Li Jin

Genetic Diversity of Mumps Virus in Oral Fluid Specimens: Application to Mumps Epidemiological Study

Three hundred nine mumps virus (MuV) strains detected in the United Kingdom during 1995-2002 were characterized by partial sequencing of the small hydrophobic gene and were shown to belong to at least 6 different genotypes. A strain representing a new genotype was isolated from a seminal fluid specimen. Identical strains belonging to genotypes G and C were found to circulate for up to 3 years. One genotype H strain reappeared after an absence of 4 years. Distinct lineages (G1-G7) within genotype G were recognizable in the present study, and this level of characterization proved to be very useful for tracking MuV importations and subsequent transmission. We propose, here, a preliminary standardization of international nomenclature for genetic characterization of MuV strains, to facilitate future molecular epidemiological studies of mumps. Oral fluid (OF) specimens (n=1441) were used to detect both anti-MuV IgM and MuV genome, and the results indicate that OF specimens can be used successfully for diagnosis and have the potential to play a valuable role in diagnosis and surveillance of mumps.

Laboratory confirmation of congenital rubella syndrome in infants: an eye hospital based investigation.

A total of 190 specimens from South Indian children aged 0–59 months with ocular anomalies consistent with suspected congenital rubella syndrome (CRS) were investigated. Twenty‐six of the 65 infants (40%) were confirmed as CRS by detection of rubella specific IgM. Rubella RNA was detected in 41 samples from 26 infants by both real‐time and block based PCR. The PCR results correlated well with the presence of anti‐rubella IgM/IgG (23/27 cases with rubella IgM were PCR positive). Whereas, only 17 of 26 infants met the WHO CRS case definition. Amongst the various specimens tested from the sero‐confirmed cases (nu2009=u200927), a high percentage of positives were detected in lens (92%) and oral fluid (60%) specimens, when compared to other samples. The quantification of viral load by real‐time PCR demonstrated higher copy number of virus in lens samples of 0–11 months infants. The rubella viruses were characterized and revealed the circulation of genotype 2B in three South Indian states. The integrated analysis of clinical manifestations, serological and molecular data in the study has generated baseline information of rubella infection and CRS in infants with ocular anomalies. J. Med. Virol. 80:536–546, 2008.

Development of a focus reduction neutralization test (FRNT) for detection of mumps virus neutralizing antibodies.

Although the plaque reduction neutralization test (PRNT) is considered the gold-standard assay for measuring neutralizing antibodies for mumps, it is technically demanding, slow and requires large serum volumes, which limits its use for investigating mumps vaccine efficacy and population susceptibility. Therefore, an immunocolourimetric-based focus reduction neutralization test (FRNT) was developed and validated against PRNT using 30 blood donor plasma samples (16 positive, 5 equivocal, and 9 negative for mumps IgG by EIA). The samples were tested in triplicate by FRNT and PRNT in 10 and 4 separate assay runs, respectively, and 50% neutralizing antibody titres calculated using the Kärber formula. There was good correlation between the two neutralization assays (R(2)=0.88). Inter-assay variation for FRNT titres was 2-fold, compared to a 3-fold variation for PRNT titres. From the distribution of results, a positive cut-off for FRNT was defined as 1:4. In conclusion, FRNT has similar sensitivity to the PRNT and offers the advantage of speed (2 days vs. 7 days), reduced sample volume (40 microL vs. 150 microL), and the possibility of automation using 96-well plates. FRNT appears to be a good substitute for PRNT for characterising the immune response to mumps and for vaccine efficacy studies.

Analysis of the genetic variability of the mumps SH gene in viruses circulating in the UK between 1996 and 2005.

Genetic analysis (genotyping) of mumps viruses has been applied to the molecular epidemiology of mumps for over 10 years in the UK. To explore further the variation of mumps strains over time, in total, 965 sequences of the entire SH gene were analysed and compared, including 954 mumps virus strains collected in the UK between 1996 and 2005 were characterised as genotypes G2 (426), G5 (369), J (157) and F (2), which were compared with 11 F sequences found in China. Phylogenetic trees drawn for G2, G5 and J sequences showed that the diversities were greater between the sequences in earlier years (before 2001/2002) than those in later years and could be divided into two clusters within each of the three genotypes over the 10-year period. One transmission of G2, G5 and a J strain was sustained from earlier years with mutations and eventually became predominant strains. Divergences amongst the same genotype or sub-genotype was up to 4.6% for G2, 5.3% for G5 and 4.9% for J. Mutation rates per site per year based on the 316nt of SH gene were 0.94, 1.3, 0.96 and 1.86 x 10(-2) for G2, G5, J and F respectively. The ratio of d(N)/d(S) was 0.556, 0.909, 0.357 and 0.811 calculated based on the sequences of G2, G5, J and F respectively. The results revealed that the possible mumps evolution process based on the SH gene was not driven by positive selection during the 10 years between 1996 and 2005.

Genetic Variation in the HN and SH Genes of Mumps Viruses: A Comparison of Strains from Mumps Cases with and without Neurological Symptoms

Background It is known that mumps virus (MuV) strains may vary in their neurovirulent capacity, and certain MuV strains may be highly neurotropic. In animal models and epidemiological studies, mutations at specific amino acids (aa) have been proposed to be associated with neurovirulence. To assess whether these genetic variations can be observed in clinical samples from patients and if they correlate with neurovirulence as determined by clinical symptoms, 39 mumps patients with or without neurological symptoms were investigated. Principal Findings Respiratory samples, oral fluids, throat swabs, and neurological and cerebrospinal fluid samples were tested by RT-PCR and products sequenced. Sequences of the entire small hydrophobic (SH) gene and the partial hemagglutinin-neuraminidase (HN) gene were compared. Conclusions The results showed there was no significant difference between the samples of the two groups of patients at the aa sites in either the HN protein or the SH protein, which have previously been hypothesized to be associated with neurovirulence or antigenicity. The occurrence of neurological symptoms of mumps does not appear to be due to a single point mutation in either the HN or SH gene.

Application of new assays for rapid confirmation and genotyping of isolates of rubella virus.

Rubella virus (RV) isolation is recommended by the WHO Measles and Rubella Labnet for studying the etiology and epidemiology of rubella. However, the absence of cytopathologic effects (CPE) in many of the cell lines used commonly makes it difficult to confirm RV growth. In this study, two assays amplifying RV cDNA were developed and validated in order to confirm and genotype RV isolates after cell culture. A SYBR Green I‐based real‐time PCR (Rtime‐SGE317) was established for initial rapid detection of RV in Vero cells and a nested PCR (PCR‐E860) was used for amplifying further the 739 nt window of the E1 gene for the identification of RV genotype as recommended by the WHO. Sensitivities of the two assays were evaluated using eight RV isolates, two from infants with the congenital rubella syndrome (CRS) and six from patients with acute rubella. All the isolates had cycle threshold (Ct) values <37 after the third passage, which is recommended as the cut‐off for the confirmation of a viable RV isolate. Phylogenetic analysis based on the 739 nt window generated by the PCR‐E860 showed that the eight RV isolates belonged to genotypes 1E, 1G, and 2B. The Rtime‐SGE317 assay can be carried out in local public health laboratories, which would extend the molecular surveillance of rubella and contribute to the WHO goal of eradicating rubella worldwide. J. Med. Virol. 83:170–177, 2011.