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.
Measles vaccination programs would benefit from delivery methods that decrease cost, simplify logistics, and increase safety. Conventional subcutaneous injection is limited by the need for skilled healthcare professionals to reconstitute and administer injections, and by the need for safe needle handling and disposal to reduce the risk of disease transmission through needle re-use and needlestick injury. Microneedles are micron-scale, solid needles coated with a dry formulation of vaccine that dissolves in the skin within minutes after patch application. By avoiding the use of hypodermic needles, vaccination using a microneedle patch could be carried out by minimally trained personnel with reduced risk of blood-borne disease transmission. The goal of this study was to evaluate measles vaccination using a microneedle patch to address some of the limitations of subcutaneous injection. Viability of vaccine virus dried onto a microneedle patch was stabilized by incorporation of the sugar, trehalose, and loss of viral titer was less than 1 log10(TCID50) after storage for at least 30 days at room temperature. Microneedle patches were then used to immunize cotton rats with the Edmonston-Zagreb measles vaccine strain. Vaccination using microneedles at doses equaling the standard human dose or one-fifth the human dose generated neutralizing antibody levels equivalent to those of a subcutaneous immunization at the same dose. These results show that measles vaccine can be stabilized on microneedles and that vaccine efficiently reconstitutes in vivo to generate a neutralizing antibody response equivalent to that generated by subcutaneous injection.
Measles vaccination; Microneedle patch; Vaccine stability; Cotton rat
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.
Mumps virus (MuV) causes acute infections in humans. In recent years, MuV has caused epidemics among highly vaccinated populations. The largest outbreak in the U.S. in the past 20 years occurred in 2005–2006 with over reported 5,000 cases which the majority of the cases was in vaccinated young adults. We sequenced the complete genome of a representative strain from the epidemic (MuV-IA). MuV-IA is a member of genotype G, the same genotype of MuV that was associated with the outbreak in the UK in 2004–2005. We constructed a reverse genetics system for MuV-IA (rMuV-IA), and rescued a virus lacking the open reading frame (ORF) of the SH gene (rMuVΔSH). rMuVΔSH infection in L929 cells induced increased NF-κB activation, TNF-α production and apoptosis compared to rMuV-IA. rMuVΔSH was attenuated in an animal model. These results indicated that the SH ORF of MuV plays a significant role in interfering with TNF-α signaling and viral pathogenesis during virus infection.
Respiratory viruses (including adenovirus, influenza virus, respiratory syncytial virus, coronavirus, and rhinovirus) cause a broad spectrum of disease in humans, ranging from mild influenza-like symptoms to acute respiratory failure. While species D adenoviruses and subtype H7 influenza viruses are known to possess an ocular tropism, documented human ocular disease has been reported following infection with all principal respiratory viruses. In this review, we describe the anatomical proximity and cellular receptor distribution between ocular and respiratory tissues. All major respiratory viruses and their association with human ocular disease are discussed. Research utilizing in vitro and in vivo models to study the ability of respiratory viruses to use the eye as a portal of entry as well as a primary site of virus replication is highlighted. Identification of shared receptor-binding preferences, host responses, and laboratory modeling protocols among these viruses provides a needed bridge between clinical and laboratory studies of virus tropism.
In 2012, a female wildlife biologist experienced fever, malaise, headache, generalized myalgia and arthralgia, neck stiffness, and a sore throat shortly after returning to the United States from a 6-week field expedition to South Sudan and Uganda. She was hospitalized, after which a maculopapular rash developed and became confluent. When the patient was discharged from the hospital on day 14, arthralgia and myalgia had improved, oropharynx ulcerations had healed, the rash had resolved without desquamation, and blood counts and hepatic enzyme levels were returning to reference levels. After several known suspect pathogens were ruled out as the cause of her illness, deep sequencing and metagenomics analysis revealed a novel paramyxovirus related to rubula-like viruses isolated from fruit bats.
Paramyxoviridae; metagenomics; zoonosis; diagnostics; rash; South Sudan; Uganda; viruses; Sosuga virus; bats; rubula-like virus
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.
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.
Here, we provide direct evidence that the receptor-binding site of measles virus (MV) hemagglutinin protein itself forms an effective conserved neutralizing epitope (CNE). Several receptor-interacting residues constitute the CNE. Thus, viral escape from neutralization has to be associated with loss of receptor-binding activity. Since interactions with both the signaling lymphocyte activation molecule (SLAM) and nectin4 are critical for MV pathogenesis, its escape, which results from loss of receptor-binding activity, should not occur in nature.
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.
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.
Effective vaccination programs have dramatically reduced the number of measles-related deaths globally. Although all the available data suggest that measles eradication is biologically feasible, a structural and biochemical basis for the single serotype nature of measles virus (MV) remains to be provided. The hemagglutinin (H) protein, which binds to two discrete proteinaceous receptors, is the major neutralizing target. Monoclonal antibodies (MAbs) recognizing distinct epitopes on the H protein were characterized using recombinant MVs encoding the H gene from different MV genotypes. The effects of various mutations on neutralization by MAbs and virus fitness were also analyzed, identifying the location of five epitopes on the H protein structure. Our data in the present study demonstrated that the H protein of MV possesses at least two conserved effective neutralizing epitopes. One, which is a previously recognized epitope, is located near the receptor-binding site (RBS), and thus MAbs that recognize this epitope blocked the receptor binding of the H protein, whereas the other epitope is located at the position distant from the RBS. Thus, a MAb that recognizes this epitope did not inhibit the receptor binding of the H protein, rather interfered with the hemagglutinin-fusion (H-F) interaction. This epitope was suggested to play a key role for formation of a higher order of an H-F protein oligomeric structure. Our data also identified one nonconserved effective neutralizing epitope. The epitope has been masked by an N-linked sugar modification in some genotype MV strains. These data would contribute to our understanding of the antigenicity of MV and support the global elimination program of measles.
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.
Viruses; electron microscopy; cell culture; emerging diseases; SARS coronavirus; Nipah virus; lymphocytic choriomeningitis virus; West Nile virus; Cache Valley virus; Heartland virus
Particularly vulnerable are health care workers who do not use personal protective equipment and persons who do not wash hands after traditional burial practices.
Active Nipah virus encephalitis surveillance identified an encephalitis cluster and sporadic cases in Faridpur, Bangladesh, in January 2010. We identified 16 case-patients; 14 of these patients died. For 1 case-patient, the only known exposure was hugging a deceased patient with a probable case, while another case-patient’s exposure involved preparing the same corpse for burial by removing oral secretions and anogenital excreta with a cloth and bare hands. Among 7 persons with confirmed sporadic cases, 6 died, including a physician who had physically examined encephalitis patients without gloves or a mask. Nipah virus–infected patients were more likely than community-based controls to report drinking raw date palm sap and to have had physical contact with an encephalitis patient (29% vs. 4%, matched odds ratio undefined). Efforts to prevent transmission should focus on reducing caregivers’ exposure to infected patients’ bodily secretions during care and traditional burial practices.
Nipah; encephalitis; outbreak; Nipah virus; NiV; nosocomial; healthcare-associated infection; corpse; Bangladesh; viruses; burial practices; PPE; personal protective equipment; health care workers; transmission
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.
Mumps virus (MuV) causes an acute infection in humans characterized by a wide array of symptoms ranging from relatively mild manifestations, such as parotitis, to more-severe complications, such as meningitis and encephalitis. Widespread mumps vaccination has reduced mumps incidence dramatically; however, outbreaks still occur in vaccinated populations. The V protein of MuV, when expressed in cell culture, blocks interferon (IFN) expression and signaling and interleukin-6 (IL-6) signaling. In this work, we generated a recombinant MuV incapable of expressing the V protein (rMuVΔV). The rescued MuV was derived from a clinical wild-type isolate from a recent outbreak in the United States (MuVIowa/US/06, G genotype). Analysis of the virus confirmed the roles of V protein in blocking IFN expression and signaling and IL-6 signaling. We also found that the rMuVIowa/US/06ΔV virus induced high levels of IL-6 expression in vitro, suggesting that V plays a role in reducing IL-6 expression. In vivo, the rMuVIowa/US/06ΔV virus was highly attenuated, indicating that the V protein plays an essential role in viral virulence.
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.
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.
Nipah; Nipah virus; outbreak; encephalitis; phylogeny; viruses; Bangladesh
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.
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.
Hendra and Nipah viruses are recently emerged zoonotic paramyxoviruses for which there is no vaccine or protective therapy available. While a number of experimental therapeutics and vaccines have recently been reported, all of these will require lengthy approval processes, limiting their usefulness in the short term. To address the urgent need for henipavirus therapeutics, a number of currently licensed pharmaceuticals have been evaluated for off label efficacy against henipavirus replication in vitro. Initially it was observed that compounds which released intracellular calcium stores induced a potent inhibition of henipaviruses replication, prompting the evaluation of known drugs with a similar effect on calcium mobilisation. Of the eight compounds randomly selected based on existing literature, seven inhibited virus replication in the micromolar range while the remaining compound also inhibited virus replication but only at millimolar concentrations. Pretreatment experiments with various calcium chelators, channel antagonists or endoplasmic reticulum release inhibitors supported a calcium mediated mechanism of action for five of these compounds. The mechanism of antiviral action for the remaining three compounds is currently unknown. Additionally, a number of other modulators of calcium flux, including calcium channel and calmodulin antagonists also exhibited potent antiviral activity in vitro providing a broad range of potential therapeutic options for the treatment of henipavirus infections. Importantly, as many of these compounds are currently licensed drugs, regulatory approval should be a much more streamlined process, with the caveat that appropriate in vivo efficacy can be demonstrated in animal models.
Nipah virus; Hendra virus; Henipaviruses; Antiviral; Calcium
Outbreaks of Hendra (HeV) and Nipah (NiV) viruses have been reported starting in 1994 and 1998, respectively. Both viruses are capable of causing fatal disease in humans and effecting great economical loss in the livestock industry.
Through screening of hybridomas derived from mice immunized with γ-irradiated Nipah virus, we identified two secreted antibodies; one reactive with the nucleocapsid (N) protein and the other, the phosphoprotein (P) of henipaviruses. Epitope mapping and protein sequence alignments between NiV and HeV suggest the last 14 amino acids of the carboxyl terminus of the N protein is the target of the anti-N antibody. The anti-P antibody recognizes an epitope in the amino-terminal half of P protein. These monoclonal antibodies were used to develop two antigen capture ELISAs, one for virus detection and the other for differentiation between NiV and HeV. The lower limit of detection of the capture assay with both monoclonal antibodies was 400 pfu. The anti-N antibody was used to successfully detect NiV in a lung tissue suspension from an infected pig.
The antigen capture ELISA developed is potentially affordable tool to provide rapid detection and differentiation between the henipaviruses.
Better understanding of transmission patterns will enhance control and elimination programs.
To determine the origin of the virus associated with a measles outbreak in Menglian County, Yunnan Province, People’s Republic of China, in 2009, we conducted genetic analyses. Phylogenetic analyses based on nucleoprotein (N) and hemagglutinin (H) gene sequences showed that these Menglian viruses were not closely related to sequences of any World Health Organization (WHO) reference strains representing the 23 currently recognized genotypes. The minimum nucleotide divergence between the Menglian viruses and the most closely related reference strain, genotype D7, was 3.3% for the N gene and 3.0% for the H gene. A search of the databases of GenBank, WHO, and the Health Protection Agency Measles Nucleotide Surveillance showed that the Menglian viruses, together with the 2 older non-Menglian viruses, could be members of a new proposed measles genotype, d11. The new genotype designation will allow for better description of measles transmission patterns, especially in the Southeast Asian and Western Pacific regions.
Measles virus; new genotype; phylogenetic analysis; viruses; research
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.
No effective therapeutic is currently in place for improved case management of severe measles or the rapid control of outbreaks. Through high-throughput screening, we recently identified a novel small-molecule class that potently blocks activity of the measles virus (MeV) RNA-dependent RNA polymerase (RdRp) complex in transient replicon assays. However, the nature of the block in RdRp activity and the physical target of the compound remained elusive. Through real-time reverse transcription-PCR analysis, we demonstrate that the lead compound AS-136A blocks viral RNA synthesis in the context of an infection. Adaptation of different MeV strains to growth in the presence of the compound identified three candidate hot spots for resistance that are located in conserved domains of the viral polymerase (L protein) subunit of the RdRp complex. Rebuilding of individual mutations in RdRp-driven reporter assays and recombinant MeV traced the molecular basis for resistance to specific mutations in L. Mutations responsible for resistance cluster in the immediate vicinity of the proposed catalytic center for phosphodiester bond formation and neighboring conserved domains of L, providing support for effective inhibition of a paramyxovirus RdRp complex through interaction of a nonnucleoside small-molecule inhibitor with the L protein. Resistance mutations are located in regions of L that are fully conserved among viral isolates, and recombinant MeV harboring individual resistance mutations show some delay in the onset of viral growth in vitro. Taken together, these data support the hypothesis that acquiring mutations in these L domains may reduce virus fitness.
Genetic analyses of viral samples from 74 laboratory confirmed measles cases occurring in Taiwan during 1992-2008 identified six viral genotypes D3, D5, D9, G2, H1 and H2. The most frequently detected genotype, H1, was associated with outbreaks in 1994 and 2002, and was the likely indigenous genotype in 1992. In response to the outbreaks, two catch-up campaigns were launched and a routine second dose of measles, mumps, and rubella vaccine at entry to elementary school was introduced. The vaccination campaigns successfully reduced the number of measles cases in Taiwan, and many of the more recent cases can be traced to importations, primarily from other Asian countries. A number of measles genotypes which were associated with outbreaks in other Asian countries were detected among the more recent cases. The more recent genotype H1 viruses had sequences that were identical to those currently circulating in China or associated with international importation of virus.