Chris Birch

Emergence and spread of oseltamivir-resistant A(H1N1) influenza viruses in Oceania, South East Asia and South Africa

The neuraminidase inhibitors (NAIs) are an effective class of antiviral drugs for the treatment of influenza A and B infections. Until recently, only a low prevalence of NAI resistance (<1%) had been detected in circulating viruses. However, surveillance in Europe in late 2007 revealed significant numbers of A(H1N1) influenza strains with a H274Y neuraminidase mutation that were highly resistant to the NAI oseltamivir. We examined 264 A(H1N1) viruses collected in 2008 from South Africa, Oceania and SE Asia for their susceptibility to NAIs oseltamivir, zanamivir and peramivir in a fluorescence-based neuraminidase inhibition assay. Viruses with reduced oseltamivir susceptibility were further analysed by pyrosequencing assay. The frequency of the oseltamivir-resistant H274Y mutant increased significantly after May 2008, resulting in an overall proportion of 64% (168/264) resistance among A(H1N1) strains, although this subtype represented only 11.6% of all isolates received during 2008. H274Y mutant viruses demonstrated on average a 1466-fold reduction in oseltamivir susceptibility and 527-fold reduction in peramivir sensitivity compared to wild-type A(H1N1) viruses. The mutation had no impact on zanamivir susceptibility. Ongoing surveillance is essential to monitor how these strains may spread or persist in the future and to evaluate the effectiveness of treatments against them.

Laboratory diagnosis and surveillance of human respiratory viruses by PCR in Victoria, Australia, 2002–2003

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.

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 ≥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.

Utility of a Multiplex PCR Assay for Detecting Herpesvirus DNA in Clinical Samples

ABSTRACT A multiplex PCR was designed to amplify herpes simplex virus types 1 and 2, cytomegalovirus, and varicella-zoster virus DNA present in a diverse range of clinical material. The susceptibility of these viruses to in vivo inhibition by at least one antiviral drug was an important consideration in their inclusion in the multiplex detection system. An aliquot of equine herpesvirus was introduced into each specimen prior to extraction and served as an indicator of potential inhibitors of the PCR and a detector of suboptimal PCR conditions. Compared to virus isolation and immunofluorescence-based antigen detection, the multiplex assay yielded higher detection rates for all viruses represented in the assay. The turnaround time for performance of the assay was markedly reduced compared to those for the other techniques used to identify these viruses. More than 21,000 tests have been performed using the assay. Overall, the multiplex PCR enabled the detection of substantially increased numbers of herpesviruses, in some cases in specimens or anatomical sites where previously they were rarely if ever identified using traditional detection methods.

Antiviral chemotherapy for chronic hepatitis B infection: lessons learned from treating HIV-infected patients

AFTER YEARS of modest treatment success with /---]kthe use of interferon alpha, antiviral chemotherapy for chronic hepatitis B virus (HBV) infection with nucleoside analogues now provides an alternative means for interrupting the progression of disease and even conceivably eradicating the virus from the infected host. Three factors have converged to provide important advances in anti-hepadnaviral chemotherapy: (iS improved understanding of the immunopathogenesis of HBV infection; (ii) the availability of standardised, reliable quantitative assays for serum HBV DNA; and (iii) the range and type of potent anti-hepadnaviral drugs. Almost identical statements were made about therapeutic options for the human immunodeficiency virus (HIV) over 2 years ago (1). Since then, confidence and optimism regarding the likelihood of suppressing and even eliminating HIV from infected individuals has waxed and waned. However, extremely important lessons have been learned from the HIV experience, and these are likely to have a significant impact on the treatment of HBV infection. Several reviews describing antiviral agents active against the HBV have recently been published (2-4) and this material will not be re-presented here. The focus and purpose of this review is to compare and contrast key virological, pathogenetic and chemotherapeutic issues in the treatment of these two viral infections so that new paradigms for managing chronic hepatitis B infection may be developed.

Phylogenetic Investigation of Transmission Pathways of Drug-Resistant HIV-1 Utilizing Pol Sequences Derived From Resistance Genotyping

Objectives:To investigate the nature of transmission links existing between patients recently infected with HIV strains containing transmitted drug resistance (TDR) mutations. Methods:Virus from 63 individuals recently infected with HIV-1 containing TDR mutations was analyzed phylogenetically to determine virological links. Phylogenetic trees were reconstructed using maximum likelihood and distance-based methods. Monophyletic clusters detected on the basis of pol sequences were confirmed using env and gag sequences. Potential bias caused by the presence of drug resistance mutations was assessed by reanalyzing the pol sequence set after the omission of 16 drug resistance codons identified in the TDR population. Results:Phylogenetic analysis revealed 9 apparent transmission clusters involving 24 of the 63 (38%) TDR patients. Each cluster was supported by high bootstrap values and low intracluster genetic distances. The 9 transmission clusters were confirmed in separate analyses using env and gag sequences and in pol sequences after the removal of codons associated with drug resistance. Conclusions:Pol sequences generated during baseline resistance genotyping for newly HIV-infected patients provide the opportunity for real-time phylogenetics to identify sources of multiple HIV transmission events. This study demonstrated the existence of several distinct clusters of patients whose TDR strains were linked. Several discrete clusters involving transmission of K103N- and/or M41L-resistant virus to multiple recipients were detected, suggesting that multiple transmission pathways can exist for viruses with the same resistance mutations.

Induction of Apoptosis by the Severe Acute Respiratory Syndrome Coronavirus 7a Protein Is Dependent on Its Interaction with the Bcl-XL Protein

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) 7a protein, which is not expressed by other known coronaviruses, can induce apoptosis in various cell lines. In this study, we show that the overexpression of Bcl-XL, a prosurvival member of the Bcl-2 family, blocks 7a-induced apoptosis, suggesting that the mechanism for apoptosis induction by 7a is at the level of or upstream from the Bcl-2 family. Coimmunoprecipitation experiments showed that 7a interacts with Bcl-XL and other prosurvival proteins (Bcl-2, Bcl-w, Mcl-1, and A1) but not with the proapoptotic proteins (Bax, Bak, Bad, and Bid). A good correlation between the abilities of 7a deletion mutants to induce apoptosis and to interact with Bcl-XL was observed, suggesting that 7a triggers apoptosis by interfering directly with the prosurvival function of Bcl-XL. Interestingly, amino acids 224 and 225 within the C-terminal transmembrane domain of Bcl-XL are essential for the interaction with the 7a protein, although the BH3 domain of Bcl-XL also contributes to this interaction. In addition, fractionation experiments showed that 7a colocalized with Bcl-XL at the endoplasmic reticulum as well as the mitochondria, suggesting that they may form complexes in different membranous compartments.

Evaluation of twenty rapid antigen tests for the detection of human influenza A H5N1, H3N2, H1N1, and B viruses.

Twenty rapid antigen assays were compared for their ability to detect influenza using dilutions of virus culture supernatants from human isolates of influenza A H5N1 (clade 1 and 2 strains), H3N2 and H1N1 viruses, and influenza B. There was variation amongst the rapid antigen assays in their ability to detect different influenza viruses. Six of the 12 assays labeled as distinguishing between influenza A and B had comparable analytical sensitivities for detecting both influenza A H5N1 strains, although their ability to detect influenza A H3N2 and H1N1 strains varied. The two assays claiming H5 specificity did not detect either influenza A H5N1 strains, and the two avian influenza‐specific assays detected influenza A H5N1, but missed some influenza A H3N2 virus supernatants. Clinical trials of rapid antigen tests for influenza A H5N1 are limited. For use in a pandemic where novel influenza strains are circulating (such as the current novel influenza A H1N1 09 virus), rapid antigen tests should ideally have comparable sensitivity and specificity for the new strains as for co‐circulating seasonal influenza strains. J. Med. Virol. 81:1918–1922, 2009.

SARS–associated Coronavirus Replication in Cell Lines

Virus can replicate in several common cell lines, sometimes without cytopathic effect.

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

Abstract 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.