Longitudinal antigenic and seroepidemiological analyses of parechovirus A1 in Yamagata, Japan.

To investigate the antigenic changes in parechovirus 1 (PeVA1), seroepidemiological analyses were performed against the Harris strain (Harris), isolated in 1956, and PeVA1/Yamagata.JPN/2021-4785, isolated in 2021, using immune sera and 207 and 237 human serum specimens collected in 2021 and 1976, respectively. Although rabbit immune sera showed the highest neutralization antibody (NT-Ab) titers against the immunized viruses at 1:12 800-1:102 400, they were cross-reactive at 1:400-1:800. All 62 Yamagata isolates obtained between 2001 and 2021 (Yamagata strains), belonging to phylogenetic lineage 1B, reacted more strongly (mostly 4-64 times) to antiserum against PeVA1/Yamagata.JPN/2021-4785 than to antiserum against Harris, belonging to phylogenetic lineage 1 A. Human serum specimens obtained in 2021 showed higher NT-Ab titers against PeVA1/Yamagata.JPN/2021-4785, whereas those obtained in 1976 had similar NT-Ab titers against both strains. These findings suggested that Yamagata strains and Harris were antigenically cross-reactive, although there were differences. There are still high NT-Abs titers present against Harris in 2021 in particular, indicating that PeVA1 has been in circulation with high immunity in the population. In conclusion, this study suggested that PeVA1 has been endemically perpetuated with only minor antigenic changes as well as with high immunity over several decades in the community.

Virulence of Enterovirus A71 Fluctuates Depending on the Phylogenetic Clade Formed in the Epidemic Year and Epidemic Region.

Although epidemics of hand, foot, and mouth disease (HFMD) caused by enterovirus A71 (EV-A71) have occurred worldwide, the Asia-Pacific region has seen large sporadic outbreaks with many severe neurological cases. This suggests that the virulence of the circulating viruses fluctuates in each epidemic and that HFMD outbreaks with many severe cases occur when highly virulent viruses are circulating predominantly, which has not been experimentally verified. Here, we analyzed 32 clinically isolated strains obtained in Japan from 2002 to 2013, along with 27 Vietnamese strains obtained from 2015 to 2016 that we characterized previously using human SCARB2 transgenic mice. Phylogenetic analysis of the P1 region classified them into five clades belonging to subgenogroup B5 (B5-I to B5-V) and five clades belonging to subgenogroup C4 (C4-I to C4-V) according to the epidemic year and region. Interestingly, clades B5-I and B5-II were very virulent, while clades B5-III, B5-IV, and B5-V were less virulent. Clades C4-II, C4-III, C4-IV, and C4-V were virulent, while clade C4-I was not. The result experimentally showed for the first time that several clades with different virulence levels emerged one after another. The experimental virulence evaluation of circulating viruses using SCARB2 transgenic mice is helpful to assess potential risks of circulating viruses. These results also suggest that a minor nucleotide or amino acid substitution in the EV-A71 genome during circulation causes fluctuations in virulence. The data presented here may increase our understanding of the dynamics of viral virulence during epidemics. IMPORTANCE Outbreaks of hand, foot, and mouth disease (HFMD) with severe enterovirus A71 (EV-A71) cases have occurred repeatedly, mainly in Asia. In severe cases, central nervous system complications can lead to death, making it an infectious disease of importance to public health. An unanswered question about this disease is why outbreaks of HFMD with many severe cases sometimes occur. Here, we collected EV-A71 strains that were prevalent in Japan and Vietnam over the past 20 years and evaluated their virulence in a mouse model of EV-A71 infection. This method clearly revealed that viruses belonging to different clades have different virulence, indicating that the method is powerful to assess the potential risks of the circulating viruses. The results also suggested that factors in the virus genome cause an outbreak with many severe cases and that further studies facilitate the prediction of large epidemics of EV-A71 in the future.

Heparan sulfate attachment receptor is a major selection factor for attenuated enterovirus 71 mutants during cell culture adaptation.

Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease (HFMD). However, this infection is sometimes associated with severe neurological complications. Identification of neurovirulence determinants is important to understand the pathogenesis of EV71. One of the problems in evaluating EV71 virulence is that its genome sequence changes rapidly during replication in cultured cells. The factors that induce rapid mutations in the EV71 genome in cultured cells are unclear. Here, we illustrate the population dynamics during adaptation to RD-A cells using EV71 strains isolated from HFMD patients. We identified a reproducible amino acid substitution from glutamic acid (E) to glycine (G) or glutamine (Q) in residue 145 of the VP1 protein (VP1-145) after adaptation to RD-A cells, which was associated with attenuation in human scavenger receptor B2 transgenic (hSCARB2 tg) mice. Because previous reports demonstrated that VP1-145G and Q mutants efficiently infect cultured cells by binding to heparan sulfate (HS), we hypothesized that HS expressed on the cell surface is a major factor for this selection. Supporting this hypothesis, selection of the VP1-145 mutant was prevented by depletion of HS and overexpression of hSCARB2 in RD-A cells. In addition, this mutation promotes the acquisition of secondary amino acid substitutions at various positions of the EV71 capsid to increase its fitness in cultured cells. These results indicate that attachment receptors, especially HS, are important factors for selection of VP1-145 mutants and subsequent capsid mutations. Moreover, we offer an efficient method for isolation and propagation of EV71 virulent strains with minimal selection pressure for attenuation.

Seroprevalence of coxsackievirus A21 neutralizing antibodies in Yamagata, Japan, between 1976 and 2019; coxsackievirus A21 has rarely affected young children.

Although coxsackievirus A21 (CV-A21) has been associated with an acute respiratory infection (ARI) as well as poliomyelitis-like paralysis, reports of CV-A21 detection have been quite limited both globally and in Japan. CV-A21 strains were isolated from five sporadic pediatric cases with ARI in 2019 in Yamagata, Japan. Neutralizing antibodies (NT Abs) were then measured against CV-A21 using sera collected in 1976, 1985, 1999, 2009, and 2019 in Yamagata, to clarify the longitudinal epidemiology of CV-A21. The total Ab-positive rate in each year was 15.2% (35/233), 10.7% (30/281), 14.3% (28/196), 3.1% (7/236), and 1.3% (3/226), respectively. Ab-positive rates generally increased with age, especially between 1976 and 1999. Among the total Ab-positive cases, the Ab titers were relatively low; 50 cases belonged to the 1:8-1:16, 40 to 1:32-1:64, 12 to 1:128-1:256, and 1 to 1:1024< groups, respectively. No Ab-positive cases under the age of 10 were observed in any of the years analyzed. In conclusion, this study and previous works suggested that CV-A21 is a unique enterovirus, which is not transmitted readily among young children but causes sporadic ARI cases mainly among those ≥15 years of age in the community.

Suitability of NIID-MDCK cells as a substrate for cell-based influenza vaccine development from the perspective of adventitious virus susceptibility.

The practical use of cell-based seasonal influenza vaccines is currently being considered in Japan. From the perspective of adventitious virus contamination, we assessed the suitability of NIID-MDCK cells (NIID-MDCK-Cs) as a safe substrate for the isolation of influenza viruses from clinical specimens. We first established a sensitive multiplex real-time PCR system to screen for 27 respiratory viruses and used it on 34 virus samples that were isolated by passaging influenza-positive clinical specimens in NIID-MDCK-Cs. Incidentally, the limit of detection (LOD) of the system was 100 or fewer genome copies per reaction. In addition to influenza viruses, human enterovirus 68 (HEV-D68) genomes were detected in two samples after two or three passages in NIID-MDCK-Cs. To further investigate the susceptibility of NIID-MDCK-Cs to adventitious viruses, eight common respiratory viruses were subjected to passages in NIID-MDCK-Cs. The genome copy numbers of seven viruses other than parainfluenza 3 decreased below the LOD by passage 4. By passaging in NIID-MDCK-Cs, the genome numbers of the input HEV-D68, 1 × 108 copies, declined to 102 at passage 3 and to under the LOD at passage 4, whereas those of the other six viruses were under the LOD by passage 3. These results implied that during the process of isolating influenza viruses with NIID-MDCK-Cs, contaminating viruses other than parainfluenza 3 can be efficiently removed by passages in NIID-MDCK-Cs. NIID-MDCK-Cs could be a safe substrate for isolating influenza viruses that can be used to develop cell-based influenza vaccine candidate viruses.

A seroepidemiologic study of a measles outbreak, Yamagata Prefecture, Japan, 2017: The estimation of spreaders using serological assays in a measles elimination setting.

INTRODUCTION: In regions where the endemic measles virus has been eliminated, early detection of contagious patients is important for preventing the spread of measles and sustaining elimination. To investigate whether serological assays can be used for the estimation of highly infectious patients with measles, we performed a seroepidemiologic study of a measles outbreak in Yamagata Prefecture, Japan, in 2017. METHODS: We tested plaque reduction neutralization (PRN), IgG avidity, and gelatin particle agglutination (PA) assays in 31 patients with measles, subdivided into two super-spreaders, three spreaders, and 26 non-spreaders. Simultaneously, these results were compared with the cycle threshold (Ct) of a semi-quantitative real-time reverse transcription PCR for the measles virus from throat swab specimens. RESULTS: In the PRN assay, one super-spreader and two spreaders lacked protective antibodies. The IgG avidity assay showed that two super-spreaders and one spreader had low avidity. The PA assay indicated that two super-spreaders and two spreaders lacked protective antibodies. Comparison of the results of the three serological assays and Ct revealed that patients whose antibody titers were judged as low in the IgG avidity and PA assays showed low Ct (i.e., high viral load), whereas non-spreaders tended to show low viral load. CONCLUSIONS: Our preliminary seroepidemiologic analysis of a population of 31 patients with measles suggests that PA and IgG avidity assays may be used for the identification of super-spreader/spreader candidates. However, further investigations are necessary to validate the robustness of these serological assays in detecting contagious measles cases.

Development of an Enterovirus 71 Vaccine Efficacy Test Using Human Scavenger Receptor B2 Transgenic Mice.

Enterovirus 71 (EV71) is a causative agent of hand-foot-mouth disease, and it sometimes causes severe neurological disease. Development of effective vaccines and animal models to evaluate vaccine candidates are needed. However, the animal models currently used for vaccine efficacy testing, monkeys and neonatal mice, have economic, ethical, and practical drawbacks. In addition, EV71 strains prepared for lethal challenge often develop decreased virulence during propagation in cell culture. To overcome these problems, we used a mouse model expressing human scavenger receptor B2 (hSCARB2) that showed lifelong susceptibility to EV71. We selected virulent EV71 strains belonging to the subgenogroups B4, B5, C1, C2, and C4 and propagated them using a culture method for EV71 without an apparent reduction in virulence. Here, we describe a novel EV71 vaccine efficacy test based on these hSCARB2 transgenic (Tg) mice and these virulent viruses. Adult Tg mice were immunized subcutaneously with formalin-inactivated EV71. The vaccine elicited sufficient levels of neutralizing antibodies in the immunized mice. The mice were subjected to lethal challenge with virulent viruses via intravenous injection. Survival, clinical signs, and body weight changes were observed for 2 weeks. Most immunized mice survived without clinical signs or histopathological lesions. The viral replication in immunized mice was much lower than that in nonimmunized mice. Mice immunized with the EV71 vaccine were only partially protected against lethal challenge with coxsackievirus A16. These results indicate that this new model is useful for in vivo EV71 vaccine efficacy testing.IMPORTANCE The development of new vaccines for EV71 relies on the availability of small animal models suitable for in vivo efficacy testing. Monkeys and neonatal mice have been used, but the use of these animals has several drawbacks, including high costs, limited susceptibility, and poor experimental reproducibility. In addition, the related ethical issues are considerable. The new efficacy test based on hSCARB2 Tg mice and virulent EV71 strains propagated in genetically modified cell lines presented here can overcome these disadvantages and is expected to accelerate the development of new EV71 vaccines.

Longitudinal epidemiology of human coronavirus OC43 in Yamagata, Japan, 2010-2017: Two groups based on spike gene appear one after another.

Human coronavirus OC43 (HCoV-OC43) is divided into genotypes A to H based on genetic recombination including the spike (S) gene. To investigate the longitudinal transition of the phylogenetic feature of the HCoV-OC43 S gene in a community, phylogenetic analysis of the S1 region of the S gene was conducted using 208 strains detected in Yamagata during 2010 to 2017 with reference strains of the genotype. The S1 sequences were divisible into four groups: A to D. All Yamagata strains belonged to either group B or group D. In group B, 46 (90.2%) out of 51 Yamagata strains were clustered with those of genotype E reference strains (cluster E). In group D, 28 (17.8%) and 122 (77.7%) out of 157 Yamagata strains were clustered, respectively, with genotype F and genotype G reference strains. In cluster G, 28 strains formed a distinct cluster. Monthly distributions of HCoV-OC43 in Yamagata in 2010 to 2017 revealed that group B and group D appeared one after another. In group B, the cluster E strains were prevalent recurrently. In conclusion, epidemics of HCoV-OC43 in Yamagata, Japan might be attributable to two genetically different groups: group B showed a recurrent epidemic of strains belonging to a single phylogenetic cluster and group D showed epidemic strains belonging to multiple clusters.

VP1 Amino Acid Residue 145 of Enterovirus 71 Is a Key Residue for Its Receptor Attachment and Resistance to Neutralizing Antibody during Cynomolgus Monkey Infection.

Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and sometimes causes severe or fatal neurological complications. The amino acid at VP1-145 determines the virological characteristics of EV71. Viruses with glutamic acid (E) at VP1-145 (VP1-145E) are virulent in neonatal mice and transgenic mice expressing human scavenger receptor B2, whereas those with glutamine (Q) or glycine (G) are not. However, the contribution of this variation to pathogenesis in humans is not fully understood. We compared the virulence of VP1-145E and VP1-145G viruses of Isehara and C7/Osaka backgrounds in cynomolgus monkeys. VP1-145E, but not VP1-145G, viruses induced neurological symptoms. VP1-145E viruses were frequently detected in the tissues of infected monkeys. VP1-145G viruses were detected less frequently and disappeared quickly. Instead, mutants that had a G-to-E mutation at VP1-145 emerged, suggesting that VP1-145E viruses have a replication advantage in the monkeys. This is consistent with our hypothesis proposed in the accompanying paper (K. Kobayashi, Y. Sudaka, A. Takashino, A. Imura, K. Fujii, and S. Koike, J Virol 92:e00681-18, 2018, https://doi.org/10.1128/JVI.00681-18) that the VP1-145G virus is attenuated due to its adsorption by heparan sulfate. Monkeys infected with both viruses produced neutralizing antibodies before the onset of the disease. Interestingly, VP1-145E viruses were more resistant to neutralizing antibodies than VP1-145G viruses in vitro A small amount of neutralizing antibody raised in the early phase of infection may not be sufficient to block the dissemination of VP1-145E viruses. The different resistance of the VP1-145 variants to neutralizing antibodies may be one of the reasons for the difference in virulence.IMPORTANCE The contribution of VP1-145 variants in humans is not fully understood. In some studies, VP1-145G/Q viruses were isolated more frequently from severely affected patients than from mildly affected patients, suggesting that VP1-145G/Q viruses are more virulent. In the accompanying paper (K. Kobayashi, Y. Sudaka, A. Takashino, A. Imura, K. Fujii, and S. Koike, J Virol 92:e00681-18, 2018, https://doi.org/10.1128/JVI.00681-18), we showed that VP1-145E viruses are more virulent than VP1-145G viruses in human SCARB2 transgenic mice. Heparan sulfate acts as a decoy to specifically trap the VP1-145G viruses and leads to abortive infection. Here, we demonstrated that VP1-145G was attenuated in cynomolgus monkeys, suggesting that this hypothesis is also true in a nonhuman primate model. VP1-145E viruses, but not VP1-145G viruses, were highly resistant to neutralizing antibodies. We propose the difference in resistance against neutralizing antibodies as another mechanism of EV71 virulence. In summary, VP1-145 contributes to virulence determination by controlling attachment receptor usage and antibody sensitivity.

Scrub typhus caused by Shimokoshi type Orientia tsutsugamushi showing variant 56-kDa type-specific antigen gene sequence in Tohoku region, Japan.

In 2018, a patient was diagnosed with Shimokoshi type scrub typhus in Yamagata Prefecture, Japan. The causative pathogen was likely a variant type because 43 (8.3%) of 521 deduced amino acid sequences of the 56-kDa type-specific antigen (TSA) were different from those of the Shimokoshi prototype strain. The patient's paired sera showed low antibody titers against the Shimokoshi prototype strain. Two cases of scrub typhus reported in the Tohoku region during 2011-2012 also involved the same 56-kDa TSA gene sequence. These findings suggest the presence of diversity in Shimokoshi type Orientia tsutsugamushi, which may impede the laboratory diagnosis of scrub typhus.

Detection of Saffold viruses from children with acute respiratory infections in Yamagata, Japan, between 2008 and 2015.

Although Saffold virus (SAFV) was reported as a novel human cardiovirus in 2007, no causative association between SAFV and clinical disease has been proven and the longitudinal epidemiology of SAFVs is not available. To establish the relationship between SAFVs and acute respiratory infections (ARIs) and to clarify the longitudinal epidemiology of SAFVs, 7258 nasopharyngeal specimens were collected from children with ARIs in Yamagata, Japan between 2008 and 2015. The specimens were inoculated on a microplate including six cell lines as part of routine surveillance, and molecular screening was performed for SAFVs using a reverse transcription (RT)-PCR method. Throughout the study period, 95 (1.3%) SAFV genotype 2 (SAFV2), and 28 (0.4%) SAFV3 were detected, mainly between September and November. There were two outbreaks of SAFV2 in 2009 and 2013, and one outbreak of SAFV3 in 2012 and the positive rates during these outbreaks were 12.1% (53/439), 11% (35/319), and 4.4% (20/453), respectively. Sixty-three SAFV2 and 28 SAFV3 strains were detected as a single virus from children with ARIs such as pharyngitis, herpangina, and tonsillitis. These results suggested that SAFV2 and SAFV3 are possible causative agents of ARIs among children and their infections occur mainly in the autumn season in Japan.

Mycobacterium tuberculosis Transmission among Elderly Persons, Yamagata Prefecture, Japan, 2009-2015.

In many countries with low to moderate tuberculosis (TB) incidence, cases have shifted to elderly persons. It is unclear, however, whether these cases are associated with recent Mycobacterium tuberculosis transmission or represent reactivation of past disease. During 2009-2015, we performed a population-based TB investigation in Yamagata Prefecture, Japan, using in-depth contact tracing and 24-loci variable-number tandem-repeat typing optimized for Beijing family M. tuberculosis strains. We analyzed 494 strains, of which 387 (78.3%) were derived from elderly patients. Recent transmission with an epidemiologic link was confirmed in 22 clusters (70 cases). In 17 (77.3%) clusters, the source patient was elderly; 11 (64.7%) of the 17 clusters occurred in a hospital or nursing home. In this setting, the increase in TB cases was associated with M. tuberculosis transmissions from elderly persons. Prevention of transmission in places where elderly persons gather will be an effective strategy for decreasing TB incidence among predominantly elderly populations.

Genetic Lineage and Reassortment of Influenza C Viruses Circulating between 1947 and 2014.

Since influenza C virus was first isolated in 1947, the virus has been only occasionally isolated by cell culture; there are only four strains for which complete genome sequences are registered. Here, we analyzed a total of 106 complete genomes, ranging from the first isolate from 1947 to recent isolates from 2014, to determine the genetic lineages of influenza C virus, the reassortment events, and the rates of nucleotide substitution. The results showed that there are six lineages, named C/Taylor, C/Mississippi, C/Aichi, C/Yamagata, C/Kanagawa, and C/Sao Paulo. They contain both antigenic and genetic lineages of the hemagglutinin-esterase (HE) gene, and the internal genes PB2, PB1, P3, NP, M, and NS are divided into two major lineages, a C/Mississippi/80-related lineage and a C/Yamagata/81-related lineage. Reassortment events were found over the entire period of 68 years. Several outbreaks of influenza C virus between 1990 and 2014 in Japan consisted of reassortant viruses, suggesting that the genomic constellation is related to influenza C virus epidemics. The nucleotide sequences were highly homologous to each other. The minimum percent identity between viruses ranged from 91.1% for the HE gene to 96.1% for the M gene, and the rate of nucleotide substitution for the HE gene was the highest, at 5.20 × 10(-4) substitutions/site/year. These results indicate that reassortment is an important factor that increases the genetic diversity of influenza C virus, resulting in its ability to prevail in humans. IMPORTANCE Influenza C virus is a pathogen that causes acute respiratory illness in children and results in hospitalization of infants. We previously demonstrated (Y. Matsuzaki et al., J Clin Virol 61:87-93, 2014, http://dx.doi.org/10.1016/j.jcv.2014.06.017) that periodic epidemics of this virus occurred in Japan between 1996 and 2014 and that replacement of the dominant antigenic group occurred every several years as a result of selection by herd immunity. However, the antigenicity of the HE glycoprotein is highly stable, and antigenic drift has not occurred for at least 30 years. Here, we analyzed a total of 106 complete genomes spanning 68 years for the first time, and we found that influenza C viruses are circulating worldwide while undergoing reassortment as well as selection by herd immunity, resulting in an increased ability to prevail in humans. The results presented in this study contribute to the understanding of the evolution, including reassortment events, underlying influenza C virus epidemics.

Seroepidemiology of human parechovirus types 1, 3, and 6 in Yamagata, Japan, in 2014.

To clarify the seroepidemiology of human parechovirus type 1 (HPeV1), 3 and 6, neutralizing antibodies (NT Abs) were measured in 214 serum specimens collected in 2014 in Yamagata, Japan. The seroprevalence against HPeV1 was 100% in all age groups, while that against HPeV3 and HPeV6 was 79.4% and 66.8%, respectively, overall. The geometric mean titers of NT Abs against HPeV1, 3 and 6 were 755.2, 255.0 and 55.9, respectively, overall. Our findings indicate that HPeV1 is the most prevalent HPeV circulating in Yamagata, followed by HPeV3 and HPeV6.

Antigenic and receptor binding properties of enterovirus 68.

UNLABELLED: Increased detection of enterovirus 68 (EV68) among patients with acute respiratory infections has been reported from different parts of the world in the late 2000s since its first detection in pediatric patients with lower-respiratory-tract infections in 1962. However, the underlying molecular mechanisms for this trend are still unknown. We therefore aimed to study the antigenicity and receptor binding properties of EV68 detected in recent years in comparison to the prototype strain of EV68, the Fermon strain. We first performed neutralization (NT) and hemagglutination inhibition (HI) tests using antisera generated for EV68 strains detected in recent years. We found that the Fermon strain had lower HI and NT titers than recently detected EV68 strains. The HI and NT titers were also significantly different between strains of different genetic lineages among recently detected EV68 strains. We further studied receptor binding specificities of EV68 strains for sialyloligosaccharides using glycan array analysis. In glycan array analysis, all tested EV68 strains showed affinity for α2-6-linked sialic acids (α2-6 SAs) compared to α2-3 SAs. Our study demonstrates that emergence of strains with different antigenicity is the possible reason for the increased detection of EV68 in recent years. Additionally, we found that EV68 preferably binds to α2-6 SAs, which suggests that EV68 might have affinity for the upper respiratory tract. IMPORTANCE: Numbers of cases of enterovirus 68 (EV68) infection in different parts of the world increased significantly in the late 2000s. We studied the antigenicity and receptor binding properties of recently detected EV68 strains in comparison to the prototype strain of EV68, Fermon. The hemagglutination inhibition (HI) and neutralization (NT) titers were significantly different between strains of different genetic lineages among recently detected EV68 strains. We further studied receptor binding specificities of EV68 strains for sialyloligosaccharides using glycan array analysis, which showed affinity for α2-6-linked sialic acids (α2-6 SAs) compared to α2-3 SAs. Our study suggested that the emergence of strains with different antigenicities was the possible reason for the increased detections of EV68 in recent years. Additionally, we revealed that EV68 preferably binds to α2-6 SAs. This is the first report describing the properties of EV68 receptor binding to the specific types of sialic acids.

Epitope mapping of the hemagglutinin molecule of A/(H1N1)pdm09 influenza virus by using monoclonal antibody escape mutants.

UNLABELLED: We determined the antigenic structure of pandemic influenza A(H1N1)pdm09 virus hemagglutinin (HA) using 599 escape mutants that were selected using 16 anti-HA monoclonal antibodies (MAbs) against A/Narita/1/2009. The sequencing of mutant HA genes revealed 43 amino acid substitutions at 24 positions in three antigenic sites, Sa, Sb, and Ca2, which were previously mapped onto A/Puerto Rico/8/34 (A/PR/8/34) HA (A. J. Caton, G. G. Brownlee, J. W. Yewdell, and W. Gerhard, Cell 31:417-427, 1982), and an undesignated site, i.e., amino acid residues 141, 142, 143, 171, 172, 174, 177, and 180 in the Sa site, residues 170, 173, 202, 206, 210, 211, and 212 in the Sb site, residues 151, 154, 156, 157, 158, 159, 200, and 238 in the Ca2 site, and residue 147 in the undesignated site (numbering begins at the first methionine). Sixteen MAbs were classified into four groups based on their cross-reactivity with the panel of escape mutants in the hemagglutination inhibition test. Among them, six MAbs targeting the Sa and Sb sites recognized both residues at positions 172 and 173. MAb n2 lost reactivity when mutations were introduced at positions 147, 159 (site Ca2), 170 (site Sb), and 172 (site Sa). We designated the site consisting of these residues as site Pa. From 2009 to 2013, no antigenic drift was detected for the A(H1N1)pdm09 viruses. However, if a novel variant carrying a mutation at a position involved in the epitopes of several MAbs, such as 172, appeared, such a virus would have the advantage of becoming a drift strain. IMPORTANCE: The first influenza pandemic of the 21st century occurred in 2009 with the emergence of a novel virus originating with swine influenza, A(H1N1)pdm09. Although HA of A(H1N1)pdm09 has a common origin (1918 H1N1) with seasonal H1N1, the antigenic divergence of HA between the seasonal H1N1 and A(H1N1)pdm09 viruses gave rise to the influenza pandemic in 2009. To take precautions against the antigenic drift of the A(H1N1)pdm09 virus in the near future, it is important to identify its precise antigenic structure. To obtain various mutants that are not neutralized by MAbs, it is important to neutralize several plaque-cloned parent viruses rather than only a single parent virus. We characterized 599 escape mutants that were obtained by neutralizing four parent viruses of A(H1N1)pdm09 in the presence of 16 MAbs. Consequently, we were able to determine the details of the antigenic structure of HA, including a novel epitope.

Characteristics of Mycoplasma pneumoniae infection identified on culture in a pediatric clinic.

BACKGROUND: The appropriate choice of antibiotics against Mycoplasma pneumoniae infection has become difficult, as the prevalence of macrolide-resistant M. pneumoniae has increased. METHODS: Throat swab specimens were collected from children with clinically suspected M. pneumoniae infection while visiting an outpatient clinic. Cultures for M. pneumoniae were done, and all isolates were sequenced for the presence of a mutation in 23S rRNA. RESULTS: Of the 80 specimens collected between February 2012 and March 2013, 27 (34%) were positive for M. pneumoniae on culture. Macrolide-resistant mutation was detected in 24 isolates (89%): 23 isolates had an A2063G transition, and one had a C2617G mutation. Both the median age and the prevalence of pneumonia were significantly higher in M. pneumoniae-positive than in M. pneumoniae-negative children (median, 7 years vs 4 years; 88.9% vs 60.4%, respectively). The percentage of serum samples with particle agglutination titer ≥ 1:160 was 69.6% in M. pneumoniae-positive cases and 17.6% in M. pneumoniae-negative cases when the serum was collected ≥ 4 days after the onset of fever. Defervescence within 72 h after the initiation of macrolides never occurred in M. pneumoniae-positive children and also did not occur in 54% of M. pneumoniae-negative children. Switching to either minocycline or tosufloxacin resulted in fever resolution within 48 h in M. pneumoniae-positive children. CONCLUSIONS: The described clinical and laboratory characteristics of M. pneumoniae infection may be useful in guiding appropriate treatment in an outpatient clinic.

TMPRSS2 is an activating protease for respiratory parainfluenza viruses.

Here, we show that human parainfluenza viruses and Sendai virus (SeV), like other respiratory viruses, use TMPRSS2 for their activation. The membrane fusion proteins of respiratory viruses often possess serine and glutamine residues at the P2 and P3 positions, respectively, but these residues were not critical for cleavage by TMPRSS2. However, mutations of these residues affected SeV growth in specific epithelial cell lines, suggesting the importance of these residues for SeV replication in epithelia.

A Case of Legionella pneumophila Serogroup 13 Pneumonia Based on the Detection of Serogroup-Specific Genes in Culture-Negative Sputum.

Legionella pneumophila serogroup (SG) 1, the main cause of Legionnaires' disease, can be diagnosed using urinary antigen testing kits. However, lower respiratory tract specimen cultures are required to identify L. pneumophila SG 2-15. We attempted to detect L. pneumophila SG-specific genes in a culture-negative sputum specimen from a patient with pneumonia who was suspected to have Legionnaires' disease. Two multiplex PCR methods targeting L. pneumophila were modified and amplicons considered to be SG13 specific were detected. Direct sequencing revealed that the amplicons were identical to the nucleotide sequence of L. pneumophila SG13. Based on the presentation and clinical course (fever, muscle pain, disturbance of consciousness, high C-reactive protein titer, rhabdomyolysis, hypophosphatemia, and symptomatic improvement with levofloxacin treatment), in combination with the detection of L. pneumophila SG-specific genes, we suspected L. pneumophila SG13 pneumonia. L. pneumophila non-SG1 pneumonia is thought to be underestimated because of its difficult laboratory diagnosis. The modified multiplex PCR system for lower respiratory tract specimens revealed in this study is likely to improve the diagnosis of Legionnaires' disease caused by L. pneumophila SG13 and other SGs.