Genetic Comparison of Human Parainfluenza Virus Type 3 Detected in Respiratory Samples from Patients with Encephalopathy and Airway Inflammation in Aichi Prefecture, Japan.

Human parainfluenza virus type 3 (HPIV-3, human respirovirus 3) is the second most frequently detected virus in lower respiratory tract infections in children after human respiratory syncytial virus (HRSV). HPIV-3, similar to related respiratory viruses such as HRSV and influenza virus, may cause encephalopathy; however, the relevance of HPIV-3 as a pathogenic factor in encephalopathy is unknown. We attempted to detect HPIV-1, HPIV-2, HPIV-3, HPIV-4, HRSV, and human metapneumovirus (HMPV) in 136 patients with encephalitis/encephalopathy or suspected encephalitis/encephalopathy during a 6-year period from 2014 to 2019. HPIV-3 was detected in 6 patients, followed by HRSV in 3 patients. The HPIV-3 strains detected were closely related to those detected in a patient with respiratory disease during the same period. Although HPIV-3 is less widely recognized than HRSV as a triggering virus of encephalopathy, our results suggest that HPIV-3 is as important as HRSV. Surveillance of the causative viruses of encephalopathy, including HPIV-3, would help clarify the causes of encephalopathy in Japan, as the cause is currently reported in less than half of cases in Japan.

Impact of the Coronavirus Disease 2019 Pandemic on the Clinical Features of Pediatric Respiratory Syncytial Virus Infection in Japan.

Background: Mitigation measures implemented during the coronavirus disease 2019 (COVID-19) pandemic remarkably reduced the incidence of infectious diseases among children. However, a re-emergence of respiratory syncytial virus (RSV) infection was observed in 2021 in Japan. We compared the clinical characteristics of hospitalized patients with RSV infection before and during COVID-19. Methods: We retrospectively enrolled children aged <6 years who were hospitalized with RSV infection in 18 hospitals and compared their clinical characteristics before (January 2019 to April 2020, 1675 patients) and during COVID-19 (September 2020 to December 2021, 1297 patients). Results: The mean age of patients with RSV infection was significantly higher during COVID-19 than before (17.4 vs 13.7 months, P < .001). Compared with before COVID-19, a 2.6-fold increase in RSV cases in the 2-5 years age group was observed from sentinel surveillance during COVID-19, whereas a 1.2-fold increase was noted in the same age group among hospitalized patients. On average for all patients, consolidation shadows obtained on radiography were less frequently observed (26.1 vs 29.6%, P = .04), and reduced respiratory assistance (42.2% vs 48.7%, P < .001) and hospitalization stay (5.7 vs 6.0 days, P < .001) was required in patients with RSV infection during COVID-19. Conclusions: Coronavirus disease 2019 and social activity restriction caused epidemiological changes in pediatric RSV infections, and a majority of patients with RSV infection aged ≥2 years did not develop severe symptoms requiring hospitalization. The RSV symptoms during the COVID-19 outbreak were equivalent to or milder than in the previous seasons.

Two Cases of Dengue Virus Type 2 (DENV-2) Infection in a Japanese Couple Returning from the Maldives during the 2018 Dengue Outbreak.

Annually, more than 1.2 million travelers from other countries visit the Maldives for sightseeing, business, and honeymoon. In 2018, the largest dengue fever outbreak occurred, affecting more than 3,200 people. During this outbreak, we encountered a newly married Japanese couple returning from the Maldives on their honeymoon in October 2018, both were infected by the dengue virus type 2 during the travel. The number of imported dengue fever cases from the Maldives may increase; hence, physicians should stay up to date on dengue outbreak information worldwide.

Molecular Epidemiology of Rubella Virus Strains Detected Around the Time of the 2012-2013 Epidemic in Japan.

A nationwide rubella epidemic occurred from 2012 to 2013 in Japan, resulting in around 17,000 rubella cases and the birth of 45 infants with congenital rubella syndrome. The aim of this study was to genetically characterize the rubella viruses (RVs) circulating around the time of the epidemic in Japan. In total, 221 RV strains detected from 14 prefectures in Japan between 2010 and 2014 were sequenced in the 739 nucleotide-window region within the E1 gene. The virus strains were chronologically and geographically characterized into groups based on phylogenetic analysis. Among the 221 strains analyzed, 192 (87%), 26 (12%), and 3 (1%) strains were classified into genotypes 2B, 1E, and 1J, respectively. The majority (n = 184) of the genotype 2B strains belonged to lineage 2B-L1 and shared nucleotide homology with the strains detected in Southeast and East Asian countries. Phylogenetic analyses demonstrated that at least six distinct clusters of RV strains (clusters 1-6) induced outbreaks in Japan between 2010 and 2014. Among them, strains from clusters 3, 4, and 6 circulated almost simultaneously during 2012-2013. The cluster 3 strains circulated locally, whereas strains from cluster 4 spread nationwide. The findings suggest that RVs were introduced into Japan many times from neighboring countries. The 2012-2013 epidemic was a complex of outbreaks induced by at least three clusters of RV strains.

Evaluation of sensitivity of TaqMan RT-PCR for rubella virus detection in clinical specimens.

BACKGROUND: An easy and reliable assay for detection of the rubella virus is required to strengthen rubella surveillance. Although a TaqMan RT-PCR assay for detection of the rubella virus has been established in Japan, its utility for diagnostic purposes has not been tested. OBJECTIVES: To allow introduction of the TaqMan RT-PCR into the rubella surveillance system in Japan, the sensitivity of the assay was determined using representative strains for all genotypes and clinical specimens. STUDY DESIGN: The detection limits of the method for individual genotypes were examined using viral RNA extracted from 13 representative strains. The assay was also tested at 10 prefectural laboratories in Japan, designated as local reference laboratories for measles and rubella, to allow nationwide application of the assay. RESULTS: The detection limits and amplification efficiencies of the assay were similar among all the representative strains of the 13 genotypes. The TaqMan RT-PCR could detect approximately 90% of throat swab and urine samples taken up to 5days of illness. These samples were determined positive by a highly sensitive nested RT-PCR. CONCLUSIONS: The TaqMan RT-PCR could detect at least 10 pfu of rubella virus. Although the sensitivity was somewhat lower than that of the conventional nested RT-PCR, the TaqMan RT-PCR could be more practical to routine tests for rubella laboratory diagnosis and detection in view of the rapid response and reducing risks of contamination.

Case-based surveillance enhanced with measles virus detection/genotyping is essential to maintain measles elimination in Aichi Prefecture, Japan.

BACKGROUND: Japan was verified as having achieved measles elimination by the Measles Regional Verification Commission in the Western Pacific Region in March 2015. Verification of measles elimination implies the absence of continuous endemic transmission. After the last epidemic in 2007 with an estimated 18,000 cases, Japan introduced nationwide case-based measles surveillance in January 2008. Laboratory diagnosis for all suspected measles cases is essentially required by law, and virus detection tests are mostly performed by municipal public health institutes. Despite relatively high vaccination coverage and vigorous response to every case by the local health center staff, outbreak of measles is repeatedly observed in Aichi Prefecture, Japan. METHODS: Measles virus N and H gene detection by nested double RT-PCR was performed with all specimens collected from suspected cases and transferred to our institute. Genotyping and further molecular epidemiological analyses were performed with the direct nucleotide sequence data of appropriate PCR products. RESULTS: Between 2010 and 2014, specimens from 389 patients suspected for measles were tested in our institute. Genotypes D9, D8, H1 and B3 were detected. Further molecular epidemiological analyses were helpful to establish links between patients, and sometimes useful to discriminate one outbreak from another. All virus-positive cases, including 49 cases involved in three outbreaks without any obvious epidemiological link with importation, were considered as import-related based on the nucleotide sequence information. Chain of transmission in the latest outbreak in 2014 terminated after the third generations, much earlier than the 2010-11 outbreak (6th generations). CONCLUSION: Since 2010, almost all measles cases reported in Aichi Prefecture are either import or import-related, based primarily on genotypes and nucleotide sequences of measles virus detected. In addition, genotyping and molecular epidemiological analyses are indispensable to prove the interruption of endemic transmission when the importations of measles are repeatedly observed.

Detection and genotyping of rubella virus from exanthematous patients suspected of having measles using reverse transcription-PCR.

Between July 2012 and March 2013, a total of 133 clinical specimens from 47 patients suspected of having measles were collected for virological surveillance in Aichi Prefecture, Japan. Facing the rubella epidemic, the reverse transcription (RT)-PCR protocol for measles virus (MeV) was modified to simultaneously detect rubella virus (RUBV) in these clinical specimens. As a result, 30 specimens from 15 patients were positive for RUBV and 8 specimens from 3 patients were positive for MeV. The RUBV genotype analysis for the samples from 13 patients revealed 12 samples as 2B and 1 sample as 1E. The results provided additional evidence for the difficulty in the diagnosis of exanthematous diseases based on clinical manifestations alone and the necessity of virological diagnosis to maintain the accuracy of case-based surveillance. Furthermore, the results indicated that the modified RT-PCR protocol could be useful as a routine procedure to simultaneously detect MeV and RUBV in clinical specimens of patients suspected of having exanthematous disease caused by these viruses.

Molecular detection and nucleotide sequence analysis of a new Aichi virus closely related to canine kobuvirus in sewage samples.

Between 2001 and 2005, 207 raw sewage samples were collected at the inflow of a sewage treatment plant in Aichi Prefecture, Japan. Of the 207 sewage samples, 137 (66.2 %) were found to be positive for amplification of Aichi virus (AiV) nucleotide using reverse transcription (RT)-PCR with 10 forward and 10 reverse primers in the 3D region corresponding to the nucleotide sequence of all kobuviruses. AiV genotype A sequences were detected in all 137 samples. New sequences of AiV were detected in nine samples, exhibiting 83 % similarity with AiV A846/88, but 95 % similarity with canine kobuvirus (CKV) US-PC0082 in this region. The nucleotide sequences from the VP3 region to the 3' untranslated region (UTR) of sewage sample Y12/2004 were determined. The number of nucleotides in each region was the same as that of CKV. The similarity of the nucleotide (amino acid) identity of a complete VP1 region was 90.5 % (94.8 %) between Y12/2004 and CKV US-PC0082. The phylogenic analyses based on the nucleotide and the deduced amino acid sequences of VP1 and 3D showed that Y12/2004 was independent from AiV, but closely related to CKV. These results suggested that CKV is present in Aichi Prefecture, Japan.

A foodborne outbreak of sapovirus linked to catered box lunches in Japan.

Sapovirus (SaV) is a common cause of acute viral gastroenteritis worldwide, and SaV outbreaks have become more frequent in recent years. In January 2010, an outbreak of acute gastroenteritis due to SaV occurred in Aichi, Gifu and Mie Prefectures, Japan. The illness was strongly associated with eating a delivered box lunch prepared by one catering company. In total, 655 (17.1 %) of 3827 individuals developed gastroenteritic symptoms. SaV was detected in seven of the nine people who became ill and in seven of the 52 food handlers at the catering company, but all the tested samples were negative for norovirus and enteropathogenic bacteria. Sequence analysis of RT-PCR products indicated that the nucleotide sequences of SaV strains from the people who became ill and the food handlers were identical. The detected SaV strains were genogrouped as SaV genotype I.2. This was the largest foodborne outbreak of sapovirus in Japan.

One-step real-time reverse transcription-PCR assays for detecting and subtyping pandemic influenza A/H1N1 2009, seasonal influenza A/H1N1, and seasonal influenza A/H3N2 viruses.

Pandemic influenza A/H1N1 2009 (A/H1N1pdm) virus has caused significant outbreaks worldwide. A previous one-step real-time reverse transcription-PCR (rRT-PCR) assay for detecting A/H1N1pdm virus (H1pdm rRT-PCR assay) was improved since the former probe had a low melting temperature and low tolerance to viral mutation. To help with the screening of the A/H1N1pdm virus, rRT-PCR assays were also developed for detecting human seasonal A/H1N1 (H1 rRT-PCR assay) and A/H3N2 influenza viruses (H3 rRT-PCR assay). H1pdm, H1, and H3 rRT-PCR assays were evaluated using in vitro-transcribed control RNA, isolated viruses, and other respiratory pathogenic viruses, and were shown to have high sensitivity, good linearity (R(2)=0.99), and high specificity. In addition, the improved H1pdm rRT-PCR assay could detect two viral strains of A/H1N1pdm, namely, A/Aichi/472/2009 (H1N1)pdm and A/Sakai/89/2009 (H1N1)pdm, which have mutation(s) in the probe-binding region of the hemagglutinin gene, without loss of sensitivity. Using the three rRT-PCR assays developed, 90 clinical specimens collected between May and October 2009 were then tested. Of these, 26, 20, and 2 samples were identified as positive for A/H1pdm, A/H3, and A/H1, respectively, while 42 samples were negative for influenza A viruses. The present results suggest that these highly sensitive and specific H1pdm, H1, and H3 rRT-PCR assays are useful not only for diagnosing influenza viruses, but also for the surveillance of influenza viruses.

Tetrameric ring formation of Epstein-Barr virus polymerase processivity factor is crucial for viral replication.

The Epstein-Barr virus BMRF1 DNA polymerase processivity factor, which is essential for viral genome replication, exists mainly as a C-shaped head-to-head homodimer but partly forms a ring-shaped tetramer through tail-to-tail association. Based on its molecular structure, several BMRF1 mutant viruses were constructed to examine their influence on viral replication. The R256E virus, which has a severely impaired capacity for DNA binding and polymerase processivity, failed to form replication compartments, resulting in interference of viral replication, while the C95E mutation, which impairs head-to-head contact in vitro, unexpectedly hardly affected the viral replication. Also, surprisingly, replication of the C206E virus, which is expected to have impairment of tail-to-tail contact, was severely restricted, although the mutant protein possesses the same in vitro biochemical activities as the wild type. Since the tail-to-tail contact surface is smaller than that of the head-to-head contact area, its contribution to ring formation might be essential for viral replication.

AT cells show dissimilar hypersensitivity to heavy-ion and X-rays irradiation.

Ataxia telangiectasia (AT) cells, with their defective double-strand break (DSB) repair processes, exhibit high sensitivity to low-LET radiation such as X-rays irradiation and gamma beams. Since heavy ion beam treatment for cancer is becoming increasingly common in Japan and elsewhere, it is important to also determine their sensitivity to high-LET radiation. For this purpose we irradiated AT and normal human cells immortalized with the human telomerase gene using high- (24-60 keV/microm carbon and 200 keV/microm iron ions) or low-LET (X-rays) radiation in non-proliferative conditions. In normal cells the RBE (relative biological effectiveness) of carbon and iron ions increased from 1.19 to 1.81 in proportion to LET. In contrast, their RBE in AT cells increased from 1.32 at 24 keV/microm to 1.59 at 40 keV/microm, and exhibited a plateau at over 40 keV/microm. In normal cells most gamma-H2AX foci induced by both carbon- and iron-ion beams had disappeared at 40 h. In AT cells, however, a significant number of gamma-H2AX foci were still observed at 40 h. The RBEs found in the AT cells after heavy-ion irradiation were consistent with the effects predicted from the presence of non-homologous end joining defects. The DSBs remaining after heavy-ion irradiation suggested defects in the AT cells' DSB repair ability.

Epstein-Barr virus polymerase processivity factor enhances BALF2 promoter transcription as a coactivator for the BZLF1 immediate-early protein.

The Epstein-Barr virus (EBV) BMRF1 protein is an essential replication protein acting at viral replication forks as a viral DNA polymerase processivity factor, whereas the BALF2 protein is a single-stranded DNA-binding protein that also acts at replication forks and is most abundantly expressed during viral productive replication. Here we document that the BMRF1 protein evidently enhances viral BZLF1 transcription factor-mediated transactivation of the BALF2 gene promoter. Mutagenesis and electrophoretic mobility shift assays demonstrated the BALF2 promoter to harbor two BZLF1 protein-binding sites (BZLF1-responsive elements). Direct binding of the BZLF1 protein to BZLF1-responsive elements and physical interaction between BZLF1 and BMRF1 proteins are prerequisite for the BMRF1 protein up-regulation of the BALF2 gene promoter. A monomeric mutant, C95E, which is defective in homodimerization, could still interact and enhance BZLF1-mediated transactivation. Furthermore although EBV protein kinase phosphorylates BMRF1 protein extensively, it turned out that phosphorylation of the protein by the kinase is inhibitory to the enhancement of the BZLF1-mediated transactivation of BALF2 promoter. Exogenous expression of BMRF1 protein augmented BALF2 expression in HEK293 cells harboring the EBV genome but lacking BMRF1 and BALF5 genes, demonstrating functions as a transcriptional regulator in the context of viral infection. Overall the BMRF1 protein is a multifunctional protein that cannot only act as a DNA polymerase processivity factor but also enhances BALF2 promoter transcription as a coactivator for the BZLF1 protein, regulating the expression level of viral single-stranded DNA-binding protein.

Identification of phosphorylation sites on transcription factor Sp1 in response to DNA damage and its accumulation at damaged sites.

DNA damage induces hyper-phosphorylation of the Sp1 transcriptional factor. We have demonstrated that ionizing radiation-associated DNA double-strand breaks (DSBs) induce phosphorylation of at least Ser-56 and Ser-101 residues on Sp1 in an ATM-dependent manner. UV irradiation- or hydroxyurea (HU)-induced replicative stress results in phosphorylation of only the Ser-101 residue. Furthermore, silencing of the ATM- and Rad3-related protein (ATR) in ATM-deficient cells treated with HU abrogated the Ser-101 phosphorylation. Thus, phosphorylation of Ser-101 on Sp1 appears to be a general response to DNA damage dependent on both ATM and ATR. Although silencing of Sp1 expression by siRNA targeting resulted in an increase in sensitivity to ionizing radiation (IR), the Ser-101 phosphorylation did not affect transcriptional activity from the Sp1 responsive promoter. Confocal laser microscopy analysis revealed co-localization of phosphorylated Sp1 at Ser-101 with phosphorylated ATM at Ser-1981, the affected sites representing DSBs. These observations suggest that phosphorylated Sp1 might play a role in DNA repair at damage sites rather than functioning in transcriptional regulation.

Pretreatment with 5-FU enhances cisplatin cytotoxicity in head and neck squamous cell carcinoma cells.

PURPOSE: In the treatment of head and neck malignancy, cisplatin and 5-FU have been used the most as chemotherapeutic agents. The difference in efficacies of these is unclear and controversial. To investigate more effective schedule, we analyzed the cytotoxicity in different treatment sequence with two agents in vitro and the mechanism for different effectiveness. METHODS: UM-SCC-23 and UM-SCC-81B, head and neck squamous cell carcinoma cell lines, were analyzed for cellular killing in alternative sequence treatment with cisplatin and 5-FU. The treatment schedule was designed based on the clinical regimen. To determine the mechanism for the difference of cytotoxicity with each schedule, cell cycle distributions of both cells after 5-FU treatment with various durations were analyzed by flow-cytometry and immunostaining with anti-PCNA and anti-BrdU. RESULTS: 5-FU pretreatment followed by cisplatin treatment showed higher cell killing in both types of cells than the reverse treatment schedule. In the cell cycle analysis and immunostaining after the treatment of 5-FU, the rate of PCNA-positive cells was increased from 24 to 144 h in both cells. The rate of BrdU-positive cells of UM-SCC-81B in flow-cytometry was also increased, while that of UM-SCC-23 was gradually decreased. These data suggested that the cells treated with 5-FU for more than 144 h were still in the S-phase with or without DNA synthesis. CONCLUSIONS: In head and neck carcinoma cells, we showed 5-FU pretreatment enhanced cisplatin cytotoxicity. The result of cell cycle analysis and immunostaining showed S-phase arrest by treatment of prolonged 5-FU treatment. The very long arrest in S-phase might be a mechanism to enhance cisplatin cytotoxicity by 5-FU pretreatment. We thus suggest pretreatment with 5-FU to enhance the effectiveness of cisplatin-based chemotherapy.

Hypersensitivity to cisplatin after hRev3 mRNA knockdown in head and neck squamous cell carcinoma cells.

Resistance to platinum-based chemotherapy frequently poses a significant problem in the treatment of head and neck squamous cell carcinomas (HNSCCs). In this study, we isolated cisplatin-resistant UM-SCC-23 CDDP/R cells from a UM-SCC-23 head and neck squamous cell carcinoma cell line. The UM-SCC-23 CDDP/R cells were approximately 3.5-fold more resistant to cisplatin than the UM-SCC-23 cells. Translesion DNA synthesis (TLS) is one of the major pathways involved in post-replication repair. To ascertain whether TLS is involved in cisplatin resistance in human cancer cells, we analyzed expression of the Rev3 gene, which encodes the catalytic subunit of DNA polymerase ζ (Polζ), using quantitative PCR. Expression of hRev3 mRNA in the UM-SCC-23 CDDP/R cells was approximately 1.4-fold higher than in the UM-SCC-23 cells. In both types of cells, expression of hRev3 mRNA was suppressed using siRNAs. Cisplatin sensitivity after hRev3 mRNA knockdown increased approximately 2-fold in UM-SCC-23 cells and approximately 3-fold in UM-SCC-23 CDDP/R cells. This study suggests that hRev3 knockdown with siRNAs increases sensitivity to cisplatin. Inhibition of the hRev3 gene may therefore prove to be a novel clinical strategy for reducing resistance to platinum-based chemotherapy in HNSCC.