Archives of Virology

, Volume 158, Issue 11, pp 2359–2363

Genotyping and subtyping of mumps virus isolates from the Indian subcontinent

Authors

    • Department of VirologyPostgraduate Institute of Medical Education and Research
    • Departmet of MicrobiologyAll India Institute of Medical Sciences
  • Sujit Kumar Pujhari
    • Department of VirologyPostgraduate Institute of Medical Education and Research
  • Vandana Dhiman
    • Department of VirologyPostgraduate Institute of Medical Education and Research
  • P. MahaLakshmi
    • Department of School of Community Medicine and Public HealthPostgraduate Institute of Medical Education and Research
  • Abhinav Bharadwaj
    • Department of VirologyPostgraduate Institute of Medical Education and Research
  • Sandeep Pokhrel
    • Department of VirologyPostgraduate Institute of Medical Education and Research
  • Deepak Sharma
    • Department of School of Community Medicine and Public HealthPostgraduate Institute of Medical Education and Research
  • Mirnalini Sharma
    • Department of VirologyPostgraduate Institute of Medical Education and Research
  • Deepak Bhatia
    • District Surveillance Unit of Punjab under Integrated Disease Surveillance Project (IDSP) Government of India
  • Radha Kanta Ratho
    • Department of VirologyPostgraduate Institute of Medical Education and Research
Brief Report

DOI: 10.1007/s00705-013-1717-4

Cite this article as:
Mishra, B., Pujhari, S.K., Dhiman, V. et al. Arch Virol (2013) 158: 2359. doi:10.1007/s00705-013-1717-4

Abstract

Mumps is a vaccine-preventable disease that usually occurs as a self-limiting parotitis, but it can also lead to several life-threatening complications, including pancreatitis, meningitis, and encephalitis. The molecular epidemiology of the virus is poorly understood. The present study describes an outbreak of mumps virus infection in Punjab, India. The etiology was confirmed by serology and RNA detection to be mumps virus in 72 % of the cases and 50 % of contacts. This study, for the first time, revealed the mumps virus genotypes circulating in the Indian subcontinent as subtype G2 of genotype G.

Mumps is a vaccine-preventable disease that is endemic in most parts of the world [6]. Following natural infection, 20 % of infected individuals remain asymptomatic; 40-50 % develop a nonspecific respiratory prodrome, and the remaining 30-40 % manifest with self-limited uni- or bilateral parotitis. However, the virus can cause severe complications including meningitis/encephalitis, deafness, pancreatitis and orchitis [8].

Mumps virus (MuV) is spread by respiratory droplets. Although an effective live attenuated mumps vaccine has been available for routine use since 1977, only 57 % (110/193) member states of WHO had included a mumps vaccine in their measles and rubella routine vaccination programme as MMR vaccine by 2005 [8].

Based on the sequence of the SH gene, WHO has recognized 12 distinct genotypes of MuV [1]. MuV shows a distinct geographic distribution where more than one genotype may circulate simultaneously in a region [2]. WHO recommends monitoring of MuV circulation. The magnitude and epidemiology of mumps infection in India are poorly understood. There is scanty literature documented in PubMed related to mumps virus circulation in India [3, 4]. This study describes diagnostic tests performed for acute mumps cases and their contacts from an outbreak in school children in Fatehgarh Sahib, Punjab, India. Molecular characterization of MuV was done for the first time in India, associated with the present outbreak.

A spurt of cases with fever and salivary gland swelling occurred in a school of Fatehgarh Sahib, Punjab, India. The local civil surgeon reported the suspected mumps outbreak to the nodal officer of the District Surveillance Unit of Punjab. A team including members of the departments of Virology, Community Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh and IDSP, Punjab, was sent for detailed investigation of the outbreak. Twenty students were diseased, and samples were collected from 11 of them whose parents gave consent for testing. Nineteen school children from a clinically suspected mumps outbreak in a school in Punjab, India, were enrolled in this study. The inclusion criteria for 11 cases and 8 contacts were followed as per the “Guidelines for the Prevention and Control of Mumps Outbreaks in Canada” [5]. A total of 69 samples (19 blood, 13 saliva, 18 urine and 19 throat swab) were collected from all of the cases and contacts in the month of August 2011. All of the samples were transported in a cold chain to the laboratory and stored in a −80 °C deep freezer until processing.

All the serum samples were screened for MuV-specific IgM and IgG antibody using a commercial ELISA kit (Demeditec Diagnostics, Germany). Virus isolation was attempted in the Vero cell line from serum, throat swab, saliva and urine samples following the standard protocol. All of the culture supernatants were tested by RT-PCR for confirmation.

Viral RNA was extracted from clinical samples and culture supernatants using a QIAamp Viral RNA Mini Kit (QIAGEN, Hilden Germany), following the manufacturer’s instructions. RNA was reverse transcribed using Moloney murine leukemia virus (mMULV) reverse transcriptase (MBI Fermentas, USA). The reaction mixture excluding RNA was included as a negative control for reverse transcription.

cDNA from all of the cases and contacts were subjected to nested RT-PCR using the MuV-specific primers targeting a 639-bp fragment covering the entire small hydrophobic (SH) gene as described previously by Jin et al. [8]. A no-template control was included in the RT-PCR reactions.

Second-round amplified products of MuV-positive samples were purified using a QIAquick Gel Extraction Kit (QIAGEN, Hilden Germany). The purified products were cycle sequenced using an ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems Inc.) in an ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems Inc., USA). The partial SH gene sequences that were obtained were deposited in the database. At least two SH gene sequences of standard viral strains representing each genotype and subtypes of mumps virus, in addition to WHO reference strains, were downloaded from GenBank and were aligned along with the sequences from the present study using Clustal X [10, 14]. A phylogenetic tree was constructed using the NJ algorithm in MEGA version 4.1.

A total of 126 pupils were attending the affected school, and 20 of them were suspected to be diseased with MuV infection. The approximate male:female ratio in the affected school was 2.4:1. The mean age of the affected population was 9.7 years, and most of them were females (10/11; 91 %). The onset of illness in all cases occurred within 5 days (acute infection). None of the school children were vaccinated for MuV, as MMR vaccination is still not included in the immunization program of India. IgM antibody, the marker of acute infection, was detected in 63 % (7/11) of the cases and 25 % (2/8) of the asymptomatic contacts. Eighty-four percent (16/19) of the subjects were positive for MuV-specific IgG antibody, revealing the past exposure to MuV. Viral RNA was detected in 45.5 % (5/11) of the cases and in 25 % (2/8) contacts. RNA positivity was 18 %, 27 %, 9 % and 27 % in throat swab, saliva, blood and urine samples, respectively, among the cases. Seventy-two percent (8 out of 11) of the cases could be diagnosed as mumps infection either by IgM or RT-PCR whereas 50 % (4 out of 8; 2 IgM + 2 RT-PCR) of the contacts showed evidence of mumps virus infection. The details of mumps IgM/IgG, RNA detection, and virus isolation in cases and contacts are given in Table 1. Virus isolation was also successful from subjects for whom any one of the samples was positive for viral RNA. Virus isolation was confirmed by cytopathic effects (discrete syncytia, cell rounding or elongation, different light reflection, irregular cell shape) and RT-PCR. The SH genes of the mumps virus isolates (n: 7) were amplified, sequenced and submitted to GenBank. The accession numbers of the present study were JQ085367-JQ085373. Phylogenetic analysis revealed that the present sequences belong to genotype G and subtype G2 (Fig. 1). Sequences from the present study showed 90-95 % homology among themselves and 85-90 % to the prototype strain of genotype G.
Table 1

Details of clinical and laboratory findings

Subject no.

Age

Sex

Subject type

Clinical diagnosis

Onset of illness (days)

Serology

PCR

Virus isolation

Genotype

 

IgM

IgG

Throat swab

Saliva

Blood

Urine

Throat swab

Saliva

Blood

Urine

1

8

F

Case

Swelling parotid L, fever

1

+

+

+

+

+

+

+

+

G

2

12

F

Case

Swelling submandibular glands

2

+

+

+

+

G

3

8

F

Case

Swelling submandibular glands

4

+

+

+

+

G

4

10

F

Case

Swelling parotid

1

 

5

6

F

Case

Swelling parotid R, fever

3

+

+

+

+

+

+

+

G

6

10

M

Case

Swelling parotid area bilateral

3

+

 

7

10

F

Case

Swelling parotid area bilateral

4

+

+

 

8

12

F

Case

Swelling parotid area bilateral

2

+

+

+

G

9

11

F

Case

URI

5

+

NS

NS

 

10

10

F

Case

Swelling parotid area bilateral

4

+

+

 

11

10

F

Case

Swelling parotid area bilateral

5

+

+

 

12

8

F

Contact

Asymptomatic

NS

+

NS

+

NS

NS

G

13

9

M

Contacts

Asymptomatic

NS

+

NS

+

G

14

10

M

Contacts

Asymptomatic

+

NS

NS

 

15

12

M

Contacts

Asymptomatic

+

+

NS

NS

 

16

10

F

Contacts

Asymptomatic

+

NS

NS

 

17

12

F

Contacts

Asymptomatic

+

+

 

18

12

M

Contacts

Asymptomatic

+

 

19

12

F

Contacts

Asymptomatic

+

 

+ positive for MuV-specific IgM/IgG or MuV RNA or viral isolate positive for MuV by PCR, NS not sufficient sample, G genotype G

https://static-content.springer.com/image/art%3A10.1007%2Fs00705-013-1717-4/MediaObjects/705_2013_1717_Fig1_HTML.gif
Fig. 1

Phylogenetic relationship of mumps virus isolates from the present study. Partial sequences of the SH gene (247 nt), covering the region nt 6241-6488 of the mumps virus genome of reference strains, were compared with those of the present isolates. At least two sequences representing each genotype or subtype were included for analysis together with the WHO reference strains [10, 14] (filled diamond). MEGA version 4.1 was used for construction of the phylogenetic tree. The NJ method was implemented for tree construction with 1000 bootstrap replicates for statistical verification of each node, and the values are represented as a percentage value at each node. Sequences of the vaccines strains are indicated by filled circles, and sequences from the present study are indicated by filled rectangular boxes

Mumps virus belongs to the family Paramyxoviridae and genus Rubulavirus. It is an enveloped RNA virus with a non-segmented single-stranded negative-sense genome of approximately 15.3 kb. The genome encodes seven proteins: the fusion (F) and the hemagglutinin-neuraminidase (HN) surface glycoproteins; four core proteins, including the nucleoprotein (NP), the phosphoprotein (P), the matrix protein (M), and the large (L) protein; and the putatively membrane-associated small hydrophobic protein (SH) [12, 13]. The SH gene is the most variable segment with a missense silent mutation ratio of 2.0, compared to <0.5 for other genes [9]. Thus, for determining evolutionary relationships, the SH gene is targeted. There are 12 genotypes, designated A to L with two subtypes each for genotype: D, G and H [7, 11]. The genotypes B, F, G, H and J are distributed in Asian countries and in other parts of the world [1]. In the present study, we have, for the first time, analyzed the molecular epidemiology of mumps virus from the Indian subcontinent and shown that subtype G2 of genotype G is circulating in this part of the world. Continuous epidemiological surveillance along with inclusion of MMR vaccination in the routine immunization program is warranted to monitor the spread and effective management of mumps virus in India.

Acknowledgement

We would like to acknowledge the technical support of Mrs. Ragini and Vikas Verma for virus isolation, and Mr. Vikas Kumar for serological tests.

Conflict of interest

The authors declare that they have no competing interests.

Copyright information

© Springer-Verlag Wien 2013