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Neutralization test is the most reliable method of evaluating immunity against viral diseases but there is no standard procedure for mumps virus, with tests differing in the infectivity of the challenge virus, 50% plaque reduction or complete inhibition of cytopathic effects (CPE), and usage of complement. A reliable, easy, and simple neutralization test for mumps virus was developed in this study. A recombinant mumps virus expressing GFP was generated as a challenge virus. Complement was added to the neutralizing mixture at 1∶200 when stocked serum samples were used. Neutralizing antibody titers were expressed as the reciprocal of the highest dilution that did not exceed two-fold of FU values (GFP expression) of the cell control wells. A total of 1,452 serum samples were assayed by inhibition of GFP expression in comparison with those examined by conventional 100% inhibition of CPE. 1,367 (94.1%) showed similar neutralizing antibody titers when examined by both methods. The GFP expression inhibition assay, using a recombinant mumps virus expressing GFP, is a simple and time- saving method.

Clinically apparent mumps reinfection is considered extremely rare, but several cases have been suspected of reinfection in an out-patient clinic. In this study, virological examination, virus isolation, the reverse transcription loop-mediated isothermal amplification (RT-LAMP), and IgG and IgM EIA antibodies, were examined in order to identify mumps reinfection. Patients were divided into three categories; the reinfection group comprised 29 patients with a history of natural infection, the vaccine-failure group consisted of 37 patients with an immunization history, and two patients had histories of both immunization and mumps infection. Another 25 patients were enrolled as a primary infection group. Mumps virus was isolated in 5 (17%) and the genome was detected in 12 (41%) of 29 in the reinfection group. Reinfection was confirmed in 21/28, demonstrating high avidity of IgG EIA. Mumps virus was isolated in 15 (41%) and there was a higher positivity of genome amplification in 25 (68%) of 37 patients in the vaccine-failure group. Among these, 23 were confirmed as secondary vaccine failure by high avidity IgG EIA serology. In the primary infection group, the isolation rate and genome detection rate was higher in 16 (64%) and in 18 (72%) of 25 patients, respectively. There was no significant difference in virus load among the three groups but high mumps virus load was suspected in the IgM EIA-positive group based on the shorter amplification time on RT-LAMP. Mumps virus reinfection was confirmed by RT-LAMP and an IgG avidity test and was not a rare event.

Most mumps patients are clinically diagnosed without any virological examinations, but some diagnosed cases of mumps may be caused by other pathogens or secondary vaccine failure (SVF). To clarify these issues, a sensitive, specific, and rapid diagnostic method is required. We obtained 60 salivary swabs from 34 patients with natural infection during the course of the illness, 10 samples from patients with vaccine-associated parotitis, and 5 samples from patients with SVF. Total RNA was extracted and subjected to reverse transcription-PCR (RT-PCR) and loop-mediated isothermal amplification (LAMP) for genome amplification. We detected mumps virus RNA corresponding to 0.1 PFU by LAMP within 60 min after RNA extraction, with the same sensitivity as RT-nested PCR. Mumps virus was isolated in 30 of 33 samples within day 2, and mumps virus genome was amplified by LAMP in 32 of them. The quantity of virus titer was calculated by monitoring the time to reach the threshold of turbidity. The viral load decreased after day 3 and was lower in patients serologically diagnosed as having SVF with milder illness. Accuracy of LAMP for the detection of mumps virus genome was confirmed; furthermore, it is of benefit for calculating the viral load, which reflects disease pathogenesis.