Genetic characterization of measles virus genotype D6 subacute sclerosing panencephalitis case, Alberta, Canada.

Subacute sclerosing panencephalitis (SSPE) is a progressive and eventually fatal neurological disease arising from a persistent infection with measles virus (MV) acquired at a young age. SSPE measles virus strains are defective and unable to produce progeny virions, due to multiple and extensive mutations in a number of key genes. We sequenced the full MV genome from our recently reported SSPE case, which typed as genotype D6, and compared it with other genotype D6 wild type and SSPE sequences. The Alberta D6 strain was significantly different from other reported SSPE D6 sequences. Mutations were observed in all the genes of the Alberta strain, with the greatest sequence divergence noted in the M gene with 17.6% nucleotide and 31% amino acid variation. The L gene showed the least variation with 1.3% nucleotide and 0.7% amino acid differences respectively. The nucleotide variability for 15,672 bases of the complete genome compared to the wild type and other SSPE D6 strains was around 3%.

Mumps epidemic in orthodox religious low-vaccination communities in the Netherlands and Canada, 2007 to 2009.

We assessed the epidemiological characteristics of a mumps virus epidemic (genotype D) that occurred in the Netherlands between August 2007 and May 2009 and its association with a subsequent mumps outbreak in Canada. In the Netherlands, five data sources were used: notifications (only mandatory since the end of 2008) (56 cases), laboratory confirmation data (177 cases), a sentinel general practitioner (GP) database (275 cases), hospitalisation data (29 cases) and weekly virological reports (96 cases). The median age of cases in the notification, laboratory and GP databases ranged from 13 to 15 years. The proportion of cases that were unvaccinated ranged from 65% to 85% in the notification, laboratory and GP databases. Having orthodox Protestant beliefs was the main reason for not being vaccinated. In Canada, a mumps virus strain indistinguishable from the Dutch epidemic strain was detected between February and October 2008 in an orthodox Protestant community with historical and family links to the affected community in the Netherlands, suggesting that spread to Canada had occurred. Prevention and control of vaccine-preventable diseases among population subgroups with low vaccination coverage remains a priority.

Differentiation and quantitation of human herpesviruses 6A, 6B and 7 by real-time PCR.

The beta-herpesviruses cause considerable morbidity in immunocompromised individuals, such as transplant patients. Most notably within this group is human cytomegalovirus, although HHV-6 and -7 are a growing concern. Identifying HHV-6 and -7 as the cause of post-transplant illness can be challenging due to high seroprevalence and latency properties associated with these human herpesviruses. We have developed a sensitive and specific real-time PCR assay, which can differentiate reliably and quantify HHV-6A, -6B and -7. Using two sets of hybridization probes specific for HHV-6A or -6B and HHV-7, the assay reliably differentiates the three viruses using melting curve analysis. The lower limit of detection for all three viruses was determined to be ten viral genomes. This real-time PCR assay will be useful for differentiation and quantitation of HHV-6A, -6B and -7, especially for monitoring transplant patients.

Role of clinicians and public health professionals for measles surveillance in Canada.

The current strategy to eliminate measles in Canada requires stringent surveillance efforts including the laboratory confirmation of measles cases. Clinicians and public health professionals are involved with the events and actions surrounding the initial contact with suspected measles cases, identification of suspected measles cases, collection of the appropriate clinical information, collection of samples for laboratory confirmation and notification of the appropriate public health authority. Clinical information is necessary for the interpretation of laboratory results and is used, together with the laboratory results, to confirm measles cases. Important clinical information required includes the date of onset of symptoms, date of sample collection and measles vaccination history. A blood sample and specimen for virus isolation (urine or nasopharyngeal or throat swab) should be collected within several days of the onset of the rash. In the laboratory, blood samples are necessary for serological confirmation of measles infection, and specimens for virus isolation are necessary for molecular epidemiological purposes.

Genomic cartography of varicella-zoster virus: a complete genome-based analysis of strain variability with implications for attenuation and phenotypic differences.

In order to gain a better perspective on the true variability of varicella-zoster virus (VZV) and to catalogue the location and number of differences, 11 new complete genome sequences were compared with those previously in the public domain (18 complete genomes in total). Three of the newly sequenced genomes were derived from a single strain in order to assess variations that can occur during serial passage in cell culture. The analysis revealed that while VZV is relatively stable genetically it does posses a certain degree of variability. The reiteration regions, origins of replication and intergenic homopolymer regions were all found to be variable between strains as well as within a given strain. In addition, the terminal viral sequences were found to vary within and between strains specifically at the 3' end of the genome. Analysis of single nucleotide polymorphisms (SNPs) identified a total of 557 variable sites, 451 of which were found in coding regions and resulted in 187 different in amino acid substitutions. A comparison of the SNPs present in the two gE mutant strains, VZV-MSP and VZV-BC, suggested that the missense mutation in gE was primarily responsible for the accelerated cell spread phenotype. Some of the variations noted with high passage in cell culture are consistent with variations seen in the IE62 gene of the vaccine strains (S628G, R958G and I1260V) that may help in pinpointing variations essential for attenuation. Although VZV has been considered to be one of the most genetically stable human herpesviruses, this initial assessment of genomic VZV cartography provides insight into ORFs with previously unreported variations.

Mapping ganciclovir resistance in the human herpesvirus-6 U69 protein kinase.

Human herpesvirus-6 (HHV-6) is a growing concern in immunocompromised individuals, such as in the transplant setting. Alone, or in concert with human cytomegalovirus (HCMV), infections with HHV-6 are often severe enough to require antiviral therapy, generally in the form of ganciclovir (GCV). GCV resistance in HCMV is well documented, both clinically and in the laboratory, and has been shown to result from mutations in the UL97 protein kinase and/or UL54 DNA polymerase. GCV resistance in HHV-6 has been documented. However, to date, it has only been investigated to a limited extent. The baculovirus system has previously been shown to be useful in studying GCV resistance with respect to herpesvirus protein kinase mutations. Using the baculovirus system, we created recombinant baculoviruses expressing either a wild-type HHV-6 U69 protein kinase or a mutated form containing homologous mutations to those documented in the UL97 protein kinase of GCV resistant HCMV isolates. The recombinant baculoviruses were used to infect Sf-9 cells and cultured in the presence of GCV to determine the effect of the HHV-6 U69 protein kinase mutations on GCV susceptibility. Mutations in the HHV-6 U69 protein kinase, homologous to those in the HCMV UL97 protein kinase documented to cause GCV resistance, result in GCV resistance in the recombinant baculoviruses.

Mutation in HBV RNA-dependent DNA polymerase confers resistance to lamivudine in vivo.

The (-) enantiomer of 3'-thiacytidine (lamivudine) has been found to be a potent inhibitor of hepatitis B virus (HBV) and human immunodeficiency virus (HIV) replication. Mutation of methionine to valine or isoleucine at the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the HIV reverse transcriptase has been shown to be responsible for lamivudine resistance in HIV. The hepadnaviruses also have the YMDD motif in their DNA polymerase. Therefore, it is possible that hepadnaviruses could develop lamivudine resistance by a similar mutation at this motif. We analyzed the HBV from a liver transplantation patient who developed recurrent HBV viremia during lamivudine treatment. The polymerase gene was amplified by polymerase chain reaction (PCR), and the region coding for the YMDD motif was sequenced. The pretreatment HBV sequence coded for YMDD, while the lamivudine-resistant mutant HBV coded for YIDD (tyrosine, isoleucine, aspartate, aspartate). With the documented changes in the YMDD motif of lamivudine-resistant HIV, it is likely that the methionine-to-isoleucine mutation in the YMDD motif of the HBV polymerase contributes significantly to the lamivudine-resistance of HBV isolated from this patient.

Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Lamivudine Clinical Investigation Group.

Cirrhosis and hepatocellular carcinoma occur as long-term complications of chronic hepatitis B virus (HBV) infection. Antiviral therapy is potentially a successful approach for the treatment of patients with HBV infection, which includes the nucleoside analog, lamivudine [(-)2'-deoxy-3'-thiacytidine, 3TC]. Although resistance to lamivudine therapy has been reported in several HBV-infected patients, the pattern of resistance-associated mutations in HBV has not been fully characterized. We report a DNA sequence database that includes a 500-base pair region of the HBV polymerase gene from 20 patients with clinical manifestations of lamivudine resistance. Analysis of the database reveals two patterns of amino acid substitutions in the tyrosine, methionine, aspartate, aspartate (YMDD) nucleotide-binding locus of the HBV polymerase. HBV DNA from the sera of patients in Group I exhibits a substitution of valine for methionine at residue 552, accompanied by a substitution of methionine for leucine at residue 528. Patients in Group II had only an isoleucine-for-methionine substitution at position 552. Reconstruction of these mutations in an HBV replication-competent plasmid was performed in a transient transfection cell assay to determine the function/relevance of these mutations to lamivudine resistance. Both Group I and Group II mutations resulted in a substantial decrease in sensitivity to lamivudine treatment (> 10,000-fold shift in IC50 over wild-type [wt] IC50), strongly indicating that these mutations were involved in resistance to lamivudine. A hypothetical model of the HBV reverse transcriptase has been generated for further study of the role of these mutations in lamivudine resistance.