Respiratory virus laboratory pandemic planning and surveillance in central Viet Nam, 2008-2010.

INTRODUCTION: Laboratory capacity is needed in central Viet Nam to provide early warning to public health authorities of respiratory outbreaks of importance to human health, for example the outbreak of influenza A(H1N1) pandemic in 2009. Polymerase chain reaction (PCR) procedures established as part of a capacity-building process were used to conduct prospective respiratory surveillance in a region where few previous studies have been undertaken. METHODS: Between October 2008 and September 2010, nose and throat swabs from adults and children (approximately 20 per week) presenting with an acute respiratory illness to the Ninh Hoa General Hospital were collected. Same-day PCR testing and result reporting for 13 respiratory viruses were carried out by locally trained scientists. RESULTS: Of 2144 surveillance samples tested, 1235 (57.6%) were positive for at least one virus. The most common were influenza A strains (17.9%), with pandemic influenza A(H1N1) 2009 and seasonal H3N2 strain accounting for 52% and 43% of these, respectively. Other virus detections included: rhinovirus (12.4%), enterovirus (8.9%), influenza B (8.3%), adenovirus (5.3%), parainfluenza (4.7%), respiratory syncytial virus (RSV) (3.9%), human coronavirus (3.0%) and human metapneumovirus (0.3%). The detection rate was greatest in the 0-5 year age group. Viral co-infections were identified in 148 (6.9%) cases. DISCUSSION: The outbreak in 2009 of the influenza A(H1N1) pandemic strain provided a practical test of the laboratory's pandemic plan. This study shows that the availability of appropriate equipment and molecular-based testing can contribute to important individual and public health outcomes in geographical locations susceptible to emerging infections.

Evaluation of swabs, transport media, and specimen transport conditions for optimal detection of viruses by PCR.

Depletion of swabs and viral transport medium during epidemics may prompt the use of unvalidated alternatives. Swabs collected and transported dry or in saline were compared to commercially available swab/medium combinations for PCR detection of influenza, enterovirus, herpes simplex virus, and adenovirus. Each was detected at an ambient temperature (22°C) and 4°C for 7 days. Detection of influenza on dry or saline swabs is important because of its capacity to cause outbreaks involving large numbers of cases.

Community epidemiology of human metapneumovirus, human coronavirus NL63, and other respiratory viruses in healthy preschool-aged children using parent-collected specimens.

OBJECTIVES: The purpose of this work was to assess the impact of recently described human metapneumovirus and human coronavirus NL63 compared with other respiratory viruses by using sensitive molecular techniques in a cohort of healthy preschool-aged children. We also aimed to assess the use of parent collection to obtain an adequate respiratory specimen from acutely unwell children in the community. PATIENTS AND METHODS: The community epidemiology and burden of human metapneumovirus and other respiratory viruses (influenza A, influenza B, respiratory syncytial virus, parainfluenza viruses, adenoviruses, and picornaviruses) were examined in a cohort of 234 preschool-aged children from Melbourne, Australia, over a 12-month period by using polymerase chain reaction testing. Parents collected a daily symptom diary for the duration of the study and were taught to collect a combined nose-throat swab and complete an impact diary when the study child had an acute respiratory illness. RESULTS: The average incidence of acute respiratory illness was 0.48 per child-month for the duration of the study, with a winter peak. Of 543 illnesses with > or = 1 specimen returned, 33 were positive for human metapneumovirus (6.1%) and 18 for human coronavirus NL63 (3.3%). Of all of the viruses for which we tested, human metapneumovirus and human coronavirus NL63 were most strongly linked to child care attendance, occurring in 82% and 78% of infected children, respectively. Picornaviruses were the most commonly identified virus group (269 [49.5%]). Influenza virus and adenovirus illnesses had the greatest impact, with fever in more than three quarters and requiring, on average, > 1 local doctor visit per illness. CONCLUSIONS: Recently identified human metapneumovirus and human coronavirus NL63 are important pathogens in community-based illness in children, particularly in those who attend child care. Picornaviruses were detected in half of the nose-throat swabs collected during acute respiratory illness in children but resulted in milder illnesses; influenza and adenovirus caused the highest-impact illnesses. The use of parent-collected specimens should be considered for additional community-based epidemiologic studies and vaccine trials.

Virological fitness of HIV in patients with resistance to enfuvirtide.

Resistance to the HIV fusion inhibitor enfuvirtide is associated with mutations in the first heptad repeat region of gp41, but little is known of their impact on replicative fitness in vivo. We followed seven patients undergoing salvage therapy that included enfuvirtide in order to document the temporal generation of genotypic and phenotypic resistance in parallel with replicative fitness. Resistance to enfuvirtide was not associated with decreased replicative fitness of HIV strains infecting these patients.

The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein.

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the yeast two-hybrid system to demonstrate protein-protein interactions and tested the same using a yeast genetic cross. Further the strength of the interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8-ORF6 interaction. To the best of our knowledge, this is the first report of the interaction between a SARS-CoV accessory protein and nsp8 and our findings suggest that ORF6 protein may play a role in virus replication.

Analysis of human coronavirus 229E spike and nucleoprotein genes demonstrates genetic drift between chronologically distinct strains.

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.

The causes and diagnosis of influenza-like illness.

BACKGROUND: Influenza and other respiratory viruses circulate between spring and autumn in temperate climates and all year in tropical climates. These viruses cause symptoms often referred to as influenza-like illness (ILI), but are not generally distinguishable on clinical grounds alone. OBJECTIVE: This article provides a brief review of the surveillance, viral causes and current diagnostic methods used to identify viruses causing ILI. DISCUSSION: Influenza-like illness surveillance with laboratory support, conducted in most Australian stats and territories, aims to define the impact of influenza seasons in the community and provide virus strains that may be used in future vaccine formulations. Surveillance may also be useful in the early stages of an influenza pandemic. In addition to influenza, viruses known to cause ILI include respiratory syncytial virus, rhinovirus, adenovirus, parainfluenza viruses, human coronaviruses (including the virus that causes severe acute respiratory syndrome) and the recently recognised human metapneumovirus. Polymerase chain reaction assays are the most common diagnostic tests now used for the differential diagnosis of ILI.

Limited evolution of HIV antiretroviral drug resistance-associated mutations during the performance of drug resistance testing.

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.

Novel pharmacophore-based methods reveal gossypol as a reverse transcriptase inhibitor.

In a program to identify new structural entities for the inhibition of the HIV-1 reverse transcriptase (RT) enzyme via database searching, a series of RT pharmacophores were developed. By utilising a novel filtering technique, the National Cancer Institute database of compounds was scanned producing 15 compounds to be screened for activity. A notable inclusion was a series of gossypol derivatives. The testing of a series of compounds revealed the parent compound gossypol to be an HIV-1 reverse transcriptase inhibitor. These results suggest that at least a part of its anti-HIV activity is due to gossypol targeting the non-nucleoside inhibitor binding pocket of RT.