Detection and subtyping of Herpes simplex virus in clinical samples by LightCycler PCR, enzyme immunoassay and cell culture

BACKGROUND: Prompt laboratory diagnosis of Herpes simplex virus (HSV) infection facilitates patient management and possible initiation of antiviral therapy. In our laboratory, which receives various specimen types for detection of HSV, we use enzyme immunoassay (EIA) for rapid detection and culture of this virus. The culture of HSV has traditionally been accepted as the diagnostic 'gold standard'. In this study, we compared the use of real time PCR (LightCycler) for amplification, detection and subtyping of specific DNA with our in-house developed rapid and culture tests for HSV. RESULTS: The LightCycler PCR (LC-PCR) detected and subtyped HSV in 99% (66/67) of HSV positive specimens, compared to 81% (54/67) by rapid antigen EIA or 57% (36/63) by culture. A specimen was considered positive when two or more tests yielded HSV identifications or was culture positive. Discordant results were confirmed with an in-house developed PCR-ELISA or DNA sequence analysis. The typing results obtained with the LC-PCR and by culture amplified test were completely concordant. CONCLUSIONS: This study showed that the LC-PCR provided a highly sensitive test for simultaneous detection and subtyping of HSV in a single reaction tube. In addition to increased sensitivity, the LightCycler PCR provided reduced turn-around-times (2 hours) when compared to enzyme immunoassay (4 hours) or culture (4 days)

Addressing non-motorised transport movement along and across railway lines in the city of Cape Town

Paper presented at the 33rd Annual Southern African Transport Conference 7-10 July 2014 "Leading Transport into the Future", CSIR International Convention Centre, Pretoria, South Africa.There are numerous reasons why pedestrians choose to cross railway tracks in South Africa and in Cape Town in particular. Whilst this behavior generally forms part of their desire line and is often part of their journey, the crossing of railway lines is illegal and unsafe and is causing a significant amount of injuries and fatalities within the rail network in the City. Such incidents occurring along railway lines cause a great disruption to the railway operational service with a resultant negative effect on the economic growth of the City. In this paper, an investigation into the Non-Motorised Transport (NMT) movements along and across all railway lines, within the jurisdictional area of the City of Cape Town, has been undertaken using innovative video-based techniques and assessed using GIS-mapping methods. The investigation identified several “hotspot” locations where various types of recommendations and/or intervention strategies are urgently needed to enhance public (pedestrian) safety and ensure uninterrupted train service. Intervention strategies proposed have been drawn from international best practice tailored to suit the unique South African pedestrian culture. The paper highlights the “Nomzamo rail crossing” as a special case study as it demonstrates the failure of town planning processes to consider the impacts of locating developments next to railway lines. The Nomzamo case study is an example of a large-scale, low-income urban development that has been allowed to develop alongside a rail line without due consideration of NMT desire lines that have materialised across the rail line. Currently, approx. 3,000 pedestrians cross the railway line at the Nomzamo location in the 2½-hour morning peak period, which include scholars and cyclists.This paper was transferred from the original CD ROM created for this conference. The material was published using Adobe Acrobat 10.1.0 Technology. The original CD ROM was produced by CE Projects cc. Postal Address: PO Box 560 Irene 0062 South Africa. Tel.: +27 12 667 2074 Fax: +27 12 667 2766 E-mail: [email protected]

Sequencing and analysis of globally obtained human parainfluenza viruses 1 and 3 genomes

Human Parainfluenza viruses (HPIV) type 1 and 3 are important causes of respiratory tract infections in young children globally. HPIV infections do not confer complete protective immunity so reinfections occur throughout life. Since no effective vaccine is available for the two virus subtypes, comprehensive understanding of HPIV-1 and HPIV-3 genetic and epidemic features is important for diagnosis, prevention, and treatment of HPIV-1 and HPIV-3 infections. Relatively few whole genome sequences are available for both HPIV-1 and HPIV-3 viruses, so our study sought to provide whole genome sequences from multiple countries to further the understanding of the global diversity of HPIV at a whole-genome level. We collected HPIV-1 and HPIV-3 samples and isolates from Argentina, Australia, France, Mexico, South Africa, Switzerland, and USA from the years 2003–2011 and sequenced the genomes of 40 HPIV-1 and 75 HPIV-3 viruses with Sanger and next-generation sequencing with the Ion Torrent, Illumina, and 454 platforms. Phylogenetic analysis showed that the HPIV-1 genome is evolving at an estimated rate of 4.97 × 10−4 mutations/ site/year (95% highest posterior density 4.55 × 10−4 to 5.38 × 10−4) and the HPIV-3 genome is evolving at a similar rate (3.59 × 10−4 mutations/site/year, 95% highest posterior density 3.26 × 10−4 to 3.94 × 10−4). There were multiple genetically distinct lineages of both HPIV-1 and 3 circulating on a global scale. Further surveillance and whole-genome sequencing are greatly needed to better understand the spatial dynamics of these important respiratory viruses in humans.S1 Text. HPIV-1 Sanger sequencing primers.S2 Text. HPIV-3 Sanger sequencing primers.S1 Table. The sequence information of the 40 HPIV-1 genomes.S2 Table. The sequence information of the 75 HPIV-3 genomes.S3 Table. MEME episodic selection results for HPIV-1 and HPIV-3.The National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under contract number HHSN272200900007C and grant numbers U19AI110819, with the sub-project directed by HAL, and grants U01AI070428 and U01AI077988 awarded to KJH.http://www.plosone.orgam2019Medical Virolog

Improved laboratory diagnosis of Mycoplasma pneumoniae infection

Thesis (M.Sc.) -- University of Adelaide, Division of Medical Virology, Institute of Medical & Veterinary Science, 198

Early events in the replication cycle of human immunodeficiency virus / Tuck Weng Kok.

Copy of author's previously published article on back end-paper.Bibliography: leaves 105-158.xii, 160, [58] leaves, [35] leaves of plates : ill. (some col.) ; 30 cm.Studies the early events in the synthesis of HIV RNA and integration if viral DNA using a cell-to-cell transmission of infection model.Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology & Immunology, 199

Neutralizing monoclonal antibodies to different clades of Influenza A H5N1 viruses

Four IgG1� monoclonal antibodies (mAbs) against Influenza A/Chicken/Vietnam/8/2004 (H5N1) virus are described. Three of these showed neutralizing activities against H5N1 strains from clades 1, 2 and 3 using a retroviral pseudotype or live virus microneutralization assay. In the pseudotype assay, the IC90 neutralizing titre range was >1600–51,200, and with the microneutralization was 80≥10,240. MAb 1C1 showed strong neutralizing activities in both assays. All four mAbs reacted specifically to the immunogen by immunohistochemical staining and to A/Hong Kong/483/1997 (H5N1) and A/Thailand/1(KAN-1)/2004 (H5N1)-infected MDCK cells by immunofluorescence. ELISA titrations of the mAbs showed specificity for H5N1 haemagglutinin (HA) and no cross-reactivity to 15 other Influenza A subtypes. Only mAbs 1C1 and the non-neutralizing 1F7 reacted with HA1, the cleaved subunit of HA, by Western blot. These results suggest that the mAbs recognize distinct or overlapping epitopes and will be useful reagents for construction of specific rapid point-of-care assays or for therapeutic use

Cytotoxic T Cells Are the Predominant Players Providing Cross-Protective Immunity Induced by γ-Irradiated Influenza A Viruses ▿

We previously demonstrated that a single dose of nonadjuvanted intranasal γ-irradiated influenza A virus can provide robust protection in mice against both homologous and heterosubtypic challenges, including challenge with an H5N1 avian virus strain. We investigated the mechanism behind the observed cross-protection to define which arms of the adaptive immune response are involved in mediating this protection. Studies with gene knockout mice showed the cross-protective immunity to be mediated mainly by T cells and to be dependent on the cytolytic effector molecule perforin. Adoptive transfer of memory T cells from immunized mice, but not of memory B cells, protected naïve recipients against lethal heterosubtypic influenza virus challenge. Furthermore, γ-irradiated influenza viruses induced cross-reactive Tc-cell responses but not cross-neutralizing or cross-protective antibodies. In addition, histological analysis showed reduced lung inflammation in vaccinated mice compared to that in unvaccinated controls following heterosubtypic challenge. This reduced inflammation was associated with enhanced early recruitment of T cells, both CD4+ and CD8+, and with early influenza virus-specific cytotoxic T-cell responses. Therefore, cross-protective immunity induced by vaccination with γ-irradiated influenza A virus is mediated mainly by Tc-cell responses

Preclinical efficacy studies of Influenza A haemagglutinin precursor cleavage loop peptides as a potential vaccine

A universal influenza vaccine that does not require annual reformulation would have clear advantages over the currently approved seasonal vaccine. In this study, we combined the mucosal adjuvant alpha-galactosylceramide (αGalCer) and peptides designed across the highly conserved influenza precursor haemagglutinin (HA0) cleavage loop as a vaccine. Peptides designed across the HA0 of influenza A/H3N2 viruses, delivered to mice via the intranasal route with αGalCer as an adjuvant, provided 100 % protection following H3N2 virus challenge. Similarly, intranasal inoculation of peptides across the HA0 of influenza A/H5N1 with αGalCer completely protected mice against heterotypic challenge with H3N2 virus. Our data suggest that these peptide vaccines effectively inhibited subsequent influenza A/H3N2 virus replication. In contrast, only 20 % of mice vaccinated with αGalCer-adjuvanted peptides spanning the HA0 of H5N1 survived homologous viral challenge, possibly because the HA0 of this virus subtype is cleaved by intracellular furin-like enzymes. Results of these studies demonstrated that HA0 peptides adjuvanted with αGalCer have the potential to form the basis of a synthetic, intranasal influenza vaccine.Darren S. Miller, John Finnie, Timothy R. Bowden, Anita C. Scholz, Sawyin Oh, Tuckweng Kok, Christopher J. Burrell, Lee Trinidad, David B. Boyle and Peng L