A New Method for the Detection of Neutralizing Antibodies against Mumps Virus

Abstract

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.

Fluorescent and Bioluminescent Reporter Myxoviruses

“…Several groups have found that insertion of ATUs at this site was relatively well tolerated and resulted in little to no viral attenuation [ 49 , 50 ]. However, ATUs have been inserted into several other locations within the paramyxovirus genome, including between genes N and P [ 51 ]; P and M [ 44 , 52 , 53 , 54 , 55 ]; M and F [ 52 ]; and H and L [ 56 , 57 ], which have resulted in varying levels of attenuation. Van Remmerden et al noted that insertion of an ATU encoding eGFP in the first gene position was attenuating, whereas insertion between the SH and G genes was not [ 58 ].…”

“…Neutralization assays have also been developed using bioluminescent reporter proteins, which can be automated to measure the reduction of virally-expressed luciferase [ 73 ]. Such assays have been developed for influenza viruses [ 61 , 74 ], in addition to several paramyxoviruses including measles virus [ 75 ], mumps virus [ 55 ], HMPV [ 54 ], and RSV [ 72 , 73 ].…”

Easy and Rapid Detection of Mumps Virus by Live Fluorescent Visualization of Virus-Infected Cells

“…Acute infection with mumps virus is manifested by demonstrable swelling of the parotid glands and several complications including aseptic meningitis, encephalitis, deafness, orchitis, oophoritis, and pancreatitis. Generally, clinical diagnosis is based on parotid swelling [ 1 – 3 ]. Laboratory detection and diagnosis are based on isolation of the virus, detection of the viral gene, or serological confirmation (generally the presence of anti-mumps virus IgM antibodies).…”

“…Therefore, for mumps virus, there is no standard procedure for easy detection of virus-infected cells and for evaluation of antiviral reagents. Furthermore, a period of more than 6 days after infection is required until appearances of an obvious CPE or plaque in many cases [ 3 , 6 – 8 ]. For laboratory confirmation and experimentation of mumps virus, it is useful if the method for detection of infected cells is easy, rapid, and independent of CPE and plaque formation.…”

Assessment of Mumps Virus-Specific Antibodies: Comparison of Plaque Reduction Neutralization Test and Enzyme-Linked Immunosorbent Assay Estimates

“…There are no well-defined protective serological response threshold against mumps virus infection [1–4], and serological methods for detecting mumps antibodies have varying sensitivity and specificity [2]. Functional neutralizing antibody assays, such as the plaque reduction neutralization test (PRNT), are thought to provide the best estimate of protection [2], but there is no standard procedure for mumps virus infection [5].…”