Virus Breakthrough Research Articles

Transport and removal of viruses in saturated sand columns under oxic and anoxic conditions – Potential implications for groundwater protection

To protect groundwater as a drinking water resource from microbiological contamination, protection zones are installed. While travelling through these zones, concentrations of potential pathogens should decline to levels that pose no risks to human health. Removal of viruses during subsurface passage is influenced by physicochemical conditions, such as oxygen concentration, which also affects virus survival. The aim of our study was to evaluate the effect of redox conditions on the removal of viruses during sand filtration. Experiments in glass columns filled with medium-grained sand were conducted to investigate virus removal in the presence and absence of dissolved oxygen. Bacteriophages MS2 and PhiX174, as surrogates for human enteric viruses were spiked in pulsed or in continuous mode and pumped through the columns at a filter velocity of about 1m/d. Virus breakthrough curves were analyzed by calculating total viral elimination and fitted using one-dimensional transport models (CXTFIT and HYDRUS-1D). While short-term experiments with pulsed virus application showed only small differences with regard to virus removal under oxic and anoxic conditions, a long-term experiment with continuous dosing revealed a clearly lower elimination of viruses under anoxic conditions. These findings suggest that less inactivation and less adsorption of viruses in anoxic environments affect their removal. Therefore, in risk assessment studies aimed to secure drinking water resources from viral contamination and optimization of protection zones, the oxic and anoxic conditions in the subsurface should also be considered.

Meeting report-workshop on spike characterizations and virus removal by filtration: trends and new developments.

This workshop was held on June 3, 2013, in Berlin, Germany, in conjunction with the PDA Virus & TSE Safety Forum 2013. A total of nine speakers presented on key considerations of virus filtration, including a historical overview and emerging trends in evaluating parvovirus filters. Several talks addressed understanding the mechanism of virus capture and breakthrough by filters, as well as addressing this risk by carefully controlling transmembrane pressure. Improvements to validation studies were proposed via the use of highly purified virus preparations, more relevant models such as Chinese Hamster Ovary retrovirus-like particles, as well as new assays for virus quantification. The workshop ended with a panel discussion covering a range of topics including virus breakthrough, up-stream media treatment, virus spike preparation quality control, and regulatory expectations.

Genetic and antigenic typing of seasonal influenza virus breakthrough cases from a 2008-2009 vaccine efficacy trial.

Estimations of the effectiveness of vaccines against seasonal influenza virus are guided by comparisons of the antigenicities between influenza virus isolates from clinical breakthrough cases with strains included in a vaccine. This study examined whether the prediction of antigenicity using a sequence analysis of the hemagglutinin (HA) gene-encoded HA1 domain is a simpler alternative to using the conventional hemagglutination inhibition (HI) assay, which requires influenza virus culturing. Specimens were taken from breakthrough cases that occurred in a trivalent influenza virus vaccine efficacy trial involving >43,000 participants during the 2008-2009 season. A total of 498 influenza viruses were successfully subtyped as A(H3N2) (380 viruses), A(H1N1) (29 viruses), B(Yamagata) (23 viruses), and B(Victoria) (66 viruses) from 603 PCR- or culture-confirmed specimens. Unlike the B strains, most A(H3N2) (377 viruses) and all A(H1N1) viruses were classified as homologous to the respective vaccine strains based on their HA1 domain nucleic acid sequence. HI titers relative to the respective vaccine strains and PCR subtyping were determined for 48% (182/380) of A(H3N2) and 86% (25/29) of A(H1N1) viruses. Eighty-four percent of the A(H3N2) and A(H1N1) viruses classified as homologous by sequence were matched to the respective vaccine strains by HI testing. However, these homologous A(H3N2) and A(H1N1) viruses displayed a wide range of relative HI titers. Therefore, although PCR is a sensitive diagnostic method for confirming influenza virus cases, HA1 sequence analysis appeared to be of limited value in accurately predicting antigenicity; hence, it may be inappropriate to classify clinical specimens as homologous or heterologous to the vaccine strain for estimating vaccine efficacy in a prospective clinical trial.

Optimum soil depth for removal of coliphage viruses from treated wastewater, Kuwait

A study was carried out to determine the optimum soil depth for the removal of coliphage viruses from treated wastewater. Treated wastewater containing viruses was passed through soil columns filled with soil collected from Sulaibiya area, Kuwait. The soil column experiments were under operation for eight months during the period from August 2004 to March 2005. Three soil depths were used in these experiments. These are 0.1, 0.2, 0.4, and 0.8 m of natural Sulaibiya soil. For each experimental condition, two identical columns were set up, so that the changes of the results can be evaluated, producing total of eight columns. For all columns, the coliphage tests were studied only under alternating 1 d flooding and 1 d drying conditions. Influent and effluent coliphage samples were collected and analyzed following cycles of flooding periods. The coliphage virus count in the treated wastewater ranged between 0 and 62,800 pfu/100 ml. The laboratory results revealed that coliphage removal for Sulaibiya soil ranged between 58 and 100% for soil depth of 10 cm, and the viruses were completely removed after passing 20 cm thickness of soil. Also, the results indicated that no virus breakthrough occurred at soil depths of 40 and 80 cm.

An optimized antiviral modification strategy for prevention of hepatitis B reactivation in patients undergoing prophylactic lamivudine and chemotherapy: a pilot study

In patients receiving prophylactic lamivudine (LAM) and chemotherapy, hepatitis B virus (HBV) reactivation cannot be eliminated without knowing the latent causes and optimal management. In our previous study, virus breakthrough and relapse were highly suspected as potential virologic causes for HBV reactivation. Therefore, we reviewed 24 previous studies and 447 patients who underwent chemotherapy and prophylactic LAM, with an incidence of 7.2 % HBV reactivation. Virus breakthrough and relapse were seldom investigated in these studies. In addition, 72 patients that underwent prophylactic LAM and chemotherapy at our centers were also analyzed. Among them, eight patients developed virus breakthrough, with another nine developing virus relapse after discontinuation of LAM. Eight patients received antiviral modification, which included administration of adefovir for patients with virus breakthrough or resumption of LAM for patients with virus relapse and none of them developed HBV reactivation. In contrast, of the nine patients who did not receive antiviral modification, six developed HBV reactivation and two died. In conclusion, this study demonstrated that virus breakthrough and relapse were the critical causative factors of HBV reactivation in patients receiving chemotherapy and prophylactic LAM. An optimized antiviral modification strategy could effectively prevent HBV reactivation in patients with virus breakthrough or relapse.

Removal Efficiency and Integrity Monitoring Techniques for Virus Removal by Membrane Processes

Membranes are a mechanical form of disinfection that works by physical separation of the target pathogen. In theory, an intact membrane is a barrier to pathogens that are larger in size than the pore size of the membrane. However, in practice the pore size distribution or molecular weight cutoff will provide an indication of the separation efficiency and in many cases complete rejection of the virus is not demonstrated by various membrane processes. The general mechanism of pathogen removal by membrane processes is predominantly achieved by size exclusion, influenced by the physicochemical properties of the membrane, surface properties of the pathogens, and the solution environment. However, in order to evaluate the intrinsic performance of the membranes for a specific objective, a standard protocol with defined set of operating conditions is required. Any anomaly with the membrane surface (e.g., abnormally big pores, compromised glue line, holes) and the filtration system (e.g., compromised O-rings, broken mechanical seals) will result in microbial contamination risk of the product water. Therefore, monitoring membrane treatment system integrity is essential for the protection of public health from microbial risk. The demand for real-time monitoring is increasing as the usage of membrane-processed water is on the rise. Monitoring membrane integrity can be achieved by regular noncontinuous direct integrity testing performed on the membrane itself, or continuous indirect tests carried out on the filtrate. These monitoring techniques provide ongoing assurance that the water quality objectives are continually met. Existing integrity monitoring techniques have been shown in various instances to be reliable and sensitive for detecting contaminants of 1.0 μm and larger, while enteric virus particles are in the size range 0.01–0.04 μm. Some of the indirect monitoring techniques are capable of detecting a membrane breach less than 1 μm, but the practical challenge resolution is typically around 1–2-log. Similarly, the process control monitoring parameters lack sensitivity and therefore virus breakthrough occurs even before a loss of integrity is detected. At present it is difficult to demonstrate virus removal without conducting challenge tests due to a lack of sufficiently sensitive process monitoring. On the other hand, regulators are under increasing pressure to grant log removal credits for enteric viruses to membrane treatment processes due to the inability of present integrity tests to verify ongoing removal of virus-sized particles. Therefore, finding reliable and sensitive integrity tests for viruses is a high-priority issue in the water treatment sector. The authors aim to review the present state of knowledge on the process efficiencies of virus removal by membrane processes and the associated membrane integrity monitoring practices for virus-sized particles. Some of the knowledge gaps and research needs associated with the existing guidelines and standing protocols for integrity monitoring are also identified.

Efficacy of lamivudine and adefovir de novo combination therapy or after mono-therapy in chronic hepatitis B patients

To investigate the efficacy of 104 weeks of lamivudine (LAM) and adefovir (ADV) de novo combination therapy, as compared to optimized combination therapy administered after 48 weeks of treatment with lamivudine or adefovir mono-therapy, in chronic hepatitis B (CHB) patients. A total of 174 patients with CHB were equally divided among three treatment groups: LAM mono-therapy; ADV mono-therapy; and LAM + ADV combination therapy. The patients in the LAM + ADV group were treated with LAM plus ADV for 104 consecutive weeks. The patients in the LAM or the ADV groups were first treated for 48 weeks with LAM or ADV, respectively, after which the patient's virological response was assessed. According to the results, the patient was continued on mono-therapy or switched to combination therapy for the subsequent 56 weeks. Virological and biochemical examinations were carried out at weeks 48 and 104. The rates of undetectable HBV DNA in the LAM mono-therapy, ADV mono-therapy, and LAM-ADV combination therapy groups at week 48 were 68%, 50%, and 84%, and at week 104 were 80%, 72%, and 95%, respectively. For the same groups, the virus breakthrough rates at week 48 were 15%, 0%, and 0%, and at week 104 were 18%, 2%, and 0%, respectively. Statistical analysis showed significant differences for the rate of undetectable HBV DNA between LAM + ADV group and LAM group at week 48 (x2 = 4.473, P= 0.034) and at week 104 (x2 = 5.795, P = 0.016), LAM + ADV group and ADM group at week 48 (x2 = 14.802, P less than 0.001) and week 104 (x2 = 5.547, P = 0.001). The hepatitis B e antigen (HBeAg) seroconversion rates at week 48 were 15% (x2 = 4.543, P = 0.033), 13% (x2 = 4.035, P = 0.045) and 38%, and at week 104 were 21% (x2 = 4.438, P = 0.035), 17% (x2 = 4.223, P = 0.04) and 44%, respectively, among patients positive for HBeAg. Statistical analysis showed that the differences among the three groups for each of these parameters were statistically significant (all, P less than 0.05). When compared with LAM or ADV mono-therapy followed by LAM+ADV at week 48, the LAM plus ADV de novo combination therapy for 104 weeks provided CHB patients with better virological and serological responses and a lower drug resistance rate.

Triple Combination Antiviral Drug (TCAD) Composed of Amantadine, Oseltamivir, and Ribavirin Impedes the Selection of Drug-Resistant Influenza A Virus

Widespread resistance among circulating influenza A strains to at least one of the anti-influenza drugs is a major public health concern. A triple combination antiviral drug (TCAD) regimen comprised of amantadine, oseltamivir, and ribavirin has been shown to have synergistic and broad spectrum activity against influenza A strains, including drug resistant strains. Here, we used mathematical modeling along with three different experimental approaches to understand the effects of single agents, double combinations, and the TCAD regimen on resistance in influenza in vitro, including: 1) serial passage at constant drug concentrations, 2) serial passage at escalating drug concentrations, and 3) evaluation of the contribution of each component of the TCAD regimen to the suppression of resistance. Consistent with the modeling which demonstrated that three drugs were required to suppress the emergence of resistance in influenza A, treatment with the TCAD regimen resulted in the sustained suppression of drug resistant viruses, whereas treatment with amantadine alone or the amantadine-oseltamivir double combination led to the rapid selection of resistant variants which comprised ∼100% of the population. Furthermore, the TCAD regimen imposed a high genetic barrier to resistance, requiring multiple mutations in order to escape the effects of all the drugs in the regimen. Finally, we demonstrate that each drug in the TCAD regimen made a significant contribution to the suppression of virus breakthrough and resistance at clinically achievable concentrations. Taken together, these data demonstrate that the TCAD regimen was superior to double combinations and single agents at suppressing resistance, and that three drugs at a minimum were required to impede the selection of drug resistant variants in influenza A virus. The use of mathematical modeling with multiple experimental designs and molecular readouts to evaluate and optimize combination drug regimens for the suppression of resistance may be broadly applicable to other infectious diseases.

RNA Interference as a Tool for Exploring HIV-1 Robustness

Short interfering RNAs (siRNAs) that target viral genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, there is the potential for viral escape, particularly with a highly mutable target such as HIV-1. We present a novel strategy for anticipating and preventing viral escape using second-generation siRNAs. The evolutionary capacity of HIV-1 was tested by exerting strong selective pressure on a highly conserved sequence in the HIV-1 genome. We assayed the antiviral efficacy of five overlapping siRNAs directed against an essential region of the HIV-1 protease. Serial viral transfers in U87-CD4-CXCR4 cells were performed using four of the siRNAs. This procedure was repeated until virus breakthrough was detected . After several serial culture passages, resistant virus with a single point mutation in the targeted region was detected in the culture supernatants. The emergence of resistant virus was confirmed by molecular cloning and DNA sequencing of viral RNA. The most common escape route was the D30N mutation. Importantly, the addition of a second-generation siRNA that matched the D30N mutation restored viral inhibition and delayed development of escape variants. Passages performed with both siRNAs prevented the emergence of the D30N escape mutant and forced the virus to develop new escape routes. Thus, second-generation siRNAs can be used to block escape from RNA interference (RNAi) and to search for new RNAi escape routes. The protocol described here may be useful for exploring the sequence space available for HIV-1 evolution and for producing attenuated or deleterious viruses.

Detection of lamivudine- or adefovir-resistant hepatitis B virus mutations by a liquid array

A novel polymerase chain reaction (PCR)-Luminex assay was developed for rapid, accurate, and high-throughput detection of the most important hepatitis B virus (HBV) variants, including those with reverse transcriptase (RT) domain L180M, M204I/V, A181T/V/S, I233V and N236T mutations associated with resistance to lamivudine (LAM) or adefovir (ADV). Using mixtures of mutant and wild-type HBV, this method was sufficiently sensitive for detecting 103HBVml−1 and could detect minor mutants when they comprised 5% of the total viral population. Comparison of the PCR-Luminex assay with INNO-LiPA for detecting clinical LAM- or ADV-resistant chronic hepatitis B virus infection in 64 patients confirmed the following: the 2 methods were 97.9% (48 of 49) and 93.3% (14 of 15) concordant for detecting LAM- or ADV-resistance mutations, respectively. The agreement with direct sequencing was 70.3% (45 of 64). The PCR-Luminex assay or multi-analyte suspension array can detect simultaneously and efficiently minor populations HBV mutants early during infection in many clinical samples. It is a simple, cost-effective method for resistance surveillance or selecting appropriate antiviral agents and initiating timely rescue treatment before the development drug-resistance related virus or biochemical breakthrough.

Anticipating and blocking HIV-1 escape by second generation antiviral shRNAs.

BackgroundRNA interference (RNAi) is an evolutionary conserved gene silencing mechanism that mediates the sequence-specific breakdown of target mRNAs. RNAi can be used to inhibit HIV-1 replication by targeting the viral RNA genome. However, the error-prone replication machinery of HIV-1 can generate RNAi-resistant variants with specific mutations in the target sequence. For durable inhibition of HIV-1 replication the emergence of such escape viruses must be controlled. Here we present a strategy that anticipates HIV-1 escape by designing 2nd generation short hairpin RNAs (shRNAs) that form a complete match with the viral escape sequences.ResultsTo block the two favorite viral escape routes observed when the HIV-1 integrase gene sequence is targeted, the original shRNA inhibitor was combined with two 2nd generation shRNAs in a single lentiviral expression vector. We demonstrate in long-term viral challenge experiments that the two dominant viral escape routes were effectively blocked. Eventually, virus breakthrough did however occur, but HIV-1 evolution was skewed and forced to use new escape routes.ConclusionThese results demonstrate the power of the 2nd generation RNAi concept. Popular viral escape routes are blocked by the 2nd generation RNAi strategy. As a consequence viral evolution was skewed leading to new escape routes. These results are of importance for a deeper understanding of HIV-1 evolution under RNAi pressure.

HIV-1 escape to CCR5 coreceptor antagonism through selection of CXCR4-using variants in vitro

HIV-1 coreceptor switch from CCR5 to CXCR4 is associated with disease progression and AIDS. Selection of resistant HIV-1 to CCR5 agents in cell culture has often occurred in the absence of coreceptor switch. With CCR5 antagonists currently in clinical trials, their impact on coreceptor use is still in doubt. Six R5 HIV-1 strains were passaged in lymphoid cells expressing high CXCR4 and low CCR5, in the absence or presence of CCR5 inhibitors (TAK-779, mAb 2D7 and CCL5). AMD3100, zidovudine and lamivudine were used as controls. Phenotype and genotype changes as well as virus coreceptor use were evaluated. In the absence of drug pressure, three out of six strains expanded their coreceptor use to CXCR4 at different times, suggesting that not all virus strains had the capacity to do so. Lowering the replication rate with a suboptimal concentration of different anti-HIV agents (reverse transcriptase inhibitors or CCR5 agents) delayed coreceptor switch. However, virus breakthrough was observed earlier in the presence of CCR5-targeting agents than in presence of reverse transcriptase inhibitors and was associated with a change in sensitivity to TAK-779 or AMD3100, virus coreceptor expansion to CXCR4 and changes in the V3 loop region of gp120. Our results suggest that HIV-1 may escape CCR5 drug pressure through coreceptor switch. Experimental conditions strongly determine the outcome of CCR5 drug pressure in cell culture. A cell culture model of the evolution of HIV-1 coreceptor use may be relevant to assess the propensity of clinical isolates to develop resistance through coreceptor change.

Characteristics of lamivudine‐resistant hepatitis B virus (HBV) strains with and without breakthrough hepatitis in patients with chronic hepatitis B evaluated by serial HBV full‐genome sequences

Lamivudine therapy often causes breakthrough of hepatitis B virus (HBV) DNA and breakthrough hepatitis. The aim of this study was to determine the viral factors that relate to HBV-DNA breakthrough with and without breakthrough hepatitis. Among 82 patients with chronic hepatitis B (CHB) who received lamivudine at a dose of 100 mg daily for more than 24 months, 23 patients had HBV-DNA breakthrough induced by a lamivudine-resistant mutant. Of these 23 patients, 16 had breakthrough hepatitis and 7 had only HBV-DNA breakthrough. Serial HBV-DNA full-genome sequences during therapy were examined in 10 (7 had breakthrough hepatitis and 3 did not) of these 23 patients by direct sequencing. Mutations in the S region were examined by cloning in representative patients. There were no significant differences in the baseline clinical backgrounds and virus marker between patients with and without breakthrough hepatitis. The HBV amino acid substitutions at breakthrough hepatitis were identical to those at HBV-DNA breakthrough. Cloning analysis revealed that monoclonal mutational strain appeared at breakthrough and no such mutations existed at baseline. Regarding HBV amino acid substitutions in the polymerase region, S region, X region, and precore-core region with breakthrough compared to baseline, there was no significant differences of the numbers of amino acid substitution between breakthrough hepatitis and non-breakthrough hepatitis. There were no common amino acid changes in patients with breakthrough hepatitis. Although monoclonal lamivudine-resistant strain emerged at HBV-DNA breakthrough in patients with CHB, there were no common amino acid changes, suggesting viral factor may have insignificant role in breakthrough hepatitis.

Novel Second Generation Anti-HIV shRNA Expressing vif and Decoy TAR Arrest the Virus-breakthrough Phenomenon Associated With siRNA-escape Variants

We describe a novel chimera RNA expressing vif shorthairpin RNA (shRNA) and decoy trans-activation response region (TAR) RNA from a human U6 Pol II promoter, which enhanced the inhibition of human immunodeficiency virus (HIV) vif small-interfering RNA (siRNA) and arrested virus breakthrough by siRNA-generated escape variants in long-term culture assays. Our strategy was based on a second-generation anti-HIV-1 shRNA vector system, in which HIV-1 vif shRNA was fused to a decoy TAR RNA by a linker UU cleavage site to generate vif shRNA-decoy TAR RNA. Upon expression, the RNA molecule was cleaved and separated into vif siRNA and decoy TAR RNA. The synergistic effect of these molecules enhanced the inhibition of HIV-1 replication in a longterm culture assay and prevented virus breakthrough associated with siRNA-mediated escape variants. Combining shRNA with decoy TAR RNA as second-generation antiHIV shRNA may provide practical basis for applying siRNA-based gene therapy to the treatment of HIV/AIDS. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Limited development and progression of resistance of HIV-1 to the nucleoside analogue reverse transcriptase inhibitor lamivudine in human primary macrophages

The aim of this study was to investigate the development and progression of phenotypic resistance to the HIV-1-reverse transcriptase (RT) inhibitor lamivudine, and genotypic variations of HIV-1-RT occurring under lamivudine treatment in HIV-1-infected human primary monocytes-macrophages (M/M). Cellular passages in the presence of lamivudine were performed every 2 weeks by transferring supernatants of infected M/M to fresh M/M. A fitness assay using wild-type virus and a lamivudine-resistant HIV-1 virus (harbouring the M184V RT mutation) was performed in peripheral blood mononuclear cells. Culture supernatants were tested for p24 antigen production and RT activity. The M184V RT mutant virus was obtained by site-directed mutagenesis on a CCR5-using HIV-1 backbone. The mutagenized M184V RT virus showed full resistance to lamivudine in M/M. However, no detectable phenotypic and genotypic resistance (neither virus breakthrough, nor RT resistance-related mutations) developed in M/M infected by HIV-1 and cultured for up to seven passages in vitro (i.e. 105 days). This inefficiency of M/M to develop M184V RT mutated virus is tightly related to the low 2'-deoxynucleotide (dNTP) pool in such cells, which in turn decreases the kinetics of HIV-1-RT. Despite this, the M184V RT mutant virus replicates in M/M, although with a 30% decreased efficiency compared with the wild-type. Our results show that the chances of development of resistance are far lower in M/M than in lymphocytes. This underlines the importance and the peculiar role of M/M as reservoirs of either wild-type or resistant strains in human organs.

Favorable efficacy of long‐term lamivudine therapy in patients with chronic hepatitis B: An 8‐year follow‐up study

The long-term efficacy of lamivudine therapy in patients with hepatitis B virus (HBV) infection is still not clear. In this study, 20 non-cirrhotic Japanese patients infected with HBV received lamivudine therapy for more than 1 year and were followed for a median period of 8.5 years (range, 6.7-8.7 years). The rates of HBe antigen (HbeAg) negative, HBV-DNA undetectable, and alanine aminotransferase (ALT) normal level at the start of lamivudine were 55%, 25%, and 20% and 85%, 80%, and were 80%, respectively, at the last visit, including patients who received additional treatment. The values at the last visit tended to and were significantly higher than those at the start. The values improved at the last visit regardless of the emergence of YMDD motif mutant and continuation of lamivudine. YMDD mutant and biochemical relapse with mutant virus (breakthrough hepatitis) appeared in 65% and 45% during follow-up, respectively, but severe breakthrough hepatitis occurred in only 5%. Furthermore, 80% of patients who received additional treatment for breakthrough hepatitis, regardless of continuation of lamivudine, were ALT normal level at the last visit, in contrast to 25% untreated. HBsAg clearance occurred in two patients of the discontinuous lamivudine group with non-vertical transmission, who were relatively young. One was infected with HBV genotype C with breakthrough hepatitis and the other had no YMDD mutant and was infected with genotype D, a rare type in Japan. None developed cirrhosis or hepatocellular carcinoma (HCC) during follow-up. Our results suggest that long-term lamivudine therapy improves long-term prognosis, especially when additional treatment for breakthrough hepatitis is used.

Genetic variability of hepatitis C virus in chronically infected patients with viral breakthrough during interferon-ribavirin therapy.

Little is known about hepatitis C virus (HCV) breakthrough during antiviral therapy, although it would help in understanding HCV resistance to current antiviral treatments. To analyse the implication of virological factors and the vigour of humoral immune responses in this phenomenon, we studied nine chronic hepatitis C patients with a viral breakthrough during IFN/ribavirin combination therapy, as well as five responders and five non-responders. The IRES and regions coding for the capsid protein, the PePHD domain of envelope glycoprotein E2 and the NS5A and 5B proteins were amplified by RT-PCR before treatment, before and during breakthrough, and after treatment. The major variant sequence was obtained by direct sequencing. The heterogeneity of quasispecies was studied by SSCP in all patients and sequencing after cloning in seven genotype 1b-infected patients. Humoral responses against HCV epitopes were also analysed. The major sequences of IRES, PePHD, and NS5B remained stable during treatment, regardless of the treatment response. However, the capsid protein and the regions flanking PePHD showed sequence variations in breakthrough patients, although no specific mutation was identified. The variable V3 region of NS5A, but not the PKR-binding domain and the ISDR, seemed to be associated with differences in response to treatment. The analysis of HCV quasispecies revealed no characteristic pattern during treatment in breakthrough patients, whose HCV genome profiles looked most similar to that of non-responders. The humoral response was similar between groups. In conclusion, viral breakthrough does not seem to be due to selection of resistant strains with signature mutations.

Streamline-based simulation of virus transport resulting from long term artificial recharge in a heterogeneous aquifer

The likelihood for viruses, protozoan oocysts, and other human pathogens to enter groundwater, and in particular, sensitive or vulnerable water supplies, has increased as the numbers of anthropogenic sources such as septic systems, leaking sewers, animal farming operations, and artificial recharge of treated wastewater have proliferated. In this paper, we utilize a detailed numerical model of groundwater flow in a region encompassing a large artificial groundwater recharge operation in Orange County, California to evaluate the potential for transport of viruses and protozoan oocysts in such a system, as dictated by a transport model that includes colloid filtration and microbial inactivation components. The purpose of the model is not oriented towards the analysis of any perceived or real microbial contamination, but rather is directed at understanding the influence of aquifer heterogeneity within the modeled system. The transport model is based upon a novel representation of geologic heterogeneity, a high-resolution flow simulator, and an efficient streamline-based transport algorithm. Example virus transport simulations illustrate a large degree of variability in virus breakthrough across water supply pumping wells, with shallower wells providing less than two orders of magnitude of virus removal, and deeper wells indicating many orders of magnitude of virus removal. Simulation results also show variability among pathogens modeled, with Cryptosporidium parvum filtered to a much greater degree than other pathogens. Comparison to transport of an abiotic colloid and a conservative chemical tracer are provided to illustrate the influence of filtration and inactivation on the transport process. The results emphasize the need for improved microbial transport models in realistic aquifer systems, more reliable virus characterization methods and monitoring networks, and their ultimate integration into a broader epidemiological and regulatory framework for aquifer management.

Long-term survival and virus production in human primary macrophages infected by human immunodeficiency virus.

The role of macrophages in the pathogenesis and progression of human immunodeficiency virus (HIV)-related infection is substantiated by in vitro and in vivo evidence. The unique ability to survive HIV infection and produce viral particles for long periods is postulated. Detailed studies of this phenomenon are lacking. The dynamics of HIV-1 replication and cumulative virus production was studied in long-term cultures of macrophages in the presence or in the absence of antiviral drugs. Multiply spliced and unspliced HIV-RNA production was assessed by quantitative PCR, and the number of infected cells was monitored by FACS analysis. Cumulative HIV-1 production was determined by a trapezoidal equation, including such parameters as times of collection and experimental values of genomic-RNA and p24 gag antigen. Unspliced and multiply spliced HIV-RNA increased linearly after macrophage infection; reached levels of 1.5 x 10(8) and 2.8 x 10(5) copies/10(5) cells, respectively, at day 10; and then remained stable throughout the course of the experiment. Cumulative production of genomic-RNA and p24 gag antigen was 10(10) copies/10(6) cells and 10(7) pg/10(6) cells, respectively, with an average of >200 virus particles produced daily by each macrophage. AZT decreased the cumulative production of both genomic-RNA and p24 gag antigen down to 2.5 x 10(9) copies and 1.1 x 10(6) pg/10(6) cells (73.8% and 88.9% inhibition, respectively) up to day 50 without virus breakthrough. Ritonavir had a limited, but consistent, efficacy on the release of mature virus proteins (about 40% inhibition), but not on HIV-RNA production. In conclusion, the long-term dynamics and the high cumulative virus production that characterize HIV-1 infection of macrophages underscore the peculiar role of these cells as a persistently infected reservoir of HIV.

Virus transport in physically and geochemically heterogeneous subsurface porous media

A two-dimensional model for virus transport in physically and geochemically heterogeneous subsurface porous media is presented. The model involves solution of the advection–dispersion equation, which additionally considers virus inactivation in the solution, as well as virus removal at the solid matrix surface due to attachment (deposition), release, and inactivation. Two surface inactivation models for the fate of attached inactive viruses and their subsequent role on virus attachment and release were considered. Geochemical heterogeneity, portrayed as patches of positively charged metal oxyhydroxide coatings on collector grain surfaces, and physical heterogeneity, portrayed as spatial variability of hydraulic conductivity, were incorporated in the model. Both layered and randomly (log-normally) distributed physical and geochemical heterogeneities were considered. The upstream weighted multiple cell balance method was employed to numerically solve the governing equations of groundwater flow and virus transport. Model predictions show that the presence of subsurface layered geochemical and physical heterogeneity results in preferential flow paths and thus significantly affect virus mobility. Random distributions of physical and geochemical heterogeneity have also notable influence on the virus transport behavior. While the solution inactivation rate was found to significantly influence the virus transport behavior, surface inactivation under realistic field conditions has probably a negligible influence on the overall virus transport. It was further demonstrated that large virus release rates result in extended periods of virus breakthrough over significant distances downstream from the injection sites. This behavior suggests that simpler models that account for virus adsorption through a retardation factor may yield a misleading assessment of virus transport in “hydrogeologically sensitive” subsurface environments.