First complete genome sequences of genogroup V, genotype 3 porcine sapoviruses: common 5'-terminal genomic feature of sapoviruses.

Sapoviruses (SaVs) are enteric viruses and have been detected in various mammals. They are divided into multiple genogroups and genotypes based on the entire major capsid protein (VP1) encoding region sequences. In this study, we determined the first complete genome sequences of two genogroup V, genotype 3 (GV.3) SaV strains detected from swine fecal samples, in combination with Illumina MiSeq sequencing of the libraries prepared from viral RNA and PCR products. The lengths of the viral genome (7494 nucleotides [nt] excluding polyA tail) and short 5'-untranslated region (14 nt) as well as two predicted open reading frames are similar to those of other SaVs. The amino acid differences between the two porcine SaVs are most frequent in the central region of the VP1-encoding region. A stem-loop structure which was predicted in the first 41 nt of the 5'-terminal region of GV.3 SaVs and the other available complete genome sequences of SaVs may have a critical role in viral genome replication. Our study provides complete genome sequences of rarely reported GV.3 SaV strains and highlights the common 5'-terminal genomic feature of SaVs detected from different mammalian species.

A False Positive Dengue Fever Rapid Diagnostic Test Result in a Case of Acute Parvovirus B19 Infection.

An outbreak of dengue fever occurred in Japan in August 2014. We herein report the case of a 63-year-old man who presented with a persistent fever in September 2014. Acute parvovirus B19 infection led to a false positive finding of dengue fever on a rapid diagnostic test (Panbio Dengue Duo Cassette(TM)). To the best of our knowledge, there are no previous reports of a false positive result for dengue IgM with the dengue rapid diagnostic test. We believe that epidemiological information on the prevalence of parvovirus B19 is useful for guiding the interpretation of a positive result with the dengue rapid diagnostic test.

Development of real-time PCR to detect oral vaccine-like poliovirus and its application to environmental surveillance.

In order to perform environmental surveillance to track oral poliovirus vaccine-like poliovirus sensitively and conveniently, real-time PCR was developed and applied to a raw sewage concentrate. The real-time PCR method detected 0.01-0.1 TCID50 of 3 serotypes of Sabin strain specifically. The method also detected the corresponding serotypes of oral poliovirus vaccine-like poliovirus specifically, but detected neither wild poliovirus, except Mahoney for type 1 and Saukett for type 3, nor other enteric viruses, as far as examined. When real-time PCR was applied to environmental surveillance, the overall agreement rates between real-time PCR and the cell culture were 83.3% for all serotypes. Since real-time PCR has the advantages of rapid detection of viruses and minimum requirement of sampling volume as compared with ordinary cell culture, it is suitable to monitor oral poliovirus vaccine-like poliovirus in the environment, especially in areas where an oral vaccine is being replaced by an inactivated vaccine.

Influenza A(H1N1)pdm09 virus and asthma.

Respiratory viral infection is a major cause of asthma exacerbations in both children and adults. Among the respiratory viruses, influenza virus is a particularly important pathogen due to its enormous morbidity and mortality in annual epidemics. The swine-origin influenza A virus, designated as A(H1N1)pdm09, emerged in the spring of 2009 and caused the first influenza pandemic in the 21st century. With the emergence of the novel A(H1N1)pdm09 virus, numerous epidemiologic studies detected asthma as a frequent comorbid condition in patients infected with this virus. Here we review recent reports regarding asthma in patients infected with influenza A(H1N1)pdm09 virus, and we discuss the utility of influenza vaccines and antivirals.

Continuity and change of Japanese encephalitis virus in Toyama Prefecture, Japan.

To determine the mechanisms of maintenance and evolution of Japanese encephalitis virus (JEV) in a temperate zone, we attempted to isolate JEV from mosquitoes and pigs in Toyama Prefecture, Japan. A total of 87 JEVs were isolated from female Culex tritaeniorhynchus mosquitoes and pigs during 2005-2009. The prevalence of JEV in Toyama Prefecture was seasonally late in comparison with that of the virus during 1966-1972. Furthermore, JEVs were isolated after the peak in the number of female Cx. tritaeniorhynchus. Among JEV strains isolated in this study, two distinct groups were observed within genotype I of the phylogeny generated from nucleotide sequence information derived from the envelope and capsid/premembrane genes: strains belonging to the major type were isolated during 2005-2009, and strains from the minor type were isolated only in 2007. The major type has exhibited gradual change in its envelope and capsid/premembrane genes, and all isolates obtained in 2008 and 2009 had a novel deletion of seven nucleotides in the variable region of the 3'-untranslated region.

Widespread circulation of echovirus type 13 demonstrated by increased seroprevalence in Toyama, Japan, between 2000 and 2003.

To confirm the magnitude of an echovirus type 13 (E13) outbreak in 2002 and to evaluate whether genetic and antigenic changes in E13 influenced the occurrence of the outbreak, we measured titers of neutralizing (NT) antibody against the Toyama, 2002-240-SF, and prototype Del Carmen E13 strains among inhabitants of Toyama before and after 2002. The rate of positivity for NT antibodies against both 2002-240-SF and Del Carmen in 2003 made a remarkable upturn in children 0 to 14 years old, compared to that in 2000. Titers of NT antibody against strain 2002-240-SF of inhabitants were slightly higher than those against Del Carmen, whereas anti-E13 rabbit serum raised against either strain Del Carmen or 2002-240-SF showed almost the same titer of NT antibody against both strains. These data indicate that the antigenic properties of the strains may be slightly different. Differences in amino acids between strains 2002-240-SF and Del Carmen in the VP4, VP2, VP3, and VP1 regions may affect both antigenic and receptor binding properties, even though they do not seem to be significant enough to escape widespread immunity. One of the factors of the outbreak was thought to be the increase in susceptibility in the young generation.

Frequent detection of noroviruses and sapoviruses in swine and high genetic diversity of porcine sapovirus in Japan during Fiscal Year 2008.

A molecular biological survey on porcine norovirus (NoV) and sapovirus (SaV) was conducted in Toyama Prefecture, Japan, during fiscal year 2008. Both NoV and SaV were detected from swine fecal samples throughout the surveillance period, indicating that these viruses were circulating in this region. NoV strains detected in this study belonged to three genotypes that are known as typical swine NoVs. Although human NoVs were occasionally detected, it was unclear whether they replicated in pigs. As for SaV, genogroup VII (GVII) and other divergent genogroups were identified in addition to the dominant genogroup, GIII, which is the prototypic porcine SaV. In addition, 3 strains genetically related to human SaV were detected. Two of these 3 strains were closely related to human SaV GV. Our study showed that genetic diversification of porcine SaV is currently progressing in the swine population.

Detection of a novel recombinant norovirus from sewage water in toyama prefecture, Japan.

Recently, the recombination event of norovirus (NoV) has been reported with high frequency, suggesting that RNA recombination is a major driving force in NoV evolution. To assess the incidence of NoV recombination in a residential area, we conducted a molecular biological survey of NoVs existing in sewage water in Toyama Prefecture, Japan. Although GII/4 was predominantly detected in sewage water that was associated with a high frequency of outbreaks caused by this genotype, other genotypes, including two types of recombinant strain, were identified during the survey period. One of the recombinants is the WUG1 type, which was first detected in Saitama Prefecture in 2000. The other recombinant is a novel type derived from two parent strains of genogroup II, GII/7 for the RNA-dependent RNA polymerase and GII/13 for the capsid. This suggests that certain NoVs circulating in the area are occasionally changing their genetic properties by recombination events.

Genetic changes of coxsackievirus A16 and enterovirus 71 isolated from hand, foot, and mouth disease patients in Toyama, Japan between 1981 and 2007.

We characterized the genetic diversity of the complete VP1 region of coxsackievirus A16 (CA16) and enterovirus 71 (EV71) isolated from patients with hand, foot, and mouth disease in Toyama from 1981 to 2007 to evaluate the relationship between epidemics and genetic changes. The predominant genogroups of CA16 changed from B to C in 1995-1998, and genogroup C further changed from subgenogroup C1 to C2 around 2002, and to C3 in 2005-2007. The subgenogroups of the EV71 isolates were classified into B1, B4, C1, and C3 in 1983-1994, and into C4 in 1997-2006. However, changes of the amino acid sequences of the VP1 regions of CA16 were restored, and those of the EV71 isolates were not observed among the same subgenogroups during this survey period, indicating that the prevalence that occurred at intervals of several years seemed to depend on an accumulating number of immunologically naive children, not viral antigenic changes.

Continuous presence of noroviruses and sapoviruses in raw sewage reflects infections among inhabitants of Toyama, Japan (2006 to 2008).

Various genotypes of norovirus (NoV) (genogroup I genotype 1 [GI.1], -2, -4, -5, -8, -11, -12, and -14; GII.3, -4, -6, -7, -10, -13, -14, and -15), and sapovirus (SaV) (GI.1 and GI.2, GII.1, and GIV.1) were detected from raw sewage from April 2006 to March 2008, while limited numbers of genotypes of NoV (GI.8, GII.4, GII.6, and GII.13) and SaV (GII.3 and GIV.1) and of NoV (GII.4, GII.7, and GII.13) were detected from clinical cases and healthy children, respectively. During the winter 2006 to 2008, a large number of sporadic gastroenteritis outbreaks and many outbreaks caused by NoV GII.4 occurred among inhabitants in Toyama, Japan. The copy number of genomes of NoV GII detected from raw sewage changed in relation to the number of outbreaks. NoV strains of the same genotypes observed in both raw sewage and human specimens belonged to the same cluster by phylogenetic analysis and had almost identical nucleotide sequences among each genotype. These data suggest that NoVs and SaVs detected from raw sewage reflect the viruses circulating in the community, irrespective of symptoms, and that subclinical infections of NoV are common in Japan. Combined surveys of raw sewage with those of clinical cases help us to understand the relationship between infection of these viruses and gastroenteritis.

Single base substitutions in the capsid region of the norovirus genome during viral shedding in cases of infection in areas where norovirus infection is endemic.

Norovirus (NoV) infections are the major cause of food- and waterborne nonbacterial gastroenteritis in Japan. Some individuals showed long-term excretion of the virus into feces in 29 outbreaks of acute nonbacterial gastroenteritis that occurred in Toyama Prefecture, Japan, in fiscal year 2006. In one of these cases, single base substitutions from A to G in the capsid region of the NoV genome were commonly detected in two individuals during virus shedding by direct sequencing of PCR products. The A-to-G substitution was accompanied by an N-to-S amino acid change. The population of clones that possessed A at the corresponding site was gradually replaced by those with G during the infectious course. Although other substitutions were observed in the complete open reading frame 2 sequence, they were not common in these two individuals. NoVs are capable of evolving in the gastroenteric tract.

Evaluation of a two-dose administration of live oral poliovirus vaccine for wild and virulent vaccine-derived poliovirus type 1, 2, 3 strains in Japan.

We evaluated the efficacy of Japan's vaccination policy, a 2-dose administration of live oral poliovirus vaccine (OPV) against wild and virulent vaccine-derived poliovirus (VDPV) type 1, 2, 3 strains, by investigating the neutralizing antibody titers of residents in Toyama Prefecture, Japan. Seropositivities against the virulent type 1 and 2 strains were more than 90%, but the values against the virulent type 3 strains were approximately 60%. Also, while geometric mean antibody titers against virulent type 1 and 2 strains were more than 180, those against the virulent type 3 strains were 58-59, and 9-12, in particular, at 10 to 19 y of age. A booster dose of the vaccine for the type 3 virus is recommended for adolescents. However, high herd immunity against type 1, 2 and 3 viruses has been maintained for these 22 y, although the seropositivity against type 3 virus was always lower than other types. Our results suggest that Japan's vaccination policy might be enough to prevent an epidemic of poliomyelitis caused by wild and virulent VDPV type 1, 2, 3 strains, even though the titers against type 3 viruses were the lowest.

Evidence for phagocytosis of influenza virus-infected, apoptotic cells by neutrophils and macrophages in mice.

Influenza virus-infected cells undergo apoptosis and become susceptible to phagocytosis by macrophages in vitro, and this leads to the propagation of the virus being inhibited. We previously showed that inhibitors of phagocytosis increased the rate of mortality among influenza virus-infected mice. However, the mode of the phagocytosis of influenza virus-infected cells in vivo has not been investigated. We, in this study, assessed this issue by histochemically analyzing bronchoalveolar lavage cells and lung tissue obtained from C57BL/6 mice infected with influenza A/WSN (H1N1) virus. Both neutrophils and macrophages accumulated in the lung soon after the viral challenge, and either type of cell was capable of phagocytosing influenza virus-infected, apoptotic cells. Changes in the level of phagocytosis and the amount of virus in lung tissue roughly correlated with each other. Furthermore, alveolar macrophages prepared from influenza virus-infected mice showed greater phagocytic activity than those from uninfected mice. The phagocytic activity of macrophages was stimulated in vitro by a heat-labile substance(s) released from influenza virus-infected cells undergoing apoptosis. These results suggested that the level of phagocytosis is augmented both quantitatively and qualitatively in the lung of influenza virus-infected animals so that infected cells are effectively eliminated. Finally, lack of TLR4 caused an increase in the rate of mortality among influenza virus-challenged mice and a decrease in the level of phagocytosis of apoptotic cells in the lung. TLR4 could thus play an important role in the host defense against influenza by positively regulating the phagocytic elimination of infected cells.

Molecular epidemiology of echoviruses 11 and 13, based on an environmental surveillance conducted in Toyama Prefecture, 2002-2003.

Nineteen echovirus 11 (E11) and 12 E13 isolates were isolated from three rivers in Toyama Prefecture, Japan, during an environmental surveillance conducted from April 2002 to March 2003. The nucleotide sequences of E13 isolates were closely related to those from patients with aseptic meningitis, with less than 1.3% divergence in the VP1 region of the viral capsid gene, and belonged to the same clade responsible for a worldwide outbreak that started in 2000. In contrast, E11 isolates were clustered into three genomic groups and were not closely related to echovirus strains isolated from patients. These results suggest that the combination of both virus isolation from environmental sources and phylogenetic analysis could be complementary assessment approaches to trace prevalent and minor circulating enteroviruses in the human population.

Augmentation of fatality of influenza in mice by inhibition of phagocytosis.

Influenza virus-infected cells undergo apoptosis and become susceptible to phagocytosis by macrophages, and this leads to the inhibition of virus propagation in vitro. To assess if this were also true in vivo, mice infected with influenza A/WSN (H1N1) virus were administered with phagocytosis inhibitors and examined for the progress of influenza. Administration of the inhibitors caused a decrease in the level of phagocytosis observed with bronchoalveolar lavage cells. We found that both the lethality in mice and the extent of inflammation in the lung were augmented in those mice. These results suggest that phagocytosis of virus-infected cells helps suppress the progress of influenza in mice.

Endoplasmic reticulum stress induces the phosphorylation of small heat shock protein, Hsp27.

There are several reports describing participation of small heat shock proteins (sHsps) in cellular protein quality control. In this study, we estimated the endoplasmic reticulum (ER) stress-induced response of Hsp27 and alphaB-crystallin in mammalian cells. Treatment targeting the ER with tunicamycin or thapsigargin induced the phosphorylation of Hsp27 but not of alphaB-crystallin in U373 MG cells, increase being observed after 2-10 h and decline at 24 h. Similar phosphorylation of Hsp27 by ER stress was also observed with U251 MG and HeLa but not in COS cells and could be blocked using SB203580, an inhibitor of p38 MAP kinase. Other protein kinase inhibitors, like Gö6983, PD98059, and SP600125, inhibitors of protein kinase C (PKC), p44/42 MAP kinase, and JNK, respectively, were without major influence. Prolonged treatment with tunicamycin but not thapsigargin for 48 h caused the second induction of the phosphorylation of Hsp27 in U251 MG cells. Under these conditions, the intense perinuclear staining of Hsp27, with some features of aggresomes, was observed in 10%-20% of the cells.

Nordihydroguaiaretic acid (NDGA) blocks the differentiation of C2C12 myoblast cells.

Addition of nordihydroguaiaretic acid (NDGA) to the differentiation medium of C2C12 mouse myoblast cells caused severe inhibition of the formation of myotubes and suppressed differentiation-dependent elevation in the levels of the creatine kinase M isozyme (CKM). Under these conditions, NDGA did not cause significant increase of damaged cells, as detected by annexin-V-FITC assay, or induction of heat shock proteins, known to be a response against extracellular stress. The results suggest that NDGA itself is not toxic but can effectively blocks the differentiation-dependent increase of CKM during C2C12 differentiation. The levels of muscle specific bHLH proteins MyoD, Myf5, and myogenin were also decreased by addition of NDGA, indicating a block of the initial step of the myogenesis through downregulation of muscle specific genes. NDGA is known to be a lipoxygenase inhibitor but other examples, like MK-886 and CDC, did not exert the same effects on differentiation of muscle cells, indicating that mechanisms of NDGA action are independent of its influence on lipoxygenase.