Mumps Virus RNA Research Articles

Mumps in Vaccinated Children and Adolescents

Source: Shepersky L, Marin M, Zhang J, et al. Mumps in vaccinated children and adolescents: 2007–2019. Pediatrics. 2021;148(6): e2021051873; doi.10.1542/peds.2021-051873Investigators from the CDC, Atlanta, GA, conducted a retrospective study to describe the incidence of mumps in children in the US and assess demographic and vaccination characteristics of those with mumps. For the study, they reviewed data in the National Notifiable Diseases Surveillance System (NNDSS) and identified cases of mumps in youths <18 years old in the US between 2007 and 2019. Data from the entire study period were analyzed to characterize pediatric mumps cases by age, state of residence, mumps vaccination status, and whether cases were outbreak-associated. Annual age-specific incidence rates were calculated. A focused analysis was conducted on pediatric mumps cases during the period 2015–2019. In addition to demographic information, data on import status (defined as a case in a child following international travel), parotitis, and complications (orchitis, deafness, meningitis, and encephalitis) were collected. Patients 1-4 years old were classified as up-to-date (UTD) for mumps vaccination if they had received 1 dose of mumps vaccine, and those 5-17 years old were classified as UTD if they had received 2 doses.Between 2007 and 2019, 28,306 mumps cases were reported to NNDSS, including 9,172 (32%) in children. Mumps was reported in all 50 states and the District of Columbia. The annual median number of pediatric mumps cases was 349, with peaks in 2009– 2010 (2,627 pediatric cases) and 2016–2017 (4,093 pediatric cases). Overall, the median annual incidence of mumps in children was 4.7 cases/1,000,000 population. Median annual incidence was 5.8 cases/1,000,000 population in children 5-10 years old and 5.8 cases/1,000,000 population in those 1-4 years of age. During the study period, 64% of pediatric mumps cases with available data were outbreak-associated. Among children with mumps, 89% had ≥1 dose of mumps vaccine.Between 2015 and 2019, 5,461 mumps cases were reported in children. During this period, 54% of pediatric mumps cases occurred in youths 11-17 years old, 33% in those 5-10 years old, 13% in children 1-4 years old, and 1% in infants <1 year old. Only 2% of pediatric cases were associated with international travel. During this period, 74% of children 1-4 years old with mumps and 86% of those 5-17 years old were UTD for mumps vaccination. Among 4,471 cases of pediatric mumps, parotitis was present in 97%. Complications occurred in 75 cases (1%), including orchitis in 2% of boys, deafness in 10 children, meningitis in 3, and encephalitis in 2.The authors conclude that since 2007, 32% of mumps cases in the US occurred in children, most of whom had been vaccinated.Dr Brady has disclosed no financial relationship relevant to this commentary. This commentary does not contain a discussion of an unapproved/investigative use of a commercial product/device.Mumps can occur in fully vaccinated individuals. (See AAP Grand Rounds. 2017;37[6]:64.)1 Two explanations have been suggested for the occurrence of mumps in vaccinated persons: waning of vaccine-induced immunity and antigenic mismatch between the vaccine strain and the circulating wild-type mumps strains.2 Rubin et al3 reported that mumps antibodies declined to near baseline levels 10 years after vaccination for children vaccinated with the second dose at age 4 to 6 years. Additionally, these vaccine-induced antibody responses were lower against the circulating wild-type strains (predominantly genotype G in the US) than against the vaccine strain (genotype A).3-4The current authors noted that 97% of children with mumps presented with parotitis. As prior mumps vaccination does not exclude mumps, diagnostic testing is a consideration when children present with parotitis or mumps complications (eg, orchitis). The preferred test is a buccal swab specimen to detect mumps virus nucleic acid by quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR).5 When buccal swab specimens were collected <2 days after symptom onset, the sensitivity of RT-qPCR was about 83%.1 Therefore, failure to detect mumps virus RNA by RT-qPCR in samples from persons with parotitis does not exclude mumps as a diagnosis. Vaccinated persons may shed small amounts of virus for short periods.5 In previously vaccinated persons who acquire mumps, the mumps-specific immunoglobulin (Ig) M response may be transient, delayed, or not detected.5 Thus, a negative IgM also does not exclude mumps as a diagnosis.With the 2-dose measles-mumps-rubella (MMR) vaccine policy, mumps cases in the US decreased by >99%.6 However, mumps still occurs in vaccinated persons. Parotitis is the hallmark of mumps, and RT-qPCR to detect mumps RNA from a buccal swab specimen is the preferred diagnostic test.A third dose of the current MMR vaccine has been administered during mumps outbreaks.4 A third dose of a mumps vaccine using an alternative mumps strain, however, is a consideration to boost and broaden immunity in vaccine recipients.4

Functional analysis of host factors involved in mumps virus propagation

Mumps virus (MuV) is the causative agent of mumps, a common childhood illness characterized by fever and swelling of the salivary glands. Like other viral infections, a number of host proteins are thought to involve in MuV infection. We have shown the function of several host factors in MuV infection. The chaperone proteins, heat shock protein 70 (Hsp70) and Hsp90, interact with the P and L proteins that form the polymerase complex and function in the protein quality control of these viral proteins, and thus they are essential host factors in MuV RNA synthesis. The R2TP complex is a host factor that contributes to effective viral propagation by precise regulation of viral RNA synthesis and evasion of host immune responses, and Rab11 is a host factor involved in viral RNP trafficking to the plasma membrane. This article summarizes the functions of host factors involved in MuV infection based on our researches.

Epidemiological and Phylogenetic Analysis of Mumps Virus Isolated from 2016 to 2019 in Henan Province, China.

Routine vaccination has proven to be highly effective in reducing the incidence of mumps. However, sporadic cases and/or mumps outbreaks have been reported in children and adolescents younger than 15 years of age, particularly among those aged 5-9 years. To explore the characteristics of such outbreaks in the Henan Province, clinical data of patients infected with mumps virus (MuV) were collected, and the isolated strains were phylogenetically analyzed. Of the total 426 samples analyzed, MuV RNA targeting the small hydrophobic (SH) gene was detected in 153 samples. MuV-positive cases in age groups <5 years, 5-9 years, 10-15 years, 16-19 years, and ≥20 years accounted for 1%, 17%, 12%, 2%, and 4% of the total number of cases, respectively. Phylogenetic analysis based on the SH gene sequences indicated that all of the isolated strains were of genotype F, and isolates in the same subcluster and with identical SH gene sequences tended to be derived from the same community or municipalities when analyzed alongside epidemiological data. In conclusion, the incidence of mumps in the Henan Province was high. The data provided in this study might promote further research in the clarification of the specific causes of mumps outbreaks, which can facilitate the implementation of effective prevention and control measures.

Evidence that a polyhexameric genome length is preferred, but not strictly required, for efficient mumps virus replication

Mumps virus (MuV) is postulated to adhere to the “rule of six” for efficient replication. To examine the requirement for MuV, minigenomes of nonpolyhexameric length (6n−1 and 6n+1) were analyzed. Expression of the reporter gene CAT was significantly reduced with minigenomes of nonpolyhexameric length compared to the wild type 6n genome, and reduction was more pronounced for the 6n−1 than for the 6n+1 minigenome. That 6n−1 genomes are impacted by nonconformance with the rule of six to a greater degree as compared to 6n+1 genomes was also suggested with MuV derived from cDNA coding for 6n+1 or 6n−1 genomes. While viruses recovered from 6n+1 cDNAs maintained a nonpolyhexameric genome length over multiple replication cycles, viruses rescued from the 6n−1 cDNAs acquired length correcting mutations rapidly following rescue. Our data indicate that polyhexameric genomes are the preferred template for the MuV RNA polymerase, but that this requirement is not absolute.

A Mumps Outbreak in Vojvodina, Serbia, in 2012 Underlines the Need for Additional Vaccination Opportunities for Young Adults.

In 2012, mumps was introduced from Bosnia and Herzegovina to Vojvodina, causing an outbreak with 335 reported cases. The present manuscript analyses the epidemiological and laboratory characteristics of this outbreak, identifies its main causes and suggests potential future preventive measures. Sera of 133 patients were tested for mumps-specific antibodies by ELISA and 15 nose/throat swabs were investigated for mumps virus RNA by RT-PCR. IgG antibodies were found in 127 patients (95.5%). Mumps infection was laboratory-confirmed in 53 patients, including 44 IgM and 9 PCR positive cases. All other 282 cases were classified as epidemiologically-confirmed. More than half of the patients (n = 181, 54%) were 20–29 years old, followed by the 15–19 age bracket (n = 95, 28.4%). Twice as many males as females were affected (67% versus 33%). Disease complications were reported in 13 cases (3.9%), including 9 patients with orchitis and 4 with pancreatitis. According to medical records or anamnestic data, 190 patients (56.7%) were immunized with two doses and 35 (10.4%) with one dose of mumps-containing vaccine. The Serbian sequences corresponded to a minor genotype G variant detected during the 2011/2012 mumps outbreak in Bosnia and Herzegovina. Vaccine failures, the initial one-dose immunization policy and a vaccine shortage between 1999 and 2002 contributed to the outbreak. Additional vaccination opportunities should be offered to young adults during transition periods in their life trajectories.

Mumps disease outbreak in Davangere district of Karnataka, India

Background: Mumps is a vaccine-preventable disease that usually occurs as a parotitis, but it can also lead to several life- threatening complications, including pancreatitis, meningitis and encephalitis. Objective: To determine and diagnosis of mumps disease, which is communicable disease usually affects childrens. Although it is seen worldwide, but outbreaks not common in India. Materials and Methods: Thirty one suspected mumps cases, who presented to the unimmunized population of Chikkahallivana village in Davangere district of Karnataka, India in January 2014, with clinical evidence of fever, cervical lymphadenitis and ear pain, manifest with self-limited uni-or bilateral parotitis. A total of 31 cases consisting of 31 blood and 31 throat swabs were tested for diagnosis of mumps disease. Results: Of the 31 suspected cases, laboratory results showed 18 positive for mumps IgM antibodies and 7 cases showed presence of mumps virus RNA by RT-PCR using MV specific nested primers. From 31 cases, 5 were positive with both the methods. Conclusion: We confirmed the cases by serological as well as a sensitive RT-nested PCR-based method and sequencing results for the molecular identification of mumps infection. Sequencing results of the SH gene identified outbreak strain as genotype C, which was consistent with other outbreaks in India.

Roles of Phosphorylation of the Nucleocapsid Protein of Mumps Virus in Regulating Viral RNA Transcription and Replication.

Mumps virus (MuV) is a paramyxovirus with a negative-sense nonsegmented RNA genome. The viral RNA genome is encapsidated by the nucleocapsid protein (NP) to form the ribonucleoprotein (RNP), which serves as a template for transcription and replication. In this study, we investigated the roles of phosphorylation sites of NP in MuV RNA synthesis. Using radioactive labeling, we first demonstrated that NP was phosphorylated in MuV-infected cells. Using both liquid chromatography-mass spectrometry (LC-MS) and in silico modeling, we identified nine putative phosphorylated residues within NP. We mutated these nine residues to alanine. Mutation of the serine residue at position 439 to alanine (S439A) was found to reduce the phosphorylation of NP in transfected cells by over 90%. The effects of these mutations on the MuV minigenome system were examined. The S439A mutant was found to have higher activity, four mutants had lower activity, and four mutants had similar activity compared to wild-type NP. MuV containing the S439A mutation had 90% reduced phosphorylation of NP and enhanced viral RNA synthesis and viral protein expression at early time points after infection, indicating that S439 is the major phosphorylation site of NP and its phosphorylation plays an important role in downregulating viral RNA synthesis. Mumps virus (MuV), a paramyxovirus, is an important human pathogen that is reemerging in human populations. Nucleocapsid protein (NP) of MuV is essential for viral RNA synthesis. We have identified the major phosphorylation site of NP. We have found that phosphorylation of NP plays a critical role in regulating viral RNA synthesis. The work will lead to a better understanding of viral RNA synthesis and possible novel targets for antiviral drug development.

Viral etiology of mumps-like illnesses in suspected mumps cases reported in Catalonia, Spain

We investigated the etiology of reported sporadic suspected mumps cases with a negative RT-PCR result for the mumps virus in the Barcelona-South region in 2007–2011. Samples from mumps virus-negative patients presenting unilateral or bilateral parotitis or other salivary gland swelling were tested for Epstein-Barr virus (EBV) by real-time PCR and for respiratory viruses by two multiplex-PCR-based assays to detect parainfluenza virus (PIV) 1–4, influenza virus (InV) A, B and C, respiratory syncytial virus (RSV), enterovirus, coronavirus 229E, coronavirus OC43, and rhinovirus. 101 samples were analyzed in persons aged 8 months to 50 years. Oral samples were collected on the first day of glandular swelling in 53 patients (52.5%), and on the first two days in 74 patients (73.3%). Viruses were detected in 52 (51.5%) of samples: one virus (25 EBV, 8 PIV3, 4 adenovirus, 4 PIV2, 1 PIV1, 1 InVA, and 1 enterovirus) was detected in 44 patients (84.6%), two viruses in 7 patients, and three viruses in one patient. In 58 patients (57.5%) whose sample was collected in the first 2 days after onset of parotitis and had received two doses of MMR vaccine and in 15 patients (14.8%) whose sample was collected on the first day, it is very likely that the cause was not the mumps virus. This would mean that 72.3% (73/101) of the reported sporadic suspected mumps cases were not mumps cases. The timing of oral-sample collection is crucial to correctly interpret the negative results for mumps virus RNA, especially when suspected cases occur in vaccinated persons.

Genetic characterization of historical epidemic mumps viruses in northern Spain, 1987–1990

The mumps virus (MuV) is genetically diverse and is divided into 12 genotypes. The World Health Organization has recommended expanding virological surveillance for MuV, and therefore molecular characterization of circulating strains (i.e. genotypes) is increasingly performed. Nevertheless, little is known about the genotypes circulating before the massive vaccination of children and adolescents. The present study analyzed the strains causing the 1988–1989 mumps epidemic in the Basque Country, northern Spain, which occurred in the early vaccination period, before the endemic circulation of mumps virus was blocked. The epidemic reached an annual incidence rate of more than 400 cases/100,000 inhabitants, and caused a large number of cases of mumps meningitis. MuV RNA was amplified from the cerebrospinal fluid of 15 infected patients during the epidemic and from three more patients affected shortly before or after this epidemic (1987, early 1988 and 1990). Genotyping of the complete small hydrophobic gene (316 nucleotides), amplified in the 18 strains, as well as of the entire hemagglutinin-neuraminidase gene (1749 nucleotides), amplified in four strains, assigned all strains to genotype K, a genotype infrequently detected at present. Although the putative HN protein sequence differed by 4.8–5.5% in relation to Jeryl Lynn 5 strain (the main strain used in the vaccination program in this region), the vaccine was effective, and dramatically reduced the incidence of mumps over the following years. The presence of genotype K strains in Spain in the 1980s, together with their contemporary detection in Scandinavia, suggests that this genotype could have caused the Spanish epidemic and was also circulating widely in Europe at that time.

Mumps-associated meningitis and encephalitis in patients with no suspected mumps infection

Mumps virus (MuV) was detected in the cerebrospinal fluid (CSF) of 6 of 158 patients with meningitis or encephalitis in absence of clinical mumps in the context of mumps epidemics. Our results suggest the need for the study of MuV RNA in the CSF of neurological patients in this context.

Mumps-virus-associated Clinically Mild Encephalopathy With a Reversible Splenial Lesion

Mumps virus infection primarily involves the parotid glands and most frequently affects young children during the winter and spring months. It is known that mumps virus infection may be accompanied by neurological manifestations, including meningitis and encephalitis. However, few cases of acute encephalopathy have been reported to date. We report a case of a 4-year-old girl with clinically mild encephalopathy with a reversible splenial lesion associated with mumps virus infection. She had swelling of the bilateral parotid glands. She was admitted to our hospital because of reduced level of consciousness, seizures, and vomiting. Cell counts in the cerebrospinal fluid (CSF) were normal. She had a low serum sodium level on admission. Brain computed tomography showed mild cerebral edema. Electroencephalography showed partial high-voltage slow waves at the occipital lesion, and diffusion-weighted magnetic resonance imaging demonstrated a transient abnormality in the splenium of the corpus callosum. We diagnosed clinically mild encephalopathy with a reversible splenial lesion associated with mumps virus infection. She recovered well, and exhibited no neurological sequelae. Mumps virus RNA was not detected in the CSF, suggesting that the reversible splenial change was caused by indirect effects on the central nervous system subsequent to viral infection. Her low serum sodium level also indicates that this change can occur with hyponatremia. doi: http://dx.doi.org/10.4021/ijcp51w

Detection of mumps virus RNA in cerebrospinal fluid of patients with neuromyelitis optica

Neuromyelitis optica (NMO) is an acute inflammatory disease that preferentially involves the optic nerves and spinal cord. Although many infectious agents, including mumps virus, are postulated to have a role in the pathogenesis of multiple sclerosis (MS), the relationship between NMO and infectious agents remains uncertain. To investigate the relationship between NMO and viruses that have special affinity for the central nervous system, we performed a nested polymerase chain reaction (PCR) to detect mumps virus or enterovirus RNA in cerebrospinal fluid samples from 13 patients with MS, 8 with NMO and 20 with other neurological diseases (ONDs). Nested PCR was positive for mumps virus in 2 (25%) of NMO patients, but in none of those with MS and ONDs. Moreover, nested PCR results became negative in the remission phase in the two PCR-positive NMO patients. Mumps virus may have some role in the pathogenesis of NMO.

Molecular Epidemiological Evaluation of the Recent Resurgence in Mumps Virus Infections in Ireland

Mumps is a vaccine-preventable disease; however, outbreaks have been reported in a number of countries with childhood immunization programs, particularly among young adults at the tertiary stage of education. We have retrospectively investigated the epidemiological, virological, and serological factors associated with mumps cases identified in Ireland from 2004 to 2009. Genetic analysis of mumps virus strain variability demonstrated that a single genotype, genotype G, was circulating, and it was also detected in cerebrospinal fluid samples obtained from patients with meningitis. We observed that younger individuals were disproportionately affected with neurological sequelae following mumps virus infection, and the average age of patients with mumps virus RNA detected in cerebrospinal fluid was 19.25 years (median, 19 years; range, 14 to 24 years). Our analysis showed a 4-fold rise in mumps cases in 2008-2009 and an increased incidence in infection in those >or=30 years of age. Over a 6-year period (2004 to 2009), a total of 7,805 serum samples were investigated; of this number, 1,813 (23%) were positive for mumps virus-specific IgM. We observed a strong bias for acute mumps virus infection in males compared to females (P < 10(-32)) that was independent of vaccination status.

Development and evaluation of a rapid immunochromatographic test for mumps‐specific IgM in oral fluid specimens and use as a matrix for preserving viral nucleic acid for RT‐PCR

The use of oral fluid specimens for diagnosis of mumps by detection of virus-specific IgM or nucleic acid is now well established. The utility of oral fluids would be improved further if testing could be performed closer to the patient. A near patient test (NPT) for the detection of mumps-specific IgM in oral fluid specimens was developed and evaluated using 196 oral fluid specimens from suspected cases of mumps and measles. Compared to enzyme immunoassay (EIA), the sensitivity, specificity, positive and negative predictive value of the mumps IgM NPT were 79.5%, 100%, 100%, and 72.6%, respectively. On oral fluid specimens with a test to negative control optical density ratio (T/N) > or =10 in the EIA, sensitivity of the NPT increased to 95.2%. To determine whether viral nucleic acid from oral fluid was preserved on the NPT strips used for IgM detection, reverse transcription-polymerase chain reaction (RT-PCR) was performed on 58 oral fluids before and after testing on NPT strips. In RT-PCR-positive oral fluids, mumps virus was detected in 19/21 (90.5%) specimens extracted from NPT strips. Mumps virus RNA was not detected in 37/37 (100%) NPT strips from mumps RT-PCR-negative oral fluid specimens. Mumps IgM NPT is rapid and simple to perform, with an acceptable level of performance for confirmation of a clinical diagnosis outside of the laboratory. The NPT strip is also a suitable matrix for preserving nucleic acid, enabling virus-specific RT-PCR to be performed. J. Med. Virol. 82:485-493, 2010. (c) 2010 Wiley-Liss, Inc.

Investigation of a mumps outbreak among university students with two measles–mumps–rubella (MMR) vaccinations, Virginia, September–December 2006

Following the clinical diagnosis of the first case of mumps on September 22, 2006 at the University of Virginia (UVA), 52 suspected cases were identified through active surveillance for mumps by the end of December 2006. Samples were collected from 47 students who presented with parotitis despite a documented history of two doses of measles, mumps, and rubella (MMR) vaccine. Six of 47 serum samples (13%) were positive for mumps IgM, and 46/47 specimens were positive for mumps IgG. Endpoint titration of acute phase serum samples from laboratory-confirmed cases did not provide evidence that elevated serum IgG is a consistent marker for infection among cases due to secondary vaccine failure. Buccal swab samples from 39 of the 47 students were tested by real-time reverse transcription-polymerase chain reaction (RT-PCR) and/or viral culture. Mumps virus or mumps RNA was detected in 12 of 39 buccal samples (31%). Genetic analysis of the virus from the outbreak at UVA indicated that the outbreak was not linked to the large mumps outbreak in the Midwestern US that occurred earlier in 2006. Our findings support the use of viral detection to improve laboratory diagnosis of mumps among persons who have received two doses of MMR.

Characterization of Large Mumps Outbreak among Vaccinated Palestinian Refugees

During a large mumps virus (MuV) outbreak which occurred in the Palestinian refugee camps of the West Bank, 68.1% (2,636/3,871) of the cases were vaccinated with one dose of trivalent measles, mumps, and rubella (MMR) vaccine. Attack rates by camp ranged from less than 1 case per 1,000 people in the population to 43/1,000 (overall, 11/1,000). The outbreak lasted from December 2003 to June 2005, with two peaks, one from April to May 2004 and the other from March to April 2005. To control the outbreak, a mass MMR vaccination campaign was conducted in May 2005. Evaluation of the immune status of cases (n=59) and healthy controls (n=51) revealed high levels of mumps immunoglobulin G (IgG) and a low MuV-specific IgM in clinical cases indicative of a booster immune response. This suggested a secondary rather than a primary infection due to the insufficient protection conferred by the single vaccine dose included in the vaccination program. This prediction was further confirmed by the low seroprevalence (68.6%) found in the healthy control group, which was below the threshold level required for MuV herd immunity. Mumps diagnosis was established mainly by reverse transcription-PCR in clinical samples obtained within 48 h from the onset of disease. Of the parotid fluids and nasopharyngeal aspirates analyzed, 92% were positive for MuV RNA, while only 33% of the urine samples were positive. Phylogenetic analysis of the MuV SH gene identified the outbreak strain as the H genotype, which has been in circulation worldwide at least since 1989.

Detection of Mumps Virus RNA by Real-Time One-Step Reverse Transcriptase PCR Using the LightCycler Platform

A real-time reverse transcriptase PCR (RT-PCR) assay that targeted both the mumps virus F gene and human RNase P in clinical samples was adapted for use with the LightCycler platform. LightCycler RT-PCR is as sensitive as conventional nested RT-PCR and significantly less expensive and labor-intensive, making it ideal for mumps diagnosis during outbreaks.

Detection of RNA of Mumps Virus during an Outbreak in a Population with a High Level of Measles, Mumps, and Rubella Vaccine Coverage

The duration of mumps virus RNA detection was studied during a mumps outbreak in a highly vaccinated university population. Seven of the eight reverse transcription-PCR-positive specimens were collected during the first 3 days of parotitis, suggesting that viral shedding is minimal after the first 3 days of symptoms.

Epidemiologic and diagnostic evaluation of a recent mumps outbreak using oral fluid samples

ObjectiveTo determine the optimal strategy to investigate mumps virus infection in a partially vaccinated cohort. Study design122 oral fluid and serum samples were collected in a recent outbreak in Ireland. The largest age cohort, students aged 18–21 years old attending third level institutions, were investigated using virus isolation, detection of mumps specific IgM, IgG, RT-PCR and molecular genotyping. Results97% of patients had both detectable serum IgM and IgG. Mumps virus RNA was detected in 17 oral fluid samples and 14 of these originated from a single geographic location. Only 6 of the IgM positive samples had detectable mumps virus RNA whereas this could be detected in 11 IgM negative samples. Genotyping studies revealed that genotypes G and J were co-circulating during this outbreak. ConclusionsThe use of an oral fluid sample to detect mumps virus RNA and IgM offers a major improvement over serological diagnosis in acute infection in both non-vaccinated or partially vaccinated individuals, and has the advantage that specimens are collected non-invasively.

Real‐time PCR and its application to mumps rapid diagnosis

A real-time polymerase chain reaction assay was initially developed in China to detect mumps genome. The primers and TaqMan-MGB probe were selected from regions of the hemagglutinin gene of mumps virus. The primers and probe for the real-time PCR were evaluated by both laboratories in China and in the UK using three different pieces of equipment, LightCycler (Roche), MJ DNA Engine Option 2 (BIO-RAD) and TaqMan (ABI Prism) on different samples. The reaction was performed with either a one-step (China) or two-step (UK) process. The sensitivity (10 copies) was estimated using a serial dilution of constructed mumps-plasmid DNA and a linear standard curve was obtained between 10 and 10(7) DNA copies/reaction, which can be used to quantify viral loads. The detection limit on cell culture-grown virus was approximately 2 pfu/ml with a two-step assay on TaqMan, which was equivalent to the sensitivity of the nested PCR routinely used in the UK. The specificity was proved by testing a range of respiratory viruses and several genotypes of mumps strains. The concentration of primers and probe is 22 pmol and 6.25 or 7 pmol respectively for a 25 microl reaction. The assay took 3 hr from viral RNA extraction to complete the detection using any of the three pieces of equipment. Three hundred forty-one (35 in China and 306 in the UK) clinical specimens were tested, the results showing that this real-time PCR assay is suitable for rapid and accurate detection of mumps virus RNA in various types of clinical specimens.