Epilepsia Partialis Continua as a Sequelae of Measles Infection in Children With Hematolymphoid Malignancies.

PURPOSE: To share our clinical experience with the diagnosis and management of children with hematolymphoid malignancies presenting with epilepsia partialis continua (EPC) as a sequelae of measles infection. MATERIALS AND METHODS: In December 2022, a series of children in our hemato-oncology unit presented with focal status epilepticus with no conclusive evidence pointing toward any underlying etiology. One such child had a typical measles rash a few weeks before the onset of this focal status epilepticus. After a series of cases with a similar presentation, a clinical pattern suspicious for measles became evident. cerebrospinal fluid polymerase chain reaction was positive for measles virus with measles immunoglobin M detected in the serum. This led to the diagnosis of measles inclusion-body encephalitis in a series of children who presented with EPC over a period of 3 months. EPC is a rare manifestation of measles that is seen only in immunocompromised patients. RESULTS: Among the 18 children reported in this series, only 10 had a history of rashes. The rash was mostly transient and elicited only on retrospective history taking. Five of the 18 children who did not lose consciousness during the prolonged seizure episode survived the disease but had residual neurologic sequelae. Among the 18 children, two were unimmunized and immunization status could not be confirmed in three other children. CONCLUSION: This case series highlights the threats posed by measles infection in children with cancer who are immunosuppressed because of the underlying disease and ongoing chemotherapy. Loss of herd immunity because of declining measles immunization rates secondary to vaccine hesitancy and COVID-19 lockdown pose a greater risk of measles infection and its complications for patients with deficient immune systems.

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.

Immuno-Colorimetric Neutralization Test: A Surrogate for Widely Used Plaque Reduction Neutralization Tests in Public Health Virology.

Since their first documentation in 1952, plaque reduction neutralization tests (PRNTs) have become the choice of test for the measurement of neutralizing antibodies against a particular virus. However, PRNTs can be performed only against viruses that cause cytopathic effects (CPE). PRNTs also require skilled personnel and can be time-consuming depending on the time required for the virus to cause CPE. Hence, their application limits large-scale studies or epidemiological and laboratory investigations. Since 1978, many surrogate PRNTs or immunocolorimetric assay (ICA)-based focus reduction neutralization tests (FRNT) have been developed. In this article, ICAs and their utility in FRNTs for the characterization of neutralizing antibodies, homologous or heterologous cross-neutralization, and laboratory diagnosis of viruses of public health importance have been discussed. Additionally, possible advancements and automations have been described that may help in the development and validation of novel surrogate tests for emerging viruses.

Genome Sequences of Measles Virus D4 and D8 Genotypes from India.

The genomes of 15 measles viruses isolated in 2006 to 2017 from patients <16 years of age with fever and skin rashes from four states and two union territories of India were sequenced. Study genomes were phylogenetically analyzed using 143 Indian and global genomes. The study reconfirms two lineages of D4 isolates and three lineages of D8 isolates from India.

Varicella Outbreak in Children from Silvassa, Dadra and Nagar Haveli, India.

Study describes epidemiological and laboratory findings of the fever with skin-rash cases (n=247) reported from Dadra and Nagar Haveli during 2018-19. For laboratory diagnosis, 33 sera and 5 blister swabs were obtained from 36 suspected cases. Varicella-zoster-virus DNA PCR and IgM EIA confirmed 33 cases and sequencing revealed circulation of clade-1 viruses.

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.

Characterization of diversity of measles viruses in India: Genomic sequencing and comparative genomics studies.

OBJECTIVE: To map genomic diversity of Measles virus (MeV) isolates collected during 2009-2017 from ten states of India. METHODS: Genome sequencing of Indian isolates and comparative genomics with global MeV using phylogeny, population stratification and selection pressure approaches were performed. RESULTS: The first report of complete genome sequences of forty-three Indian MeV isolates belonging to genotypes D4 (eight) and D8 (thirty-five). Three Indian isolates mapped to named strains D4-Enfield, D8-Villupuram and D8-Victoria. Indian D4 isolates deviate from standard genome length due to indels in M-F intergenic region. Estimated nucleotide substitution rates of Indian MeV derived using genome and individual genes are lower than that of global isolates. Phylogeny revealed genotype-based temporal clustering, suggesting existence of two lineages of D4 and three lineages of D8 in India. Absence of spatial clustering suggests role of cross-border travel in MeV transmission. CONCLUSIONS: Evolutionary analyses suggest the need for surveillance of MeV in India, particularly in view of diversified trajectories of D4 and D8 isolates. This study contributes to global measles epidemiology and indicates no major impact on antigenicity in Indian isolates, thereby substantiating the use of current vaccines to meet measles elimination target of 2023 set by World Health Organization for South-East Asia Region.

Molecular mechanism by which residues at position 481 and 546 of measles virus hemagglutinin protein define CD46 receptor binding using a molecular docking approach.

The hemagglutinin (H) protein of measles viruses (MeV) mediates binding to the cellular receptors, CD46,human signaling lymphocyte activation molecule and nectin-4. Vaccine strains primarily contain H-proteins possessing MeV-H: Y481 and can utilize CD46. Reports suggest that a single amino acid change in MeV-H at position 481 in wild type strains renders them inefficient in utilizing CD46. The in-depth molecular mechanism by which substitutions at 481 and another reported critical residue position 546 affects CD46 binding affinity however remains elusive. We used molecular docking studies of CD46 with MeV-H possessing Y481 N/D to understand the in-depth molecular mechanism involved. It was found that loss in either of the hydrogen bond (H-bond) contacts (MeV-H:481-CD46:65, MeV-H:546-CD46:63) in the central contact region prevented efficient CD46 binding. Y481 N could form the specific H-bond, while G546S H-bond could be formed only in conjunction with Y481, revealing the significance of these residues in determining CD46 receptor binding potential. Elucidating the underlying molecular mechanism of receptor usage by the MeV has implications to understanding cellular tropism, viral pathogenesis and therapy.

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.

Global spatiotemporal transmission dynamics of measles virus clade D genotypes in the context of the measles elimination goal 2020 in India.

Measles viruses (MeV) circulating in India mainly belong to genoypes D4 and D8 of clade D. In the context of measles elimination goal 2020 in India, molecular clock and phylogeography studies would help to identify the timescales of evolution and track the transmission pathways of MeV. We used nucleoprotein gene sequences (n = 756) from GenBank, representing 86 countries (1973-2016), to study the spatiotemporal transmission dynamics of clade D. Genotype D4 was introduced into India around 1991 and genotype D8 around 1994. Recent transmissions of the D4 genotype of measles virus (MeV) were noted from India to the United States of America and East Asia region while D8 genotype importations from North America were noted in recent years.

Outbreak of mumps virus genotype G infection in tribal individuals during 2016-17 in India.

Tribal individuals presented with fever and uni- or bi-lateral parotitis in Galonda and Silli villages (Dadra and Nagar Haveli, India) between 2 October 2016 and 19 March 2017. Consequently, the magnitude and epidemiological characteristics of the outbreak were investigated. Overall, 139 cases of suspected mumps were identified in both the above villages. Most of the suspected cases were 5-15 years old, the exceptions being three adults who had no noticeable complications. Specimens were collected from 42 of the suspected cases and their close contacts (n = 39) for laboratory investigation. Mumps infection was laboratory-confirmed in 73.8% and 20.5% of the suspected cases and contacts, respectively. Mumps was confirmed in seven adults aged 17-42 years, including three suspected cases and four contacts. To the best of our knowledge, this is the first report of a complete virus genome circulating among tribal individuals. Sequencing and phylogenetic studies revealed circulation of mumps virus genotype G in these tribal villages with 99% identity to a mumps virus detected in the UK (1996) and Canada (2009). Comparison with Indian mumps viruses revealed 99% and 98% identity to previously reported isolates from Pune during 2012 and 1986, respectively. Although the outbreak was large, no major complications were reported in the tribal villages. Detection of asymptomatic mumps in numerous close contacts indicates the importance of laboratory investigations in an outbreak setting.

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.

Mumps outbreak in a tribal population from the Union Territory of Dadra and Nagar Haveli, India.

A cluster of parotitis cases (n = 13) were observed in a tribal population of Vansda village from the Union Territory of Dadra and Nagar Haveli, India between 20th and 22nd week of 2016. Primary information was received by the local Infectious Disease Surveillance Program team, and subsequently field investigations were carried out in the affected area. Active surveillance was conducted till twice the incubation period from onset of the last surveyed case. For the laboratory investigations, 19 serum samples were collected from 11-suspected cases and their close contacts (n = 8). All samples were transported within 12 h on icepacks to the main laboratory at Pune. Majority of the suspected mumps cases were children except four adults. Mumps infection was confirmed in 8 of 11 suspected cases with post-onset ranging from 28 to 43 days and none from the close contacts. Both mumps specific IgM and IgG antibodies were detected in nine cases (including one equivocal) and single contact (equivocal result). Overall, ten cases and eight contacts (including one equivocal) showed mumps specific IgG antibodies. Present investigation provides information about the characteristics of mumps outbreak in a tribal community that resides in the remote areas. In addition, introduction of mumps containing vaccine in the tribal population may have added advantages in the tribal health program.

Complete Genome Sequence of Mumps Virus Isolated from Karnataka State, India.

We report the first whole-genome sequence of mumps virus isolated from a two-year-old girl with bilateral parotitis from a Chikkahallivana village in the Davangere district of Karnataka State, India. The genome of the Davangere mumps isolate was 15,384 bp in length and identical to previously published mumps virus (MuV) genomes from India. BLAST results show 99.1% identity with previously sequenced genotype C viruses isolated from the states of Maharashtra, Tamil Nadu, and Uttar Pradesh.

Measles virus genotypes circulating in India, 2011-2015.

The Government of India is accepted to participate in the measles elimination and rubella control goal 2020, hence genetic characterization of measles viruses (MeV) becomes essential. At National Reference Laboratory (National Institute of Virology, Pune), the throat swabs/urine specimens (n = 380) or PCR products (n = 219) obtained from the suspected measles cases were referred for the molecular testing and subsequently, MeV nucleoprotein (N) gene sequencing/genotyping. In addition, 2,449 suspected measles cases, mainly from the Maharashtra state were referred for the laboratory diagnosis. A detailed study was performed on N gene sequences obtained during last two decades. Indian MeV sequences obtained during 2011-2015 were compared with 1996-2010 sequences and genetic divergence was studied. Circulation of measles genotypes B3 (n = 3), D4 (n = 49), and D8 (n = 351) strains were observed in 19 States and three Union Territories of India. In addition, 64 measles viruses were isolated from 253 throat swab or urine specimens obtained from the suspected measles cases. During 2011-2015, 67.9% (1,663/2,449) suspected measles cases were laboratory confirmed. Molecular studies showed circulation of measles genotype B3 in India along with prominently circulating genotypes D4 and D8 except D7 strains. The genetic diversion within Indian B3, D4, and D8 genotypes was 0.3%, 1.1%, and 2.1%, respectively. The genetic divergence of Indian B3, D4, and D8 measles strains with the WHO reference sequences was 2.5%, 2.6%, and 1.8%, respectively. It is crucial data for national immunization program. More measles/rubella genotyping studies are necessary to track transmission and to support measles elimination and rubella control. J. Med. Virol. 89:753-758, 2017. © 2016 Wiley Periodicals, Inc.

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.

Laboratory confirmation of rubella infection in suspected measles cases.

As a part of measles outbreak based surveillance undertaken by the World Health Organization India, suspected measles cases were referred for the laboratory diagnosis at National Institute of Virology (NIV) Pune and NIV Unit Bengaluru. Altogether, 4,592 serum samples were referred during 2010-2015 from the States of Karnataka (n = 1,173), Kerala (n = 559), and Maharashtra (n = 2,860). Initially, serum samples were tested in measles IgM antibody EIA and samples with measles negative and equivocal results (n = 1,954) were subjected to rubella IgM antibody detection. Overall, 62.9% (2,889/4,592) samples were laboratory confirmed measles, 27.7% (542/1,954) were laboratory confirmed rubella and remaining 25.2% (1,161/4,592) were negative for measles and rubella. The measles vaccination status was available for 1,206 cases. Among the vaccinated individuals, 50.7% (612/1,206) were laboratory confirmed measles. The contribution of laboratory confirmed measles was 493 (40.8%) from Maharashtra, 90 (7.5%) from Karnataka, and 29 (2.4%) from Kerala. Since, 1/3rd of suspected measles cases were laboratory confirmed rubella, an urgent attention needed to build rubella surveillance in India. Additional efforts are required to rule out other exanthematous disease including Dengue and Chikungunya in measles and rubella negatives. J. Med. Virol. 88:1685-1689, 2016. © 2016 Wiley Periodicals, Inc.

Cross-neutralization between three mumps viruses & mapping of haemagglutinin-neuraminidase (HN) epitopes.

BACKGROUND & OBJECTIVES: The reports from the countries where mumps vaccine is given as routine immunization suggest differences in mumps virus neutralizing antibody titres when tested with vaccine and wild type viruses. Such reports are unavailable from countries like India where mumps vaccine is not included in routine immunization. We, therefore, undertook this study to understand the cross-neutralization activity of Indian mumps viruses. METHODS: By using commercial mumps IgG enzyme immunoassay (EIA) and a rapid focus reduction neutralization test (FRNT), a panel of serum samples was tested. The panel consisted of 14 acute and 14 convalescent serum samples collected during a mumps outbreak and 18 archived serum samples. Two wild types (genotypes C and G) and Leningrad-Zagreb vaccine strain (genotype N) were used for the challenge experiments and FRNT titres were determined and further compared. The HN protein sequence of three mumps viruses was analyzed for the presence of key epitopes. RESULTS: All serum samples effectively neutralized mumps virus wild types and a vaccine strain. However, significantly lower FRNT titres were noted to wild types than to vaccine strain (P<0.05). The comparison between EIA and FRNT results revealed 95.6 per cent agreement. No amino acid changes were seen in the epitopes in the Indian wild type strains. All potential N-linked glycosylation sites were observed in Indian strains. INTERPRETATION & CONCLUSIONS: Good cross-neutralization activity was observed for three mumps virus strains, however, higher level of FRNT titres was detected for mumps virus vaccine strain compared to Indian wild type isolates.

Complete genome sequence of mumps viruses isolated from patients with parotitis, pancreatitis and encephalitis in India.

Limited information is available regarding epidemiology of mumps in India. Mumps vaccine is not included in the Universal Immunization Program of India. The complete genome sequences of Indian mumps virus (MuV) isolates are not available, hence this study was performed. Five isolates from bilateral parotitis and pancreatitis patients from Maharashtra, a MuV isolate from unilateral parotitis patient from Tamil Nadu, and a MuV isolate from encephalitis patient from Uttar Pradesh were genotyped by the standard protocol of the World Health Organization and subsequently complete genomes were sequenced. Indian MuV genomes were compared with published MuV genomes, including reference genotypes and eight vaccine strains for the genetic differences. The SH gene analysis revealed that five MuV isolates belonged to genotype C and two belonged to genotype G strains. The percent nucleotide divergence (PND) was 1.1% amongst five MuV genotype C strains and 2.2% amongst two MuV genotype G strains. A comparison with widely used mumps Jeryl Lynn vaccine strain revealed that Indian mumps isolates had 54, 54, 53, 49, 49, 38, and 49 amino acid substitutions in Chennai-2012, Kushinagar-2013, Pune-2008, Osmanabad-2012a, Osmanabad-2012b, Pune-1986 and Pune-2012, respectively. This study reports the complete genome sequences of Indian MuV strains obtained in years 1986, 2008, 2012 and 2013 that may be useful for further studies in India and globally.