Analysis of Suspected Measles Cases with Discrepant Measles-Specific IgM and rRT-PCR Test Results, Japan.

We investigated clinically suspected measles cases that had discrepant real-time reverse transcription PCR (rRT-PCR) and measles-specific IgM test results to determine diagnoses. We performed rRT-PCR and measles-specific IgM testing on samples from 541 suspected measles cases. Of the 24 IgM-positive and rRT-PCR--negative cases, 20 were among children who received a measles-containing vaccine within the previous 6 months; most had low IgG relative avidity indexes (RAIs). The other 4 cases were among adults who had an unknown previous measles history, unknown vaccination status, and high RAIs. We detected viral nucleic acid for viruses other than measles in 15 (62.5%) of the 24 cases with discrepant rRT-PCR and IgM test results. Measles vaccination, measles history, and contact history should be considered in suspected measles cases with discrepant rRT-PCR and IgM test results. If in doubt, measles IgG avidity and PCR testing for other febrile exanthematous viruses can help confirm or refute the diagnosis.

A mutant H3N2 influenza virus uses an alternative activation mechanism in TMPRSS2 knockout mice by loss of an oligosaccharide in the hemagglutinin stalk region.

The host protease TMPRSS2 plays an essential role in proteolytic activation of the influenza A virus (IAV) hemagglutinin (HA) protein possessing a monobasic cleavage site. However, after passages in TMPRSS2 knockout mice, an H3N2 subtype IAV began to undergo cleavage activation of HA, showing high virulence in the mice due to the loss of an oligosaccharide at position 8 in the HA stalk region. Thus, the H3N2 IAV acquired cleavability by an alternative HA activation mechanism/protease(s).

The host protease TMPRSS2 plays a major role in in vivo replication of emerging H7N9 and seasonal influenza viruses.

UNLABELLED: Proteolytic cleavage of the hemagglutinin (HA) protein is essential for influenza A virus (IAV) to acquire infectivity. This process is mediated by a host cell protease(s) in vivo. The type II transmembrane serine protease TMPRSS2 is expressed in the respiratory tract and is capable of activating a variety of respiratory viruses, including low-pathogenic (LP) IAVs possessing a single arginine residue at the cleavage site. Here we show that TMPRSS2 plays an essential role in the proteolytic activation of LP IAVs, including a recently emerged H7N9 subtype, in vivo. We generated TMPRSS2 knockout (KO) mice. The TMPRSS2 KO mice showed normal reproduction, development, and growth phenotypes. In TMPRSS2 KO mice infected with LP IAVs, cleavage of HA was severely impaired, and consequently, the majority of LP IAV progeny particles failed to gain infectivity, while the viruses were fully activated proteolytically in TMPRSS2+/+ wild-type (WT) mice. Accordingly, in contrast to WT mice, TMPRSS2 KO mice were highly tolerant of challenge infection by LP IAVs (H1N1, H3N2, and H7N9) with ≥1,000 50% lethal doses (LD50) for WT mice. On the other hand, a high-pathogenic H5N1 subtype IAV possessing a multibasic cleavage site was successfully activated in the lungs of TMPRSS2 KO mice and killed these mice, as observed for WT mice. Our results demonstrate that recently emerged H7N9 as well as seasonal IAVs mainly use the specific protease TMPRSS2 for HA cleavage in vivo and, thus, that TMPRSS2 expression is essential for IAV replication in vivo. IMPORTANCE: Influenza A virus (IAV) is a leading pathogen that infects and kills many humans every year. We clarified that the infectivity and pathogenicity of IAVs, including a recently emerged H7N9 subtype, are determined primarily by a host protease, TMPRSS2. Our data showed that TMPRSS2 is the key host protease that activates IAVs in vivo through proteolytic cleavage of their HA proteins. Hence, TMPRSS2 is a good target for the development of anti-IAV drugs. Such drugs could also be effective for many other respiratory viruses, including the recently emerged Middle East respiratory syndrome (MERS) coronavirus, because they are also activated by TMPRSS2 in vitro. Consequently, the present paper could have a large impact on the battle against respiratory virus infections and contribute greatly to human health.

Intracellular transport of the measles virus ribonucleoprotein complex is mediated by Rab11A-positive recycling endosomes and drives virus release from the apical membrane of polarized epithelial cells.

Many viruses use the host trafficking system at a variety of their replication steps. Measles virus (MV) possesses a nonsegmented negative-strand RNA genome that encodes three components of the ribonucleoprotein (RNP) complex (N, P, and L), two surface glycoproteins, a matrix protein, and two nonstructural proteins. A subset of immune cells and polarized epithelial cells are in vivo targets of MV, and MV is selectively released from the apical membrane of polarized epithelial cells. However, the molecular mechanisms for the apical release of MV remain largely unknown. In the present study, the localization and trafficking mechanisms of the RNP complex of MV were analyzed in detail using recombinant MVs expressing fluorescent protein-tagged L proteins. Live cell imaging analyses demonstrated that the MV RNP complex was transported in a manner dependent on the microtubule network and together with Rab11A-containing recycling endosomes. The RNP complex was accumulated at the apical membrane and the apical recycling compartment. The accumulation and shedding of infectious virions were severely impaired by expression of a dominant negative form of Rab11A. On the other hand, recycling endosome-mediated RNP transport was totally dispensable for virus production in nonpolarized cells. These data provide the first demonstration of the regulated intracellular trafficking events of the MV RNP complex that define the directional viral release from polarized epithelial cells.

The receptor-binding site of the measles virus hemagglutinin protein itself constitutes a conserved neutralizing epitope.

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.

Functional and structural characterization of neutralizing epitopes of measles virus hemagglutinin protein.

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.

Lethal canine distemper virus outbreak in cynomolgus monkeys in Japan in 2008.

Canine distemper virus (CDV) has recently expanded its host range to nonhuman primates. A large CDV outbreak occurred in rhesus monkeys at a breeding farm in Guangxi Province, China, in 2006, followed by another outbreak in rhesus monkeys at an animal center in Beijing in 2008. In 2008 in Japan, a CDV outbreak also occurred in cynomolgus monkeys imported from China. In that outbreak, 46 monkeys died from severe pneumonia during a quarantine period. A CDV strain (CYN07-dV) was isolated in Vero cells expressing dog signaling lymphocyte activation molecule (SLAM). Phylogenic analysis showed that CYN07-dV was closely related to the recent CDV outbreaks in China, suggesting continuing chains of CDV infection in monkeys. In vitro, CYN07-dV uses macaca SLAM and macaca nectin4 as receptors as efficiently as dog SLAM and dog nectin4, respectively. CYN07-dV showed high virulence in experimentally infected cynomolgus monkeys and excreted progeny viruses in oral fluid and feces. These data revealed that some of the CDV strains, like CYN07-dV, have the potential to cause acute systemic infection in monkeys.

Canine distemper virus associated with a lethal outbreak in monkeys can readily adapt to use human receptors.

A canine distemper virus (CDV) strain, CYN07-dV, associated with a lethal outbreak in monkeys, used human signaling lymphocyte activation molecule as a receptor only poorly but readily adapted to use it following a P541S substitution in the hemagglutinin protein. Since CYN07-dV had an intrinsic ability to use human nectin-4, the adapted virus became able to use both human immune and epithelial cell receptors, as well as monkey and canine ones, suggesting that CDV can potentially infect humans.

Simple method for differentiating measles vaccine from wild-type strains using loop-mediated isothermal amplification.

Because of increasing measles vaccine coverage, the proportion of patients with modified measles has been increasing. Such patients have low-grade fever with very mild eruptions similar to vaccine-related adverse events. Differentiation between these two pathogenic conditions is required to improve the quality of laboratory-based measles surveillance. In this study, vaccine-specific and wild-type specific primer sets were designed for loop-mediated isothermal amplification in the N gene, and vaccine strains, C1, D3, D4, D5, D8, D9, G3 and H1 wild strains were examined. Three vaccine strains were efficiently amplified using a vaccine-specific primer set with an approximately 10-times higher sensitivity than wild-type primer. Modified measles was differentiated from vaccine-associated cases by this system, but limitations were encountered with the other genotypes.

Epitope mapping of neutralizing monoclonal antibody in avian influenza A H5N1 virus hemagglutinin.

The global spread of highly pathogenic avian influenza A H5N1 viruses raises concerns about more widespread infection in the human population. Pre-pandemic vaccine for H5N1 clade 1 influenza viruses has been produced from the A/Viet Nam/1194/2004 strain (VN1194), but recent prevalent avian H5N1 viruses have been categorized into the clade 2 strains, which are antigenically distinct from the pre-pandemic vaccine. To understand the antigenicity of H5N1 hemagglutinin (HA), we produced a neutralizing monoclonal antibody (mAb12-1G6) using the pre-pandemic vaccine. Analysis with chimeric and point mutant HAs revealed that mAb12-1G6 bound to the loop (amino acid positions 140-145) corresponding to an antigenic site A in the H3 HA. mAb12-1G6 failed to bind to the mutant VN1194 HA when only 3 residues were substituted with the corresponding residues of the clade 2.1.3.2 A/Indonesia/5/05 strain (amino acid substitutions at positions Q142L, K144S, and S145P), suggesting that these amino acids are critical for binding of mAb12-1G6. Escape mutants of VN1194 selected with mAb12-1G6 carried a S145P mutation. Interestingly, mAb12-1G6 cross-neutralized clade 1 and clade 2.2.1 but not clade 2.1.3.2 or clade 2.3.4 of the H5N1 virus. We discuss the cross-reactivity, based on the amino acid sequence of the epitope.

The SI strain of measles virus derived from a patient with subacute sclerosing panencephalitis possesses typical genome alterations and unique amino acid changes that modulate receptor specificity and reduce membrane fusion activity.

Subacute sclerosing panencephalitis (SSPE) is a fatal sequela associated with measles and is caused by persistent infection of the brain with measles virus (MV). The SI strain was isolated in 1976 from a patient with SSPE and shows neurovirulence in animals. Genome nucleotide sequence analyses showed that the SI strain genome possesses typical genome alterations for SSPE-derived strains, namely, accumulated amino acid substitutions in the M protein and cytoplasmic tail truncation of the F protein. Through the establishment of an efficient reverse genetics system, a recombinant SI strain expressing a green fluorescent protein (rSI-AcGFP) was generated. The infection of various cell types with rSI-AcGFP was evaluated by fluorescence microscopy. rSI-AcGFP exhibited limited syncytium-forming activity and spread poorly in cells. Analyses using a recombinant MV possessing a chimeric genome between those of the SI strain and a wild-type MV strain indicated that the membrane-associated protein genes (M, F, and H) were responsible for the altered growth phenotype of the SI strain. Functional analyses of viral glycoproteins showed that the F protein of the SI strain exhibited reduced fusion activity because of an E300G substitution and that the H protein of the SI strain used CD46 efficiently but used the original MV receptors on immune and epithelial cells poorly because of L482F, S546G, and F555L substitutions. The data obtained in the present study provide a new platform for analyses of SSPE-derived strains as well as a clear example of an SSPE-derived strain that exhibits altered receptor specificity and limited fusion activity.

Phylogenetic analysis of rubella viruses in Vietnam during 2009-2010.

Rubella virus (RV) usually causes a mild disease. However, infection during the first trimester of pregnancy often leads to severe birth defects known as congenital rubella syndrome (CRS). Although wild-type RVs exist and circulate worldwide, their genotypes remain unknown in many countries. The aim of this study was to identify the molecular characteristics of RVs found in Vietnam during the years 2009-2010 and to provide the first data concerning RV genotypes in this country. Throat swab samples were collected between 2009 and 2010 from four CRS cases and nine rubella infection cases visiting one Children's Hospital and one outpatient clinic in Ho Chi Minh City. The 739-nucleotide coding region of the RV E1 gene recommended by the World Health Organization was amplified by reverse transcriptase PCR, and the resulting DNA fragments were then sequenced. Sequences were assigned to genotypes by phylogenetic analysis with RV reference strains. RV RNA was detected in 11 clinical specimens. Phylogenetic analysis of the sequences showed that all 11 strains belonged to 2B genotype. Several variations in amino acids were found, among which five changes were involved in the B and T cell epitopes. These data indicate that viruses of genotype 2B were circulating in Vietnam. The increasing information about RV genotype in Vietnam should aid in the control of rubella infection and CRS in this country.

Expansion of the global measles and rubella laboratory network 2005-09.

Enhancing measles surveillance with integration of epidemiologic and laboratory information is one of the key strategies for accelerated measles control and elimination. The World Health Organization (WHO) Global Measles and Rubella Laboratory Network (LabNet) has been developed since 2000 to currently include 690 laboratories serving 183 countries. The LabNet testing strategy follows well-validated, standardized procedures for confirming suspected cases and for monitoring measles and rubella virus transmission patterns. The strength of the LabNet is a strong quality assurance program that monitors the performance of all laboratories through annual proficiency testing and continuous assessment. In the 5-year period 2005-2009, the results of >1 million measles immunoglobulin M (IgM) tests have been reported by the LabNet and, in addition, sequence information on >7000 measles and 600 rubella viruses has been shared. Progress with the development of the LabNet during 2005-2009 is discussed.

Global distribution of measles genotypes and measles molecular epidemiology.

A critical component of laboratory surveillance for measles is the genetic characterization of circulating wild-type viruses. The World Health Organization (WHO) Measles and Rubella Laboratory Network (LabNet), provides for standardized testing in 183 countries and supports genetic characterization of currently circulating strains of measles viruses. The goal of this report is to describe the lessons learned from nearly 20 years of virologic surveillance for measles, to describe the global databases for measles sequences, and to provide regional updates about measles genotypes detected by recent surveillance activities. Virologic surveillance for measles is now well established in all of the WHO regions, and most countries have conducted at least some baseline surveillance. The WHO Global Genotype Database contains >7000 genotype reports, and the Measles Nucleotide Surveillance (MeaNS) contains >4000 entries. This sequence information has proven to be extremely useful for tracking global transmission patterns and for documenting the interruption of transmission in some countries. The future challenges will be to develop quality control programs for molecular methods and to continue to expand virologic surveillance activities in all regions.

Status of global virologic surveillance for rubella viruses.

The suspected measles case definition captures rubella cases. Therefore, measles surveillance will be improved in the course of the control and eventual elimination of rubella transmission. One aspect of rubella control, virologic surveillance, is reviewed here. A systematic nomenclature for rubella viruses (RVs) based on 13 genotypes has been established and is updated when warranted by increases in information about RVs. From 2005 through 2010, the genotypes of RVs most frequently reported were 1E, 1G, and 2B, and genotypes 1a, 1B, 1C, 1h, 1j, and 2C were less frequently reported. Virologic surveillance can support rubella control and elimination. Synopses of rubella virologic surveillance in various countries, regions, and globally are given, including characterization of viruses from imported cases in a country that has eliminated rubella and studies of endemic viruses circulating in countries without rubella control objectives. Current challenges are discussed.

Characterization of mumps viruses circulating in Mongolia: identification of a novel cluster of genotype H.

Although mumps virus is still causing annual epidemics in Mongolia, very few epidemiological and virological data have been reported. We describe here the first phylogenetic analysis data on the mumps viruses circulated in Mongolia in 2009. We detected 21 mumps virus cDNAs and obtained a virus isolate from 32 throat swabs of mumps patients in Ulaanbaatar, the capital of Mongolia. The phylogenetic analyses based on the 316 nucleotides of the small hydrophobic gene show that these sequences form a single cluster, with the closest relatedness to the viruses belonging to genotype H. According to the recommendation of the World Health Organization, Mongolian mumps viruses could be classified into a novel genotype because the divergence between new sequences and genotype H reference viruses is >5% (6.3 to 8.2%). However, additional analyses based on the fusion gene, the hemagglutinin-neuraminidase gene, and the whole-genome indicate that the divergences between the Mongolian isolate and other genotype H strains never exceed the within-genotype divergences of other genotypes. These results suggest that Mongolia strains should be included in genotype H and that the current criteria for mumps virus genotyping should be revised. We propose here that the Mongolian viruses should be classified as a new subgenotype termed H3. Since previous epidemiological studies suggested that genotypes H may be associated with central nervous system diseases, we evaluated the neurovirulence of the Mongolian isolate in the neonatal rat system. However, the virus does not exhibit prominent neurovirulence in rats.

Amino Acid substitutions in matrix, fusion and hemagglutinin proteins of wild measles virus for adaptation to vero cells.

BACKGROUND: Wild-type measles virus (MV) is isolated in B95a but not in Vero cells. Through an adaptation process of wild-type MV to Vero cells, several amino acid substitutions were reported. METHODS: Six strains were adapted to Vero cells and membrane (M), fusion (F) and hemagglutinin (H) genes were sequenced. Cell fusion was assessed and recombinant MVs were constructed, having wild-type H or M gene with or without mutations. RESULTS: No F gene substitution was noted. Amino-acid substitutions at positions 481 from Asn to Tyr (N481Y) and 546 from Ser to Gly (S546G) were observed in the H protein. Glu at position 89 of the M protein was substituted for Gly (E89G) and two mutations were noted at positions 62 (S62R) and 83 (S83P) in M protein. Recombinant viruses with mutation(s) detected in Vero-adapted strains induced a cytopathic effect and grew well in Vero cells, but those with the wild type did not. Recombinant viruses with mutation(s) demonstrated lower viral growth in B95a cells. CONCLUSIONS: Substitutions of E89G, S62R and S83P of the M protein were newly observed through adaptation to Vero cells, besides the mutations described in previous reports, with varying adaptation for each strain.

Remarkable similarity in genome nucleotide sequences between the Schwarz FF-8 and AIK-C measles virus vaccine strains and apparent nucleotide differences in the phosphoprotein gene.

The Schwarz FF-8 (FF-8) and AIK-C measles virus vaccine strains are currently used for vaccination in Japan. Here, the complete genome nucleotide sequence of the FF-8 strain has been determined and its genome sequence found to be remarkably similar to that of the AIK-C strain. These two strains are differentiated only by two nucleotide differences in the phosphoprotein gene. Since the FF-8 strain does not possess the amino acid substitutions in the phospho- and fusion proteins which are responsible for the temperature-sensitivity and small syncytium formation phenotypes of the AIK-C strain, respectively, other unidentified common mechanisms likely attenuate both the FF-8 and AIK-C strains.

Viral molecular characterization of the first congenital rubella syndrome case in Vietnam.

BACKGROUND: Rubella virus (RV) infection during the first trimester of pregnancy often leads to severe birth defects known as congenital rubella syndrome (CRS). METHODS: A premature newborn male was clinically diagnosed as CRS with cataracts, congenital heart defects, microcephaly, hepatosplenomegaly, and thrombocytopenia. The infection was confirmed serologically and molecularly. RESULTS: The RV was characterized and clustered with the 2B genotype. CONCLUSIONS: The integrated description of clinical features, serological and molecular data provide a baseline for diagnosis and control of rubella and CRS in Vietnam. This is the first report of molecular investigation of wildtype RV strain in Vietnam, thus contributing to the documentation of RV's worldwide distribution.

Seroprevalence of rubella and measles antibodies in Lao PDR.

BACKGROUND: Nowadays, measles and rubella epidemics still occur worldwide, particularly in developing countries. METHODS: The present study investigated seroprevalence of rubella and measles IgG antibodies by using a comercial enzyme immunoassay. The findings helped to understand the current status of these infections in school children in Vientiane Capital, Lao PDR. RESULTS: The seropositive rate for measles antibody was 97.6%. Most importantly, more than half of 411 healthy children (56.4%) were rubella seronegative and susceptible to acquired rubella infection. CONCLUSIONS: The results highlight a high proportion of rubella-seronegative children, suggesting that they are susceptible to rubella and face the risk of congenital rubella syndrome. It was the first study to report on seroprevalence of rubella in Vientiane Capital, Lao PDR.