Broadly Reactive Real-Time RT-PCR Assay for the Detection of Hepatitis E Virus and Simultaneous Genotyping by Single Nucleotide Polymorphism Analysis.

Hepatitis E virus (HEV) infection is a global public health concern. Although HEV infection is usually asymptomatic and self-limiting, extrahepatic manifestations and chronic infections in immunocompromised patients have been described. HEV strains infecting humans have been classified into four main genotypes. In this study we have developed and validated a novel sensitive real-time RT-PCR assay for the detection of all four HEV genotypes. Simultaneous discrimination of genotypes 1, 2, and 4 from genotype 3 by single nucleotide polymorphism (SNP) analysis was possible. In all, 201 serum samples from cases and carriers previously tested for HEV by nested RT-PCR were analyzed. Twenty-seven HEV-positive samples could not be typed by the nested RT-PCR and nucleotide sequencing, but were newly typed by SNP analysis. As polymorphisms were present at the primer or probe binding site, we adopted a degenerate primer and mixed probes. When a mixed probe was added, the fluorescence intensity increased, facilitating genotype determination. IMPORTANCE The distribution of HEV-3 and HEV-4 has been changing. HEV-4, which had been predominantly found in Asia, is now being detected in other parts of the world, and there are now reports of chronic infections. Additionally, neurological disorders have frequently been reported in patients with acute or chronic HEV infections. HEV-4 has also been shown to lead to a higher severity in terms of acute hepatitis than does HEV-3. Early typing can provide useful information regarding the route of infection and for tailoring treatment to the expected course of the disease. The present method afforded a good detection rate even when polymorphisms were present within the target region for viral gene detection. We believe that this method can be applied to the analysis of mutation-prone viral genes in the future.

Genetic characterization of a novel recombinant echovirus 30 strain causing a regional epidemic of aseptic meningitis in Hokkaido, Japan, 2017.

A regional epidemic of aseptic meningitis caused by echovirus 30 (E30) occurred in Hokkaido, Japan, during the period of August-December 2017. To investigate their phylogenetic relationship to other human enteroviruses, we determined the complete genomic nucleotide sequences of isolates from this outbreak. Phylogenetic analysis of the viral capsid protein 1 gene showed that the strains were most closely related to E30 strains detected in Germany, France, and Russia in 2013. In contrast, the region encoding the viral protease and the RNA-dependent RNA polymerase had a close phylogenetic relationship to non-E30 enteroviruses detected in the United Kingdom and Switzerland in 2015-2017, suggesting that a recombination event had occurred.

Genetic characterization of hemagglutinin protein of measles viruses in Hokkaido district, Japan, 2006-2015.

Strains of measles virus of genotypes D5, H1, D4, D8, and B3 were detected among epidemic, endemic, imported and import-associated cases in Hokkaido district, Japan, during 2006-2015. In the present study, their antigenic features were evaluated by determining the complete nucleotide sequences of their hemagglutinin proteins, which are a major target for neutralizing antibodies, and their amino acid sequences deduced. It was found that the hemagglutinin proteins of these strains had several novel amino acid changes in some functional regions. Although these strains have not caused further infections thus far, these antigenic changes should continue to be monitored to maintain their elimination status.

Hepatitis E outbreak at a nursing home for aged people in Hokkaido, Japan, between February and March 2016.

BACKGROUND: Infection with hepatitis E virus (HEV) genotypes 3 and 4 are usually asymptomatic but can occasionally result in life-threatening acute hepatitis. To date, only sporadic cases together with a few outbreaks have been documented. Seroprevalence studies with assays for the detection of HEV IgG antibodies, suggest that HEV is more prevalent than previously thought, even in non-endemic regions. OBJECTIVES: The aim of this study was to characterize an outbreak of hepatitis E (HE) in a nursing home for aged people between February and March 2016. STUDY DESIGN: After the identification of two cases living in the same nursing home, the presence of antibodies against HEV and HEV RNA were examined in serum samples collected from the other residents and staff members to identify any additional cases. An epidemiological investigation was also carried out. RESULTS: Only 4 patients showed mild symptoms such as anorexia, abdominal pain and fatigue. Among the 125 persons tested, 28 residents and one dietitian were confirmed positive for anti-HEV IgA or IgM antibodies, and/or HEV RNA. Eight samples had only IgG antibodies. Finally, 22 cases were notified with HE on the basis of the presence of IgA antibodies. All HEV isolates obtained were 99.8-100% identical and belonged to genotype 3. CONCLUSION: HEV infections seem to be under-reported or underestimated possibly due to cases being generally asymptomatic. Testing for the presence of both anti-HEV antibodies and HEV RNA would be beneficial for both the comprehensive diagnosis of HE infections and the prevention of further infections.

Detection of Measles Virus Genotypes B3, D4, D5, D8, and H1 in the Surveillance System in Hokkaido, Japan, 2006-2015, the Last Decade toward the Elimination.

Measles is an acute and highly contagious disease caused by measles virus (MeV). The government of Japan, following the last epidemic in 2007 and 2008, which was caused by genotype D5 strains, introduced a catch-up-vaccination program for teenagers during Japan fiscal years 2008-2012 and a mandatory case-based reporting system for the nationwide elimination. Furthermore, laboratory confirmation of measles cases by genotyping of isolates has been performed to clarify the source of infection and support the interruption of measles cases. Owing to these preventive measures, the number of measles cases has been steadily decreasing after the last epidemic. In March 2015, Japan was internationally verified as having achieved measles elimination by the World Health Organization Regional Office for the Western Pacific. The continuous elimination of measles and high levels of vaccination coverage for MeV have been maintained nationally. However, imported or import-associated cases of measles have sporadically occurred during this time. After the last nationwide epidemic, 17 imported or import-associated measles cases (MeV strains identified as genotypes H1, D4, D8, and B3) were reported in Hokkaido, the northern islands of Japan. In this study, we present the occurrence of measles and surveillance activities in Hokkaido during 2006-2015.

Epidemiology and laboratory diagnoses of rubella in Hokkaido district during the Nationwide Outbreak in Japan, 2011-2013.

We report the epidemiology and laboratory diagnostic results of rubella cases from 2011 to 2013 in Hokkaido district, Japan. A total of 150 cases were officially reported as rubella; 102 (68%) involved males and 48 (32%) involved females. The highest proportion of cases were notified in 40-49-year-old age group among males and the 20-29-years-old age group among females. Forty-six cases (25 males and 21 females) had not been vaccinated, and 17 had been vaccinated, whereas 87 had the unknown vaccination status. Eighty-three cases (55.3%) showed the 3 typical principal rubella symptoms (fever, rash, and lymphadenopathy). Seven, 11, 92, and 40 cases were identified in the northern, eastern, central, and southern areas of Hokkaido district, respectively. In the central and southern areas of Hokkaido district, endemic rubella transmissions were indicated by both the epidemiological survey and molecular analyses. However, these outbreaks terminated spontaneously and did not expand to other areas of Hokkaido district. Fortunately, no congenital rubella syndrome (CRS) cases were reported during this observation period. However, to control virus transmission, prevent CRS, and maintain the routine vaccination program, the immediate introduction of an immunization strategy is required for susceptible individuals, particularly young adults.

Recent progress toward measles elimination in Hokkaido, Japan, during 2011-2012.

Laboratory diagnoses for measles were performed in a total of 97 cases in Hokkaido, Japan, during 2011-2012. Two patients were confirmed to be positive for measles virus (MV), both of whom lived in the Iburi district of Hokkaido. Molecular analysis of the nucleotide sequences of the nucleoprotein (N) gene revealed that these 2 strains had high homology with each other and belonged to the genotype D8. The onset interval of these cases and epidemiological data suggested that MV transmission had occurred between them and then terminated. Phylogenetic analysis of the N gene revealed that the strains identified in Hokkaido were classified into a cluster that contained many genotype D8 strains that were detected within a large area of Japan. Eventually, 9 cases were officially reported as measles. However, other than the abovementioned 2 cases, no genetic information regarding MV was obtained. In future, further active surveillance combined with the genetic investigation should be required in all suspected measles cases to verify the elimination status.

Genomic characterization of echovirus 6 causing aseptic meningitis in Hokkaido, Japan: a novel cluster in the nonstructural protein coding region of human enterovirus B.

We determined four complete nucleotide sequences of echovirus 6 (E6) isolated from an epidemic of aseptic meningitis (AM) in Hokkaido, Japan, in 2011. Phylogenetic analysis of the genes encoding viral capsid protein 1 revealed that the strains were closely related to E6 strains isolated in China in recent years, but they were distantly related to E6 strains isolated from patients with AM in Osaka Prefecture, Japan, in 2011. The genes encoding the viral protease and RNA-dependent RNA polymerase (3CD) were closely related to those of several non-E6 strains of the species Human enterovirus B isolated in China, South Korea, and Australia from 1999 to 2010, resulting in a novel cluster in the phylogenetic tree. These results suggest that the incidence of AM in Japan in 2011 was caused by at least two lineages of E6 strains, and a lineage of the 3CD gene was interspersed among different serotypic strains isolated in Western Pacific countries.

Detection and molecular characterization of hepatitis E virus in clinical, environmental and putative animal sources.

Putative animal reservoirs and environmental samples were studied to investigate potential routes of transmission for indigenous hepatitis E virus (HEV) infection in Hokkaido, Japan. A total of 468 liver samples and 954 environmental samples were collected from 2003 to 2011 for this study. Four swine livers (1 %) were positive for HEV RNA; two strains belonged to genotype 3 and the other two strains were genotype 4. Genotype 3 HEV was detected in a sewage sample and a seawater sample. HEV strains derived from swine liver, seawater and raw sewage samples shared 93-100 % sequence similarity with human HEV strains.

Usefulness of the rapid determination system of viral genome sequences in human stool specimens.

The rapid determination system of viral genome sequences (the RDV method) consists of detecting and determining the nucleotide sequences of viral genomes without using specific primers. To evaluate the usefulness of the RDV method, the detection of human norovirus (NV) genomes in stool specimens was investigated. In addition, the effect of nuclease treatment of the process was examined. A total of 23 human stool specimens were used, all of which were collected from patients with acute viral gastroenteritis, and were shown to contain NV genomes and also determined the cDNA copy numbers by the real-time reverse transcriptase-polymerase chain reaction. NV genomes were detected by the RDV method with nuclease treatment in nine specimens containing cDNA copies ranging between 6.2×10(9) and 9.8×10(11)/g stool. In contrast, NV genome was found by the method in 15 specimens without nuclease treatment and the number of NV cDNA copies ranged between 1.2×10(6) and 9.8×10(11)/g stool. These results suggest that the RDV method has potential for detecting viral genomes in stool specimens. The procedure without a step of nuclease treatment appears to be sensitive.

Detection of noroviruses in fecal specimens by direct RT-PCR without RNA purification.

Noroviruses are important human pathogens which cause epidemic acute viral gastroenteritis. Current techniques used for detection of noroviruses in fecal specimens involve multi-step viral RNA extraction and purification followed by reverse transcriptase-polymerase chain reaction (RT-PCR). This study demonstrates a method for easy detection of norovirus in fecal specimens, involving one-step RNA release and direct use of the released RNA for RT-PCR (direct RT-PCR). For one-step RNA release, a simple method was adopted based on addition of the sample treatment reagent from a commercialized Norovirus GI and GII RNA Detection Kit to suspended fecal specimens, followed by a brief heat treatment. The released RNA was then added directly to the RT mixture from the same kit. After reverse transcription and PCR, the product was detected by agarose gel electrophoresis. Direct RT-PCR was evaluated with 275 fecal specimens comprising 230 norovirus-positive and 45 norovirus-negative samples as assessed by real-time RT-PCR, considered to be the "gold standard" for norovirus detection. Direct RT-PCR was sufficiently specific and sensitive for norovirus detection, and eliminated the RNA extraction and purification step. Use of this method should facilitate detection of norovirus in fecal specimens and provide valuable information regarding the incidence of the virus. In addition, this method should be applicable for other RNA viruses.

Sensitive and rapid detection of norovirus using duplex TaqMan reverse transcription-polymerase chain reaction.

Conventional reverse transcription-polymerase chain reaction (RT-PCR) to detect norovirus (NV) is a complex of multi-step procedure that requires gel electrophoresis as well as hybridization or sequencing to confirm the final diagnosis. A duplex TaqMan RT-PCR was developed to detect and classify genogroup (G) I and GII of NV. The primers and TaqMan probes for this assay were selected from the region of open reading frame (ORF) 1-ORF2 junction. A total of 796 stool specimens from 103 outbreaks of gastroenteritis, and a series of 46 stool specimens containing most NV genotypes was used for this study. For these specimens from 103 outbreaks, NV was detected and classified by the duplex TaqMan RT-PCR in 536 of the 541 specimens tested previously to be positive using the conventional RT-PCR. Two hundred fifty-one of the 255 specimens that were negative by the conventional RT-PCR were also negative by the TaqMan RT-PCR. No false positive result was observed for other enteric RNA viruses such as rotavirus and sapovirus. This is the first report on the development of a duplex TaqMan RT-PCR end-point assay for detection and differentiation of GI and GII of NV strains simultaneously followed by genotyping. These results suggest the practical application of this duplex TaqMan RT-PCR is useful for the detection of NV in clinical specimens.

Adenovirus E1A negatively regulates E1AF, an ets family of the protein.

E1AF was first identified as a transcription factor that binds to enhancer motifs of the adenovirus E1A gene and is thought to be a human homologue of mouse PEA3, one of the ets oncoprotein families. Here we show the effect of E1A on the gene expression and function of E1AF. E1A repressed the activity of E1AF promoter, and the N-terminal region of E1A, which is involved in the oncogenic activity of E1A, was essential for this repression. The ability as a transcription factor of E1AF, as well as those of the other PEA3 subfamily members ER81 and ERM, was also repressed by E1A via the same oncogenic domain. Furthermore, E1AF repressed the transformation activity of E1A cooperating with E1B, whereas the other ets family Ets-1 enhanced this activity. These results suggest that E1AF is one of the targets of E1A.