We assessed the in vivo efficacy of surgical and N95 (respirator) masks to filter reverse transcription-polymerase chain reaction (RT-PCR)-detectable virus when worn correctly by patients with laboratory-confirmed acute influenza. Of 26 patients with a clinical diagnosis of influenza, 19 had the diagnosis confirmed by RT-PCR, and 9 went on to complete the study. Surgical and N95 masks were equally effective in preventing the spread of PCR-detectable influenza.

Historically, coronaviruses have been recognized as a cause of minor respiratory infections in humans. However, the recent identification of three novel human coronaviruses, one causing severe acute respiratory syndrome (SARS), has prompted further examination of these viruses. Previous studies of geographically and chronologically distinct Human coronavirus 229E (HCoV-229E) isolates have found only limited variation within S gene nucleotide sequences. In contrast, analysis of the S genes of contemporary Human coronavirus OC43 variants identified in Belgium revealed two distinct viruses circulating during 2003 and 2004. Here, the S and N gene sequences of 25 HCoV-229E variants identified in Victoria, Australia, between 1979 and 2004 in patients with symptomatic infections were determined. Phylogenetic analysis showed clustering of the isolates into four groups, with evidence of increasing divergence with time. Evidence of positive selection in the S gene was also established.

Respiratory viruses were identified by the polymerase chain reaction (PCR) in more than 4,200 specimens collected during 2002 and 2003 in Victoria, Australia from patients admitted to hospitals or participating in an influenza surveillance program. Influenza viruses and picornaviruses were important causes of morbidity in both years. Additional testing of picornavirus‐positive samples suggested that rhinoviruses but not enteroviruses were more likely to be associated with respiratory symptoms, irrespective of the season in which they circulated. Detection of influenza viruses was strongly associated with the clinical symptoms of cough, fever, and fatigue; but each of the other respiratory viruses occasionally caused these symptoms or was responsible for symptoms severe enough to require hospitalization. Human coronaviruses HCoV‐OC43 and HCoV‐229E circulated at low levels throughout the study period with peak activity in winter, but overall did not circulate as widely as has often been reported for these agents. Evidence for the human metapneumovirus (hMPV) was only sought in the second year of the study and revealed low‐level circulation of this virus, mainly in the cooler months among the very young and adult populations. The detection rate of all viruses declined with increasing age of the patient, particularly in hospital patients. Infection with more than one respiratory virus occurred in a small number of patients; picornaviruses were most commonly implicated in these dual infections. J. Med. Virol. 75:122–129, 2005. © 2005 Wiley‐Liss, Inc.

We investigated the evolution of HIV reverse transcriptase (RT)- and protease-associated antiretroviral (ARV) drug resistance mutations during the time taken to perform genotypic drug resistance testing. Thirty treatment-experienced patients who were adherent to therapy and who underwent genotypic drug resistance testing provided blood samples at randomization and when reviewing the test results (baseline). Patients remained on their existing therapy between randomization and baseline. The predominant HIV strains in 10 patients (33%) either lost and/or gained primary RT inhibitor (RTI)- or protease inhibitor (PI)-associated resistance mutations during the testing period. Of the 9 patients with RT mutations, 2 lost, 5 gained, and 2 both lost and gained RTI resistance mutations. One patient gained a significant PI-associated resistance mutation on an existing PI-resistant background. The evolution that occurred in the RT may have altered the effectiveness of subsequent ARV therapy in some patients. Neither viral load at randomization, ARV drug class used at randomization, time between collection of blood samples, duration of current therapy, nor number of ARV drugs used influenced gain or loss of resistance mutations. There was a significant association between duration of previous ARV therapy and gain of RTI-associated resistance mutations ( p =.02), however. In general, our results suggest that patients should continue current therapy until test results are available. A few patients would be expected to gain ARV drug-associated resistance mutations during this time, however.

Background: Mutations in the thymidine kinase (TK) and DNA polymerase (pol) genes of herpes simplex virus (HSV) may confer resistance to antiviral drugs, particularly in the context of immunosuppression induced by infection with the human immunodeficiency virus (HIV). Objectives: To characterise the HSV type 2 (HSV-2) TK and DNA pol genes in an immunocompromised patient with clinical resistance to both acyclovir and foscarnet. Study design: The TK and DNA pol genes of isolates obtained over a 2-year period from an AIDS patient with severe genital herpes infection were characterised both phenotypically and genotypically. Results: HSV strains that were acyclovir resistant/foscarnet sensitive, acyclovir sensitive/foscarnet sensitive and acyclovir resistant/foscarnet resistant were isolated during this time. The TK gene of all the acyclovir resistant isolates contained a large 969 bp deletion which extended into a downstream untranslated region. The foscarnet resistance was associated with an S725G mutation in a conserved region (region II) of the herpesvirus DNA pol gene. Conclusions: Clinical and virological suppression of the infection was not always associated with subsequent reactivation with wild-type virus. Mutations of the nature we describe have not previously been reported occurring simultaneously in HSV strains isolated from patients treated with acyclovir and foscarnet.

Measles outbreaks in 1999 in Queensland and Victoria, Australia, were caused by a novel strain of clade G virus (proposed name g3). Epidemiologic and molecular evidence supports independent circulation of this virus in Queensland, northern Australia, in addition to importation of the virus by East Timor refugees seeking safe haven in Australia.

Abstract Purpose: To relate baseline plasma HIV genotypic and virtual phenotypic antiretroviral drug susceptibility to subsequent virological response in patients receiving saquinavir (SQV)-enhancing therapy. Individuals were randomized to receive stavudine (d4T), SQV, and one of ritonavir, nelfinavir, or delavirdine to enhance SQV blood levels. Method: The protease and reverse transcriptase baseline sequences of 31 treatment-experienced patients were analyzed by genotype and virtual phenotype and were related to viral load at weeks 12 and 24. Genotypic resistance to SQV was defined by the presence of G48V and/or L90M mutations in the protease gene. Potential cross-resistance to d4T in zidovudine (ZDV)-experienced individuals was defined by the presence of thymidine-associated mutations in the reverse transcriptase gene. Results: ZDV-associated mutations did not affect the virological response at 24 weeks. Individuals who were sensitive to SQV at baseline as determined by either genotyping or virtual phenotyping showed a greater decrease in viral load at week 24 than those resistant to SQV, irrespective of treatment arm. By genotyping, SQV-sensitive individuals had a median log decrease of 1.12 compared to 0.32 for those individuals who were SQV resistant. By virtual phenotyping, SQV-sensitive individuals had a median log decrease of 1.0 compared to a rise of 0.08 in resistant individuals. Conclusion: Thymidine analogue-associated mutations at baseline did not influence the response to subsequent therapy involving d4T. Individuals who were sensitive or resistant to SQV by genotyping or virtual phenotyping responded to SQV-enhancing regimens, but the virological response was greater in those who were sensitive.

Objective To determine whether HIV-1 can be recovered from blood monocytes as well as resting, memory CD4 T lymphocytes of patients on highly active antiretroviral therapy (HAART) with undetectable plasma viraemia and whether infection is active or latent. Design Five patients with plasma HIV-1-RNA levels of less than 500 copies/ml for at least 3 months and less than 50 copies/ml at the time of sampling were initially selected, followed by an additional five patients with viral loads of less than 50 copies/ml for 3 months or more. Methods Monocytes were isolated from blood by plastic adherence, then further purified by a second adherence step or CD3 depletion before co-culture with CD8-depleted donor peripheral blood mononuclear cells. Virus isolates were examined for mutations conferring resistance to reverse transcriptase or protease inhibitors and for genotype. The highly purified monocytes were also analysed for the presence of proviral and unintegrated viral DNA and multiply spliced (MS) viral mRNA by polymerase chain reaction. Results Virus was recovered from monocytes of five patients. Sequencing of the recovered viruses did not reveal multiple drug resistance, and was consistent with a non-syncytium-inducing/CCR5 phenotype. Proviral DNA was detectable in monocytes from all subjects, and unintegrated HIV-1 DNA and MS RNA was found in four out of five populations examined. Conclusion Recovery of replication-competent virus from some HAART patients indicates that monocytes can also harbour HIV-1. Detection of circular, viral DNA and spliced RNA, albeit at very low levels, in these cells suggests that their infection is recent and transcriptionally active rather than latent.

We investigated the effects of conditions often encountered during handling, transit, and storage of blood specimens on the quantity of detectable human immunodeficiency virus (HIV) RNA in plasma. HIV RNA copy numbers were measured with a commercially available assay (the Amplicor HIV-1 Monitor test kit). Variables examined were the time to processing of blood and plasma, the holding temperature of blood and plasma prior to processing, the effect of freezing and thawing of plasma, and the use of different anticoagulants. The relationship between the HIV RNA copy number and the HIV isolation rate by peripheral blood mononuclear cell (PBMC) coculture was also examined. We found that RNA copy numbers were maintained to within 0.5 log10 (approximately threefold) in blood and plasma samples held at room temperature or 4 degrees C for up to 3 days and remained stable despite (limited) freezing and thawing of the plasma. HIV RNA copy numbers were also maintained after long-term storage of plasma at -70 degrees C. The ability to isolate HIV from PBMCs was directly proportional to the HIV RNA copy number.

The protease of human immunodeficiency virus (HIV) is an important target for antiretroviral drug therapy. The synthesis and in vitro antiviral activity of a novel protease inhibitor, DG-35-VIII, which contains an hydroxyethylhydrazide core unit, is described. DG-35-VIII had potent activity against HIV-1 and related viruses (HIV-2 and simian immunodeficiency virus) in an acutely infected T lymphocyte line (MT-2) and was also active in cells chronically infected with HIV-1, where it inhibited processing of the Pr55(gag) and Pr160(gag-pal) precursor proteins.

Sequencing of the reverse transcriptase (RT) region of 26 human immunodeficiency virus type 1 (HIV-1) isolates from eight patients treated with 3'-azido-3'-deoxythymidine (AZT) revealed a mutation at codon 210 from TTG (leucine) to TGG (tryptophan) exclusively in association with resistance to AZT. The mutation Trp-210 was observed in 15 of the 20 isolates phenotypically resistant to AZT, being more commonly observed than resistance-associated mutations at codons 67, 70, and 219. Trp-210 was never observed before the emergence of resistance-associated mutations Leu-41 and Tyr-215, and in a sequential series of five isolates from one patient the order of emergence of mutations was found to be Tyr-215, Leu-41, and then Trp-210. Trp-210 was also found in association with the Leu-41, Asn-67, Arg-70, and Tyr-215 resistance genotype. To define the role of Trp-210 in AZT resistance, molecular HIV-1 clones were constructed with various combinations of RT mutations at codons 41, 67, 70, 210, and 215 and tested for susceptibility to AZT. In clones with polymerase genes derived either from HXB2-D or clinical isolates, Trp-210 alone did not increase AZT resistance, whereas in conjunction with Leu-41 and Tyr-215, Trp-210 contributed to high-level resistance (50% inhibitory concentration of >1 microM). In HXB2-D, Trp-210 with Tyr-215 generated a virus with resistance comparable to one with Leu-41, Tyr-215, and Trp-210. Inserting Trp-210 into the genetic context of mutations at codons 41, 67, 70, and 215 further enhanced resistance from a 50% inhibitory concentration of 1.44 microM to 8.41 microM. Molecular modeling of the tertiary structure of HIV-1 RT revealed that the distance between the side chains of Trp-210 (in helix alphaF) and Tyr-215 (in strand beta11a) approximated 4 A (1 A = 0.1 nm), sufficiently close to result in significant energetic interaction between these two aromatic side chains. In conclusion, Trp-210 contributes significantly to phenotypic AZT resistance of HIV-1 by augmenting resistance at least three- to sixfold in the context of two resistant genotypes, and its effect may require an interaction with an aromatic amino acid at position 215.

Both foscarnet (PFA) and zidovudine (AZT) select for drug-resistant variants of human immunodeficiency virus type 1 (HIV-1), but the interactions between the mutations causing such resistance are unknown. The introduction of the previously identified PFA resistance mutation W to G at codon 88 (W88G), E89K, L92I, or Q161L into an HIV-1 strain having the four known AZT resistance mutations completely reversed high-level AZT resistance. Two additional PFA resistance mutations, W88S and S156A, partially suppressed AZT resistance. Phenotypic reversion of AZT resistance by W88S, W88G, E89K, L92I, and S156A was associated with a concomitant suppression of PFA resistance. The degree to which PFA resistance mutations reversed AZT resistance was directly correlated with each mutation's ability to confer high-level PFA resistance (greater than or equal to 5.0-fold) and AZT hypersusceptibility in a wild-type genetic background. Highly PFA-resistant HIV-1 strains were hypersusceptible to AZT; conversely, AZT-resistant strains with M41L and T215Y; M41L, L210W, and T215Y; or M41L, D67N, K70R, and T215Y mutations were 2.2- to 2.5-fold hypersusceptible to PFA. Prolonged in vitro selection of wild-type or AZT-resistant HIV-1 strains with the combination AZT and PFA failed to generate coresistant virus, indicating that dual resistance was relatively difficult to achieve. Strains selected by passage in PFA plus AZT were phenotypically PFA resistant and AZT susceptible despite multiple reverse transcriptase mutations known to confer AZT resistance, These data show that PFA resistance mutations can phenotypically reverse AZT resistance and that AZT and PFA resistance might be mutually exclusive. The reciprocal interactions between AZT and PFA resistance-conferring mutations have implications for structure-function studies of the HIV-1 reverse transcriptase.

Foscarnet (PFA) is a pyrophosphate analogue antiviral active against human immunodeficiency virus (HIV-1) and herpesviruses, Strains of HIV-1 resistant to PFA have mutations in the HIV-1 reverse transcriptase (RT), We examined the influence of PFA resistance mutations, in different genetic backgrounds, on HIV-1 replication competency in both replication kinetics and growth competition assays. In replication kinetics assays, the recombinant strains HX89K, HX92I, and HX156A (encoding RT mutations E89K, L92I, and S156A, respectively, in the HXB2-D genetic background) replicated to lower titers than the wild-type parent in the absence of drug, and the degree of replication impairment increased as PFA resistance increased. PFA-resistant strains LAI 92I and LAI 156A (encoding RT mutations L92I and S156A, respectively) were replication impaired in comparison to the wild-type parent LAI to a similar degree as observed for strains in the HXB2-D background. In growth competition assays with wild-type LAI, strains LAI 92I and LAI 156A had relative fitness values of 0.5 and 0.8, respectively. These results show that the RT mutations E89K, L92I and S156A, observed in PFA-resistant strains selected in cell culture, reduce replication competence. Furthermore, these data show a correlation of increasing PFA resistance and decreasing replication competence mediated by single amino acid substitutions in the RT.

Sequential human-immunodeficiency virus type 1 (HIV-1) isolates from an acquired immunodeficiency syndrome (AIDS) patient on long-term foscarnet and zidovudine therapy were examined for the emergence of foscarnet resistance. One isolate obtained before therapy, and three post-foscarnet therapy HIV isolates had similar foscarnet sensitivity profiles, despite the emergence of foscarnet-resistant herpes simplex virus type 2 (HSV-2) during the period of therapy. Virion reverse transcriptases from these isolates were also equally inhibited by foscarnet. In contrast, sequential HIV isolates taken pre- and postzidovudine therapy showed a gradual increase in IC50 to this drug. In this patient, long-term foscarnet and zidovudine therapy selected foscarnet-resistant HSV and zidovudine-resistant HIV; in contrast, HIV remained susceptible to foscarnet. Concurrent administration of other anti-HIV drugs (zidovudine and interferon-alpha) may have hindered the development of foscarnet resistant HIV-1 in vivo.

We have developed an assay using flavivirus RNA-dependent RNA polymerase to test the inhibitory activity of potential antiviral agents. The effects of antiviral agents on RNA synthesis were examined in this assay using extracts of Vero cells infected with dengue virus type 2 or Kunjin virus. Several different classes of known polymerase inhibitors were tested. The synthesis of double-stranded replicative form RNA was inhibited in a dose-dependent fashion in the presence of the polyoxometalate HPA-23 [(NH4)18(NaW21Sb9O86)17].14 H2O and several structurally related compounds.

Topoisomerase I activity was detected in detergent-disrupted human immunodeficiency virus type 1 (HIV-1) particles. The enzyme did not require ATP for its conversion of SC DNA to an RC form, had divalent cation requirements similar to those of eukaryotic topoisomerase I, and was significantly inhibited by the specific topoisomerase I inhibitor camptothecin. However, camptothecin failed to inhibit replication of HIV in infected cells at nontoxic concentrations, and an active site motif for topoisomerase I could not be detected on the HIV genome. These results suggests that HIV does not encode a novel topoisomerase I, but rather packages the cellular enzyme.