Rubella seroprevalence among pregnant women in Burkina Faso.

BACKGROUND: Despite the serious consequences of rubella infection during early pregnancy, very little is known about the rubella seroprevalence in a number of African countries including Burkina Faso. METHODS: Between December 2007 and March 2008 serum samples were collected from 341 pregnant women in Bobo (n = 132, urban area) and Houndé (n = 209, rural area) and were tested for rubella-specific IgG antibodies with a commercial ELISA kit. RESULTS: An overall seropositivity rate of 95.0% (324/341) was found, with a higher percentage in the urban population and in the oldest age group. Considering an antibody titer of at least 10 International Units per ml as protective, the overall immunity rate in the cohort of pregnant women was 93.3% (318/341). CONCLUSIONS: The high overall seropositivity rate in the absence of routine immunization suggests a continuous transmission of endemic rubella virus in Burkina Faso, posing a threat to non-immune pregnant women.

Molecular genotyping and epidemiology of measles virus transmission in the World Health Organization European Region, 2007-2009.

BACKGROUND: In 2002, the World Health Organization (WHO) adopted a goal to eliminate measles in the European Region by 2010. Measles elimination is defined as the interruption of indigenous measles virus (MV) transmission. The molecular epidemiology of MV transmission in the WHO European Region was studied through the investigation of reported cases and outbreaks to monitor the region's progress toward its measles elimination goal. METHODS: National and regional laboratories performed molecular characterization of MV detected between 2007 and 2009 in the WHO European Region. To document indigenous transmission and importations into the region, we analyzed genotyping results and epidemiological data on measles outbreaks reported by the member states. RESULTS: Since 2007, MV genotype D6 has not been reported in the WHO European Region, suggesting that its chains of transmission have been interrupted, whereas several other MV genotypes are still circulating. Although several European countries have already interrupted indigenous MV transmission, genotyping showed that 3 endemic MV transmission chains have been reestablished in other countries. CONCLUSIONS: The WHO European Region 2010 goal will not be met, as indigenous transmission of MV has not been interrupted. As the region begins to document its process of elimination verification to monitor progress toward the goal, countries will need to ensure that genotyping is performed in all measles outbreaks.

Swine influenza virus antibodies in humans, western Europe, 2009.

Serologic studies for swine influenza viruses (SIVs) in humans with occupational exposure to swine have been reported from the Americas but not from Europe. We compared levels of neutralizing antibodies against 3 influenza viruses--pandemic (H1N1) 2009, an avian-like enzootic subtype H1N1 SIV, and a 2007-08 seasonal subtype H1N1--in 211 persons with swine contact and 224 matched controls in Luxembourg. Persons whose profession involved contact with swine had more neutralizing antibodies against SIV and pandemic (H1N1) 2009 virus than did the controls. Controls also had antibodies against these viruses although exposure to them was unlikely. Antibodies against SIV and pandemic (H1N1) 2009 virus correlated with each other but not with seasonal subtype H1N1 virus. Sequential exposure to variants of seasonal influenza (H1N1) viruses may have increased chances for serologic cross-reactivity with antigenically distinct viruses. Further studies are needed to determine the extent to which serologic responses correlate with infection.

Revealing new measles virus transmission routes by use of sequence analysis of phosphoprotein and hemagglutinin genes.

With improved measles virus (MV) control, the genetic variability of the MV-nucleoprotein hypervariable region (NP-HVR) decreases. Thus, it becomes increasingly difficult to determine the origin of a virus using only this part of the genome. During outbreaks in Europe and Africa, we found MV strains with identical NP-HVR sequences. However, these strains showed considerable diversity within a larger sequencing window based on concatenated MV phosphoprotein and hemagglutinin genes (P/H pseudogenes). In Belarus, Germany, Russia, and the Democratic Republic of Congo, the P/H pseudogenes provided insights into chains of transmission, whereas identical NP-HVR provided none. In Russia, for instance, the P/H pseudogene identified temporal clusters rather than geographical clusters, demonstrating the circulation and importation of independent variants rather than large local outbreaks lasting for several years, as suggested by NP-HVR. Thus, by extending the sequencing window for molecular epidemiology, a more refined picture of MV circulation was obtained with more clearly defined links between outbreaks and transmission chains. Our results also suggested that in contrast to the P gene, the H gene acquired fixed substitutions that continued to be found in subsequent outbreaks, possibly with consequences for its antigenicity. Thus, a longer sequencing window has true benefits both for the epidemiological surveillance of measles and for the better monitoring of viral evolution.

Concentration and diversity of noroviruses detected in Luxembourg wastewaters in 2008-2009.

Noroviruses (NoV) in 78 wastewater samples from Luxembourg were quantified, cloned, and sequenced in 2008-2009. The concentrations of NoV genogroup II and the relative occurrences of certain genotypes changed significantly during the winter season. NoV genogroup I was frequently detected by real-time reverse transcription-PCR (RT-PCR), albeit at 30-fold lower concentrations than for genogroup II, hampering attempts to assess overall genetic diversity by the cloning/sequencing approach.

Extensive phosphorylation flanking the C-terminal functional domains of the measles virus nucleoprotein.

The measles virus nucleoprotein (vNP) is the first and most abundant protein in infected cells. It plays numerous important roles including the encapsidation of genomic viral RNA and the transcription of viral proteins. Intricate interactions with host cell proteins rely on the structural integrity of its functional domains. Although some of these functional domains are known, their structural features are still poorly understood. Here we identified multiple isoforms of measles vNP by two-dimensional differential gel electrophoresis (2D-DIGE) and 2D Western blot. These isoforms were further analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)/TOF using MS (PMF) and MSMS (PSD) and electrospray ionization (ESI)-ion trap using LC-ESI-ion trap MS(1), MS(2) (neutral loss), MS(3) (phosphosite). Both recombinant NP (rNP) and vNP were α-acetylated at the N-terminus. After tryptic or chymotryptic digestion, phosphopeptides were enriched and nine phosphorylation sites were identified and localized in the rNP, seven of which were also phosphorylated in vNP, probably by casein kinase 2. The phosphosites were all found within the intrinsically unstructured C-terminal domain. They clustered around functional domains involved in transcription and replication, as well as in sequences interacting with host-cell proteins. This underlines the importance of these post-translational modifications.

Measles virus strain diversity, Nigeria and Democratic Republic of the Congo.

We investigated the genetic diversity of measles virus (MV) in Nigeria (2004-2005) and the Democratic Republic of the Congo (DRC) (2002-2006). Genotype B3 strains circulating in Kinshasa, DRC, in 2002-2003 were fully replaced by genotype B2 in 2004 at the end of the second Congo war. In Nigeria (2004-2005), two genetic clusters of genotype B3, both of which were most closely related to 1 variant from 1998, were identified. Longitudinal analysis of MV strain diversity in Nigeria suggested that only a few of the previously described 1997-1998 variants had continued to circulate, but this finding was concomitant with a rapid restoration of genetic diversity, probably caused by low vaccination coverage and high birth rates. In contrast, the relatively low genetic diversity of MV in DRC and the genotype replacement in Kinshasa reflect a notable improvement in local measles control.

Newcastle disease virus in West Africa: new virulent strains identified in non-commercial farms.

Forty-four Newcastle disease virus (NDV) strains, obtained between 2002 and 2007 from different poultry species in Nigeria, Niger, Burkina Faso and Cameroon, were phylogenetically analysed based on partial F sequences. Lineage 2 viruses were genetically identical or similar to the locally used LaSota vaccine strain and were mostly detected in commercial farms. Lineage 1, 3 and 4 strains were only sporadically found, and their origin was less clear. Twenty-one strains from backyard farms and live bird markets formed three new clusters within lineage 5, tentatively named 5f, 5g and 5h. All of these strains were predicted to be virulent based on their F protein cleavage site sequence. Minimal genetic distances between new and previously established sublineages ranged from 9.4 to 15.9%, and minimal distances between the new sublineages were 11.5 to 17.3%. Their high genetic diversity and their presence in three different Sub-Saharan countries suggest that these new sublineages represent the NDV variants indigenous to West Africa.

Replacement of sublineages of avian influenza (H5N1) by reassortments, sub-Saharan Africa.

Eight new full-length sequences from highly pathogenic avian influenza viruses (H5N1) from 4 states in southwest Nigeria were analyzed. All gene sequences were more closely related to the first strains found in Nigeria in 2006 than to any strain found outside the country. Six viruses had evolved by at least 3 reassortment events (AC HA/NS, AC NS) from previously identified sublineages A (EMA 2) and C (EMA 1). Our results suggest that highly pathogenic avian influenza viruses (H5N1) initially imported into Nigeria in 2006 have been gradually replaced by various reassortments. In all reassortants, nonstructural genes were derived from sublineage C with 2 characteristic amino acids (compared with sublineage A). If the high prevalence of reassortants was typical for West Africa in 2007, the absence of such reassortants anywhere else suggests that reintroductions of influenza A (H5N1) from Africa into Eurasia must be rare.

A multiplex TaqMan PCR assay for the detection of measles and rubella virus.

Measles and rubella virus cause fever/rash diseases that are difficult to differentiate clinically. Both viruses can be detected in the same clinical specimens and are propagated on the same cell cultures. A single-tube multiplex TaqMan assay is described for the simultaneous and rapid detection of the full spectrum of known genetic variants. The performance of the assay is similar to a conventional nested PCR and generates cDNA with random primers which can be used directly for virus genotyping.

Re-exposure to wild-type virus stabilizes measles-specific antibody levels in late convalescent patients.

BACKGROUND: Infection with wild-type (wt) measles virus strains induces high antibody levels believed to provide life-long protection against disease. OBJECTIVES: Humoral immunity was followed up in convalescent measles patients to assess the persistence of specific antibodies after measles disease in individuals without and with documented re-exposure to wt virus. STUDY DESIGN: Paired sera were collected from 43 late convalescents (LC) before re-exposure and 3.7-4.8 years after re-exposure to at least one measles patient (LC+ group). Antibody persistence in this group was compared to paired sera from 43 age- and sex-matched controls without documented exposure to wt virus (LC- group). Paired sera were also obtained from 26 measles patients 1.3-1.7 and 3.8-4.1 years after they had recovered from measles to observe the waning of antibodies in early convalescents (EC group). RESULTS: Antibody levels decreased by 12.1% (CI: 3.2-20.3%, p=0.01) within 6.3 years in the LC- group of late convalescent measles patients. In contrast, in the LC+ group GMT of first and second sera were virtually identical, indicating that exposure to wt virus stabilizes antibody levels even in absence of a detectable secondary immune response. In a subset of late convalescents of group LC+ with a secondary immune response, antibody waning after re-exposure was as high as 15.6%/year (CI: 13.0-17.7%/year), corresponding to a half-life of 4.1 years (CI: 3.5-5.0 years), but antibodies were still higher than before re-exposure. In the EC group GMT decreased by 6.5% (95% CI: -13.3% to +0.1%) during 2.5 years but significance was low (p=0.08). CONCLUSION: The maintenance of antibody levels in convalescent measles patients is at least partially dependant on recurrent exposure to circulating wt virus.

Interplay of measles virus with early induced cytokines reveals different wild type phenotypes.

Differential effects of measles virus (MV) on the innate immune response may influence virus spread and severity of disease. Using a representative panel of 22 MV strains including 14 different genotypes, we found that wild-type (wt) differ considerably in their sensitivity to type I interferon (IFN). The wt virus production was 2-47-fold lower in IFN-alpha treated Vero/hSLAM cells, whereas vaccine virus production was reduced only 2-3-fold. Sequence analysis of the MV-P/C/V gene, revealed no obvious amino acid mutations that correlated with the different phenotypes. Strains also widely differed in their ability to induce type I IFN, tumor necrosis factor (TNF) alpha and other cytokines in human A549/hSLAM cells. Some wt strains that were highly sensitive to type I IFN induced only low levels of these and other cytokines. In vitro wt strains that produced the 5' copy-back defective interfering RNAs (5'cb-diRNA) characterized by Shingai et al. (2007), induced high levels of cytokines that otherwise were only reached by vaccine strains. These 5'cb-diRNAs emerged only in virus cultures during multiple passaging and were not detectable in clinical samples of measles patients. These subgenomic RNAs are an important confounding parameter in passaged wt viruses which must be carefully assessed in all in vitro studies. The present data show that MV wt strains differ in their sensitivity and their ability to temper with the innate immune response, which may result in differences in virulence.

High genetic diversity of measles virus, World Health Organization European Region, 2005-2006.

During 2005-2006, nine measles virus (MV) genotypes were identified throughout the World Health Organization European Region. All major epidemics were associated with genotypes D4, D6, and B3. Other genotypes (B2, D5, D8, D9, G2, and H1) were only found in limited numbers of cases after importation from other continents. The genetic diversity of endemic D6 strains was low; genotypes C2 and D7, circulating in Europe until recent years, were no longer identified. The transmission chains of several indigenous MV strains may thus have been interrupted by enhanced vaccination. However, multiple importations from Africa and Asia and virus introduction into highly mobile and unvaccinated communities caused a massive spread of D4 and B3 strains throughout much of the region. Thus, despite the reduction of endemic MV circulation, importation of MV from other continents caused prolonged circulation and large outbreaks after their introduction into unvaccinated and highly mobile communities.

Reducing global disease burden of measles and rubella: report of the WHO Steering Committee on research related to measles and rubella vaccines and vaccination, 2005.

The WHO Steering Committee reviewed and evaluated the progress towards global control of measles and rubella and provided guidelines for future research activities concerning both diseases during its meeting in New Delhi, in April 2005. Global measles vaccination coverage increased from 71% in 1999 to 76% in 2004 and indigenous transmission was interrupted or kept at very low levels in many countries. However, Africa and Southeast Asia continue to experience endemic transmission and high mortality rates, despite a global mortality reduction of 39% between 1999 and 2003. On the basis of reports from countries with continued indigenous measles virus transmission, future control strategies as well as advantages and potential drawbacks of global measles eradication were discussed. Similarly the burden of rubella and congenital rubella syndrome (CRS) as well as the cost-effectiveness of rubella vaccination was assessed using different methods in several countries without vaccination programs. As measles and rubella viruses continue to circulate surveillance and control strategies need further optimization. RT-PCR was considered as an alternative method for laboratory diagnosis of CRS. The value of dried blood spots and oral fluid as alternative samples for measles and rubella IgG and IgM detection and genotype determination was evaluated. However further validation of these methods in different settings is required before their routine use can be recommended.

Genotyping of recent measles virus strains from Russia and Vietnam by nucleotide-specific multiplex PCR.

The nucleoprotein genes of 49 measles virus (MV) strains circulating in Russia between 2000 and 2006 and in Vietnam in 2003 were analyzed by genotype-specific PCR and the results were compared with their sequences. The sequences revealed the presence of genotypes H1 and H2 in the center (Nha Trang) and the north (Hanoi) of Vietnam, respectively. The relative diversity of the H2 strains suggested an endemic circulation of these viruses in the capital. In contrast genotype H1 strains from Nha Trang were homogenous genetically, which may indicate a recent importation. The strains obtained from 12 different regions of the Russian Federation were assigned to the genotypes H1, D4, and D6. Most strains (81.4%) were correctly genotyped by a multiplex PCR method which was sensitive to genotype-specific mutations [Kremer et al. (2004): J Clin Microbiol 42: 3017-3022]. Ambiguous or negative results for some clade H and genotype D6 strains were due to point mutations in the type-specific primer binding sites. After exchanging a single nucleotide in both the clade H- and the genotype D6-specific primers, all strains were assigned correctly to their genotype. A simplified procedure for use in Vietnam was developed to distinguish directly between genotypes H1 and H2 and any non-H genotype. These results demonstrate that our multiplex PCR method can be adapted easily to new sequence variants or specific epidemiological situations, and thus be very useful for rapid genotyping of large number of samples even in laboratories which do not have sequencing facilities.

Waning antibodies in measles and rubella vaccinees--a longitudinal study.

The evolution of measles- and rubella-specific serum IgG was followed in a longitudinal study in 224 young adolescent vaccinees, with or without boost vaccination before or during the 6.8-year observation period. Antibody titres were monitored by enzyme immuno assay (Enzygnost, Dade-Behring). After revaccination (second dose) rubella seropositivity rate increased from 92.1 to 100%, whereas measles seroprevalence (about 90%) did not significantly change between the paired sera. Significantly higher IgG (> three-fold) in the second serum of 5.2% (measles) and 7.8% (rubella) of participants with low antibodies (measles: < 1500 mIU; rubella < 40 IU) in first serum, suggest a secondary immune response (SIR) during the study period, only partially explained by revaccination. Excluding individuals with SIR, minimal annual antibody decay rates of -2.9% (confidence interval, CI: -0.7 to -4.8%) for rubella and -1.6% (CI: -0.1 to -3%) for measles were determined in participants with single dose vaccination. Thus, two-dose vaccination was adequate to protect women from rubella infection at least during childbearing age. Similarly only few individuals may become seronegative for measles again after successful vaccination due to minimal waning of low antibody levels (< 1500 mIU). However, as a result of a more rapid decay of high-titre (> 1500 mIU) antibodies (-2.4%/year), many vaccinees may eventually become susceptible to vaccine-modified measles (VMM) and consequently complicate measles control strategies.

Evaluation of commercial assay detecting specific immunoglobulin g in oral fluid for determining measles immunity in vaccinees.

A commercial assay for detection of measles immunoglobulin G (IgG) in oral fluid was evaluated in a highly vaccinated cohort using serum IgG as gold standard. In contrast to previous studies from cohorts protected by natural immunity, antibody prevalence was significantly underestimated (-7.4%; confidence interval: -1.5 to -13.2%; P = 0.01) due to a reduced sensitivity when antibody levels were low.

Measles virus genotyping by nucleotide-specific multiplex PCR.

A simple genotyping method based on multiplex PCR has been developed to discriminate between all active measles virus (MV) clades and genotypes (A, B3.1, B3.2, C2, D2-D9, G2-G3, and H1-H2). The sequencing reaction was replaced by six multiplex PCRs: one to identify the clade and five to identify the respective genotype. Primers were sensitive to clade- and genotype-specific nucleotides and generated fragments of type-specific sizes that were analyzed by conventional agarose gel electrophoresis. On the basis of all published MV sequences, positive and negative predictive values of 99.2% and 98.6% were calculated. Variability in the primer binding sites, which could potentially reduce sensitivity, was very limited among published sequences. As new genotypes are described, additional specific primers can be included in the multiplex PCR with relative ease. Although sequencing remains the "gold standard," the present method should facilitate MV genotyping especially in developing countries and will therefore contribute to enhanced MV control and elimination strategies as recommended by the World Health Organization.