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1.  Estimates of Mumps Seroprevalence May Be Influenced by Antibody Specificity and Serologic Method 
Neutralizing antibodies are assumed to be essential for protection against mumps virus infection, but their measurement is labor- and time-intensive. For this reason, enzyme-linked immunosorbent assays (ELISAs) are typically used to measure mumps-specific IgG levels. However, since there is poor correlation between mumps neutralization titers and ELISAs that measure the presence of mumps-specific IgG levels, ELISAs that better correlate with neutralization are needed. To address this issue, we measured mumps antibody levels by plaque reduction neutralization, by a commercial ELISA (whole-virus antigen), and by ELISAs specific for the mumps nucleoprotein and hemagglutinin. The results indicate that differences in the antibody response to the individual mumps proteins could partially explain the lack of correlation among various serologic tests. Furthermore, the data indicate that some seropositive individuals have low levels of neutralizing antibody. If neutralizing antibody is important for protection, this suggests that previous estimates of immunity based on whole-virus ELISAs may be overstated.
PMCID: PMC3957677  PMID: 24371258
2.  Seroprevalence of Measles, Mumps, Rubella and Varicella Antibodies in the United States Population, 2009–2010 
Open Forum Infectious Diseases  2015;2(1):ofv006.
Background. In the United States, measles, mumps, rubella, and varicella immunity is now primarily achieved through vaccination. Monitoring population immunity is necessary.
Methods. We evaluated seroprevalence of antibodies to measles, mumps, rubella, and varicella using the National Health and Nutrition Examination Survey during 2009–2010.
Results. Measles, mumps, rubella, and varicella seroprevalence was 92.0% (95% confidence interval [CI], 90.9%−93.0%), 87.6% (CI, 85.8%−89.2%), 95.3% (CI, 94.3%−96.2%), and 97.8% (CI, 97.1%−98.3%), respectively. United States (US)-born persons had lower mumps seroprevalence and higher varicella seroprevalence than non-US born persons.
Conclusions. Seroprevalence was high (88%–98%) for all 4 viruses in the US population during 2009−2010.
PMCID: PMC4438887  PMID: 26034757
antibodies; chickenpox; measles; mumps; NHANES; rubella; seroepidemiologic studies.
3.  Evolutionary genetics of genotype H1 measles viruses in China from 1993 to 2012 
The Journal of General Virology  2014;95(Pt 9):1892-1899.
Virologic surveillance is a critical component of measles management. One of the criteria for verification of elimination of endemic measles is genetic analysis of wild-type viruses to demonstrate lack of an indigenous genotype. Measles is yet to be eliminated in China, and genotype H1 has been detected continuously since virologic surveillance was initiated in 1993. Virologic surveillance has been very active in China, providing a unique opportunity to conduct a detailed study of the evolution of a single, endemic genotype over a timespan of nearly two decades. Phylogenetic analysis performed on the 450 nt coding sequence for the C-terminal 150 amino acids of the nucleoprotein (N-450), fusion (F) gene and haemagglutinin (H) gene confirmed the continued circulation of genotype H1 viruses for 19 years. No evidence of selective pressure for the H protein was found. The substitution rates ranged from 0.75×10−3 substitutions site−1 year−1 for H to 1.65×10−3 substitutions site−1 year−1 for N-450. The time of most recent common ancestor (TMRCA) for genotype H1 was estimated as approximately 1985 (95 % highest probability density, 1979–1989). Finally, the overall diversity of measles sequences from China decreased from 2005 to 2012, coincident with a substantial decrease in measles cases. The results suggest that detailed evolutionary analyses should facilitate the documentation of eventual measles elimination in China. Moreover, the molecular approaches used in this study can be applied in other countries approaching measles elimination.
PMCID: PMC4135087  PMID: 24914068
4.  Wild-Type Measles Viruses with Non-Standard Genome Lengths 
PLoS ONE  2014;9(4):e95470.
The length of the single stranded, negative sense RNA genome of measles virus (MeV) is highly conserved at 15,894 nucleotides (nt). MeVs can be grouped into 24 genotypes based on the highly variable 450 nucleotides coding for the carboxyl-terminus of the nucleocapsid protein (N-450). Here, we report the genomic sequences of 2 wild-type viral isolates of genotype D4 with genome lengths of 15,900 nt. Both genomes had a 7 nt insertion in the 3′ untranslated region (UTR) of the matrix (M) gene and a 1 nt deletion in the 5′ UTR of the fusion (F) gene. The net gain of 6 nt complies with the rule-of-six required for replication competency of the genomes of morbilliviruses. The insertions and deletion (indels) were confirmed in a patient sample that was the source of one of the viral isolates. The positions of the indels were identical in both viral isolates, even though epidemiological data and the 3 nt differences in N-450 between the two genomes suggested that the viruses represented separate chains of transmission. Identical indels were found in the M-F intergenic regions of 14 additional genotype D4 viral isolates that were imported into the US during 2007–2010. Viral isolates with and without indels produced plaques of similar size and replicated efficiently in A549/hSLAM and Vero/hSLAM cells. This is the first report of wild-type MeVs with genome lengths other than 15,894 nt and demonstrates that the length of the M-F UTR of wild-type MeVs is flexible.
PMCID: PMC3991672  PMID: 24748123
5.  Genetic Characterization of the Hemagglutinin Genes of Wild-Type Measles Virus Circulating in China, 1993–2009 
PLoS ONE  2013;8(9):e73374.
China experienced several large measles outbreaks in the past two decades, and a series of enhanced control measures were implemented to achieve the goal of measles elimination. Molecular epidemiologic surveillance of wild-type measles viruses (MeV) provides valuable information about the viral transmission patterns. Since 1993, virologic surveillnace has confirmed that a single endemic genotype H1 viruses have been predominantly circulating in China. A component of molecular surveillance is to monitor the genetic characteristics of the hemagglutinin (H) gene of MeV, the major target for virus neutralizing antibodies.
Principal Findings
Analysis of the sequences of the complete H gene from 56 representative wild-type MeV strains circulating in China during 1993–2009 showed that the H gene sequences were clustered into 2 groups, cluster 1 and cluster 2. Cluster1 strains were the most frequently detected cluster and had a widespread distribution in China after 2000. The predicted amino acid sequences of the H protein were relatively conserved at most of the functionally significant amino acid positions. However, most of the genotype H1 cluster1 viruses had an amino acid substitution (Ser240Asn), which removed a predicted N-linked glycosylation site. In addition, the substitution of Pro397Leu in the hemagglutinin noose epitope (HNE) was identified in 23 of 56 strains. The evolutionary rate of the H gene of the genotype H1 viruses was estimated to be approximately 0.76×10−3 substitutions per site per year, and the ratio of dN to dS (dN/dS) was <1 indicating the absence of selective pressure.
Although H genes of the genotype H1 strains were conserved and not subjected to selective pressure, several amino acid substitutions were observed in functionally important positions. Therefore the antigenic and genetic properties of H genes of wild-type MeVs should be monitored as part of routine molecular surveillance for measles in China.
PMCID: PMC3779233  PMID: 24073194
6.  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.
PMCID: PMC3592341  PMID: 23324519
7.  Immunogenicity, Immunologic Memory, and Safety Following Measles Revaccination in HIV-Infected Children Receiving Highly Active Antiretroviral Therapy 
The Journal of Infectious Diseases  2012;206(4):512-522.
Background. Response rates and immunologic memory following measles vaccination are reduced in human immunodeficiency virus (HIV)–infected children in the absence of highly active antiretroviral therapy (HAART).
Methods. HIV-infected children 2 to <19 years old receiving HAART and with HIV loads <30 000 copies/mL, CD4% ≥15, and ≥1 prior measles-mumps-rubella vaccination (MMR) were given another MMR. Measles antibody concentrations before and 8, 32, and 80 weeks postvaccination were determined by plaque reduction neutralization (PRN). A subset was given another MMR 4–5 years later, and PRN antibody was measured before and 7 and 28 days later.
Results. At entry, 52% of 193 subjects were seroprotected (PRN ≥120 mIU/mL). Seroprotection increased to 89% 8 weeks postvaccination, and remained at 80% 80 weeks postvaccination. Of 65 subjects revaccinated 4–5 years later, 85% demonstrated memory based on seroprotection before or 7 days after vaccination. HIV load ≤400 copies/mL at initial study vaccination was associated with higher seroprotection rates, greater antibody concentrations, and memory. Grade 3 fever or fatigue occurred in 2% of subjects.
Conclusions. Measles revaccination induced high rates of seroprotection and memory in children receiving HAART. Both endpoints were associated with HIV viral load suppression.
Clinical Trials Registration: NCT00013871 (
PMCID: PMC3491735  PMID: 22693229
8.  Poor Immune Responses of Newborn Rhesus Macaques to Measles Virus DNA Vaccines Expressing the Hemagglutinin and Fusion Glycoproteins 
A vaccine that would protect young infants against measles could facilitate elimination efforts and decrease morbidity and mortality in developing countries. However, immaturity of the immune system is an important obstacle to the development of such a vaccine. In this study, DNA vaccines expressing the measles virus (MeV) hemagglutinin (H) protein or H and fusion (F) proteins, previously shown to protect juvenile macaques, were used to immunize groups of 4 newborn rhesus macaques. Monkeys were inoculated intradermally with 200 μg of each DNA at birth and at 10 months of age. As controls, 2 newborn macaques were similarly vaccinated with DNA encoding the influenza virus H5, and 4 received one dose of the current live attenuated MeV vaccine (LAV) intramuscularly. All monkeys were monitored for development of MeV-specific neutralizing and binding IgG antibody and cytotoxic T lymphocyte (CTL) responses. These responses were poor compared to the responses induced by LAV. At 18 months of age, all monkeys were challenged intratracheally with a wild-type strain of MeV. Monkeys that received the DNA vaccine encoding H and F, but not H alone, were primed for an MeV-specific CD8+ CTL response but not for production of antibody. LAV-vaccinated monkeys were protected from rash and viremia, while DNA-vaccinated monkeys developed rashes, similar to control monkeys, but had 10-fold lower levels of viremia. We conclude that vaccination of infant macaques with DNA encoding MeV H and F provided only partial protection from MeV infection.
PMCID: PMC3571263  PMID: 23239799
9.  Cell Culture and Electron Microscopy for Identifying Viruses in Diseases of Unknown Cause 
Emerging Infectious Diseases  2013;19(6):864-869.
During outbreaks of infectious diseases or in cases of severely ill patients, it is imperative to identify the causative agent. This report describes several events in which virus isolation and identification by electron microscopy were critical to initial recognition of the etiologic agent, which was further analyzed by additional laboratory diagnostic assays. Examples include severe acute respiratory syndrome coronavirus, and Nipah, lymphocytic choriomeningitis, West Nile, Cache Valley, and Heartland viruses. These cases illustrate the importance of the techniques of cell culture and electron microscopy in pathogen identification and recognition of emerging diseases.
PMCID: PMC3713842  PMID: 23731788
Viruses; electron microscopy; cell culture; emerging diseases; SARS coronavirus; Nipah virus; lymphocytic choriomeningitis virus; West Nile virus; Cache Valley virus; Heartland virus
10.  Measles Virus IgG Avidity Assay for Use in Classification of Measles Vaccine Failure in Measles Elimination Settings 
Clinical and Vaccine Immunology : CVI  2012;19(11):1810-1817.
In regions where endemic measles virus has been eliminated, diagnostic assays are needed to assist in correctly classifying measles cases irrespective of vaccination status. A measles IgG avidity assay was configured using a commercially available measles-specific IgG enzyme immunoassay by modifying the protocol to include three 5-min washes with diethylamine (60 mM; pH 10.25) following serum incubation; serum was serially diluted, and the results were expressed as the end titer avidity index. Receiver operating characteristic analysis was used for evaluation and validation and to establish low (≤30%) and high (≥70%) end titer avidity thresholds. Analysis of 319 serum specimens expected to contain either high- or low-avidity antibodies according to clinical and epidemiological data indicated that the assay is highly accurate, with an area under the curve of 0.998 (95% confidence interval [CI], 0.978 to 1.000), sensitivity of 91.9% (95% CI, 83.2% to 97.0%), and specificity of 98.4% (95% CI, 91.6% to 100%). The assay is rapid (<2 h) and precise (standard deviation [SD], 4% to 7%). In 18 samples from an elimination setting outbreak, the assay identified 2 acute measles cases with low-avidity results; both were IgM-positive samples. Additionally, 11 patients (15 samples) with modified measles who were found to have high-avidity IgG results were classified as secondary vaccine failures; one sample with an intermediate-avidity result was not interpretable. In elimination settings, measles IgG avidity assays can complement existing diagnostic tools in confirming unvaccinated acute cases and, in conjunction with adequate clinical and epidemiologic investigation, aid in the classification of vaccine failure cases.
PMCID: PMC3491540  PMID: 22971778
11.  Distinct and Overlapping Roles of Nipah Virus P Gene Products in Modulating the Human Endothelial Cell Antiviral Response 
PLoS ONE  2012;7(10):e47790.
Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that causes fatal encephalitis in up to 75% of infected humans. Like other paramyxoviruses, NiV employs co-transcriptional mRNA editing during transcription of the phosphoprotein (P) gene to generate additional mRNAs encoding the V and W proteins. The C protein is translated from the P mRNA, but in an alternative reading frame. There is evidence from both in vitro and in vivo studies to show that the P gene products play a role in NiV pathogenesis. We have developed a reverse genetic system to dissect the individual roles of the NiV P gene products in limiting the antiviral response in primary human microvascular lung endothelial cells, which represent important targets in human NiV infection. By characterizing growth curves and early antiviral responses against a number of recombinant NiVs with genetic modifications altering expression of the proteins encoded by the P gene, we observed that multiple elements encoded by the P gene have both distinct and overlapping roles in modulating virus replication as well as in limiting expression of antiviral mediators such as IFN-β, CXCL10, and CCL5. Our findings corroborate observations from in vivo hamster infection studies, and provide molecular insights into the attenuation and the histopathology observed in hamsters infected with C, V, and W-deficient NiVs. The results of this study also provide an opportunity to verify the results of earlier artificial plasmid expression studies in the context of authentic viral infection.
PMCID: PMC3477106  PMID: 23094089
12.  Subclinical reactivation of varicella zoster virus in all stages of HIV infection 
Analysis of 200 paired serum and cerebrospinal fluid (CSF) samples from 180 HIV-positive individuals, 136 of whom had AIDS, revealed intrathecal synthesis of antibodies specific for varicella zoster virus (VZV) in 28 (16%) individuals, measles virus in 15 (8%), herpes simplex virus-1 (HSV-1) in 1 (0.6%), and HSV-2 in none. Of the 28 subjects with a positive VZV antibody specificity index, only 1 had zoster rash at the time of serum and CSF sampling; of the total 180 HIV-positive subjects, 146 (81%) had no history of zoster. Based on an estimated 33.4 million HIV-positive individuals worldwide, subclinical reactivation of VZV in even less than 16% of HIV-positive people suggests the possibility that millions of people have active VZV infection of the central nervous system. In cases of VZV vasculopathy, myelopathy and even zoster sine herpete, the CSF is often positive for anti-VZV antibody, but negative for VZV DNA. To rule out VZV infection of the nervous system, CSF must be tested for VZV DNA and anti-VZV IgG and IgM antibody.
PMCID: PMC3176733  PMID: 21419427
VZV; HIV; subclinical reactivation; CSF; intrathecal synthesis
13.  Single Endemic Genotype of Measles Virus Continuously Circulating in China for at Least 16 Years 
PLoS ONE  2012;7(4):e34401.
The incidence of measles in China from 1991 to 2008 was reviewed, and the nucleotide sequences from 1507 measles viruses (MeV) isolated during 1993 to 2008 were phylogenetically analyzed. The results showed that measles epidemics peaked approximately every 3 to 5 years with the range of measles cases detected between 56,850 and 140,048 per year. The Chinese MeV strains represented three genotypes; 1501 H1, 1 H2 and 5 A. Genotype H1 was the predominant genotype throughout China continuously circulating for at least 16 years. Genotype H1 sequences could be divided into two distinct clusters, H1a and H1b. A 4.2% average nucleotide divergence was found between the H1a and H1b clusters, and the nucleotide sequence and predicted amino acid homologies of H1a viruses were 92.3%–100% and 84.7%–100%, H1b were 97.1%–100% and 95.3%–100%, respectively. Viruses from both clusters were distributed throughout China with no apparent geographic restriction and multiple co-circulating lineages were present in many provinces. Cluster H1a and H1b viruses were co-circulating during 1993 to 2005, while no H1b viruses were detected after 2005 and the transmission of that cluster has presumably been interrupted. Analysis of the nucleotide and predicted amino acid changes in the N proteins of H1a and H1b viruses showed no evidence of selective pressure. This study investigated the genotype and cluster distribution of MeV in China over a 16-year period to establish a genetic baseline before MeV elimination in Western Pacific Region (WPR). Continuous and extensive MeV surveillance and the ability to quickly identify imported cases of measles will become more critical as measles elimination goals are achieved in China in the near future. This is the first report that a single endemic genotype of measles virus has been found to be continuously circulating in one country for at least 16 years.
PMCID: PMC3332093  PMID: 22532829
14.  Characterization of Nipah Virus from Outbreaks in Bangladesh, 2008–2010 
Emerging Infectious Diseases  2012;18(2):248-255.
New genotyping scheme facilitates classification of virus sequences.
Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes fatal encephalitis in humans. The initial outbreak of NiV infection occurred in Malaysia and Singapore in 1998–1999; relatively small, sporadic outbreaks among humans have occurred in Bangladesh since 2001. We characterized the complete genomic sequences of identical NiV isolates from 2 patients in 2008 and partial genomic sequences of throat swab samples from 3 patients in 2010, all from Bangladesh. All sequences from patients in Bangladesh comprised a distinct genetic group. However, the detection of 3 genetically distinct sequences from patients in the districts of Faridpur and Gopalganj indicated multiple co-circulating lineages in a localized region over a short time (January–March 2010). Sequence comparisons between the open reading frames of all available NiV genes led us to propose a standardized protocol for genotyping NiV; this protcol provides a simple and accurate way to classify current and future NiV sequences.
PMCID: PMC3310473  PMID: 22304936
Nipah; Nipah virus; outbreak; encephalitis; phylogeny; viruses; Bangladesh
15.  Enzyme-Linked Immunospot Assay Detection of Mumps-Specific Antibody-Secreting B Cells as an Alternative Method of Laboratory Diagnosis ▿  
Although high measles, mumps, and rubella (MMR) vaccination coverage has been successful in dramatically reducing mumps disease in the United States, mumps (re)infections occasionally occur in individuals who have been either previously vaccinated or naturally infected. Standard diagnostics that detect virus or virus-specific antibody are dependable for confirming primary mumps infection in immunologically naïve persons, but these methods perform inconsistently for individuals with prior immune exposure. We hypothesized that detection of activated mumps-specific antibody-secreting B cells (ASCs) by enzyme-linked immunospot (ELISPOT) assay could be used as a more reliable diagnostic. To test this, a time course of virus-specific ASC responses was measured by ELISPOT assay following MMR vaccination of 16 previously vaccinated or naturally exposed adult volunteers. Mumps-specific ASCs were detectable in 68% of these individuals at some point during the first 3 weeks following revaccination. In addition, mumps-specific ASCs were detected in 7/7 previously vaccinated individuals who recently had been infected as part of a confirmed mumps outbreak. These data suggest that ELISPOT detection of mumps-specific ASCs has the potential for use as an alternative method of diagnosis when suspect cases cannot be confirmed by detection of IgM or virus. In addition, it was determined that mumps-specific memory B cells are detected at a much lower frequency than measles- or rubella-specific cells, suggesting that mumps infection may not generate robust B-cell memory.
PMCID: PMC3019787  PMID: 21047998
16.  Development of a Neutralization Assay for Nipah Virus Using Pseudotype Particles 
Journal of virological methods  2009;160(1-2):1-6.
Nipah virus (NiV) and Hendra virus (HeV) are zoonotic paramyxoviruses capable of causing severe disease in humans and animals. These viruses require biosafety level 4 (BSL-4) containment. Like other paramyxoviruses, the plaque reduction neutralization test (PRNT) can be used to detect antibodies to the surface glycoproteins, fusion (F) and attachment (G), and PRNT titers give an indication of protective immunity. Unfortunately, for NiV and HeV, the PRNT must be performed in BSL-4 containment and takes 5–7 days to complete. Thus, we have developed a neutralization assay using VSV pseudotype particles expressing the F and G proteins of NiV (pVSV-NiV-F/G) as target antigens. This rapid assay, which can be performed at BSL-2, was evaluated using serum samples from outbreak investigations and more than 300 serum samples from an experimental NiV vaccination study in swine. The results of the neutralization assays with pVSV-NiV-F/G as antigen showed a good correlation with those of standard PRNT. Therefore, this new method has the potential to be a rapid and cost-effective diagnostic method, especially in locations that lack high containment facilities, and will provide a valuable tool for basic research and vaccine development.
PMCID: PMC2704486  PMID: 19559943
17.  Genetic characterization of wild-type measles viruses isolated in China, 2006-2007 
Virology Journal  2010;7:105.
Molecular characterization of wild-type measles viruses in China during 1995-2004 demonstrated that genotype H1 was endemic and widely distributed throughout the country. H1-associated cases and outbreaks caused a resurgence of measles beginning in 2005. A total of 210,094 measles cases and 101 deaths were reported by National Notifiable Diseases Reporting System (NNDRS) and Chinese Measles Laboratory Network (LabNet) from 2006 to 2007, and the incidences of measles were 6.8/100,000 population and 7.2/100,000 population in 2006 and 2007, respectively. Five hundred and sixty-five wild-type measles viruses were isolated from 24 of 31 provinces in mainland China during 2006 and 2007, and all of the wild type virus isolates belonged to cluster 1 of genotype H1. These results indicated that H1-cluster 1 viruses were the predominant viruses circulating in China from 2006 to 2007. This study contributes to previous efforts to generate critical baseline data about circulating wild-type measles viruses in China that will allow molecular epidemiologic studies to help measure the progress made toward China's goal of measles elimination by 2012.
PMCID: PMC2887432  PMID: 20500809
18.  Measles Resurgence Associated with Continued Circulation of Genotype H1 Viruses in China, 2005 
Virology Journal  2009;6:135.
Measles morbidity and mortality decreased significantly after measles vaccine was introduced into China in 1965. From 1995 to 2004, average annual measles incidence decreased to 5.6 cases per 100,000 population following the establishment of a national two-dose regimen. Molecular characterization of wild-type measles viruses demonstrated that genotype H1 was endemic and widely distributed throughout the country in China during 1995-2004. A total of 124,865 cases and 55 deaths were reported from the National Notifiable Diseases Reporting System (NNDRS) in 2005, which represented a 69.05% increase compared with 2004. Over 16,000 serum samples obtained from 914 measles outbreaks and the measles IgM positive rate was 81%. 213 wild-type measles viruses were isolated from 18 of 31 provinces in China during 2005, and all of the isolates belonged to genotype H1. The ranges of the nucleotide sequence and predicted amino acid sequence homologies of the 213 genotype H1 strains were 93.4%-100% and 90.0%-100%, respectively. H1-associated cases and outbreaks caused the measles resurgence in China in 2005. H1 genotype has the most inner variation within genotype, it could be divided into 2 clusters, and cluster 1 viruses were predominant in China throughout 2005.
PMCID: PMC2759936  PMID: 19737391
19.  Broadening of Neutralization Activity to Directly Block a Dominant Antibody-Driven SARS-Coronavirus Evolution Pathway 
PLoS Pathogens  2008;4(11):e1000197.
Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection. While several studies support that some amino acid changes between animal and human viruses are the result of inter-species adaptation, the role of neutralizing antibodies (nAbs) in driving SARS-CoV evolution, particularly during intra-species transmission, is unknown. A detailed examination of SARS-CoV infected animal and human convalescent sera could provide evidence of nAb pressure which, if found, may lead to strategies to effectively block virus evolution pathways by broadening the activity of nAbs. Here we show, by focusing on a dominant neutralization epitope, that contemporaneous- and cross-strain nAb responses against SARS-CoV spike protein exist during natural infection. In vitro immune pressure on this epitope using 2002/03 strain-specific nAb 80R recapitulated a dominant escape mutation that was present in all 2003/04 animal and human viruses. Strategies to block this nAb escape/naturally occurring evolution pathway by generating broad nAbs (BnAbs) with activity against 80R escape mutants and both 2002/03 and 2003/04 strains were explored. Structure-based amino acid changes in an activation-induced cytidine deaminase (AID) “hot spot” in a light chain CDR (complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs. These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV. Somatic hypermutation (SHM) of a single VL CDR can markedly broaden the activity of a strain-specific nAb. The strategies investigated in this study, in particular the use of structural information in combination of chain-shuffling as well as hot-spot CDR mutagenesis, can be exploited to broaden neutralization activity, to improve anti-viral nAb therapies, and directly manipulate virus evolution.
Author Summary
The SARS-CoV caused a worldwide epidemic of SARS in 2002/03 and was responsible for this zoonotic infectious disease. The role of neutralizing antibody (nAb) mediated immune pressure in the evolution of SARS-CoV during the 2002/03 outbreak and a second 2003/04 zoonotic transmission is unknown. Here we demonstrate nAb responses elicited during natural infection clearly have strain-specific components which could have been the driving force for virus evolution in spike protein during intra-species transmission. In vitro immune pressure using 2002/03 strain-specific nAb 80R recapitulate a dominant escape mutation that was present in all 2003/04 animal and human viruses. We investigated how to generate a single broad nAb (BnAb) with activity against various natural viral variants of the 2002/03 and 2003/04 outbreaks as well as nAb escape mutants. Remarkably, amino acid changes in an activation-induced cytidine deaminase (AID) “hot spot” of somatic hypermutation and localized to a single VL CDR were successful in generating BnAbs. These results provide an effective strategy for generating BnAbs that should be generally useful for improving immune based anti-viral therapies as well as providing a foundation to directly manipulate virus evolution by blocking escape pathways.
PMCID: PMC2572002  PMID: 18989460
20.  Adaptation to cell culture induces functional differences in measles virus proteins 
Virology Journal  2008;5:129.
Live, attenuated measles virus (MeV) vaccine strains were generated by adaptation to cell culture. The genetic basis for the attenuation of the vaccine strains is unknown. We previously reported that adaptation of a pathogenic, wild-type MeV to Vero cells or primary chicken embryo fibroblasts (CEFs) resulted in a loss of pathogenicity in rhesus macaques. The CEF-adapted virus (D-CEF) contained single amino acid changes in the C and matrix (M) proteins and two substitutions in the shared amino terminal domain of the phosphoprotein (P) and V protein. The Vero-adapted virus (D-VI) had a mutation in the cytoplasmic tail of the hemagglutinin (H) protein.
In vitro assays were used to test the functions of the wild-type and mutant proteins. The substitution in the C protein of D-CEF decreased its ability to inhibit mini-genome replication, while the wild-type and mutant M proteins inhibited replication to the same extent. The substitution in the cytoplasmic tail of the D-VI H protein resulted in reduced fusion in a quantitative fusion assay. Co-expression of M proteins with wild-type fusion and H proteins decreased fusion activity, but the mutation in the M protein of D-CEF did not affect this function. Both mutations in the P and V proteins of D-CEF reduced the ability of these proteins to inhibit type I and II interferon signaling.
Adaptation of a wild-type MeV to cell culture selected for genetic changes that caused measurable functional differences in viral proteins.
PMCID: PMC2582235  PMID: 18954437
21.  Lack of Association between Measles Virus Vaccine and Autism with Enteropathy: A Case-Control Study 
PLoS ONE  2008;3(9):e3140.
The presence of measles virus (MV) RNA in bowel tissue from children with autism spectrum disorders (ASD) and gastrointestinal (GI) disturbances was reported in 1998. Subsequent investigations found no associations between MV exposure and ASD but did not test for the presence of MV RNA in bowel or focus on children with ASD and GI disturbances. Failure to replicate the original study design may contribute to continued public concern with respect to the safety of the measles, mumps, and rubella (MMR) vaccine.
Methodology/Principal Findings
The objective of this case-control study was to determine whether children with GI disturbances and autism are more likely than children with GI disturbances alone to have MV RNA and/or inflammation in bowel tissues and if autism and/or GI episode onset relate temporally to receipt of MMR. The sample was an age-matched group of US children undergoing clinically-indicated ileocolonoscopy. Ileal and cecal tissues from 25 children with autism and GI disturbances and 13 children with GI disturbances alone (controls) were evaluated by real-time reverse transcription (RT)-PCR for presence of MV RNA in three laboratories blinded to diagnosis, including one wherein the original findings suggesting a link between MV and ASD were reported. The temporal order of onset of GI episodes and autism relative to timing of MMR administration was examined. We found no differences between case and control groups in the presence of MV RNA in ileum and cecum. Results were consistent across the three laboratory sites. GI symptom and autism onset were unrelated to MMR timing. Eighty-eight percent of ASD cases had behavioral regression.
This study provides strong evidence against association of autism with persistent MV RNA in the GI tract or MMR exposure. Autism with GI disturbances is associated with elevated rates of regression in language or other skills and may represent an endophenotype distinct from other ASD.
PMCID: PMC2526159  PMID: 18769550
22.  Detection of RNA of Mumps Virus during an Outbreak in a Population with a High Level of Measles, Mumps, and Rubella Vaccine Coverage▿  
Journal of Clinical Microbiology  2008;46(3):1101-1103.
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.
PMCID: PMC2268349  PMID: 18184850
23.  Real-Time Reverse Transcription-PCR Assay for Detection of Mumps Virus RNA in Clinical Specimens▿  
Journal of Clinical Microbiology  2007;45(9):2902-2908.
The mumps virus is a negative-strand RNA virus in the family Paramyxoviridae. Mumps infection results in an acute illness with symptoms including fever, headache, and myalgia, followed by swelling of the salivary glands. Complications of mumps can include meningitis, deafness, pancreatitis, orchitis, and first-trimester abortion. Laboratory confirmation of mumps infection can be made by the detection of immunoglobulin M-specific antibodies to mumps virus in acute-phase serum samples, the isolation of mumps virus in cell culture, or by detection of the RNA of the mumps virus by reverse transcription (RT)-PCR. We developed and validated a multiplex real-time RT-PCR assay for rapid mumps diagnosis in a clinical setting. This assay used oligonucleotide primers and a TaqMan probe targeting the mumps SH gene, as well as primers and a probe that targeted the human RNase P gene to assess the presence of PCR inhibitors and as a measure of specimen quality. The test was specific, since it did not amplify a product from near-neighbor viruses, as well as sensitive and accurate. Real-time RT-PCR results showed 100% correlation with results from viral culture, the gold standard for mumps diagnostic testing. Assay efficiency was over 90% and displayed good precision after performing inter- and intraassay replicates. Thus, we have developed and validated a molecular method for rapidly diagnosing mumps infection that may be used to complement existing techniques.
PMCID: PMC2045251  PMID: 17652480
24.  Measles Immunization Coverage Determined by Serology and Immunization Record from Children in Two Chicago Communities 
Public Health Reports  2006;121(3):262-269.
We compared the prevalence of measles immunization determined by serology with the prevalence of measles immunization determined by immunization records, and identified factors predictive of measles immunization among a sample of children from two Chicago communities.
We collected demographic information and blood specimens from a sample of children aged 12–71 months in two Chicago communities at risk for low measles immunization coverage levels. We collected immunization information from provider records, parent-held records, and the statewide immunization registry. We compared evidence of immunization determined by serology with evidence of immunization from these three sources of immunization records.
The sample of children from the two communities had serologic measles immunity levels of 85% and 90%. Significantly fewer children had evidence of immunization by record in both communities (45% and 63%, respectively).
Immunization coverage levels determined using immunization records were significantly lower than immunization coverage determined using serology. A fully populated immunization registry used by all immunization providers could prevent the problems of record loss and scatter.
PMCID: PMC1525293  PMID: 16640148
25.  Molecular epidemiology of measles viruses in China, 1995–2003 
Virology Journal  2007;4:14.
This report describes the genetic characterization of 297 wild-type measles viruses that were isolated in 24 provinces of China between 1995 and 2003. Phylogenetic analysis of the N gene sequences showed that all of the isolates belonged to genotype H1 except 3 isolates, which were genotype A. The nucleotide sequence and predicted amino acid homologies of the 294-genotype H1 strains were 94.7%–100% and 93.3%–100%, respectively. The genotype H1 isolates were divided into 2 clusters, which differed by approximately 2.9% at the nucleotide level. Viruses from both clusters were distributed throughout China with no apparent geographic restriction and multiple co-circulating lineages were present in many provinces. Even though other measles genotypes have been detected in countries that border China, this report shows that genotype H1 is widely distributed throughout the country and that China has a single, endemic genotype. This important baseline data will help to monitor the progress of measles control in China.
PMCID: PMC1802751  PMID: 17280609

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