Abstract Background Between January 2005 and April 2006, six patients of influenza A/H5N1 virus infection were reported in Cambodia, all with fatal outcome. Objectives We describe the virological ...findings of these six H5N1 patients in association with clinical and epidemiologic findings. Study design Broncho-alveolar lavage, nasopharyngeal, throat and rectal swabs and sera were cultured for virus isolation and viral load quantified in clinical specimens by real-time RT-PCR. We compared sequences obtained from different body sites within the same patient to detect viral quasi-species. Results H5N1 virus strains isolated in Cambodia belong to genotype Z, clade 1 viruses. H5N1 viruses were isolated from serum and rectal swab specimens in two patients. The haemagglutinin gene sequences of the virus in different body sites did not differ. Amino acid substitutions known to be associated with a change in virus binding were not observed. Conclusion The high frequency of virus isolation from serum and faecal swabs highlights that H5N1 is likely to be a disseminated infection in humans and this has implications for antiviral treatment, biosafety in clinical laboratories and on risks for nosocomial and human-to-human transmission. There were no tissue-specific adaptive mutations in the HA gene from viruses isolated from different organs.
Human B19 erythrovirus replicates in erythroid progenitors present in bone marrow and fetal tissues where partial oxygen tension is low. Here we show that infected human primary erythroid progenitor ...cells exposed to hypoxia (1% O
2) in vitro increase viral capsid protein synthesis, virus replication, and virus production. Hypoxia-inducible factor-1 (HIF-1), the main transcription factor involved in the cellular response to reduced oxygenation, is shown to bind an HIF binding site (HBS) located in the distal part of the B19 promoter region, but the precise mechanism involved in the oxygen-sensitive upregulation of viral gene expression remains to be elucidated.
Prions can be detected and quantified currently by using either immunoassays such as Western-blot, ELISA or conformation dependent immunoassay, or an infectivity assay in laboratory animals ...(bioassay). While immunoassays are inexpensive and rapid, they are based on the detection of PrP
Sc, the abnormal isoform of the prion protein, a surrogate marker for prion infectivity. The bioassay is considered the gold-standard analytical method for measuring prion infectivity, but it is very costly and time-consuming, involving the destruction of large numbers of animals. The use of the transgenic MovS6 cell line is described for the development of an
in vitro tissue culture infectivity assay (TCIA) for prion detection and quantitation. Compared to a bioassay, the TCIA is rapid (∼8 weeks), easy to implement, much less expensive, and requires far fewer animals. After titrating concomitantly a prion-infected brain homogenate sample by Western-blot, TCIA and bioassay, data show that the sensitivity of the TCIA is close to that of the bioassay, since 1 TCID
50 corresponds to 4 ID
50, and 80-fold more sensitive than the Western-blot. The application of the TCIA to the evaluation of prion removal in biological products manufacturing processes is described using a 15
nm-nanofiltration step of human albumin as a model.
Highly pathogenic avian influenza (HPAI) virus was first detected in Cameroon in February 2006. Analysis of NA sequences of the virus demonstrated that it is closely related to the H5N1 isolates from ...Northern Nigeria, Sudan and Ivory Coast, suggesting a common virus ancestor.
Laboratoire de Virologie, UPRES EA 2387, CERVI 1 , Laboratoire de Génétique Moléculaire, Service de Biochimie Médicale 2 and Service des Maladies Infectieuses et Tropicales 3 , Groupe Hospitalier ...Pitié-Salpêtrière, 83 Bld de lHôpital, 75651 Paris Cedex 13, France
Author for correspondence: Henri Agut. Fax +33 1 42 17 74 11. e-mail henri.agut{at}psl.ap-hop-paris.fr
After serial passage in the presence of increasing concentrations of ganciclovir (GCV) in vitro , a human herpesvirus-6 (HHV-6) mutant exhibiting a decreased sensitivity to the drug was isolated. Analysis of drug susceptibility showed that the IC 50 of this mutant was 24-, 52- and 3-fold higher than that of the wild-type (wt) IC 50 in the case of GCV, cidofovir and foscarnet, respectively. Genotypic analysis showed two single nucleotide changes as compared to the wild-type: an A G substitution of the U69 protein kinase (PK) gene resulted in an M 318 V amino acid substitution and the other change, located in the C-terminal part of the U38 gene, resulted in an A 961 V amino acid substitution within the DNA polymerase. The M 318 V change was located within the consensus sequence DISPMN of the putative catalytic domain VI of the PK. This change was homologous to the M 460 V and M 460 I changes that had been reported previously within the consensus sequence DITPMN of the human cytomegalovirus (HCMV) UL97 PK and associated with the resistance of HCMV to GCV. The M 318 V change was also detected by PCR in HHV-6-infected PBMCs from an AIDS patient who had been treated with GCV for a long period of time and exhibited a clinically GCV-resistant HCMV infection. These findings provide strong circumstantial evidence that the M 318 V change of the PK gene is associated with resistance to GCV and raise the question of cross resistance to this drug among different betaherpesviruses.
Wild aquatic birds are considered to be the natural reservoir for influenza A viruses, and previous studies have focused mainly on species in the orders Anseriformes and Charadriiformes. In this ...study, we surveyed a larger spectrum of potential hosts belonging to 10 avian orders. Cloacal swabs (n=1,044) from 72 free-living bird species, were analysed by reverse transcription-polymerase chain reaction for the presence of avian influenza virus. Only two Mediterranean Gulls (Larus melanocephalus) tested positive; one of these viruses was identified as an H9N2 subtype. The absence of infection among passerine birds supports the idea that the prevalence of avian influenza virus infection in terrestrial species is low.
Evolution of H5N1 avian influenza viruses in Asia World Health Organization Global Influenza Program Surveillance Network, /; Aubin, Jean-Thierry; Azebi, Saliha ...
Emerging infectious diseases,
10/2005, Volume:
11, Issue:
10
Journal Article
Peer reviewed
Open access
An outbreak of highly pathogenic avian influenza A (H5N1) has recently spread to poultry in 9 Asian countries. H5N1 infections have caused > or =52 human deaths in Vietnam, Thailand, and Cambodia ...from January 2004 to April 2005. Genomic analyses of H5N1 isolates from birds and humans showed 2 distinct clades with a nonoverlapping geographic distribution. All the viral genes were of avian influenza origin, which indicates absence of reassortment with human influenza viruses. All human H5N1 isolates tested belonged to a single clade and were resistant to the adamantane drugs but sensitive to neuraminidase inhibitors. Most H5N1 isolates from humans were antigenically homogeneous and distinct from avian viruses circulating before the end of 2003. Some 2005 isolates showed evidence of antigenic drift. An updated nonpathogenic H5N1 reference virus, lacking the polybasic cleavage site in the hemagglutinin gene, was produced by reverse genetics in anticipation of the possible need to vaccinate humans.
During fall 2005, the rapid and wide spread of highly pathogenic (HP) H5N1 avian influenza viruses (AIV) outside Asia alerted European health authorities. Because of abnormal and recurrent field ...mortality, wild migratory birds were considered to be the main dispersing agent of the virus at an intercontinental scale. European wintering wetlands, such as the Camargue (Rhône delta, France), are identified as potential hot spots for the risk of introduction and transmission of bird-borne diseases. In this study, we investigated the role of migratory waterbirds (mainly ducks) in the spread of HP H5N1 viruses. We combined molecular analysis of living and freshly killed birds with population surveillance (aerial censuses and death surveillance). We sampled 1345 birds belonging to 17 waterbird species (3 orders) in the Camargue between September 2005 and March 2006. The prevalence of AIV was 1.8%. We did not detect HP H5N1 virus. Population censuses did not reveal any population decreases nor abnormal mortalities. We discuss, in the light of these results, the implication of wild migratory ducks in the arrival of HP H5N1 AIV in Europe.
We have compared the sensitivity and specificity of four PCR methods of RHD gene detection using different sets of primers located in the regions of highest divergence between the RHD and RHCE genes, ...notably exon 10 (method I), exon 7 (method II), exon 4 (method III) and intron 4 (method IV). Methods I–III were the most sensitive and gave a detectable signal with D‐pos/D‐neg mixtures containing only 0.001% D‐positive cells. Moreover, method II could detect the equivalent DNA amount present in only three nucleated cells in the assay without hybridization of PCR products, whereas the sensitivity of the other methods was 10–50 times less. Investigation of D variants indicated that false‐negative results were obtained with method II (DIVb variant), method III (DVI and DFR variants) and method IV (DVI variants), but not method I. Weak D (Du) was correctly detected as D‐positive by all methods, but most cases of Rhnull appeared as false‐positives, as they carry normal RH genes that are not phenotypically expressed. Some false‐positive results were obtained with method I in a few Caucasian DNA samples serotyped as RhD‐neg but carrying a C‐ or E‐allele, whereas a high incidence of false‐positives was found among non‐Caucasian Rh‐negative samples by all methods. In the Caucasian population, however, we found a full correlation between the predicted genotype and observed phenotype at birth of 92 infants. Although we routinely use the four methods for RHD genotyping, a PCR strategy based on at least two methods is recommended.
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