Neutralizing Antibody Response to Genotypically Diverse Measles Viruses in Clinically Suspected Measles Cases.

The neutralizing antibody (Nt-Ab) response to vaccine and wild-type measles viruses (MeV) was studied in suspected measles cases reported during the years 2012-2016. The neutralization activity against MeV A, D4 and D8 genotypes was studied on sera (Panel A; n = 68 (measles-immunized) and Panel B; n = 50 (unvaccinated)) that were either laboratory confirmed or not confirmed by the presence of IgM antibodies. Additionally, the Nt-Ab response in Panel A was measured against the MeV vaccine and four wild-type viruses. Neutralization results were compared using homology modeling and molecular dynamics simulation (MDS) of MeV-hemagglutinin (H) and fusion (F) proteins. Overall, the Nt-Ab titres for MeV-A were found to be significantly lower than MeV-D4 and MeV-D8 viruses for Panel A. No major difference was noted in Nt-Ab titres between MeV-D8 viruses (Jamnagar and New Delhi), whereas MeV-D4 (Sindhudurg and Bagalkot (BGK) viruses) showed significant differences between Nt-Ab titres for Panel B. Interestingly, the substitutions observed in epitopes of H-protein, L249P and G316A are observed to be unique to MeV-BGK. MDS of H-protein revealed significant fluctuations in neutralizing epitopes due to L249P substitution. The majority of the clinically suspected cases showed Nt-Abs to MeV wild-types. Higher IgG antibody avidity and Nt-Ab titres were noted in IgM-negatives than in IgM-positives cases, indicating reinfection or breakthrough. MDS revealed reduced neutralization due to decreased conformational flexibility in the H-epitope.

Genetic and antigenic characterization of wild type rubella viruses isolated from India.

Rubella, is a contagious disease caused by Rubella virus (RuV) that manifests as fever with skin-rashes in children and adults along with complications in pregnant women. WHO-SEAR has set a target for Rubella elimination by 2023. This is the first report of antigenic characterization and genome sequencing of nine RuVs sampled during 1992, 2007-9, and 2015-17 from four Indian states. Comparative analysis of Indian RuVs (2B) with that of global isolates and vaccine strain RA 27/3 (1a) revealed that the observed mutations in structural proteins have no major impact on the 3D structure, function and antigenicity. Indian RuVs formed three major clusters (Pune-1992, Kannur-2009 and Chitradurg-2007) in genome-based phylogeny of global isolates. Neutralizing antibody titers in a panel of serum samples from measles negative cases were significantly higher to the vaccine strain compared to a wild-type 2B isolate (Kannur) with concordance of 91.9%, thereby substantiating the use of current vaccines.

Laboratory-based investigation of fever with rash cases in the Maharashtra State - India, 2014 to 2017.

The Maharashtra State (MS), India, launched measles surveillance in the year 2013. From 2014 to 2017, specimens from the patients presenting with fever and skin rashes were received at the National Institute of Virology, Pune. From 36 districts of the MS, 2795 cases (1428 males and 1367 females) were referred for the laboratory diagnosis of measles and rubella using immunoglobulin M enzyme immunoassay and/or RT-PCRs. The majority of the cases (93.3%, n = 2609) were under 15 years of age. About 17.7% (494) cases had a history of measles immunization (one dose) during their childhood. Virus isolation was attempted from 107 throat swabs and 84 urine samples obtained from 191 cases using Vero hSLAM cells. The results confirmed measles in 1756 and rubella in 282 fever with rash cases by serological and molecular tools. Precisely, 170 of 382 and 35 of 149 specimens were positive for measles and rubella RT-PCRs, respectively. Sequencing of the representative PCR products showed the circulation of measles virus genotypes D4 (n = 26) and D8 (n = 107), and rubella virus genotype 2B (n = 1). Twenty-three measles viruses were isolated and genotyped, of which, 6 were D4 and 17 were D8 genotypes. Amongst the measles-immunized individuals, 51.2% (253/494) had laboratory-confirmed measles. Overall, 72.9% fever with skin rash cases (n = 2038) from the MS was laboratory confirmed for either measles or rubella. The contribution of measles was higher than rubella in the fever with rash cases. As expected, more fever with rash cases were documented in children compared with adults and highlighted the need to increase measles-rubella immunization coverage.

Utility of neutralization test for laboratory diagnosis of suspected mumps.

Mumps is an infectious disease caused by mumps virus (MuV), which belongs to the family Paramyxoviridae and genus Rubulavirus. Typical symptoms of mumps include fever and swelling of the parotid glands; however, mumps can be asymptomatic. Mumps is diagnosed by molecular and serological methods (i.e., PCR and Enzyme Immunoassay [EIA]); however, both methods have pros and cons. This study was performed to compare the diagnostic utility of a focus reduction neutralization test (FRNT) to that of MuV-specific commercial IgM and IgG antibody EIA in patients suspected of having mumps. One hundred-eighty six samples collected during mumps outbreak in 2012-16 were studied. Samples (n = 80) were tested by all the three serological assays and showed 70.4%, 83% and 92.5% positivity by IgM EIA, IgG and FRNT, respectively. In all, 58.8% samples (n = 47) tested positive in all three assays. Concordance between mumps RT-PCR and IgM EIA was highest during the first 2-5 days and decreased with increasing time post-onset. Mumps FRNT results agreed with those of RT-PCR/IgM EIA from the second week onwards, whereas the results of mumps IgG EIA agreed with those of RT-PCR/IgM EIA from post-onset days 3-10. These findings suggest the utility of a FRNT for laboratory diagnosis of mumps in countries whose populations are not immunized against this infection.

Measles & rubella outbreaks in Maharashtra State, India.

BACKGROUND & OBJECTIVES: Under the outbreak-based measles surveillance in Maharashtra State the National Institute of Virology at Pune receives 3-5 serum samples from each outbreak and samples from the local hospitals in Pune for laboratory diagnosis. This report describes one year data on the measles and rubella serology, virus isolation and genotyping. METHODS: Maharashtra State Health Agencies investigated 98 suspected outbreaks between January-December 2013 in the 20 districts. Altogether, 491 serum samples were received from 20 districts and 126 suspected cases from local hospitals. Samples were tested for the measles and rubella IgM antibodies by commercial enzyme immunoassay (EIA). To understand the diagnostic utility, a subset of serum samples (n=53) was tested by measles focus reduction neutralization test (FRNT). Further, 37 throat swabs and 32 urine specimens were tested by measles reverse transcription (RT)-PCR and positive products were sequenced. Virus isolation was performed in Vero hSLAM cells. RESULTS: Of the 98 suspected measles outbreaks, 61 were confirmed as measles, 12 as rubella and 21 confirmed as the mixed outbreaks. Four outbreaks remained unconfirmed. Of the 126 cases from the local hospitals, 91 were confirmed for measles and three for rubella. Overall, 93.6 per cent (383/409) confirmed measles cases were in the age group of 0-15 yr. Measles virus was detected in 18 of 38 specimens obtained from the suspected cases. Sequencing of PCR products revealed circulation of D4 (n=9) and D8 (n=9) strains. Four measles viruses (three D4 & one D8) were isolated. INTERPRETATION & CONCLUSIONS: Altogether, 94 measles and rubella outbreaks were confirmed in 2013 in the State of Maharasthra indicating the necessity to increase measles vaccine coverage in the State.

Detection of measles, mumps and rubella viruses by immuno-colorimetric assay and its application in focus reduction neutralization tests.

Measles, mumps and rubella are vaccine-preventable diseases; however limited epidemiological data are available from low-income or developing countries. Thus, it is important to investigate the transmission of these viruses in different geographical regions. In this context, a cell culture-based rapid and reliable immuno-colorimetric assay (ICA) was established and its utility studied. Twenty-three measles, six mumps and six rubella virus isolates and three vaccine strains were studied. Detection by ICA was compared with plaque and RT-PCR assays. In addition, ICA was used to detect viruses in throat swabs (n = 24) collected from patients with suspected measles or mumps. Similarly, ICA was used in a focus reduction neutralization test (FRNT) and the results compared with those obtained by a commercial IgG enzyme immuno assay. Measles and mumps virus were detected 2 days post-infection in Vero or Vero-human signaling lymphocytic activation molecule cells, whereas rubella virus was detected 3 days post-infection in Vero cells. The blue stained viral foci were visible by the naked eye or through a magnifying glass. In conclusion, ICA was successfully used on 35 virus isolates, three vaccine strains and clinical specimens collected from suspected cases of measles and mumps. Furthermore, an application of ICA in a neutralization test (i.e., FRNT) was documented; this may be useful for sero-epidemiological, cross-neutralization and pre/post-vaccine studies.

Circulation of two mumps virus genotypes in an unimmunized population in India.

Two separate outbreaks of fever with parotitis were reported from the Apsinga and Pimpla villages in the Osmanabad district of the Maharashtra State, India during February and March 2012. Meningo-encephalitis was noted in two patients resulting in the death of an 11-year male. Samples of blood and throat swabs were collected from patients with fever and parotitis. Serum samples from suspected (n=62) and convalescent (n=19) patients were tested for mumps virus specific IgM and/or IgG antibodies. Mumps virus specific IgM antibodies were detected in 44 of 62 serum samples (71%). Of the 19 convalescent phase sera 16 had both, anti-mumps virus IgM and IgG antibodies. Twenty-eight throat swabs collected from patients with parotitis were tested by RT-PCR for the SH gene. Twenty-three specimens were found to be positive and nucleotide sequencing of the amplified PCR products revealed circulation of two distinct genotypes that were village specific. Mumps virus genotype C (n=18) was detected in Apsinga village and genotype G (n=5) in Pimpla village. Two mumps virus isolates were also obtained using Vero cells. This is the first report from India confirming simultaneous circulation of mumps virus genotype C in one village and the G genotype in another village only 37 km away.