Pauline Schipper

Simultaneous Detection of Influenza Viruses A and B Using Real-Time Quantitative PCR

ABSTRACT Since influenza viruses can cause severe illness, timely diagnosis is important for an adequate intervention. The available rapid detection methods either lack sensitivity or require complex laboratory manipulation. This study describes a rapid, sensitive detection method that can be easily applied to routine diagnosis. This method simultaneously detects influenza viruses A and B in specimens of patients with respiratory infections using a TaqMan-based real-time PCR assay. Primers and probes were selected from highly conserved regions of the matrix protein gene of influenza virus A and the hemagglutinin gene segment of influenza virus B. The applicability of this multiplex PCR was evaluated with 27 influenza virus A and 9 influenza virus B reference strains and isolates. In addition, the specificity of the assay was assessed using eight reference strains of other respiratory viruses (parainfluenza viruses 1 to 3, respiratory syncytial virus Long strain, rhinoviruses 1A and 14, and coronaviruses OC43 and 229E) and 30 combined nose and throat swabs from asymptomatic subjects. Electron microscopy-counted stocks of influenza viruses A and B were used to develop a quantitative PCR format. Thirteen copies of viral RNA were detected for influenza virus A, and 11 copies were detected for influenza virus B, equaling 0.02 and 0.006 50% tissue culture infective doses, respectively. The diagnostic efficacy of the multiplex TaqMan-based PCR was determined by testing 98 clinical samples. This real-time PCR technique was found to be more sensitive than the combination of conventional viral culturing and shell vial culturing.

Increased fitness of drug resistant HIV-1 protease as a result of acquisition of compensatory mutations during suboptimal therapy.

OBJECTIVE It is thought as a consequence of continuous replication, HIV-1 has acquired an optimal fitness state and that suboptimal antiretroviral therapy selects for drug resistant variants which show impaired fitness in the absence of the drug. In this paper we studied the evolution and fitness of viral populations appearing in a patient who received protease monotherapy. METHODS Two factors contributing to fitness, drug resistance and protease catalytic activity, were studied at the enzymatic and virological level. RESULTS The first drug resistant viral variants that were selected in vivo harboured one to three protease substitutions. These mutants showed reduced protease activity and consequently a reduction in viral replication capacity. During continued in vivo replication of these viruses in the presence of the drug, novel variants harbouring additional substitutions in the viral protease appeared. These variants did not display any further increase in drug resistance but demonstrated clearly increased protease activity. Consequently the replication capacity of these viruses was raised to a level at which they replicated better than the original wild-type virus. CONCLUSION This study indicates that the viral population in the patient does not have to represent the fittest possible variants, and thus antiretroviral therapy may drive the viral population first through a lower fitness level and then to a higher fitness level.

Frequent Detection of Human Coronaviruses in Clinical Specimens from Patients with Respiratory Tract Infection by Use of a Novel Real-Time Reverse-Transcriptase Polymerase Chain Reaction

During the past years, human coronaviruses (HCoVs) have been increasingly identified as pathogens associated with more-severe respiratory tract infection (RTI). Diagnostic tests for HCoVs are not frequently used in the routine setting. It is likely that, as a result, the precise role that HCoVs play in RTIs is greatly underestimated. We describe a rapid, sensitive, and highly specific quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) for the detection of HCoV that can easily be implemented in the routine diagnostic setting. HCoV was detected in 28 (11%) of the 261 clinical specimens obtained from patients presenting with symptoms of RTI ranging from common cold to severe pneumonia. Only 1 (0.4%) of the 243 control specimens obtained from patients without symptoms of RTI showed the presence of HCoV. We conclude that HCoVs can be frequently detected in patients presenting with RTI. Real-time RT-PCR provides a tool for large-scale epidemiological studies to further clarify the role that coronavirus infection plays in RTI in humans.

A Novel Substrate-Based HIV-1 Protease Inhibitor Drug Resistance Mechanism

Background HIV protease inhibitor (PI) therapy results in the rapid selection of drug resistant viral variants harbouring one or two substitutions in the viral protease. To combat PI resistance development, two approaches have been developed. The first is to increase the level of PI in the plasma of the patient, and the second is to develop novel PI with high potency against the known PI-resistant HIV protease variants. Both approaches share the requirement for a considerable increase in the number of protease mutations to lead to clinical resistance, thereby increasing the genetic barrier. We investigated whether HIV could yet again find a way to become less susceptible to these novel inhibitors. Methods and Findings We have performed in vitro selection experiments using a novel PI with an increased genetic barrier (RO033-4649) and demonstrated selection of three viruses 4- to 8-fold resistant to all PI compared to wild type. These PI-resistant viruses did not have a single substitution in the viral protease. Full genomic sequencing revealed the presence of NC/p1 cleavage site substitutions in the viral Gag polyprotein (K436E and/or I437T/V) in all three resistant viruses. These changes, when introduced in a reference strain, conferred PI resistance. The mechanism leading to PI resistance is enhancement of the processing efficiency of the altered substrate by wild-type protease. Analysis of genotypic and phenotypic resistance profiles of 28,000 clinical isolates demonstrated the presence of these NC/p1 cleavage site mutations in some clinical samples (codon 431 substitutions in 13%, codon 436 substitutions in 8%, and codon 437 substitutions in 10%). Moreover, these cleavage site substitutions were highly significantly associated with reduced susceptibility to PI in clinical isolates lacking primary protease mutations. Furthermore, we used data from a clinical trial (NARVAL, ANRS 088) to demonstrate that these NC/p1 cleavage site changes are associated with virological failure during PI therapy. Conclusions HIV can use an alternative mechanism to become resistant to PI by changing the substrate instead of the protease. Further studies are required to determine to what extent cleavage site mutations may explain virological failure during PI therapy.

Effects of discontinuation of zidovudine treatment on zidovudine sensitivity of human immunodeficiency virus type 1 isolates.

Zidovudine treatment of individuals infected with human immunodeficiency virus type 1 (HIV-1) results in HIV-1 isolates with a reduced zidovudine sensitivity in vitro. This reduction is due to mutations causing amino acid substitutions at five codons (41, 67, 70, 215, and 219) on the reverse transcriptase enzyme of HIV. HIV-1 isolates were obtained 8 to 69 weeks after therapy discontinuation from 10 patients at different stages of disease. Zidovudine sensitivity was determined by the HeLa CD4+ plaque assay. The presence of the resistance-conferring mutations was determined by using a selective polymerase chain reaction. Sensitivity could be determined for six isolate pairs: one showed a decline in the 50% inhibitory zidovudine concentration after therapy discontinuation; four pairs did not show a change. The majority of changes in the five codons in isolates from all 10 patients were the result of a relative increase in the wild-type sequence. Complete changes from mutant to the wild type were seen for only two codons in isolates from two patients. This study of isolates from a small group of individuals at different stages of disease, who had been taking zidovudine for 1 to 2 years, shows that a period of 1 year without zidovudine may be required to achieve a change from a mutant or mixed virus population to a wild-type virus population.

Enhanced severity of virus associated lower respiratory tract disease in asthma patients may not be associated with delayed viral clearance and increased viral load in the upper respiratory tract.

Abstract Background Viral respiratory infections, particularly human rhinovirus (HRV) infections, are the most common cause of asthma exacerbation. HRV infections usually lead to more severe and longer duration of lower respiratory tract (LRT) symptoms in asthmatics than in otherwise healthy individuals. However, the exact mechanism by which viruses contribute to exacerbation of asthma is unknown. Objectives The main objective of our study was to investigate the relationship of the enhanced severity of LRT symptoms to viral dynamics or cytokine responses in the upper respiratory tract (URT). Study design Therefore, we conducted a longitudinal study in which asthmatics and healthy controls were followed during natural viral respiratory tract infections. Results Our study confirmed that viral respiratory tract infections caused more severe problems of the LRT in asthma patients as compared to healthy controls. However, for all subjects, the severity of LRT symptoms were not related to viral load or prolonged viral shedding in the URT. In addition, we did not detect differences in proinflammatory cytokines in the URT between asthmatics and controls. Conclusion Persistence of the virus, as well as viral load in the URT, may not be associated with the induction and/or persistence of asthmatic symptoms.

HIV-1 protease inhibitor mutations affect the development of HIV-1 resistance to the maturation inhibitor bevirimat

BackgroundMaturation inhibitors are an experimental class of antiretrovirals that inhibit Human Immunodeficiency Virus (HIV) particle maturation, the structural rearrangement required to form infectious virus particles. This rearrangement is triggered by the ordered cleavage of the precursor Gag polyproteins into their functional counterparts by the viral enzyme protease. In contrast to protease inhibitors, maturation inhibitors impede particle maturation by targeting the substrate of protease (Gag) instead of the protease enzyme itself. Direct cross-resistance between protease and maturation inhibitors may seem unlikely, but the co-evolution of protease and its substrate, Gag, during protease inhibitor therapy, could potentially affect future maturation inhibitor therapy. Previous studies showed that there might also be an effect of protease inhibitor resistance mutations on the development of maturation inhibitor resistance, but the exact mechanism remains unclear. We used wild-type and protease inhibitor resistant viruses to determine the impact of protease inhibitor resistance mutations on the development of maturation inhibitor resistance.ResultsOur resistance selection studies demonstrated that the resistance profiles for the maturation inhibitor bevirimat are more diverse for viruses with a mutated protease compared to viruses with a wild-type protease. Viral replication did not appear to be a major factor during emergence of bevirimat resistance. In all in vitro selections, one of four mutations was selected: Gag V362I, A364V, S368N or V370A. The impact of these mutations on maturation inhibitor resistance and viral replication was analyzed in different protease backgrounds. The data suggest that the protease background affects development of HIV-1 resistance to bevirimat and the replication profiles of bevirimat-selected HIV-1. The protease-dependent bevirimat resistance and replication levels can be explained by differences in CA/p2 cleavage processing by the different proteases.ConclusionsThese findings highlight the complicated interactions between the viral protease and its substrate. By providing a better understanding of these interactions, we aim to help guide the development of second generation maturation inhibitors.

Modulation of HIV-1 Gag NC/p1 cleavage efficiency affects protease inhibitor resistance and viral replicative capacity

BackgroundMutations in the substrate of HIV-1 protease, especially changes in the NC/p1 cleavage site, can directly contribute to protease inhibitor (PI) resistance and also compensate for defects in viral replicative capacity (RC) due to a drug resistant protease. These NC/p1 changes are known to enhance processing of the Gag protein. To investigate the capacity of HIV-1 to modulate Gag cleavage and its consequences for PI resistance and RC, we performed a detailed enzymatic and virological analysis using a set of PI resistant NC/p1 variants (HXB2431V, HXB2436E+437T, HXB2437T and HXB2437V).ResultsHere, we demonstrate that single NC/p1 mutants, which displayed only a slight increase in PI resistance did not show an obvious change in RC. In contrast, the double NC/p1 mutant, which displayed a clear increase in processing efficiency and PI resistance, demonstrated a clear reduction in RC. Cleavage analysis showed that a tridecameric NC/p1 peptide representing the double NC/p1 mutant was cleaved in two specific ways instead of one.The observed decrease in RC for the double NC/p1 mutant (HXB2436E+437T) could (partially) be restored by either reversion of the 436E change or by acquisition of additional changes in the NC/p1 cleavage site at codon 435 or 438 as was revealed during in vitro evolution experiments. These changes not only restored RC but also reduced PI resistance levels. Furthermore these changes normalized Gag processing efficiency and obstructed the novel secondary cleavage site observed for the double NC/p1 mutant.ConclusionsThe results of this study clearly demonstrate that HIV-1 can modulate Gag processing and thereby PI resistance. Distinct increases in Gag cleavage and PI resistance result in a reduced RC that can only be restored by amino acid changes in NC/p1 which reduce Gag processing to an optimal rate.

Mutation Q95K enhances N155H-mediated integrase inhibitor resistance and improves viral replication capacity

OBJECTIVES The genetic barrier to development of raltegravir resistance is considered to be low, requiring at least one primary integrase mutation: Y143C, Q148H/K/R or N155H to confer raltegravir therapy failure. However, during continued raltegravir treatment failure, additional mutations may be selected. In a patient failing raltegravir therapy, we investigated the impact of multiple integrase mutations on resistance and viral replication. Furthermore, in vivo fitness was investigated during failure of raltegravir-containing highly active antiretroviral therapy and after raltegravir was discontinued from the regimen. METHODS Patient-derived viral integrase genes were cloned into a reference strain. These recombinant viruses were used to determine the contribution of individual integrase mutations to raltegravir resistance and replication capacity in vitro. To determine in vivo fitness, the relative proportion of specific integrase mutations was monitored over time by in-depth clonal analysis of the viral integrase at baseline, during and after raltegravir treatment. RESULTS Raltegravir therapy failure was associated with the initial selection of primary resistance mutation N155H. This mutation conferred a 3.8-fold reduction in raltegravir susceptibility and a severe reduction in viral replication. Acquisition of integrase mutation Q95K increased resistance (6.2-fold) and partly restored viral replication. Selection of a third mutation, V151I, further increased raltegravir resistance (20-fold), but decreased viral replication. After prolonged raltegravir interruption, raltegravir resistance mutations were lost, demonstrating the reduced replication capacity of the resistant virus. CONCLUSIONS We describe selection of Q95K as a secondary resistance mutation during raltegravir therapy failure. In the background of N155H, Q95K enhances raltegravir and elvitegravir resistance and improves the impaired replication of the virus.

Use of dolutegravir in two INI-experienced patients with multiclass resistance resulted in excellent virological and immunological responses

Dolutegravir is a second generation integrase inhibitor with a proposed high genetic barrier to resistance. However, in clinical trials, decreased virological response was seen in a subset of patients with prior exposure to raltegravir and multiple integrase resistance mutations.