Contribution of laboratories in the WHO Eastern Mediterranean Region to the selection of candidate seasonal influenza vaccine, 2010-2015.

The World Health Organization (WHO) formulates recommendations for viruses to be included in vaccines for the influenza seasons in the northern and southern hemispheres on the basis of analyses by its collaborating centres (CCs). This report describes the contribution of influenza laboratories and national influenza centres in countries in the WHO Region for the Eastern Mediterranean to the selection process of seasonal and pre-pandemic influenza virus subtypes. Data submitted by 22 countries to FluNet and FluID between September 2010 and June 2015 were analysed. National Influenza Centres (NICs) in 12 countries (55%) reported data, 5 (23%) to both FluNet and FluID and 7 (32%) only to FluNet. The WHO CC in London characterized 78% of the samples, and the CC in Atlanta, characterized 21%. The contribution of influenza laboratories and NICs from this Region to global influenza surveillance is appreciable. However, enhancing the contribution through initiatives such as the Pandemic Influenza Preparedness Framework is still needed.

Contribution of laboratories in the WHO Eastern Mediterranean Region to the selection of candidate seasonal influenza vaccine, 2010-2015

The World Health Organization [WHO] formulates recommendations for viruses to be included in vaccines for the influenza seasons in the northern and southern hemispheres on the basis of analyses by its collaborating centres [CCs]. This report describes the contribution of influenza laboratories and national influenza centres in countries in the WHO Region for the Eastern Mediterranean to the selection process of seasonal and pre-pandemic influenza virus subtypes. Data submitted by 22 countries to FluNet and FluID between September 2010 and June 2015 were analysed. National Influenza Centres [NICs] in 12 countries [55%] reported data, 5 [23%] to both FluNet and FluID and 7 [32%] only to FluNet. The WHO CC in London characterized 78% of the samples, and the CC in Atlanta, characterized 21%. The contribution of influenza laboratories and NICs from this Region to global influenza surveillance is appreciable. However, enhancing the contribution through initiatives such as the Pandemic Influenza Preparedness Framework is still needed

L'Organisation mondiale de la Santé [OMS] émet des recommandations quant aux virus à inclure dans les vaccins contre les grippes saisonnières des hémisphères nord et sud, en fonction des analyses réalisées par ses centres collaborateurs. Le présent article décrit la contribution des laboratoires de la grippe et des centres nationaux de la grippe [CNG] des pays de la Région OMS de la Méditerranée orientale au processus de sélection des sous-types du virus de la grippe saisonnière et pré-pandémique. Les données transmises par 22 pays à FluNet et à FluID entre septembre 2010 et juin 2015 ont été analysées. Les CNG de 12 pays [55%] ont transmis leurs données, dont 5 [23%] à la fois à FluNet et à FluID, et 7 [32%] à FluNet uniquement. Les centres collaborateurs de l'OMS de Londres et d'Atlanta ont caractérisé 78% et 21% des échantillons respectivement. La contribution des laboratoires de la grippe et des CNG de cette Région à la surveillance mondiale de la grippe est appréciable. Cependant, il est nécessaire de renforcer cette contribution en tirant parti d'opportunités telles que celle du Cadre de préparation en cas de grippe pandémique

The role of glycoprotein H in herpesvirus membrane fusion.

The mechanism by which herpesviruses fuse with cellular membranes to permit virus entry is still relatively poorly understood. This process is proving difficult to unravel, largely due to the fact that multiple viral envelope proteins appear to function in concert to mediate the fusion event. For Herpes Simplex Virus Type 1 (HSV1), glycoproteins B, D and the gHL heterodimer are all required for fusion, and gHL counterparts are involved in the fusion process of all other members of the herpesvirus family. An understanding of the functional domains of gH that are critical for fusion may offer the possibility of designing specific peptide inhibitors of virus entry, and recent progress has highlighted the potential usefulness of this approach. This review discusses these advances and outlines some of the similarities and differences between gH homologues of the different members of this diverse family of viruses.

Characterization of herpes simplex virus type 1 recombinants with mutations in the cytoplasmic tail of glycoprotein H.

Herpes simplex virus (HSV) type 1 glycoprotein H is essential for fusion of virus envelopes with cellular membranes and for the fusion of an infected cell membrane with an uninfected neighbour. Previous studies have pointed to a requirement for certain amino acid residues of the cytoplasmic tail of gH in these processes. Results from transient transfection experiments suggested that the serine-valine-proline (SVP) motif in the cytoplasmic tail may be important for gH-mediated fusion. HSV recombinants expressing gH molecules with mutations in the cytoplasmic tail were constructed and analysed in terms of their abilities to fuse cellular membranes and to function in virus entry. Viruses containing a deletion of the SVP motif, or in which the valine residue of this triplet was replaced by alanine, entered cells less efficiently than wild-type virus and were unable to induce syncytium formation on Vero cells.

CCD imaging of luciferase gene expression in single mammalian cells.

Quantitative and sensitive imaging of chemiluminescence, bioluminescence and fluorescence emissions is emerging as an increasingly important technique for a range of biomedical applications (Hooper et al., 1990). A brief review of low-light-level imaging is presented, with particular reference to charge-coupled devices (CCD). Detectors for sensitive imaging are described and compared, including various CCDs and photon-counting devices. Image analysis techniques based on digital image processing, may be applied to quantify luminescent processes with these detectors. Images of luciferase gene expression in single mammalian cells have been obtained using a particular high-sensitivity intensified CCD camera. The method is illustrated using cell monolayers infected with recombinant vaccinia virus encoding the firefly luciferase, luc gene (Rodriguez et al., 1988). The CCD camera has been used to detect luciferase expression in single, recombinant infected cells amongst over one million non-infected cells. The rapid detection of luciferase-expressing viruses may be used for the selection of virus deletion mutants into which the luciferase gene has been cloned at specific sites. This is particularly useful in the case of viruses such as cytomegalovirus which have slow replication cycles. This direct imaging technique is simple and versatile. It offers a rapid, non-invasive method for the sensitive detection of luciferase activity in single, luciferase-expressing cells. One can envisage the use of luciferase as a sensitive and convenient co-selection marker gene in the analysis of both gene expression and protein function. These methods offer tremendous potential in the fields of molecular and cellular biology.

Properties of a non-tumorigenic human cervical keratinocyte cell line.

A human cervical keratinocyte cell line, W12, has been initiated from a low-grade cervical lesion histologically diagnosed as CIN I. This cell line has, to date, undergone over 300 generations in vitro with an average doubling time of 30 hr: an aneuploid karyotype has developed with progressive in vitro growth. W12 contains HPV16 DNA present at approximately 100 copies and the state of the viral DNA over a number of passages has been examined. The HPV16 DNA is stably maintained at high copy number over several passages and restriction enzyme analysis together with electrophoresis of uncleaved viral DNA indicate that it is present predominantly as the episomal molecule. W12 cells exhibit a typical keratinocyte morphology and, when transplanted into the flank of the nude mouse, form an epithelial lesion with the histological features of CIN I/II.

Studies on mitochondrial Ca2+-transport and matrix Ca2+ using fura-2-loaded rat heart mitochondria.

Rat heart mitochondria were incubated for 5 min at 30 degrees C and at approx. 40 mg protein.ml-1 and in the presence of 10 microM fura-2/AM. This allowed the entrapment of free fura-2 within the mitochondrial matrix and its use as a probe for Ca2+, but without affecting the apparent viability of the mitochondria. Parallel measurements of the activities of the intramitochondrial Ca2+-sensitive enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase, allowed an assessment of their sensitivity to measured free Ca2+ within intact mitochondria incubated under different conditions; the enzymes responded to matrix Ca2+ over the approximate range 0.02-2 microM with half-maximal effects at about 0.3-0.6 microM Ca2+. Effectors of Ca2+-transport across the inner membrane (e.g., Na+, Mg2+, Ruthenium red, spermine) did not appear to affect these ranges, but did bring about expected changes in Ca2+ distribution across this membrane. Significantly, when mitochondria were incubated in the presence of physiological concentrations of both Na+ and Mg2+, and at low extramitochondrial Ca2+ (less than 400 nM), there was a gradient of Ca2+ (in:out) of less than unity; at higher extramitochondrial [Ca2+] (but still within the physiological range) the gradient was greater than unity indicating a highly cooperative nature of transmission of the Ca2+ signal into the matrix under such conditions.

Analysis of the L1 gene product of human papillomavirus type 16 by expression in a vaccinia virus recombinant.

The L1 open reading frame of human papillomavirus type 16 (HPV16) has been expressed in vaccinia virus under the control of both the 7.5K early and late promoter, and the 4b major late promoter. Antibodies to a beta-galactosidase fusion protein containing a C-terminal portion of the HPV16 L1 gene product were used to compare the levels of L1 expression in the two recombinants, and showed that greater levels of expression were obtained when the gene was placed under the control of the 4b late promoter. Immunofluorescence studies revealed a nuclear location of the L1 gene product when expressed in vaccinia virus. Antibodies to the beta-galactosidase fusion protein detected a major polypeptide species of 57K and a minor species of 64K in Western blots of recombinant-infected cell lysates. The 64K species was not detected when cells were infected in the presence of tunicamycin, indicating that the primary translation product of the HPV16 L1 open reading frame is modified by N-linked glycosylation when expressed in vaccinia virus. Whereas antibodies to HPV16 L1 fusion proteins and to a peptide containing amino acids from the C terminus of HPV16 L1 reacted well in Western blots with the HPV16 L1 target expressed in vaccinia virus, no reactivity was observed with antibodies to bovine papillomavirus type 1 particles or to a HPV6b fusion protein.