Molecular characterization of measles virus strains causing subacute sclerosing panencephalitis in France in 1977 and 2007.

Measles virus strains from two subacute sclerosing panencephalitis (SSPE) cases diagnosed in 1977 (Laine strain) and in 2007 (Hoedts strain) were studied. Phylogenetic analysis based on C-terminal part of the nucleoprotein and the entire H gene showed that Hoedts strain, circulating in France presumably in the 1980s, belonged to genotype C2. However, Laine strain, suspected to have circulated between 1940s and 1960s, could not be assigned to any known measles virus genotypes. Sequences analysis of the Laine strain suggested that it originated from a measles virus that may have circulating at the same period as the Edmonston strain. The analysis of the whole genome of both SSPE strains revealed biased hypermutations in M, F, and H gene. Some of these mutations like the L165P found in the M protein sequence of the Laine strain, the amino acid position 94, where a mutation M94V was found in the F protein sequence of the Hoedts strain are known to play an important role in the glycoprotein interaction and to impair the ability of measles virus strain to produce cell-free infectious viral particles.This is the first study on molecular characterization of the entire coding region of measles virus isolated from SSPE cases in France.

NucliSENS EasyQ HPV v1 test - Testing for oncogenic activity of human papillomaviruses.

BACKGROUND: Analytical sensitivity of DNA-based assays to detect infection with human papillomaviruses is very high, but clinical specificity for cervical cancer strongly depends on the age of the patient and case classification. To solve the dilemma between sensitivity and specificity, a new generation of assays focuses on the pathogenic factors that underlie the development of HPV-associated tumors: the expression of the viral oncogenes E6 and E7. Demonstration of persistent expression of these mRNAs or expression in the context of relevant clinical symptoms has a strong positive predictive value for the development of HPV-associated carcinomas and strongly warrants further diagnostic action. OBJECTIVES: The NucliSENS EasyQ HPV v1 test was designed to test cervical scrapes for the expression of the oncogenic E6/E7 mRNA from the five most common carcinogenic HPV types (16, 18, 31, 33 and 45). The test can be used for confirmation and risk stratification of individuals with proven infection with high risk papillomaviruses. STUDY DESIGN: In order to establish performance of the assay, sensitivity, specificity, repeatability, and reproducibility were determined with artificial and clinical specimens. Further, a total of 420 cervical scrapes were tested and the results directly compared to the CE-market device PreTect HPV-Proofer assay (NorChip, Klokkarstua, Norway). For arbitration of discordant clinical results, the specimens were rated according to Pap-classification and the presence of HPV DNA was determined. RESULTS: The limit of detection for the five HPV types 16, 18, 31, 33, and 45 ranged from 2.3x10(2) to 3.0x10(4) copies/mL on a background of 5x10(3) HPV-negative HS67 cells. No cross-reactivity for other viral, bacterial, or fungal agents known to be potentially present in cervical fluids was detected. Repeatability and reproducibility were shown by testing panels of HPV-spiked artificial and clinical samples. A comparative analysis of 420 cervical scrapes demonstrated an overall concordance of >90% between the NucliSENS EasyQ HPV test and the technologically related PreTect HPV-Proofer assay.

Genotyping measles virus by real-time amplification refractory mutation system PCR represents a rapid approach for measles outbreak investigations.

Real-time PCR has been developed to genotype measles virus (MV) isolates. MV strains circulating in epidemics in Gabon in 1984, Cameroon in 2001, Morocco in 2003, and France in 2004 were investigated. We developed a real-time amplification refractory mutation system PCR (RT-AMRS PCR) using SYBR green fluorescent dye. Six pairs of primers for RT-ARMS PCR were designed to specifically amplify genotypes A, B2, B3.1, B3.2, C2, and D7. Genotypes could be differentiated by melting curve analysis. All strains were also confirmed by direct sequencing. Using the result obtained by direct sequencing and phylogenetic analysis as the reference, the accuracy of MV by RT-ARMS PCR and melting curve analysis was 97%. However, the latter method is more rapid and sensitive than the former method. This method could be a useful tool for molecular epidemiological studies of MV, providing an efficient alternative for large-scale studies.

Re-emergence of measles among young adults in Marseilles, France.

A total of 89 cases of measles were diagnosed in southeastern France between January and June 2003. Nation-wide epidemiological investigations suggested that this outbreak was restricted to southeastern France, and most likely reflected the endemic circulation of measles virus due low vaccination coverage. Genetic analysis identified genotype D7 strains as the cause of the outbreak.

Cloning and expression of surface antigens from occult chronic hepatitis B virus infections and their recognition by commercial detection assays.

Occult hepatitis B virus (HBV) infections show little or no serological markers of viral infection, including the absence of hepatitis B surface antigen (HBsAg) which is the main marker of ongoing HBV infection. Such infections can be important in the context of blood and/or organ donations. To study whether mutations contribute to HBsAg seronegativity, S gene sequences from such patients were amplified and cloned. Sequencing revealed 12 clones from seven different patients which contained potentially important mutations. The sequences were subcloned into an expression vector and mutant HBsAgs were expressed in cell culture. The capacity of three HBsAg detection assays to recognise the mutant HBsAgs was studied. Three categories were found: mutant HBsAgs that are not recognised by the assays, those that are recognised as well as wild-type (WT) antigen and an intermediate category where detection of the mutant HBsAgs is reduced with respect to WT. Most of the isolates fall into the second category. Mutations can therefore contribute to HBsAg seronegativity in occult HBV infections, but in most cases the explanation is probably the low level of viral replication.

Characterization of two hepatitis B virus populations isolated from a hepatitis B surface antigen-negative patient.

In a study of surface antigen-negative, but weakly hepatitis B virus (HBV) DNA-positive, patients, we were able to amplify and clone whole HBV genomes from the serum of a cirrhotic patient. Sequencing showed that the patient harbored two different HBV populations, one of genotype A and the other of genotype D, with the genotype D genome apparently predominating. The surface antigen of the genotype A virus is heavily mutated, especially in the extracellular << determinant a >> region, with several mutations that have not been previously described. The genotype D virus is a precore mutant. Both genomes possess the common A1762T-G1764A double mutation of the basal core promoter (BCP), and the genotype D virus is also mutated in the << TATA box >> of the large surface antigen promoter. Biological characterization showed that the genotype A genome was fully replication-competent, whereas the genotype D genome replicated poorly. The small surface antigen of the genotype A virus was only very weakly recognized by commercial tests. The small surface antigen of the genotype D virus could be recognized by the tests, but it was mainly retained within transfected cells, probably because of an excess of large surface antigen. In conclusion, the cryptic nature of this double HBV infection is characterized by the predominance of the replication-deficient genotype D virus over the replication-competent genotype A virus.