Between January 1995 and September 2009, 91 congenital infections were reported to the French National Institute for Public Health Surveillance after prenatal diagnosis. For three cases, prenatal diagnosis relied only on the detection of rubella virus-specific IgM in fetal blood. For 88 cases (97% of the reported cases), the rubella virus genome was detected in AF (Fig. ). Two main peaks of cases were observed—one in 1997 (22 cases) and the other in 2000 (15 cases)—while no cases were reported in 2006 and 2008 (Fig. ). Out of 88 CRI cases detected by prenatal diagnosis on AF in France during the study period, 78 (88.6%) were in fact diagnosed in our laboratory and were therefore available for the study. Two other AF samples were addressed to our laboratory from abroad (Portugal and Tunisia) during the study period, and we also included these in the study. The 26 other clinical samples (urine samples, cerebrospinal fluid samples, and a lens aspirate) were available from French children/newborns with CRS.
FIG. 1. Numbers of CRI cases in pregnant women in France between 1995 and 2009 that were reported (shaded bars), investigated by Antoine Béclère's laboratory (filled bars), and genotyped (open bars). (Only CRI cases detected by prenatal diagnosis (more ...)
All these rubella cases were detected using a diagnostic PCR different from the PCR generating the fragments for E1 gene sequencing and genotyping. During storage, partial RNA degradation may have taken place, no longer allowing the amplification of the larger fragments needed for genotyping. Therefore, complete or nearly complete E1 gene sequences (1,437 to 1,446 nt) were obtained from 49 cases. From seven additional samples (samples 9, 30, 53, 78, 84, 104, and 121), regions between nt 848 (sample 53) and nt 1280 (sample 121) of the E1 gene, including the complete window region, were amplified. Of these 56 sequences, 1 was derived from the lens sample (sample 2), 2 from cerebrospinal fluid (samples 84 and 92), 3 from urine (samples 66, 73, and 97), and 50 from amniotic fluid.
Phylogenetic analysis of these sequences showed that at least five genotypes (1E, 1B, 1G, 2B, and 1h) were present in France during the years 1995 to 2009 (Fig. ). Of the 56 sequences obtained, 49 (87.50%) clustered with genotype 1E reference sequences. The sequences within genotype 1E were quite similar (within-group distance, 0.70%) and clustered interspersed with sequences from America and Europe (Fig. ). Several pairs of 1E sequences were identical over the total length of sequence information obtained (samples 115 and 120, 3 and 42, 48 and 53, 58 and 61, 78 and 81, and 85 and 97). The two most distant viruses within genotype 1E (samples 104 and 26) differed by 1.93% over the E1 gene region. Besides genotype 1E sequences, two representatives each of genotypes 1G (samples 9 and 51; Kimura distance, 1.43%) and 2B (samples 114 and 121; Kimura distance, 1.58%) were found (Fig. ). One sequence each grouped with 1B (sample 92) and 1h (sample 5), while another single strain (sample 2) clustered separately from all reference sequences as an outlier to the 1G/1h/1i group of sequences, close to the 1B reference strain RVi/Jerusalem.ISR/75 (Fig. ). Analysis based on the complete E1 gene region showed a similar picture, with strain 2 again an outlier to the 1G/1h/1i group of viruses, again with no bootstrap support (data not shown). When all available window sequences from GenBank were included in the analysis, strain 2 still clustered separately from all other viruses (Fig. ).
The sequence most similar to strain 2 identified by BLAST was a Japanese vaccine strain first isolated in 1967 (GenBank accession number D50676; Kimura distance, 2.48%), which clusters with the 1B viruses (Fig. ). Different sequences from Italy showed the highest similarity to the genotype 1G viruses (GenBank accession number AY161361, collected in 1993, to strain 9 [Kimura distance, 0.35%]; GenBank accession numbers AY161371, AY161372, and AY161373, all collected in 1995, to strain 51 [Kimura distance, 0.71%]), while a sequence determined in 2000 in the United States from a virus considered to have been imported from India was the closest match of both 2B strains (GenBank accession number AY968220; Kimura distances to strains 114 and 121, 1.77% and 2.15%, respectively). The closest fit to the 1B sequence (sample 92) was a virus from Israel collected in 1979 (GenBank accession number AY968208; Kimura distance, 3.22%). The 1h sequence was most similar to a sequence from Western Siberia from 2004 (GenBank accession number EF421977; Kimura distance, 1.62%). The 1E viruses obtained during this study showed the highest similarity with viruses from Italy collected in 1997 (GenBank accession numbers DQ085343 [Kimura distance, 0.28 to 1.36%] and AY161378 [Kimura distance, 0.12 to 0.85%]).
Several unique amino acid changes relative to 169 complete or nearly complete E1 gene sequences from GenBank were identified in single sequences: 28L (sample 26), 50V (sample 100), 103T (sample 68), 138A (sample 99), 142L (sample 34), 152R (sample 35), 181K (sample 68), 196A (sample 71), 215R (sample 84), 285L (sample 84), 316R (sample 104), 340L (sample 99), 347A (sample 62), and 405T (sample 26). At least one of these mutations (215R) occurs in the epitope of a neutralizing monoclonal antibody (33