Functional Characterization of Circulating Mumps Viruses with Stop Codon Mutations in the Small Hydrophobic Protein.

Between 2015 and 2017, routine molecular surveillance in the United States detected multiple mumps viruses (MuVs) with mutations in the small hydrophobic (SH) gene compared to a reference virus of the same genotype. These mutations include an unusual pattern of uracil-to-cytosine hypermutations and other mutations resulting in the generation of premature stop codons or disruption of the canonical stop codon. The mumps virus SH protein may serve as a virulence factor, based on evidence that it inhibits apoptosis and innate immune signaling in vitro and that recombinant viruses that do not express the SH protein are attenuated in an animal model. In this study, mumps viruses bearing variant SH sequences were isolated from contemporary outbreak samples to evaluate the impact of the observed mutations on SH protein function. All isolates with variant SH sequences replicated in interferon-competent cells with no evidence of attenuation. Furthermore, all SH-variant viruses retained the ability to abrogate induction of NF-κB-mediated innate immune signaling in infected cells. Ectopic expression of variant mumps SH genes is consistent with findings from infection experiments, indicating that the observed abrogation of signaling was not mediated by other viral factors that may modulate innate immune signaling. Molecular surveillance is an important public health tool for monitoring the diversity of circulating mumps viruses and can provide insights into determinants of disease. These findings, in turn, will inform studies employing reverse genetics to elucidate the specific mechanisms of MuV pathogenesis and potential impacts of observed sequence variants on infectivity, fitness, and virulence.IMPORTANCE Mumps virus (MuV) outbreaks occur in the United States despite high coverage with measles, mumps, rubella (MMR) vaccine. Routine genotyping of laboratory-confirmed mumps cases has been practiced in the United States since 2006 to enhance mumps surveillance. This study reports the detection of unusual mutations in the small hydrophobic (SH) protein of contemporary laboratory-confirmed mumps cases and is the first to describe the impact of such mutations on SH protein function. These mutations are predicted to profoundly alter the amino acid sequence of the SH protein, which has been shown to antagonize host innate immune responses; however, they were neither associated with defects in virus replication nor attenuated protein function in vitro, consistent with detection in clinical specimens. A better understanding of the forces governing mumps virus sequence diversity and of the functional consequences of mutations in viral proteins is important for maintaining robust capacity for mumps detection and disease control.

Genetic characterization of mumps viruses associated with the resurgence of mumps in the United States: 2015-2017.

Despite high coverage with measles, mumps, and rubella vaccine in the United States, outbreaks of mumps occur in close contact settings such as schools, colleges, and camps. Starting in late 2015, outbreaks were reported from several universities, and by the end of 2017, greater than 13,800 cases had been reported nation-wide. In 2013, the CDC and the Association of Public Health Laboratories contracted four Vaccine Preventable Diseases Reference Centers (VPD-RCs) to perform real-time reverse transcription PCR (RT-qPCR) to detect mumps RNA in clinical samples and to determine the genotype. Twelve genotypes of mumps virus are currently recognized by the World Health Organization, and the standard protocol for genotyping requires sequencing the entire gene coding for the small hydrophobic (SH) protein. Phylogenetic analysis of the 1862 mumps samples genotyped from 2015 through 2017 showed that the overall diversity of genotypes detected was low. Only 0.8 % of the sequences were identified as genotypes C, H, J, or K, and 0.5 % were identified as vaccine strains in genotypes A or N, while most sequences (98.7 %) were genotype G. The majority of the genotype G sequences could be included into one of two large groups with identical SH sequences. Within genotype G, a small number of phylogenetically significant outlier sequences were associated with epidemiologically distinct chains of transmission. These results demonstrate that molecular and epidemiologic data can be used to track transmission pathways of mumps virus; however, the limited diversity of the SH sequences may be insufficient for resolving transmission in all outbreaks.

Use of FTA Cards To Transport Throat Swabs and Oral Fluid Samples for Molecular Detection and Genotyping of Measles and Rubella Viruses.

The genetic characterization of measles viruses is an important tool for measles surveillance. Reverse cold chain requirements for the transportation of samples to reference laboratories are challenging in resource-limited settings. FTA cards facilitate the transport of virologic samples at ambient temperature as noninfectious material; however, the utility of FTA cards for the detection and genotyping of measles virus from clinical samples has not been evaluated. Throat swabs (TS) and oral fluid (OF) samples were collected from suspected measles cases in the Democratic Republic of the Congo. Virus detection (reverse transcription-quantitative real-time PCR [RT-qPCR]) and genotyping (endpoint RT-PCR) were compared for samples from 238 suspected cases; these samples were either transported using the reverse cold chain or at ambient temperature on FTA cards. Virus detection showed excellent positive agreement for OF samples compared to TS (95.3%; confidence interval [CI], 91.6 to 97.4), in contrast to 79.4% (CI, 73.5 to 84.3) for TS on FTA, and 85.5% (CI, 80.2 to 89.6) for OF on FTA compared to OF samples. Genotyping results obtained for a subset of samples indicated that 77.3% of all TS and 71.0% of OF samples would produce genotype information compared to 41.6% of TS and 41.3% of OF on FTA cards. Similar results were found for 16 measles-negative samples that were confirmed as rubella cases. Measles genotype B3 and rubella genotype 2B were detected. FTA cards have limited utility for virologic surveillance of sporadic cases of measles; however, they can be a useful tool for the expansion of virologic surveillance in countries where the reverse cold chain is not available.

Detection of Measles Virus RNA in Air and Surface Specimens in a Hospital Setting.

Measles virus (MeV) is known to be highly contagious, with an infectious period lasting from 4 days before to 4 days after rash onset. An unvaccinated, young, female patient with measles confirmed by direct epidemiologic link was hospitalized on day 5 after rash onset. Environmental samples were collected over the 4-day period of hospitalization in a single room. MeV RNA was detectable in air specimens, on surface specimens, and on respirators on days 5-8 after rash onset. This is the first report of environmental surveillance for MeV, and the results suggest that MeV-infected fomites may be present in healthcare settings.

Improving molecular tools for global surveillance of measles virus.

BACKGROUND: The genetic characterization of wild-type measles viruses plays an important role in the description of viral transmission pathways and the verification of measles elimination. The 450 nucleotides that encode the carboxyl-terminus of the nucleoprotein (N-450) are routinely sequenced for genotype analysis. OBJECTIVES: The objectives of this study were to develop improved primers and controls for RT-PCR reactions used for genotyping of measles samples and to develop a method to provide a convenient, safe, and inexpensive means to distribute measles RNA for RT-PCR assays and practice panels. STUDY DESIGN: A newly designed, genetically defined synthetic RNA and RNA isolated from cells infected with currently circulating genotypes were used to compare the sensitivity of primer pairs in RT-PCR and nested PCR. FTA® cards loaded with lysates of measles infected cells were tested for their ability to preserve viral RNA and destroy virus infectivity. RESULTS: A new primer pair, MeV216/MeV214, was able to amplify N-450 from viruses representing 10 currently circulating genotypes and a genotype A vaccine strain and demonstrated 100-fold increased sensitivity compared to the previously used primer set. A nested PCR assay further increased the sensitivity of detection from patient samples. A synthetic positive control RNA was developed that produced PCR products that are distinguishable by size from PCR products amplified from clinical samples. FTA® cards completely inactivated measles virus and stabilized RNA for at least six months. CONCLUSIONS: These improved molecular tools will advance molecular characterization of circulating measles viruses globally and provide enhanced quality control measures.

Comparison of the sensitivity of laboratory diagnostic methods from a well-characterized outbreak of mumps in New York city in 2009.

A mumps outbreak in upstate New York in 2009 at a summer camp for Orthodox Jewish boys spread into Orthodox Jewish communities in the Northeast, including New York City. The availability of epidemiologic information, including vaccination records and parotitis onset dates, allowed an enhanced analysis of laboratory methods for mumps testing. Serum and buccal swab samples were collected from 296 confirmed cases with onsets from September through December 2009. All samples were tested using the Centers for Disease Control and Prevention (CDC) capture IgM enzyme immunoassay (EIA) and a real-time reverse transcription-PCR (rRT-PCR) that targets the short hydrophobic gene. A subset of the samples (n = 205) was used to evaluate 3 commercial mumps IgM assays and to assess the sensitivity of using an alternative target gene (nucleoprotein) in the rRT-PCR protocol. Among 115 cases of mumps with 2 documented doses of measles, mumps, and rubella (MMR) vaccine, the CDC capture IgM EIA detected IgM in 51% of serum samples compared to 9% to 24% using three commercial IgM assays. The rRT-PCR that targeted the nucleoprotein gene increased RNA detection by 14% compared to that obtained with the original protocol. The ability to detect IgM improved when serum was collected 3 days or more after symptom onset, whereas sensitivity of RNA detection by rRT-PCR declined when buccal swabs were collected later than 2 days after onset. Selection of testing methods and timing of sample collection are important factors in the ability to confirm infection among vaccinated persons. These results reinforce the need to use virus detection assays in addition to serologic tests.