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Norovirus is a major human pathogen that can cause severe gastroenteritis in vulnerable populations. The extensive viral diversity presented by human noroviruses constitutes a major roadblock for the development of effective vaccines. In addition to the large number of genotypes, antigenically distinct variants of GII.4 noroviruses have chronologically emerged over the last 3 decades. The last variant to emerge, Sydney_2012, has been circulating at high incidence worldwide for over a decade. We analyzed 1449 capsid sequences from GII.4 Sydney_2012 viruses to determine genetic changes indicative of antigenic diversification. Phylogenetic analyses show that Sydney_2012 viruses scattered within the tree topology with no single cluster dominating during a given year or geographical location. Fourteen residues presented high variability, 7 of which mapped to 4 antigenic sites. Notably, ~52% of viruses presented mutations at 2 or more antigenic sites. Mutational patterns showed that residues 297 and 372, which map to antigenic site A, changed over time. Virus-like particles (VLPs) developed from wild-type Sydney_2012 viruses and engineered to display all mutations detected at antigenic sites were tested against polyclonal sera and monoclonal antibodies raised against Sydney_2012 and Farmington_Hills_2002 VLPs. Minimal changes in reactivity were detected with polyclonal sera and only 4 MAbs lost binding, with all mapping to antigenic site A. Notably, reversion of residues from Sydney_2012 reconstituted epitopes from ancestral GII.4 variants. Overall, this study demonstrates that, despite circulating for over a decade, Sydney_2012 viruses present minimal antigenic diversification and provides novel insights on the diversification of GII.4 noroviruses that could inform vaccine design. IMPORTANCE GII.4 noroviruses are the major cause of acute gastroenteritis in all age groups. This predominance has been attributed to the continued emergence of phylogenetically discrete variants that escape immune responses to previous infections. The last GII.4 variant to emerge, Sydney_2012, has been circulating at high incidence for over a decade, raising the question of whether this variant is undergoing antigenic diversification without presenting a major distinction at the phylogenetic level. Sequence analyses that include >1400 capsid sequences from GII.4 Sydney_2012 showed changes in 4 out of the 6 major antigenic sites. Notably, while changes were detected in one of the most immunodominant sites over time, these resulted in minimal changes in the antigenic profile of these viruses. This study provides new insights on the mechanism governing the antigenic diversification of GII.4 norovirus that could help in the development of cross-protective vaccines to human noroviruses.

The genetic characterization of archival specimens is important for evaluating the evolutionary processes of noroviruses. Complete viral genome sequences, GVIII.1[GII.P28] and GIX.1[GII.P15], were determined from two archival specimens collected in Tokyo, Japan, in 1986 and 1995. In addition, complete VP1 and partial RdRp sequences of four samples collected between 1975 and 1983 were determined. Two viruses were classified as GI.5[P5] and GI.9[P9]; however, the viruses from the other two samples could not be assigned to any known genotypes using norovirus typing tools and phylogenetic analysis, suggesting that they might be untypable genotypes. Further evolutionary analysis of these viruses is warranted.

Human noroviruses are the most common viral agents of acute gastroenteritis. Recently, human intestinal enteroids were shown to be permissive for norovirus infection. We tested their suitability as a system to study norovirus neutralization. Hyperimmune sera raised against virus-like particles (VLPs) representing different genotypes showed highly specific neutralization activity against GII.4 and GII.6 noroviruses. Carbohydrate blocking assays and neutralization exhibited similar patterns in antibody responses. Notably, sera produced against chimeric VLPs that presented swapped structural shell and protruding (P) domains, from different genotypes showed that neutralization is primarily mediated by antibodies mapping to the P domain of the norovirus capsid protein. This study provides empirical information on the antigenic differences among genotypes as measured by neutralization, which could guide vaccine design.

A paradigm of RNA viruses is their ability to mutate and escape from herd immunity. Because antibody responses are a major effector for viral immunity, antigenic sites are usually under strong diversifying pressure. Here, we use norovirus as a model to study mechanisms of antigenic diversification of non-enveloped, fast-evolving RNA viruses. We comprehensively characterize all variable antigenic sites involved in virus neutralization and find that single neutralizing monoclonal antibodies (mAbs) map to multiple antigenic sites of GII.4 norovirus. Interactions of multiple epitopes on the viral capsid surface provide a broad mAb-binding repertoire with a remarkable difference in the mAb-binding profiles and immunodominance hierarchy for two distantly related GII.4 variants. Time-ordered mutant viruses confirm a progressive change of antibody immunodominance along with point mutations during the process of norovirus evolution. Thus, in addition to point mutations, switches in immunodominance that redirect immune responses could facilitate immune escape in RNA viruses.

Norovirus is a major cause of acute gastroenteritis worldwide. Over 30 different genotypes, mostly from genogroup I (GI) and II (GII), have been shown to infect humans. Despite three decades of genome sequencing, our understanding of the role of genomic diversification across continents and time is incomplete. To close the spatiotemporal gap of genomic information of human noroviruses, we conducted a large-scale genome-wide analyses that included the nearly full-length sequencing of 281 archival viruses circulating since the 1970s in over 10 countries from four continents, with a major emphasis on norovirus genotypes that are currently underrepresented in public genome databases. We provided new genome information for 24 distinct genotypes, including the oldest genome information from 12 norovirus genotypes. Analyses of this new genomic information, together with those publicly available, showed that (i) noroviruses evolve at similar rates across genomic regions and genotypes; (ii) emerging viruses evolved from transiently-circulating intermediate viruses; (iii) diversifying selection on the VP1 protein was recorded in genotypes with multiple variants; (iv) non-structural proteins showed a similar branching on their phylogenetic trees; and (v) contrary to the current understanding, there are restrictions on the ability to recombine different genomic regions, which results in co-circulating populations of viruses evolving independently in human communities. This study provides a comprehensive genetic analysis of diverse norovirus genotypes and the role of non-structural proteins on viral diversification, shedding new light on the mechanisms of norovirus evolution and transmission., Author summary Norovirus is a highly diverse enteric pathogen. The large genomic database accumulated in the last three decades advanced our understanding of norovirus diversity; however, this information is limited by geographical bias, sporadic times of collection, and missing or incomplete genome sequences. In this multinational collaborative study, we mined archival samples collected since the 1970s and sequenced nearly full-length new genomes from 281 historical noroviruses, including the first full-length genomic sequences for three genotypes. Using this novel dataset, we found evidence for restrictions in the recombination of genetically disparate viruses and that diversifying selection results in new variants with different epidemiological profiles. These new insights on the diversification of noroviruses could provide baseline information for the study of future epidemics and ultimately the prevention of norovirus infections.

Significance The GII.4 genotype of human noroviruses accounts for the majority of gastroenteritis outbreaks worldwide. This predominance is characterized by the chronological emergence of new variants in response to immune pressure. We performed a comprehensive analysis on a large panel of GII.4 viruses to investigate the genetic determinants of antigenic diversification. Using immunoassays and neutralization data, we observed major changes to antigenic properties over the course of GII.4 evolution and increased intervariant cross-reactivity across contemporary viruses. Antigenic cartography and sequence analyses indicated a minimum number of coevolving amino acid changes on the structural protein necessary for the emergence of antigenically distinct variants. These insights could facilitate the monitoring and characterization of emerging GII.4 noroviruses and the development of cross-protective vaccines., Noroviruses are the predominant cause of acute gastroenteritis, with a single genotype (GII.4) responsible for the majority of infections. This prevalence is characterized by the periodic emergence of new variants that present substitutions at antigenic sites of the major structural protein (VP1), facilitating escape from herd immunity. Notably, the contribution of intravariant mutations to changes in antigenic properties is unknown. We performed a comprehensive antigenic analysis on a virus-like particle panel representing major chronological GII.4 variants to investigate diversification at the inter- and intravariant level. Immunoassays, neutralization data, and cartography analyses showed antigenic similarities between phylogenetically related variants, with major switches to antigenic properties observed over the evolution of GII.4 variants. Genetic analysis indicated that multiple coevolving amino acid changes—primarily at antigenic sites—are associated with the antigenic diversification of GII.4 variants. These data highlight complexities of the genetic determinants and provide a framework for the antigenic characterization of emerging GII.4 noroviruses.

Human noroviruses are the most common viral cause of acute gastroenteritis worldwide. Currently, there are no approved vaccines or specific therapeutics to treat the disease. Some obstacles delaying the development of a norovirus vaccine are: (i) the extreme diversity presented by noroviruses; (ii) our incomplete understanding of immunity to noroviruses; and (iii) the lack of a robust cell culture system or animal model for human noroviruses. Recent advances in in vitro cultivation of norovirus, novel approaches applied to viral genomics and immunity, and completion of vaccine trials and birth cohort studies have provided new information toward a better understanding of norovirus immunity. Here, we will discuss the complex relationship between norovirus diversity and correlates of protection for human noroviruses, and how this information could be used to guide the development of cross-protective vaccines.

Norovirus is a major cause of acute gastroenteritis. Human noroviruses present >30 different genotypes, with a single genotype (GII.4) predominating worldwide. Concurrent outbreaks of norovirus are often associated with the emergence of new viruses. While different hypotheses have been presented, the source of new mutations in noroviruses is still unknown. In this study, we applied high-resolution sequencing to determine the intra-host viral diversity presented by noroviruses during the acute and shedding phase of infection in children. Profiling viral intra-host diversification at nearly full genome level indicated that GII.4 viruses presented dynamic intra-host variation, while non-GII.4 viruses presented minimal variation throughout the infection. Notably, the intra-host genetic variation during the shedding phase recapitulates the genetic diversity observed at the global level, particularly those mapping at the VP1 antigenic sites. Thus the intra-host evolution in healthy children explains the source of norovirus mutations that results in diversification at the global scale.

We report multiple nontypeable genotype II noroviruses circulating in South America; nucleotides differed by >25% from those of other genotypes. These viruses have been circulating in the Americas for ≈20 years and show recombination with other genotypes. Clues to norovirus natural history can guide development of treatment and prevention plans.

Background: Norovirus (NV) causes acute gastroenteritis in infants. Humoral and fecal immunoglobulin A (IgA) responses have been correlated with protection against NV; however, the role of breast milk IgA against NV infection and associated diarrhea is still unknown. This study aimed to evaluate the protective role of NV-specific IgA in breast milk. Methods: We analyzed 95 breast milk samples collected from mothers enrolled in a birth cohort study conducted in Peru during 2016–2017. Samples were collected before the first NV infection of infants and tested for total IgA and NV-specific IgA using virus-like particles (VLPs) that represented GII·4 variants and non-GII·4 genotypes. Norovirus infection in infants was assessed by testing their stool samples for NV via real-time PCR. Breast milk samples were grouped according to NV infection and diarrheal status of infants: NV positive with diarrhea (NV+D+, n=18); NV positive without diarrhea (NV+D-, n=37); and NV negative without diarrhea (NV-D-, n=40). The percent positivity and titer of NV-specific IgA (Log10) were compared among groups. Findings: Breast milk IgA had a high percent positivity against different NV VLPs, especially against GII (89–100%). Higher median titer of NV-specific IgA was observed in NV+D- when compared to the NV+D+ group against GII·4 Sydney (NV+D+:500 vs. NV+D-:100, p=0·05) and GI·1 (NV+D+:100 vs. NV+D-:50, p=0·01). Although the median titer of NV IgA against GI·9 was similar, NV+D- had higher distribution of titers when compared to NV+D+ group (NV+D-:100 [interquartile range (IQR):100–500] vs. NV+D+:100 [IQR:50–200], p=0·03). Interpretations: Breast milk IgA highly reacted to different NV VLPs. NV-specific IgA may protect infants from having NV-associated diarrhea, but not from NV infection, thus reducing NV symptoms. Funding Statement: National Institute of Allergy and Infectious Diseases, National Institute of Health: 1R01AI108695–01A1 and the Japan Society for the Promotion of Science (Fostering Joint International Research B): 19KK0241 Declaration of Interests: We declare that we have no conflicts of interest. Ethics Approval Statement: The mothers included in this study were selected from a larger birth cohort study carried out in Peru during 2016-2017. The birth cohort study was approved by the Institutional Review Boards of Asociacion Benefica PRISMA, Universidad Peruana Cayetano Heredia, Johns Hopkins University, University of California-San Francisco. Informed consent were obtained from the mothers or caretakers of the infants. The analysis of archived specimen was approved by the ethics committee of the Tohoku University, Graduate School of Medicine.

Noroviruses are an important cause of viral gastroenteritis around the world. An obstacle delaying the development of norovirus vaccines is inadequate understanding of the role of norovirus diversity in immunity. Using a population genomics approach, we identified new residues on the viral capsid protein (VP1) from GII.4 noroviruses, the predominant genotype, that appear to be involved in the emergence and antigenic topology of GII.4 variants. Careful monitoring of the substitutions in those residues involved in the diversification and emergence of new viruses could help in the early detection of future novel variants with pandemic potential. Therefore, this novel information on the antigenic diversification could facilitate GII.4 norovirus vaccine design., GII.4 noroviruses are a major cause of acute gastroenteritis. Their dominance has been partially explained by the continuous emergence of antigenically distinct variants. To gain insights into the mechanisms of viral emergence and population dynamics of GII.4 noroviruses, we performed large-scale genomics, structural, and mutational analyses of the viral capsid protein (VP1). GII.4 noroviruses exhibited a periodic replacement of predominant variants with accumulation of amino acid substitutions. Genomic analyses revealed (i) a large proportion (87%) of conserved residues; (ii) variable residues that map on the previously determined antigenic sites; and (iii) variable residues that map outside the antigenic sites. Residues in the third pattern category formed motifs on the surface of VP1, which suggested extensions of previously predicted and new uncharacterized antigenic sites. The role of two motifs (C and G) in the antigenic makeup of the GII.4 capsid protein was confirmed with monoclonal antibodies and carbohydrate blocking assays. Amino acid profiles from antigenic sites (A, C, D, E, and G) correlated with the circulation patterns of GII.4 variants, with three of them (A, C, and G) containing residues (352, 357, 368, and 378) linked with the diversifying selective pressure on the emergence of new GII.4 variants. Notably, the emergence of each variant was followed by stochastic diversification with minimal changes that did not progress toward the next variant. This report provides a methodological framework for antigenic characterization of viruses and expands our understanding of the dynamics of GII.4 noroviruses and could facilitate the design of cross-reactive vaccines.

Noroviruses are important human enteric pathogens and monitoring their genetic diversity is important for epidemiological surveillance, vaccine development, and understanding of RNA viruses evolution. Epidemiological investigations have revealed that genogroup II, genotype 6 noroviruses (GII.6) are common agents of gastroenteritis. Upon sequencing of the ORF2 (encoding the viral capsid), GII.6 viruses have been distinguished into three variants. Sentinel hospital-based surveillance in Italy revealed that GII.6 noroviruses were the second most common capsid genotype in 2015, mostly in association with a GII.P7 ORF1 (encoding the viral polymerase). Upon molecular characterization of the ORF1 and ORF2, the GII.P7_GII.6 epidemic viruses circulating in 2014–2015 (variant GII.6b) were different from those that circulated sporadically in 2011–2013 (variant GII.6a). Analysis of the ORF1 (GII.P7) and ORF2 (GII.6) sequences available in the databases unveiled marked genetic diversity and peculiarities in the phylogenetic segregation patterns, suggesting multiple recombination events. Phylogenetic analyses suggest that recent GII.P7_GII.6b viruses were circulating as early as 2008, and formed a genetically homogenous group that emerged globally.

Norovirus is a leading etiological agent of viral gastroenteritis. Because of relatively mild disease symptoms and frequent asymptomatic infections, information on the ecology of this virus is limited. Our objective was to examine the genetic diversity of norovirus circulating in the human population by means of genotyping the virus in municipal wastewater. We investigated norovirus genogroups I and II (GI and GII) in municipal wastewater in Japan by pyrosequencing and quantitative PCR (qPCR) from November 2012 to March 2013. Virological surveillance for gastroenteritis cases was concurrently conducted in the same area. A total of fourteen distinct genotypes in total (GI.1, 3, 4, 6, 7, GII.2, 4, 5, 6, 7, 12, 13, 14, and 17), with up to eight genotypes detected per sample, were observed in wastewater using pyrosequencing; only four genotypes (GI.6, GII.4, 5, and 14) were obtained from clinical samples. Seventy-eight percent of norovirus-positive stool samples contained GII.4, but this genotype was not dominant in wastewater. The norovirus GII.4 Sydney 2012 variant, which appeared and spread during our study period, was detected in both the wastewater and clinical samples. These results suggest that an environmental approach using pyrosequencing yields a more detailed distribution of norovirus genotypes/variants. Thus, wastewater monitoring by pyrosequencing is expected to provide an effective analysis of the distribution of norovirus genotypes causing symptomatic and asymptomatic infections in human populations.

Rabies is endemic in the Philippines and dog bites are a major cause of rabies cases in humans. The rabies control program has not been successful in eliminating rabies because of low vaccination coverage among dogs. Therefore, more effective and feasible strategies for rabies control are urgently required in the country. To control rabies, it is very important to know if inter-island transmission can occur because rabies can become endemic once the virus is introduced in areas that previously had no reported cases. Our molecular epidemiological study suggests that inter-island transmission events can occur; therefore, we further investigated these inter-island transmission using phylogenetic and modeling approaches.We investigate inter-island transmission between Luzon and Tablas Islands in the Philippines. Phylogenetic analysis and mathematical modeling demonstrate that there was a time lag of several months to a year from rabies introduction to initial case detection, indicating the difficulties in recognizing the initial rabies introductory event. There had been no rabies cases reported in Tablas Island; however, transmission chain was sustained on this island after the introduction of rabies virus because of low vaccination coverage among dogs.Across the islands, a rabies control program should include control of inter-island dog transportation and rabies vaccination to avoid viral introduction from the outside and to break transmission chains after viral introduction. However, this program has not yet been completely implemented and transmission chains following inter-island virus transmission are still observed. Local government units try to control dog transport; however, it should be more strictly controlled, and a continuous rabies control program should be implemented to prevent rabies spread even in rabies-free areas.

Noroviruses are extremely diverse, with ≥30 genotypes infecting humans. GII genotype 4 (GII.4) noroviruses, the most prevalent genotype, present a constant accumulation of mutations on the major capsid protein (VP1), resulting in the chronological emergence of new variants every 2-8 years. On the other hand, non-GII.4 noroviruses present a limited number of changes on the capsid protein over time. Despite limited diversification, non-GII.4 viruses can also be associated with large outbreaks. To gain insights into the evolutionary dynamics of non-GII.4 viruses, we performed variant-specific phylogenetic analyses on a comprehensive dataset of 13 genotypes. Although the genotypes with a single variant presented a linear (clock-like) evolution, maximum-likelihood analyses revealed a lack of clock-like signals for the genotypes with ≥3 variants: GI.3, GII.6 and GII.17. Notably, the evolutionary pattern of non-GII.4 viruses showed clock-like signals when each variant was analysed separately. A minimal impact on the long-term clock-like evolution of VP1 was detected due to the exchange (recombination) of the polymerase types. The linear evolution, without replacement among variants, is explained by minimal changes at the protein level due to the higher ratio of synonymous compared to non-synonymous substitutions in their evolution. Taken together, these data indicate that (i) the variants of non-GII.4 noroviruses evolve and persist in the population independently, probably due to strong evolutionary constraints on VP1, and (ii) variant-specific analyses with robust sequence databases that cover long periods of surveillance are needed to limit the potential for misinterpretation of the evolutionary dynamics of non-GII.4 noroviruses.

Norovirus, a leading cause of acute gastroenteritis in humans, is a highly diverse virus. Here, we report the complete genome sequence of a nontypeable genogroup II (GII) norovirus that was detected in a symptomatic Peruvian child in 2008. This virus showed low nucleotide sequence identities (≤82%) against all known genotypes.

Sewage samples have been investigated to study the norovirus concentrations in sewage or the genotypes of noroviruses circulating in human populations. However, the statistical relationship between the concentration of the virus and the number of infected individuals and the clinical importance of genotypes or strains detected in sewage are unclear. In this study, we carried out both environmental and clinical surveillance of noroviruses for 3 years, 2013 to 2016. We performed cross-correlation analysis of the concentrations of norovirus GI or GII in sewage samples collected weekly and the reported number of gastroenteritis cases. Norovirus genotypes in sewage were also analyzed by pyrosequencing and compared with those identified in stool samples. The cross-correlation analysis found the peak coefficient ( R = 0.51) at a lag of zero, indicating that the variation in the GII concentration, expressed as the log 10 number of copies per milliliter, was coincident with that in the gastroenteritis cases. A total of 15 norovirus genotypes and up to 8 genotypes per sample were detected in sewage, which included all of the 13 genotypes identified in the stool samples except 2. GII.4 was most frequently detected in both sample types, followed by GII.17. Phylogenetic analysis revealed that a strain belonging to the GII.17 Kawasaki 2014 lineage had been introduced into the study area in the 2012-2013 season. An increase in GI.3 cases was observed in the 2015-2016 season, and sewage monitoring identified the presence of GI.3 in the previous season (2014-2015). Our results demonstrated that monitoring of noroviruses in sewage is useful for sensitive detection of epidemic variants in human populations. IMPORTANCE We obtained statistical evidence of the relationship between the variation in the norovirus GII concentration in sewage and that of gastroenteritis cases during the 3-year study period. Sewage sample analysis by a pyrosequencing approach enabled us to understand the temporal variation in the norovirus genotypes circulating in human populations. We found that a strain closely related to the GII.17 Kawasaki 2014 lineage had been introduced into the study area at least 1 year before its appearance and identification in clinical cases. A similar pattern was observed for GI.3; cases were reported in the 2015-2016 season, and closely related strains were found in sewage in the previous season. Our observation indicates that monitoring of noroviruses in sewage is useful for the rapid detection of an epidemic and is also sensitive enough to study the molecular epidemiology of noroviruses. Applying this approach to other enteric pathogens in sewage will enhance our understanding of their ecology.

Two novel G3P[4] rotavirus strains were detected from children with acute diarrhea in Sendai, Japan, identified as a G3–P[4]–I2–R2–C2–M2–A2–N2–T2–E2–H2 genotype constellation by whole-genome sequence analysis. The VP7 gene of the two strains displayed the highest nucleotide sequence identity (91 %) and showed a close genetic relationship (99 % bootstrap value) to an equine rotavirus reported in India. The other gene segments were related to human group A rotaviruses. This report suggests a possible reassortment event between human and equine rotaviruses. Electronic supplementary material The online version of this article (doi:10.1007/s11262-014-1135-z) contains supplementary material, which is available to authorized users.

Mongolian Academy of Medical Sciences, Ulaanbaatar, Mongolia(Received December 27, 2011. Accepted April 16, 2012)SUMMARY: Human adenoviruses (HAdVs) are responsible for approximately 5z–10z of acuterespiratory infections. The serotypes of commonly detected respiratory HAdV in Asian countries arediverse. However, there are no well-documented reports of circulating HAdV serotypes in Mongolia.Between January 2010 and May 2011, 1,950 influenza-negative samples from patients with influenza-like illness, including eye swabs from patients with eye symptoms, were screened for HAdV, and 40samples (2.1z) were positive for HAdVs. Among these 40 samples, 31 samples were positive for thehexon gene used in phylogenetic analysis, as determined by PCR. We identified 7 different serotypes.We constructed the phylogenetic trees of HAdV-B7 and HAdV-B3, the 2 most commonly detected sero-types in this study. All detected HAdV-B7 and -B3 Mongolian strains had identical sequences. HAdV-D8, known to be associated with epidemic keratoconjunctivitis (EKC), was detected from nasopharyn-geal and eye swabs. There was no difference between the amino acid sequences of the hexon and fibergenes that may affect tissue tropism in Mongolian strains and those in EKC-causing strains.INTRODUCTIONHuman adenoviruses (HAdVs) are non-enveloped,double-stranded DNA viruses belonging to the genusMastadenovirusand the family Adenoviridae (1). Thereare 52 serotypes that have been classified into 7 species(A–G) (2,3). Recently, full genome sequencing ofHAdVs was performed, and some novel HAdVs(HAdV-D53, -D54, -B55, -D56, -C57, and -D58) werediscovered through phylogenetic analysis (4–9). HAdVscause variable serotype-specific diseases, such as acuterespiratory infection, conjunctivitis, and gastroenteri-tis, and are responsible for approximately 5z–10z ofacute respiratory infections (1). HAdV-C1, -C2, -B3,-E4, -C5, -C6, and -C7 are the most common serotypesthat cause respiratory infections while HAdV-D8 is theone of the major causes of epidemic keratoconjunctivi-tis (EKC) (1).The circulating respiratory HAdV serotypes varyboth geographically and temporally among Asian coun-tries. For example, HAdV-B3 and -B7 were the mostcommonly encountered serotypes in surveillance con-ducted from 1991 to 2007 in Korea (10), and HAdV-C2and -B3 were common in Japan between 2000 and 2007(11). HAdV-C, especially HAdV-C1 and -C2, werecommon in Malaysia between 1999 and 2005 (12),whereas HAdV-B3 was common in Taiwan in 2004 and2005 (13). However, there are no available data on cur-rently circulating HAdV serotypes in Mongolia. Be-tween January 2010 and May 2011, HAdVs were detect-ed in patients with influenza-like illness (ILI), andphylogenetic analysis of the hexon gene from theseHAdVs was performed to determine which serotypeswere circulating in Mongolia. To our knowledge, this isthe first report describing the serotypes of HAdVs inMongolia.MATERIALS AND METHODSFrom January 2010 to May 2011, a total of 6,774nasopharyngeal or eye swabs were collected frompatients who visited outpatient clinics and hospitals inMongolia and were clinically diagnosed with ILI within3 days of disease onset. ILI was defined as sudden onsetof fever (A38.09C) with cough or sore throat in the ab-sence of other diagnoses. Nasopharyngeal swabs werecollected from patients with ILI whereas eye swabs werecollected from patients with ILI and eye symptoms,such as tear shedding, eyelid swelling, and festering.The swabs were placed into 2 ml of Hanks (－) balancedsalt solution (pH 7.2) with bovine serum albumin(0.5z), penicillin (500 U/ml), streptomycin (500mg/ml), and amphotericin B (Fungizone) (2.5mg/ml).The specimens were transported under refrigerationwith ice packs to theNational Influenza Center, Nation-al Center of Communicable Diseases in Ulaanbaatar wi-thin 48 h of collection.At least 22 samples in each month were randomlyselected from among those samples that were collectedfrom patients o4yearsorA60 years of age and testednegative for influenza virus using real-time reverse tran-scription polymerase chain reaction (rt-RT PCR) (14).Selected samples were screened for HAdVs either using

Selecting the best quantitative PCR assay is essential to detect human norovirus genome effectively from clinical and environmental samples because no cell lines have been developed to propagate this virus. The real-time PCR methods for noroviruses GI (4 assays) and GII (3 assays) were evaluated using wastewater (n = 70) and norovirus-positive stool (n = 77) samples collected in Japan between 2012 and 2013. Standard quantitative PCR assays recommended by the U.S. Environmental Protection Agency, International Organization for Standardization, and Ministry of Health, Labour and Welfare, Japan, together with recently reported assays were included. Significant differences in positive rates and quantification cycles were observed by non-parametric analysis. The present study identifies the best assay for norovirus GI and GII to amplify norovirus genomes efficiently.

Pneumonia is a leading cause of deaths in infants and young children in developing countries, including the Philippines. However, data at the community level remains limited. Our study aimed to estimate incidence and mortality rates and to evaluate risk factors and health-seeking behavior for childhood pneumonia. A household level interview survey was conducted in Biliran Island, the Philippines. Caregivers were interviewed using a semi-structured questionnaire to check if children had symptoms suggesting pneumonia-like episodes from June 2011 to May 2012. Of 3,327 households visited in total, 3,302 (99.2%) agreed to participate, and 5,249 children less than 5 years of age were included in the study. Incidence rates of pneumonia-like episodes, severe pneumonia-like episodes, and pneumonia-associated mortality were 105, 61, and 0.9 per 1,000 person-years, respectively. History of asthma [hazard ratio (HR): 5.85, 95% confidence interval (CI): 4.83–7.08], low socioeconomic status (SES) (HR: 1.11, 95% CI: 1.02–1.20), and long travel time to the healthcare facility estimated by cost distance analysis (HR: 1.32, 95% CI: 1.09–1.61) were significantly associated with the occurrence of pneumonia-like episodes by the Cox proportional hazards model. For severe pneumonia-like episodes, a history of asthma (HR: 8.39, 95% CI: 6.54–10.77) and low SES (HR: 1.30, 95% CI: 1.17–1.45) were significant risk factors. Children who had a long travel time to the hospital were less likely to seek hospital care (Odds ratio: 0.32, 95% CI: 0.19–0.54) when they experienced severe pneumonia-like episodes. Incidence of pediatric pneumonia-like episodes was associated with a history of asthma, SES, and the travel time to healthcare facilities. Travel time was also identified as a strong indicator for health-seeking behavior. Improved access to healthcare facilities is important for early and effective management. Further studies are warranted to understand the causal relationship between asthma and pneumonia.

Rabies still remains a public health threat in the Philippines. A significant number of human rabies cases, about 200–300 cases annually, have been reported, and the country needs an effective strategy for rabies control. To develop an effective control strategy, it is important to understand the transmission patterns of the rabies viruses. We conducted phylogenetic analyses by considering the temporal and spatial evolution of rabies viruses to reveal the transmission dynamics in the Philippines.After evaluating the molecular clock and phylogeographic analysis, we estimated that the Philippine strains were introduced from China around the beginning of 20th century. Upon this introduction, the rabies viruses evolved within the Philippines to form three major clades, and there was no indication of introduction of other rabies viruses from any other country. However, within the Philippines, island-to-island migrations were observed. Since then, the rabies viruses have diffused and only evolved within each island group.The evolutionary pattern of these viruses was strongly shaped by geographical boundaries. The association index statistics demonstrated a strong spatial structure within the island group, indicating that the seas were a significant geographical barrier for viral dispersal. Strong spatial structure was also observed even at a regional level, and most of the viral migrations (79.7% of the total median number) in Luzon were observed between neighboring regions.Rabies viruses were genetically clustered at a regional level, and this strong spatial structure suggests a geographical clustering of transmission chains and the potential effectiveness of rabies control that targets geographical clustering. Dog vaccination campaigns have been conducted independently by local governments in the Philippines, but it could be more effective to implement a coordinated vaccination campaign among neighboring areas to eliminate geographically-clustered rabies transmission chains.

Influenza has a significant impact on public health when a natural disaster occurs during the influenza season. However, the epidemiological characteristics of influenza following natural disasters have not been well documented due to the difficulty of implementing laboratory-based influenza surveillance in such situations. The Great East Japan Earthquake occurred on March 11, 2011, when influenza was already circulating. Since routine influenza surveillance was not performed in Miyagi Prefecture, we initiated an ad hoc laboratory-based monitoring system immediately after the earthquake. From March 15 to May 19, we tested 277 samples for influenza virus collected around Sendai City and from evacuation centers in Miyagi Prefecture. Influenza A (H3N2) was detected in 112 cases, influenza A (H1N1) 2009 in one case, and influenza B in 92 cases. The H3N2 virus was dominant until the 14th week. However, a sudden increase in the number of influenza B cases occurred after schools were reopened. According to phylogenetic analysis, a major clade switch of the H3N2 virus took place after the earthquake. The Yamagata lineage of influenza B was detected in one patient from western Japan, indicating the importing of viruses into the affected area.

Background Rabies continues to be a major public health problem in the Philippines, where 200–300 human cases were reported annually between 2001 and 2011. Understanding the phylogeography of rabies viruses is important for establishing a more effective and feasible control strategy. Methods We performed a molecular analysis of rabies viruses in the Philippines using rabied animal brain samples. The samples were collected from 11 of 17 regions, which covered three island groups (Luzon, Visayas, and Mindanao). Partial nucleoprotein (N) gene sequencing was performed on 57 samples and complete glycoprotein (G) gene sequencing was performed on 235 samples collected between 2004 and 2010. Results The Philippine strains of rabies viruses were included in a distinct phylogenetic cluster, previously named Asian 2b, which appeared to have diverged from the Chinese strain named Asian 2a. The Philippine strains were further divided into three major clades, which were found exclusively in different island groups: clades L, V, and M in Luzon, Visayas, and Mindanao, respectively. Clade L was subdivided into nine subclades (L1–L9) and clade V was subdivided into two subclades (V1 and V2). With a few exceptions, most strains in each subclade were distributed in specific geographic areas. There were also four strains that were divided into two genogroups but were not classified into any of the three major clades, and all four strains were found in the island group of Luzon. Conclusion We detected three major clades and two distinct genogroups of rabies viruses in the Philippines. Our data suggest that viruses of each clade and subclade evolved independently in each area without frequent introduction into other areas. An important implication of these data is that geographically targeted dog vaccination using the island group approach may effectively control rabies in the Philippines., Author Summary Rabies continues to be a major public health problem in the Philippines. We conducted a molecular epidemiological study of rabies using the complete glycoprotein (G) gene from 235 animal brain samples collected in the Philippines between 2004 and 2010. We identified three major clades and two distinct genogroups in the Philippines. The three major clades L, V, and M were found specifically in the Luzon, Visayas, and Mindanao island groups, respectively. Additionally, two minor genogroups were located in the Luzon island group. These data suggest that although human mediated transmission may have occurred, these virus clades evolved independently after a single introduction into each island group. All of the analyzed Philippine strains were clustered into Asian 2b, which diverged from the Chinese strain Asian 2a. No recent introduction of rabies into the Philippines from other countries was apparent. The elimination of rabies by 2020 is a national goal in the Philippines, necessitating urgent development of a more effective and feasible strategy for controlling rabies. Our findings indicate that a geographically targeted dog vaccination campaign may effectively control rabies in island nations such as the Philippines.

Jasmonates are ubiquitously occurring plant growth regulators with high structural diversity that mediate numerous developmental processes and stress responses. We have recently identified 12-O-β-d-glucopyranosyljasmonic acid as the bioactive metabolite, leaf-closing factor (LCF), which induced nyctinastic leaf closure of Samanea saman. We demonstrate that leaf closure of isolated Samanea pinnae is induced upon stereospecific recognition of (−)-LCF, but not by its enantiomer, (+)-ent-LCF, and that the nonglucosylated derivative, (−)-12-hydroxyjasmonic acid also displays weak activity. Similarly, rapid and cell type-specific shrinkage of extensor motor cell protoplasts was selectively initiated upon treatment with (−)-LCF, whereas flexor motor cell protoplasts did not respond. In these bioassays related to leaf movement, all other jasmonates tested were inactive, including jasmonic acid (JA) and the potent derivates JA-isoleucine and coronatine. By contrast, (−)-LCF and (−)-12-hydroxyjasmonic acid were completely inactive with respect to activation of typical JA responses, such as induction of JA-responsive genes LOX2 and OPCL1 in Arabidopsis (Arabidopsis thaliana) or accumulation of plant volatile organic compounds in S. saman and lima bean (Phaseolus lunatus), generally considered to be mediated by JA-isoleucine in a COI1-dependent fashion. Furthermore, application of selective inhibitors indicated that leaf movement in S. saman is mediated by rapid potassium fluxes initiated by opening of potassium-permeable channels. Collectively, our data point to the existence of at least two separate JA signaling pathways in S. saman and that 12-O-β-d-glucopyranosyljasmonic acid exerts its leaf-closing activity through a mechanism independent of the COI1-JAZ module.

Analyzing the evolutionary pattern of the influenza A(H1N1)pdm09 strain in different regions is important for understanding its diversification. We therefore conducted this study to elucidate the genetic variability and molecular evolution of the influenza A(H1N1)pdm09 strains that circulated during the 2009–2010 and 2010–2011 influenza seasons in Sendai, Japan. Nasopharyngeal swab specimens were collected from patients with influenza-like illnesses who visited outpatient clinics in Sendai City, Japan, from September 2009 to April 2011. A total of 75 isolates were selected from September 2009 to April 2011 to analyze the genetic changes in the entire hemagglutinin 1 (HA1) segment of the HA gene and the neuraminidase (NA) gene based on sequence analysis. Bayesian coalescent Markov chain Monte Carlo analyses of HA1 and NA gene sequences were performed for further analysis. High sequence identities were observed for HA1 and NA in influenza A(H1N1)pdm09, displaying 99.06 and 99.33 % nucleotide identities, respectively, with the A(H1N1)pdm09 vaccine strain A/California/07/2009. The substitution rates of nucleotides for HA1 in the 2009–2010 and 2010–2011 were 1.5 × 10−3 and 1.6 × 10−3 substitutions per site per year, respectively. Phylogenetic tree analysis demonstrated that Sendai isolates were clustered into global clade 7, which is characterized by an S203T mutation in the HA1 gene. Moreover, two distinct circulation clusters were present in the 2010–2011 season. Mutations were present in antigenic or receptor-binding domains of the HA1 segment, including A141V, S143G, S183P, S185T, and S203T. The Bayesian skyline plot model illustrated a steady rate for the maintenance of genetic diversity, followed by a slight increase in the later part of the 2010–2011 season. Selection analysis revealed that the HA1 (position 197) and NA (position 46) sites were under positive selection; however, no known mutation conferring resistance to NA inhibitors such as H275Y was observed. The effect on control of the influenza A(H1N1)pdm09 virus, including vaccine strain selection, requires continuous monitoring of the strain by genetic surveillance. Electronic supplementary material The online version of this article (doi:10.1007/s11262-013-0980-5) contains supplementary material, which is available to authorized users.

Background The Great East Japan Earthquake of magnitude 9.0 that struck on 11 March 2011 resulted in more than 18000 deaths or cases of missing persons. The large-scale tsunami that followed the earthquake devastated many coastal areas of the Tohoku region, including Miyagi Prefecture, and many residents of the tsunami-affected areas were compelled to reside in evacuation centres (ECs). In Japan, seasonal influenza epidemics usually occur between December and March. At the time of the Great East Japan Earthquake on 11 March 2011, influenza A (H3N2) was still circulating and there was a heightened concern regarding severe outbreaks due to influenza A (H3N2). Methods After local hospital staff and public health nurses detected influenza cases among the evacuees, an outbreak investigation was conducted in five ECs that had reported at least one influenza case from 23 March to 11 April 2011. Cases were confirmed by point-of-care tests and those residues were obtained and subjected to reverse transcription PCR and/or real time RT-PCR for sub-typing of influenza. Results There were 105 confirmed cases detected during the study period with a mean attack rate of 5.3% (range, 0.8%–11.1%). An epidemiological tree for two ECs demonstrated same-room and familial links that accounted for 88.5% of cases. The majority of cases occurred in those aged 15-64 years, who were likely to have engaged in search and rescue activities. No deaths were reported in this outbreak. Familial link accounted for on average 40.5% of influenza cases in two ECs and rooms where two or more cases were reported accounted for on average 85% in those ECs. A combination of preventative measures, including case cohorting, personal hygiene, wearing masks, and early detection and treatment, were implemented during the outbreak period. Conclusions Influenza can cause outbreaks in a disaster setting when the disaster occurs during an epidemic influenza season. The transmission route is more likely to be associated with sharing room and space and with familial links. The importance of influenza surveillance and early treatments should be emphasized in EC settings for implementing preventive control measures.