To ascertain the influence of TTV (detected by polymerase chain reaction amplification of a conserved region of the viral genome) coinfection in individuals infected with hepatitis viruses (A, B, and C) and to investigate the putative role played by TTV in hepatic dysfunction in individuals with acute non-A-E hepatitis. Sixty-two patients with viral hepatitis were included in the study in addition to 18 blood donors. Viral study of 4 hepatotropic viruses (A, B, C, and E) was carried out. Study for TTV DNA was performed by nested polymerase chain reaction. The prevalence of TTV was not statistically different between hepatitis patients and blood donors, and it was not correlated to the levels of the hepatic aspartate aminotransferase and alanine aminotransferase between individuals evidencing dual infection with hepatitis B and C viruses and healthy blood donors. However, in the group of patients with viral hepatitis of unknown etiology (non-A-E), those evidencing TTV viremia had statistically significant lower levels of alanine aminotransferase (P = .03) and aspartate aminotransferase (P = .04) than those who were TTV negative. We can conclude that TTV is a frequent virus isolated from patients with various types of viral hepatitis, from cases of hepatitis without obvious viral agent, and from the healthy population. TTV has no effect on biochemical markers of associated viral hepatitis. It may be associated with a mild form of non-A-E hepatitis.

Lactoferrin inhibits early steps of human BK polyomavirus infection

Lactoferrin, a member of the transferrin family, is a bi-globular iron binding glycoprotein, found in milk, exocrine secretions of mammals, and in secondary granules of polymorphonuclear neutrophiles that plays an important role in the defence against various pathogenic microorganisms. Previous studies in different virus-cell systems showed that lactoferrin is a potent inhibitor of different enveloped and naked virus infection. In this research we studied the effect of lactoferrin on BK polyomavirus, a human naked double-stranded DNA virus responsible for productive, persistent, and latent infections of the urinary tract. Results obtained demonstrate that lactoferrin treatment prevents early steps of BK virus infection in Vero cells, at the level of the adsorption phase, probably through the interaction with capsidic structures, although a lactoferrin-BK virus competition for cell plasma-membrane receptors cannot be ruled out.

Hepatitis B virus genetic diversity

Hepatitis B virus (HBV) is a human DNA virus, which replicates through an RNA intermediate because of the reverse-transcriptase (RT) activity of its DNA polymerase. As a result, the mutation rate for HBV is higher than the rate observed for most DNA viruses. HBVs are classified into genotypes based on genomic sequencing, and antigenic subtypes based on the antigenic properties of its major surface glycoprotein, the HBV surface antigen (HBsAg). Subgenotypes have been identified within most of the HBV genotypes. The HBV groups defined by the different genotype-HBsAg subtype associations found over the world display characteristic geographical distributions, reflecting the movements of human populations and other epidemiologically significant events. Such HBV groups constitute genetically stable viral populations sharing a common evolutionary history, but additional stable changes, originating from mutation and mutant selection, are observed within all of them. These viral sub-populations are known as the HBV variants, and some of which have medical and public health relevance. Pre-core (pre-C) defective variants have been shown to make HBV infection much less susceptible to interferon treatment, and treatment failures with other antiviral drugs have been associated with selection of resistant variants that display specific mutations in the genome region encoding the viral RT activity. Since the RT region of the genome overlaps the sequence encoding the HBsAg molecule, selection of drug resistant variants involves, in some cases, the indirect selection of HBsAg variants. Viral variants displaying changes in HBsAg seem to be very common among chronic HBV carriers; and some of these variants may emerge under the pressure of the neutralizing antibody response, leading to vaccine resistance and resistance to immunotherapy. Mutations conferring resistance to immunotherapy are noted often among liver transplant recipients and among babies born to HBV-carrier mothers. In addition, some of these HBsAg variants have been associated with lack of detection by HBsAg tests used for the diagnosis of HBV infection, for the identification of chronic carriers, for screening of blood donations for transfusion, and in the manufacture of therapeutic blood products.