Search

A mixed infection of a single tick or host by Lyme disease spirochetes is common and a unique challenge for the diagnosis, treatment, and surveillance of Lyme disease. Here, we describe a novel protocol for differentiating Lyme strains on the basis of deep sequencing of the hypervariable outer surface protein C locus (ospC). Improving upon the traditional DNA-DNA hybridization method, the next-generation sequencing-based protocol is high throughput, quantitative, and able to detect new pathogen strains. We applied the method to more than one hundred infected Ixodes scapularis ticks collected from New York State, USA, in 2015 and 2016. An analysis of strain distributions within individual ticks suggests an overabundance of multiple infections by five or more strains, inhibitory interactions among coinfecting strains, and the presence of a new strain closely related to Borreliella bissettiae. A supporting bioinformatics pipeline has been developed. The newly designed pair of universal ospC primers target intergenic sequences conserved among all known Lyme pathogens. The protocol could be used for culture-free identification and quantification of Lyme pathogens in wildlife and potentially in clinical specimens.

Mixed infection of a single tick or host by Lyme disease spirochetes is common and a unique challenge for diagnosis, treatment, and surveillance of Lyme disease. Here we describe a novel protocol for differentiating Lyme strains based on deep sequencing of the hypervariable outer-surface protein C locus (ospC). Improving upon the traditional DNA-DNA hybridization method, the next-generation sequencing-based protocol is high-throughput, quantitative, and able to detect new pathogen strains. We applied the method to over one hundred infectedIxodes scapularisticks collected from New York State, USA in 2015 and 2016. Analysis of strain distributions within individual ticks suggests an overabundance of multiple infections by five or more strains, inhibitory interactions among co-infecting strains, and presence of a new strain closely related toBorreliella bissettiae. A supporting bioinformatics pipeline has been developed. With the newly designed pair of universalospCprimers targeting intergenic sequences conserved among all known Lyme pathogens, the protocol could be used for culture-free identification and quantification of Lyme pathogens in wildlife and clinical specimens across the globe.

Helioxanthin is a natural product that inhibits the replication of a number of viruses. We found that a previously undescribed helioxanthin analogue, 8-1, exhibited potent anti-hepatitis B virus (HBV) activity with little cytotoxicity. 8-1 suppressed both HBV RNA and protein expression, as well as DNA replication of both wild-type and 3TC-resistant virus. Time-course analyses revealed that RNA expression was blocked first after treatment with 8-1, followed by viral proteins, and then DNA. 8-1 inhibited the activity of all HBV promoters by decreasing the binding of hepatocyte nuclear factor 4 (HNF-4), HNF-3, and fetoprotein factor to the precore/core promoter enhancer II region. The amount of HNF-4 and HNF-3 was decreased posttranscriptionally by 8-1 in HBV-producing cells, but not in HBV-negative cells. Therefore, 8-1 suppresses HBV replication by posttranscriptional down-regulation of critical transcription factors in HBV-producing cells, thus diminishing HBV promoter activity and blocking viral gene expression and replication. This mechanism is unique and different from other anti-HBV compounds previously described.

MPA [the active metabolite of the immuno-suppressive agent CellCept] and ribavirin markedly potentiate the anti-HBV activity of the guanine-based nucleoside analogue entecavir (ETV) against both wild-type HBV and a lamivudine-resistant variant. Ribavirin (in its 5'-monophosphate form) and MPA are inhibitors of IMP-dehydrogenase and cause depletion of intracellular dGTP pools. The active triphosphorylated form of ETV may inhibit more efficiently the priming reaction, reverse transcription and DNA-dependent DNA polymerase activity of the HBV polymerase in the presence of reduced levels of dGTP. The potential for enhanced ETV activity is supported by the observation that exogenously added deoxyguanosine reversed the potentiating effect of ribavirin and MPA. Our observations may have important implications for those (liver) transplant recipients that receive MMF as part of their immunosuppressive regimen and who, because of a de novo or a persistent infection with HBV need antiviral therapy such as ETV. Further studies will need to be conducted to determine if combining ribavirin (a compound used for the treatment of HCV infections) with ETV could have an advantage for the treatment of HBV infections, in particular in patients co-infected with HCV.

All the approved chemotherapeutic drugs for the treatment of HBV hepatitis are nucleoside analogs targeting on HBV DNA polymerase. Drugs targeting on other viral unique targets are needed. A new class of chemicals with novel action against HBV replication was discovered. A brief description of their mode of action is given.

Three acyclic nucleoside phosphonates (ANPs) have been formally approved for clinical use in the treatment of 1) cytomegalovirus retinitis in AIDS patients (cidofovir, by the intravenous route), 2) chronic hepatitis B virus (HBV) infections (adefovir dipivoxil, by the oral route), and 3) human immunodeficiency virus (HIV) infections (tenofovir disoproxil fumarate, by the oral route). The activity spectrum of cidofovir {(S)- 1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine [(S)-HPMPC)]}, like that of (S)-HPMPA [(S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine) and (S)-HPMPDAP [(S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-2, 6-diaminopurine), encompasses a broad spectrum of DNA viruses, including polyoma-, papilloma-, adeno-, herpes-, and poxviruses. Adefovir {9-[2-(phosphonomethoxy)ethyl]adenine (PMEA)} and tenofovir [(R)-9-[2-(phosphonomethoxy) propyl]adenine [(R)-PMPA)]} are particularly active against retroviruses (ie., HIV) and hepadnaviruses (ie., HBV); additionally, PMEA also shows activity against herpes- and poxviruses. We have recently identified a new class of ANPs, namely 6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidines, named, in analogy with their alkylpurine counterparts, HPMPO-DAPy, PMEO-DAPy, and (R)-PMPO-DAPy. These compounds exhibit an antiviral activity spectrum and potency that is similar to that of (S)-HPMPDAP, PMEA, and (R)-PMPA, respectively. Thus, PMEO-DAPy and (R)-PMPO-DAPy, akin to PMEA and (R)-PMPA, proved particularly active against HIV- 1, HIV-2, and the murine retrovirus Moloney sarcoma virus (MSV). PMEO-DAPy and (R)-PMPO-DAPy also showed potent activity against both wild-type and lamivudine-resistant strains of HBV. HPMPO-DAPy was found to inhibit different poxviruses (ie., vaccinia, cowpox, and orf) at a similar potency as cidofovir. HPMPO-DAPy also proved active against adenoviruses. In vivo, HPMPO-DAPy proved equipotent to cidofovir in suppressing vaccinia virus infection (tail lesion formation) in immunocompetent mice and promoting healing of disseminated vaccinia lesions in athymic-nude mice. The 6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidines offer substantial potential for the treatment of a broad range of retro-, hepadna-, herpes-, adeno-, and poxvirus infections.

The use of digoxigenin-labelled probes was studied for quantitation of HBV-DNA during antiviral drug evaluation. Digoxigenin (dig)-labelled probes were generated either via incorporation of dig-dUTP in a polymerase chain reaction (PCR) or a random priming reaction. Using the PCR-labelled probe (delineating a 523 bp fragment in the core gene of the HBV) as little as 1 pg of immobilized HBV-DNA could be detected following an 8 h exposure of the hybridized membrane. A close correlation (r = 0.95) was found between the amount of HBV-DNA (range 2.5-200 pg) and the signal generated by the probe hybridized to its target DNA. By using a probe that was labelled with digoxigenin via random priming, the minimal quantity of immobilized HBV plasmid DNA that could be detected following an 8 h exposure was 4 pg, whereas a 32P-labelled probe, generated in parallel by random priming, allowed the detection of 16 pg of HBV plasmid DNA following a 4-day exposure. The PCR-generated digoxigenin-labelled probe proved to be useful for antiviral drug evaluation, i.e. to detect HBV-DNA in total cellular DNA from HBV-positive hepatoma cells (HepG2.2.15) that had either been treated with reference antiviral agents or left untreated. The 50% effective concentrations (EC50) that were calculated for inhibition of HBV-DNA production by lamivudine (3TC), penciclovir (PCV), lobucavir (LBV), adefovir (PMEA) and tenofovir (PMPA) were comparable to those reported in the literature. The use of digoxigenin-labelled probes thus appears to be a simple, convenient, rapid, reliable and non-radioactive method for use for anti-HBV screening. In addition, and in contrast to 32P-labelled probes, digoxigenin-labelled probes can be stored for >1 year without loss of specific activity, which makes these probes particularly attractive for large-scale antiviral drug evaluation purposes.

Objective: X-ray cross-complementing groups 1 (XRCC1) rs1799782 C>T polymorphisms and colorectal cancer susceptibility were not clear. The purpose of this study was to evaluate the association between XRCC1 rs1799782 C>T polymorphisms and colorectal cancer susceptibility by meta-analysis. Materials and Methods: Related databases of Medline, CNKI, and Wanfang were systematic searched for the studies related to XRCC1 rs1799782 C>T polymorphisms and colorectal cancer risk in Chinese Han population. The genotype distribution of CC, CT and TT were extracted from each included studies in the colorectal cancer patients and healthy control subjects. The odds ratio (OR) and its 95% confidence interval (95% CI) was used to assess the correlation between genetype and colorectal cancer risk. The publications for this study was evaluated by Begg's funnel plot and Egger's line regression test. Results: The median frequency of CC, CT, and TT genotype in cancer group were 48%, 41% and 11%; For control group, they were 51%, 40% and 8%; the pooled results showed that OR = 1.32 (95% CI: 1.041–1.67, P < 0.05). The pooled results indicated that XRCC1 rs1799782 C>T polymorphisms was associated with colorectal cancer susceptibility in recessive genetic model OR = 1.32 (95% CI: 1.041–1.67, P < 0.05), dominant genetic model OR = 1.21 (95% CI: 1.00–1.46, P < 0.05) and homozygous genetic model OR = 1.43 (95% CI: 1.07–1.91, P < 0.05). The funnel plot was significant asymmetric at the bottom and the Egger's test also indicated significant publication bias (t = 2.43, P = 0.04) for recessive genetic model. But, no publication bias was found in dominant and homozygous model (P > 0.05). Conclusion: Chinese Han people with rs1799782 TT/CT genotype of XRCC1 gene may have increased risk of developing colorectal.

Small interfering RNA (siRNA) is a powerful tool to silence gene expression in mammalian cells including genes of viral origin. To evaluate the therapeutic efficacy of siRNA against the hepatitis B virus (HBV), we studied the effect of transfection of the HBV-inducible cell lines HepAD38 and HepAD79 with siRNA specific for the core gene of the HBV genome. HepAD38 cells produce wild-type HBV, whereas HepAD79 cells produce the lamivudine resistant YMDD variant. Transfection of HepAD38 cells with either 1.6 or 4 microg/ml siRNA resulted in a profound inhibition (72% and 98%, respectively) of viral replication (as assessed by real-time quantitative PCR). The inhibitory effect was corroborated by a marked reduction of HBV core protein synthesis in induced HepAD38 cells. In HepAD79 cells, transfected with 1.6 or 4 microg/ml HBV-specific siRNA, virus production was reduced by 75% and 89%, respectively.

Background: Current approved anti-HBV treatment cannot completely eliminate HBV infection, and emergence of resistant virus is an important treatment issue. Effective anti-HBV agents with different mechanisms of action on novel target sites are needed for the treatment of HBV infection and for combating the resistant virus, alone or in combination with current anti-HBV strategies. Helioxanthin analogue 8-1 displayed potent anti-HBV activity in human HBV in vitro and in animal models, with a unique antiviral mechanism. Its antiviral activity in other HBV system needs further study. Methods: The anti-duck hepatitis B virus (DHBV) activity of 8-1, an analogue of a natural product, helioxanthin, was studied in the DHBV inducible cell line, dstet5, in comparison to and in combination with the nucleoside analogue, lamivudine (3TC). Results: Helioxanthin analogue 8-1 exhibited anti-DHBV activity as demonstrated by quantification of viral DNA, RNA, covalently closed circular DNA and protein synthesis. Analogue 8-1 did not affect the stability of cellular macromolecules and did not have a sustained antiviral effect after drug removal. When DHBV replication was induced, virus-harbouring cells were more susceptible to the cytotoxicity of 8-1 than non-induced cells. Conclusions: 8-1 exhibited effective inhibition on DHBV replication. The combination of 8-1 with 3TC resulted in additional anti-DHBV activity. Viral induced cells displayed higher susceptibility to 8-1 treatment than non-induced cells. HBV X protein might not be an essential factor in the initiation of the biological activity of 8-1, as demonstrated by its absence in DHBV. These findings warrant further development of 8-1 for the treatment of chronic hepatitis B and its associated diseases.

We used human hepatoma HepAD38 cells, in which HBV production is under the control of a tetracycline-regulated promotor, to investigate changes induced in the host cell by HBV replication that could contribute to malignant transformation. Parameters of oxidative stress (malondialdehyde, glutathione) and cell proliferation were determined at different times after induction (0–96 h). In HBV-producing cells, the redox status peaked at 72 h. cDNA micro array analysis at 72 h post induction revealed 3 groups of genes that were up-regulated by HBV: (i) heat shock proteins, (ii) oxidative and metabolic stress and (iii) growth and apoptosis related genes. Continuous HBV production did not accelerate karyotypic changes in cells cultured for 4 months (18 passages). In conclusion: HBV replication modulates host gene expression and induces oxidative stress. In this HepAD38 model early events (0–4 days) in the host cell after induction of HBV replication can be studied under strictly defined conditions.

Novel acyclic nucleoside phosphonates with a pyrimidine base preferentially containing an amino group at C-2 and C-4 and a 2-(phosphonomethoxy)ethoxy or ( R )-2-(phosphonomethoxy)propoxy group at C-6 selectively inhibit the replication of wild-type and lamivudine-resistant hepatitis B viruses. The activity of the most potent compounds was comparable to that of adefovir.

The initial step during hepatitis B virus (HBV) infection is the specific attachment of the virus to the hepatocyte. Here we studied whether the binding of HBV to hepatocytes can, as is the case with most other enveloped viruses, be blocked by polyanionic compounds. Viral particles produced by HepAD38 cells were used as inoculum and HBV-negative HepG2 cells, as well as primary human hepatocytes, as target cells. Three sulphated polymers, that is, PAVAS (a co-polymer of acrylic acid with vinyl alcohol sulphate), heparin and dextran sulphate (DS) (MW 5000), and the sulphonated polymer PAMPS [poly(2-acryl-amido-2-methyl-1-propanesulfonic acid] (MW approximately 7000-12000), proved strong inhibitors of the binding of HBV to HepG2 cells and primary hepatocytes. The 50% effective concentration (EC50) for inhibition of HBV binding to HepG2 cells by PAVAS, heparin, DS and PAMPS was 1.3 microg/ml, 1.6 microg/ml, 1.8 microg/ml and 3.3 microg/ml, respectively, and to primary hepatocytes 1.6 microg/ml (PAVAS), 1.6 microg/ml (heparin), 2.6 microg/ml (DS) and 4.1 microg/ml (PAMPS). These values are in the same range as the concentrations required for these compounds to prevent such viruses as herpesviruses and HIV from binding to cells. These findings may be helpful in elucidating the mechanism of the initial interaction of HBV with hepatocytes.

Several guanosine analogues, i.e. acyclovir (and its oral prodrug valaciclovir), penciclovir (in its oral prodrug form, famciclovir) and ganciclovir, are widely used for the treatment of herpesvirus [i.e. herpes simplex virus type 1 (HSV-1), and type 2 (HSV-2), varicella-zoster virus (VZV) and/or human cytomegalovirus (HCMV)] infections. In recent years, several new guanosine analogues have been developed, including the 3-membered cyclopropylmethyl and -methenyl derivatives (A-5021 and synguanol) and the 6-membered D- and L-cyclohexenyl derivatives. The activity of the acyclic/carbocyclic guanosine analogues has been determined against a wide spectrum of viruses, including the HSV-1, HSV-2, VZV, HCMV, and also human herpesviruses type 6 (HHV-6), type 7 (HHV-7) and type 8 (HHV-8), and hepatitis B virus (HBV). The new guanosine analogues (i.e. A-5021 and D- and L-cyclohexenyl G) were found to be particularly active against those viruses (HSV-1, HSV-2, VZV) that encode for a specific thymidine kinase (TK), suggesting that their antiviral activity (at least partially) depends on phosphorylation by the virus-induced TK. Marked antiviral activity was also noted with A-5021 against HHV-6 and with D- and L-cyclohexenyl G against HCMV and HBV. The antiviral activity of the acyclic/carbocyclic nucleoside analogues could be markedly potentiated by mycophenolic acid, a potent inhibitor of inosine 5'-monophosphate (IMP) dehydrogenase. The new carbocyclic guanosine analogues (i.e. A-5021 and D- and L-cyclohexenyl G) hold great promise, not only as antiviral agents for the treatment of herpesvirus infections, but also an antitumor agents for the combined gene therapy/chemotherapy of cancer, provided that (part of) the tumor cells have been transduced by the viral (HSV-1, VZV) TK gene.

The novel nucleoside analog (1 S ′,2 R ′)-9-[[1′,2′-bis(hydroxymethyl)cycloprop-1-yl]methyl]guanine (A-5021) was previously shown to be a potent inhibitor of the replication of herpes simplex virus type 1 and 2 (HSV-1 and HSV-2) and varicella zoster virus (VZV), both in vitro and in vivo (J. Med. Chem. 41, 1284–1298; Antimicrob. Agents Chemother. 42, 1666–1670). Here we demonstrate that A-5021 is also a potent inhibitor of Epstein-Barr virus (EBV) and human herpes virus 6 (HHV-6A and HHV-6B), but that the compound lacks activity against HHV-8. A-5021, in comparison to acyclovir, was also assessed for protective activity against HSV-1-induced mortality in SCID mice. The compounds were administered at 50 mg/kg per day by subcutaneous injection for four consecutive days and treatment was initiated at either 2 h, 1 or 2 days post infection (p.i.). When administered from day 0 to 4 p.i., A-5021 conferred complete protection against the infection (as assessed at 22 days p.i.), whereas acyclovir delayed virus induced mortality by only 5 days. When treatment was begun on day 1 or 2, A-5021 still afforded marked protection against the infection, whereas acyclovir was virtually devoid of any activity under these conditions. Our data underline that A-5021 may offer great promise for the treatment of herpesvirus infections.

Mycophenolic acid [the active metabolite of the immunosuppressive agent mycophenolate mofetil (MMF)] and ribavirin were found to potentiate the anti-HBV activity of the guanine-based nucleoside analogues penciclovir (PCV), lobucavir (LBV) and 3'-fluorodideoxyguanosine (FLG) and diaminopurine dioxolane (DAPD). Ribavirin and mycophenolic acid are both inhibitors of inosine 5'-monophosphate dehydrogenase and cause a depletion of intracellular dGTP levels. It may be assumed that the 5'-triphosphorylated derivatives of the guanine-based nucleoside analogues, in the presence of reduced levels of dGTP, inhibit more efficiently the priming reaction as well as the reverse transcription and DNA-dependent DNA polymerase activity of the HBV polymerase. This assumption is corroborated by the observation that exogenously added guanosine reversed the potentiating effect of ribavirin and mycophenolic acid on the anti-HBV activity of the guanosine analogues. Our observations may have implications for those (liver) transplant recipients that receive MMF as (part of their) immunosuppressive regimen and that, because of de novo or persistent infection with HBV, need specific anti-HBV therapy.