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Neutralizing antibodies (NAbs) to human cytomegalovirus (HCMV) are associated with the risk of transplacental HCMV infection of the fetus in pregnant women. The IgG-positivity rate to HCMV determined by enzyme immunoassay (EIA) or indirect immunofluorescence assay has decreased from approximately 100% to 70% over the past 30 years in Japan. We tested serum samples from 630 Japanese women aged 20-49 years whose blood samples were obtained between 1980 and 2015. IgG titer was measured using an EIA-based assay. HCMV-NAb titer was measured using a neutralization test assay with an HCMV isolate on human retinal epithelial cells. Longitudinal transitions in HCMV-NAb prevalence were clarified. The prevalence of HCMV-EIA-IgG, and HCMV-NAb at a titer of 16-fold, and HCMV-NAb at a titer of 100-fold, changed from 96.7% to 78.9%, 93.3% to 85.6%, and 35.5% to 41.1%, respectively, between 1980-1990 and 2010-2015. Prevalence of HCMV-NAb at a titer of 16-fold decreased by 7.7%, whereas that at a titer of 100-fold increased by 5.6%. A high titer of HCMV-NAb in pregnant women is expected to reduce the risk of intrauterine HCMV transmission from the mother to the fetus. The association between the risk of congenital HCMV infection and the prevalence of HCMV-NAb remains to be addressed.

Heartland bandavirus (HRTV), which is an emerging tick-borne virus first identified in Missouri in 2009, causes fever, fatigue, decreased appetite, headache, nausea, diarrhea, and muscle or joint pain in humans. HRTV is genetically close to Dabie bandavirus, which is the causative agent of severe fever with thrombocytopenia syndrome (SFTS) in humans and is known as SFTS virus (SFTSV). The generation of infectious HRTV entirely from cloned cDNAs has not yet been reported. The absence of a reverse genetics system for HRTV has delayed efforts to understand its pathogenesis and to generate vaccines and antiviral drugs. Here, we developed a reverse genetics system for HRTV, which employs an RNA polymerase I-mediated expression system. A recombinant nonstructural protein (NSs)-knockout HRTV (rHRTV-NSsKO) was generated. We found that NSs interrupted signaling associated with innate immunity in HRTV-infected cells. The rHRTV-NSsKO was highly attenuated, indicated by the apparent absence of symptoms in a mouse model of HRTV infection. Moreover, mice immunized with rHRTV-NSsKO survived a lethal dose of HRTV. These findings suggest that NSs is a virulence factor of HRTV and that rHRTV-NSsKO could be a vaccine candidate for HRTV. IMPORTANCE Heartland bandavirus (HRTV) is a tick-borne virus identified in the United States in 2009. HRTV causes fever, fatigue, decreased appetite, headache, nausea, diarrhea, and muscle or joint pain in humans. FDA-approved vaccines and antiviral drugs are unavailable. The lack of a reverse genetics system hampers efforts to develop such antiviral therapeutics. Here, we developed a reverse genetics system for HRTV that led to the generation of a recombinant nonstructural protein (NSs)-knockout HRTV (rHRTV-NSsKO). We found that NSs interrupted signaling associated with innate immunity in HRTV-infected cells. Furthermore, rHRTV-NSsKO was highly attenuated and immunogenic in a mouse model. These findings suggest that NSs is a virulence factor of HRTV and that rHRTV-NSsKO could be a vaccine candidate for HRTV.

Zika virus (ZIKV) is a mosquito-borne flavivirus that causes febrile illness. The recent spread of ZIKV from Asia to the Americas via the Pacific region has revealed unprecedented features of ZIKV, including transplacental congenital infection causing microcephaly. Amino acid changes have been hypothesized to underlie the spread and novel features of American ZIKV strains; however, the relationship between genetic changes and the epidemic remains controversial. A comparison of the characteristics of a Southeast Asian strain (NIID123) and an American strain (PRVABC59) revealed that the latter had a higher replication ability in cultured cells and higher virulence in mice. In this study, we aimed to identify the genetic region of ZIKV responsible for these different characteristics using reverse genetics. A chimeric NIID123 strain in which the E protein was replaced with that of PRVABC59 showed a lower growth ability than the recombinant wild-type strain. Adaptation of the chimeric NIID123 to Vero cells induced a Phe-to-Leu amino acid substitution at position 146 of the prM protein; PRVABC59 also has Leu at this position. Leu at this position was found to be responsible for the viral replication ability and partially, for the pathogenicity in mouse testes., (© 2021. The Author(s).)

Genotype V (GV) Japanese encephalitis virus (JEV) has emerged in Korea and China since 2009. Recent findings suggest that current Japanese encephalitis (JE) vaccines may reduce the ability to induce neutralizing antibodies against GV JEV compared to other genotypes. This study sought to produce a novel live attenuated JE vaccine with a high efficacy against GV JEV. Genotype I (GI)-GV intertypic recombinant strain rJEV-E XZ0934 -M41 (E XZ0934 ), in which the E region of the GI Mie/41/2002 strain was replaced with that of GV strain XZ0934, was introduced with the same 10 attenuation substitutions in the E region found in the live attenuated JE vaccine strain SA 14-14-2 to produce a novel mutant virus rJEV-E XZ/SA14142m -M41 (E XZ/SA14142m ). In addition, another mutant rJEV-E M41/SA14142m -M41 (E M41/SA14142m ), which has the same substitutions in the Mie/41/2002, was also produced. The neuroinvasiveness and neurovirulence of the two mutant viruses were significantly reduced in mice. The mutant viruses induced neutralizing antibodies against GV JEV in mice. The growth of E XZ/SA14142m was lower than that of E M41/SA14142m . In mouse challenge tests, a single inoculation with a high dose of the mutants blocked lethal GV JEV infections; however, the protective efficacy of E XZ/SA14142m was weaker than that of E M41/SA14142m in low-dose inoculations. The lower protection potency of E XZ/SA14142m may be ascribed to the reduced growth ability caused by the attenuation mutations.

Zika virus (ZIKV) strains are classified into the African and Asian genotypes. The higher virulence of the African MR766 strain, which has been used extensively in ZIKV research, in adult IFNα/β receptor knockout (IFNAR-/-) mice is widely viewed as an artifact associated with mouse adaptation due to at least 146 passages in wild-type suckling mouse brains. To gain insights into the molecular determinants of MR766's virulence, a series of genes from MR766 were swapped with those from the Asian genotype PRVABC59 isolate, which is less virulent in IFNAR-/- mice. MR766 causes 100% lethal infection in IFNAR-/- mice, but when the prM gene of MR766 was replaced with that of PRVABC59, the chimera MR/PR(prM) showed 0% lethal infection. The reduced virulence was associated with reduced neuroinvasiveness, with MR766 brain titers ≈3 logs higher than those of MR/PR(prM) after subcutaneous infection, but was not significantly different in brain titers of MR766 and MR/PR(prM) after intracranial inoculation. MR/PR(prM) also showed reduced transcytosis when compared with MR766 in vitro. The high neuroinvasiveness of MR766 in IFNAR-/- mice could be linked to the 10 amino acids that differ between the prM proteins of MR766 and PRVABC59, with 5 of these changes affecting positive charge and hydrophobicity on the exposed surface of the prM protein. These 10 amino acids are highly conserved amongst African ZIKV isolates, irrespective of suckling mouse passage, arguing that the high virulence of MR766 in adult IFNAR-/- mice is not the result of mouse adaptation., Competing Interests: The authors have declared that no competing interests exist.

Severe fever with thrombocytopenia syndrome (SFTS) caused by a species Dabie bandavirus (formerly SFTS virus [SFTSV]) is an emerging hemorrhagic infectious disease with a high case-fatality rate. One of the best strategies for preventing SFTS is to develop a vaccine, which is expected to induce both humoral and cellular immunity. We applied a highly attenuated but still immunogenic vaccinia virus strain LC16m8 (m8) as a recombinant vaccine for SFTS. Recombinant m8s expressing SFTSV nucleoprotein (m8-N), envelope glycoprotein precursor (m8-GPC), and both N and GPC (m8-N+GPC) in the infected cells were generated. Both m8-GPC- and m8-N+GPC-infected cells were confirmed to produce SFTSV-like-particles (VLP) in vitro, and the N was incorporated in the VLP produced by the infection of cells with m8-N+GPC. Specific antibodies to SFTSV were induced in mice inoculated with each of the recombinant m8s, and the mice were fully protected from lethal challenge with SFTSV at both 103 TCID50 and 105 TCID50. In mice that had been immunized with vaccinia virus strain Lister in advance of m8-based SFTSV vaccine inoculation, protective immunity against the SFTSV challenge was also conferred. The pathological analysis revealed that mice immunized with m8-GPC or m8-N+GPC did not show any histopathological changes without any viral antigen-positive cells, whereas the control mice showed focal necrosis with inflammatory infiltration with SFTSV antigen-positive cells in tissues after SFTSV challenge. The passive serum transfer experiments revealed that sera collected from mice inoculated with m8-GPC or m8-N+GPC but not with m8-N conferred protective immunity against lethal SFTSV challenge in naïve mice. On the other hand, the depletion of CD8-positive cells in vivo did not abrogate the protective immunity conferred by m8-based SFTSV vaccines. Based on these results, the recombinant m8-GPC and m8-N+GPC were considered promising vaccine candidates for SFTS., Competing Interests: No authors have competing interests.

Introduction: Diagnosing tuberculous pleurisy is important in Japan because it currently has a moderate tuberculosis prevalence. However, physicians often have difficulty making a diagnosis. It was reported that thoracoscopy under local anesthesia is useful for the diagnosis of tuberculous pleurisy, but there are no reports focusing on elderly patients., Methods: In this study, the usefulness of thoracoscopy under local anesthesia was evaluated in elderly patients. Among 170 patients who underwent thoracoscopy under local anesthesia at our hospital during 11 years from January 2008 to December 2018, those aged 75 years or older (n = 75) were investigated retrospectively., Results: A total of 55 patients underwent thoracoscopy under local anesthesia for detailed examination of pleural effusion of unknown cause. Of these, 18 were diagnosed as tuberculous pleurisy. The median age was 82 years (range: 75-92 years). The diagnosis of tuberculous pleurisy was made in 11 patients in whom Mycobacterium tuberculosis was detected and in four patients whose pathological findings indicated epithelioid granuloma accompanied by caseous necrosis. Clinical diagnosis was made in the remaining three patients based on thoracoscopic findings of the pleural cavity and a high level of adenosine deaminase in pleural fluid. No serious complications attributable to the examination were observed in any patient., Conclusions: Thoracoscopy under local anesthesia was useful for the diagnosis of tuberculous pleurisy in elderly patients, with useful information being also obtained for the treatment of tuberculosis., Competing Interests: Declaration of competing interest The authors have declared no conflicts of interest., (Copyright © 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Morphological changes in the structure of the herpes simplex virus 1 (HSV-1) viral thymidine kinase (vTK) polypeptide usually lead to conferring acyclovir (ACV) resistance. HSV-1 I4-2, in which a UAG stop codon is present at the 8th position between the 1st initiation AUG codon (1st position) and the 2nd initiation AUG codon (46th position) of the HSV-1 vTK gene, showed sensitivity to ACV. In contrast, HSV-1 KG111, in which a UAG stop codon was artificially inserted at the 44th position, showed resistance to ACV at 39˚C. The mechanism underlying the difference in the sensitivity profiles was elucidated. The virus recombinants HSV-1-TK(8UAG) and HSV-1-TK(44UAG) containing a UAG stop codon at the 8th and 44th positions counted from the 1st initiation codon, respectively, were generated and tested for susceptibility to antiviral compounds. HSV-1-TK(8UAG) and HSV-1-TK(44UAG) were sensitive and resistant to ACV and BVdU at 37˚C, respectively. The expression level of the truncated vTK translated from the 2nd initiation codon in Vero cells infected with HSV-1-TK(44UAG) was clearly less than that with HSV-1-TK(8UAG) in a temperature-dependent manner. The differences in the antiviral sensitivity profiles were due to the position of the UAG stop codon between the 1st and the 2nd initiation codons.

Herpesviruses exist in nature within each host animal. Ten herpesviruses have been isolated from bats and their biological properties reported. A novel bat alphaherpesvirus, which we propose to name " Pteropus lylei -associated alphaherpesvirus (PLAHV)," was isolated from urine of the fruit bat Pteropus lylei in Vietnam and characterized. The entire genome sequence was determined to be 144,008 bp in length and predicted to include 72 genes. PLAHV was assigned to genus Simplexvirus with other bat alphaherpesviruses isolated from pteropodid bats in Southeast Asia and Africa. The replication capacity of PLAHV in several cells was evaluated in comparison with that of herpes simplex virus 1 (HSV-1). PLAHV replicated better in the bat-originated cell line and less in human embryonic lung fibroblasts than HSV-1 did. PLAHV was serologically related to another bat alphaherpesvirus, Pteropodid alphaherpesvirus 1 (PtAHV1), isolated from a Pteropus hypomelanus -related bat captured in Indonesia, but not with HSV-1. PLAHV caused lethal infection in mice. PLAHV was as susceptible to acyclovir as HSV-1 was. Characterization of this new member of bat alphaherpesviruses, PLAHV, expands the knowledge on bat-associated alphaherpesvirology. IMPORTANCE A novel bat alphaherpesvirus, Pteropus lylei -associated alphaherpesvirus (PLAHV), was isolated from urine of the fruit bat Pteropus lylei in Vietnam. The whole-genome sequence was determined and was predicted to include 72 open reading frames in the 144,008-bp genome. PLAHV is circulating in a species of fruit bats, Pteropus lylei , in Asia. This study expands the knowledge on bat-associated alphaherpesvirology., (Copyright © 2020 American Society for Microbiology.)

Background: Human cytomegalovirus (HCMV) causes asymptomatic infections, but also causes congenital infections when women were infected with HCMV during pregnancy, and life-threatening diseases in immunocompromised patients. To better understand the mechanism of the neutralization activity against HCMV, the association of HCMV NT antibody titers was assessed with the antibody titers against each glycoprotein complex (gc) of HCMV., Methods: Sera collected from 78 healthy adult volunteers were used. HCMV Merlin strain and HCMV clinical isolate strain 1612 were used in the NT assay with the plaque reduction assay, in which both the MRC-5 fibroblasts cells and the RPE-1 epithelial cells were used. Glycoprotein complex of gB, gH/gL complexes (gH/gL/gO and gH/gL/UL128-131A [PC]) and gM/gN were selected as target glycoproteins. 293FT cells expressed with gB, gM/gN, gH/gL/gO, or PC, were prepared and used for the measurement of the antibody titers against each gc in an indirect immunofluorescence assay (IIFA). The correlation between the IIFA titers to each gc and the HCMV-NT titers was evaluated., Results: There were no significant correlations between gB-specific IIFA titers and the HCMV-NT titers in epithelial cells or between gM/gN complex-specific IIFA titers and the HCMV-NT titers. On the other hand, there was a statistically significant positive correlation between the IIFA titers to gH/gL complexes and HCMV-NT titers., Conclusions: The data suggest that the gH/gL complexes might be the major target to induce NT activity against HCMV.

We previously showed that the growth ability of the Japanese encephalitis virus (JEV) genotype V (GV) strain Muar is clearly lower than that of the genotype I (GI) JEV strain Mie/41/2002 in murine neuroblastoma cells. Here, we sought to identify the region in GV JEV that is involved in its low growth potential in cultured cells. An intertypic virus containing the NS1-3 region of Muar in the Mie/41/2002 backbone (NS1-3 Muar ) exhibited a markedly diminished growth ability in murine neuroblastoma cells. Moreover, the growth rate of a Muar NS2A-bearing intertypic virus (NS2A Muar ) was also similar to that of Muar in these cells, indicating that NS2A of Muar is one of the regions responsible for the Muar-specific growth ability in murine neuroblastoma cells. Sequencing analysis of murine neuroblastoma Neuro-2a cell-adapted NS1-3 Muar virus clones revealed that His-to-Tyr mutation at position 166 of NS2A (NS2A 166 ) could rescue the low replication ability of NS1-3 Muar in Neuro-2a cells. Notably, a virus harboring a Tyr-to-His substitution at NS2A 166 (NS2A Y166H ) showed a decreased growth ability relative to that of the parental virus Mie/41/2002, whereas an NS2A Muar -based mutant virus, NS2A Muar-H166Y , showed a higher growth ability than NS2A Muar in Neuro-2a cells. Thus, these results indicate that the NS2A 166 amino acid in JEV is critical for the growth and tissue tropism of JEV in vitro.

Middle East respiratory syndrome-coronavirus (MERS-CoV) is an emerging virus that causes severe disease with fatal outcomes; however, there are currently no approved vaccines or specific treatments against MERS-CoV. Here, we developed a novel bivalent vaccine against MERS-CoV and rabies virus (RV) using the replication-incompetent P-gene-deficient RV (RVΔP), which has been previously established as a promising and safe viral vector. MERS-CoV spike glycoprotein comprises S1 and S2 subunits, with the S1 subunit being a primary target of neutralizing antibodies. Recombinant RVΔP, which expresses S1 fused with transmembrane and cytoplasmic domains together with 14 amino acids from the ectodomains of the RV-glycoprotein (RV-G), was developed using a reverse genetics method and named RVΔP-MERS/S1. Following generation of RVΔP-MERS/S1 and RVΔP, our analysis revealed that they shared similar growth properties, with the expression of S1 in RVΔP-MERS/S1-infected cells confirmed by immunofluorescence and western blot, and the immunogenicity and pathogenicity evaluated using mouse infection experiments. We observed no rabies-associated signs or symptoms in mice inoculated with RVΔP-MERS/S1. Moreover, virus-specific neutralizing antibodies against both MERS-CoV and RV were induced in mice inoculated intraperitoneally with RVΔP-MERS/S1. These findings indicate that RVΔP-MERS/S1 is a promising and safe bivalent-vaccine candidate against both MERS-CoV and RV., Competing Interests: The authors have declared that no competing interests exist.

Acyclovir (ACV) resistance-associated mutations in two recombinant herpes simplex virus 1 (HSV-1) clones were compared. Recombinant HSV-1 lacking its thymidine kinase (TK) and expressing varicella-zoster virus (VZV) TK ectopically had no mutations in the VZV TK gene. In contrast, recombinant HSV-1 expressing HSV-1 TK ectopically harbored mutations in the HSV-1 TK gene. These results suggest that the relatively low frequency of ACV-resistant VZV is a consequence of the characteristics of the TK gene., (Copyright © 2019 American Society for Microbiology.)

Several cases of herpes simplex encephalitis (HSE) caused by acyclovir (ACV)-resistant herpes simplex virus type 1 (HSV-1) have been reported. Amino acid substitutions of R41H, Q125H, and A156V in the viral thymidine kinase (vTK) gene have been reported to confer ACV resistance. Recombinant HSV-1 clones, containing each amino acid substitution in the vTK gene, were generated using the bacterial artificial chromosome system. A recombinant HSV-1 with the Q125H substitution showed ACV resistance while the R41H or A156V substitutions were ACV-sensitive. Furthermore, the Q125H recombinant HSV-1 was less virulent than the repaired virus, but it maintained neurovirulence in mice at relatively high levels. Substitution of Q125H, which was detected in the neonatal HSE patient, conferred ACV resistance, but the substitutions of R41H and A156V, which were detected in immunocompetent adult HSE patients, did not. This suggests that HSE caused by ACV-resistant HSV-1 might be a very rare event to occur during the course of ACV treatment in immunocompetent patients. Showing resistance to ACV treatment does not always indicate emergence of ACV-resistant HSV-1 in HSE patients.

Purpose: Higher serum 25-hydroxyvitamin D (25(OH)D) levels are reportedly associated with better survival in early-stage non-small cell lung cancer (NSCLC). Therefore, whether vitamin D supplementation can improve the prognosis of patients with NSCLC was examined (UMIN000001869). Patients and Methods: A randomized, double-blind trial comparing vitamin D supplements (1,200 IU/day) with placebo for 1 year after operation was conducted. The primary and secondary outcomes were relapse-free survival (RFS) and overall survival (OS), respectively. Prespecified subgroup analyses were performed with stratification by stage (early vs. advanced), pathology (adenocarcinoma vs. others), and 25(OH)D levels (low, <20 ng/mL vs. high, ≥20 ng/mL). Polymorphisms of vitamin D receptor (VDR) and vitamin D-binding protein (DBP) and survival were also examined. Results: Patients with NSCLC ( n = 155) were randomly assigned to receive vitamin D ( n = 77) or placebo ( n = 78) and followed for a median of 3.3 years. Relapse and death occurred in 40 (28%) and 24 (17%) patients, respectively. In the total study population, no significant difference in either RFS or OS was seen with vitamin D compared with the placebo group. However, by restricting the analysis to the subgroup with early-stage adenocarcinoma with low 25(OH)D, the vitamin D group showed significantly better 5-year RFS (86% vs. 50%, P = 0.04) and OS (91% vs. 48%, P = 0.02) than the placebo group. Among the examined polymorphisms, DBP1 (rs7041) TT and CDX2 (rs11568820) AA/AG genotypes were markers of better prognosis, even with multivariate adjustment. Conclusions: In patients with NSCLC, vitamin D supplementation may improve survival of patients with early-stage lung adenocarcinoma with lower 25(OH)D levels. Clin Cancer Res; 24(17); 4089-97. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Pulmonary cryptococcosis is difficult to distinguish from lung cancer clinically, and is often diagnosed by surgery. A 72-year-old woman, who underwent distal pancreatectomy and splenectomy for pancreatic carcinoma. Four months after surgery, a tumor shadow was detected in the left lung as a groundglass nodule (GGN)of 12 mm in diameter, which was found to change to 15 mm with increased density by the computed tomography(CT)scan after 2 months. The nodule showed positive accumulation of fluorodeoxyglucose(FDG)by positron emission tomography(PET), and was suspected of malignant tumor. She underwent a partial resection of the left lung under thoracoscopy.

A novel system was developed for generating highly attenuated vaccinia virus LC16m8 (m8, third-generation smallpox vaccine) that expresses foreign genes. The innovations in this system are its excisable selection marker, specificity of the integration site of a gene of interest, and easy identification of clones with a fluorescent signal. Using this system, recombinant m8s, which expressed herpes simplex virus 2 (HSV-2) glycoprotein B (gB)-, gD-, or both gB and gD (gB + gD), were generated, and their efficacy was evaluated. First, the induction of a specific IgG against these HSV-2 glycoproteins in mice infected with one of these recombinant m8s was confirmed by an immunofluorescent assay. Next, mice preinfected with one of the recombinant m8s were infected with HSV-2 at a lethal dose to examine the vaccine efficacy. The fatality rate among the mice preinfected with either the recombinant gB + gD- or gD-expressing m8 significantly decreased in comparison with the control. The survival rate in male and female mice preinfected with either the recombinant gB + gD- or gD-expressing m8 increased to 100% and 60%, respectively, while most of the control mice died. In summary, this new system may be applicable to creation of a novel m8-based vaccine.

LC16m8 (m8), a highly attenuated vaccinia virus (VAC) strain, was developed as a smallpox vaccine, and its safety and immunogenicity have been confirmed. Here, we aimed to develop a system that recovers infectious m8 from a bacterial artificial chromosome (BAC) that retains the full-length viral genomic DNA (m8-BAC system). The infectious virus was successfully recovered from a VAC-BAC plasmid, named pLC16m8-BAC. Furthermore, the bacterial replicon-free virus was generated by intramolecular homologous recombination and was successfully recovered from a modified VAC-BAC plasmid, named pLC16m8.8S-BAC. Also, the growth of the recovered virus was indistinguishable from that of authentic m8. The full genome sequence of the plasmid, which harbors identical inverted terminal repeats (ITR) to that of authentic m8, was determined by long-read next-generation sequencing (NGS). The ITR contains x 18 to 32 of the 70 and x 30 to 45 of 54 base pair tandem repeats, and the number of tandem repeats was different between the ITR left and right. Since the virus recovered from pLC16m8.8S-BAC was expected to retain the identical viral genome to that of m8, including the ITR, a reference-based alignment following a short-read NGS was performed to validate the sequence of the recovered virus. Based on the pattern of coverage depth in the ITR, no remarkable differences were observed between the virus and m8, and the other region was confirmed to be identical as well. In summary, this new system can recover the virus, which is geno- and phenotypically indistinguishable from authentic m8.

Ion Torrent next-generation sequencing (NGS) technology was applied to study the mode of emergence of acyclovir (ACV)-resistant (ACVr) herpes simplex virus type 1 (HSV-1) in patients with hematopoietic stem cell transplantation (HSCT) by quantitatively detecting mutations in the viral thymidine kinase (vTK) gene in the HSV-1 isolates recovered from HSCT patients. All of the mutations detected with the Sanger sequencing method in the vTK genes of HSV-1 isolates were also detected with the NGS assay. Furthermore, different mutations, which conferred ACV resistance and were not detected with the Sanger sequencing method, were also detected in a quantitative manner by using the NGS assay. The approach described here is applicable to studying the emergence process of vTK gene mutation-associated ACVr HSV-1 more in detail than the Sanger method. The NGS assay makes it possible to make a diagnosis of vTK gene mutation-associated ACVr HSV-1 infections at the early stage, which the ratio of ACVr HSV-1 is much lower than that of ACV-sensitive (ACVs) HSV-1., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Acyclovir (ACV)-resistant (ACVr) herpes simplex virus type 1 (HSV-1) infections are concern in immunocompromised patients. Most clinical ACVr HSV-1 isolates have mutations in the viral thymidine kinase (vTK) genes. The vTK-associated ACVr HSV-1 shows reduced virulence, but the association between the level of resistance and the virulence of the vTK-associated ACVr HSV-1 is still unclear., Methods: The virulence in mice of 5 vTK-associated ACVr HSV-1 clones with a variety of ACV sensitivities, when inoculated through intracerebral and corneal routes, was evaluated in comparison with ACV-sensitive (ACVs) parent HSV-1 TAS., Results: Although all the 5 ACVr HSV-1 clones and ACVs HSV-1 TAS showed a similar single-step growth capacity in vitro, the virulence of ACVr HSV-1 clones significantly decreased. A 50% lethal dose (LD 50 ) of each clone was closely correlated with 50% inhibitory concentrations (IC 50 ), demonstrating that the higher the ACV-sensitvity, the the higher the virulence among the ACVr clones. One of the ACVr HSV-1 clones with a relatively low IC 50 value maintained similar virulence to that of the parent TAS. The infection in mice with ACVr HSV-1 due to a single amino acid substitution in vTK induced local diseases, keratitis and dermatitis, while vTK-deficient clone did not., Conclusions: A statistically significant correlation between the virulence and susceptibility to ACV among ACVr HSV-1 clones was demonstrated.

Chronic myeloid leukemia is a clonal disease characterized by the presence of the Philadelphia chromosome and its oncogenic product, BCR-ABL, which activates multiple pathways involved in cell survival, growth promotion, and disease progression. We previously reported that in murine hematopoietic Ba/F3 cells, signal transducing adaptor protein-2 (STAP-2) binds to BCR-ABL and up-regulates BCR-ABL phosphorylation, leading to enhanced activation of its downstream signaling molecules. The binding of STAP-2 to BCR-ABL also influenced the expression levels of chemokine receptors, such as CXCR4 and CCR7. For the induction of CCR7 expression, signals mediated by the MAPK/ERK pathway were critical in Ba/F3 cells expressing BCR-ABL and STAP-2. In addition, STAP-2 cooperated with BCR-ABL to induce the production of CCR7 ligands, CCL19 and CCL21. Our results demonstrate a contribution of CCR7 to STAP-2-dependent enhancement of BCR-ABL-mediated cell growth in Ba/F3 cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Here we report a case of thymic cavernous haemangioma with the left innominate vein aneurysm. A 43-year-old man presented with chest pain. Enhanced chest computed tomography revealed an anterior mediastinal tumor measuring 60 × 52 × 38 mm with multiple venous lakes and focal specks of calcification, composed of a low-density soft tissue mass along with a left innominate vein aneurysm. We preoperatively diagnosed the mass as a thymic hemangioma and subsequently performed surgical resection. Pathologic diagnosis was a thymic cavernous hemangioma with a left innominate vein aneurysm, which is very rare., (Copyright © 2015 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

The fissureless lobectomy or anterior fissureless technique is a novel surgical technique, which avoids dissection of the lung parenchyma over the pulmonary artery during lobectomy by open thoracotomy approach or direct vision thoracoscopic surgery. This technique is indicated for fused lobes. We present two cases where thoracoscopic pulmonary segmentectomy was performed using the fissureless technique simulated by three-dimensional (3D) pulmonary models. The 3D model and rapid prototyping provided an accurate anatomical understanding of the operative field in both cases. We believe that the construction of these models is useful for thoracoscopic and other complicated surgeries of the chest.

Preoperative three-dimensional (3D) imaging of a mediastinal tumor using two-dimensional (2D) axial computed tomography is sometimes difficult, and an unexpected appearance of the tumor may be encountered during surgery. In order to evaluate the preoperative feasibility of a 3D mediastinal model that used the rapid prototyping technique, we created a model and report its results. The 2D image showed some of the relationship between the tumor and the pericardium, but the 3D mediastinal model that was created using the rapid prototyping technique showed the 3D lesion in the outer side of the extrapericardium. The patient underwent a thoracoscopic resection of the tumor, and the pathological examination showed a rare middle mediastinal ectopic thymoma. We believe that the construction of mediastinal models is useful for thoracoscopic surgery and other complicated surgeries of the chest diseases.

Thoracoscopic segmentectomies and subsegmentectomies are more difficult than lobectomy because of the complexity of the procedure; therefore, preoperative decision-making and surgical procedure planning are essential. In the literature, we could successfully perform thoracoscopic anatomical subsegmentectomy of the right S2b + S3 using a 3D printing model with rapid prototyping. This innovative surgical support model is extremely useful for planning a surgical procedure and identifying the surgical margin., (© The Author 2014. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

A 64-year-old man presented with dyspnea, chest pain, cough, expectoration, and continuous low-grade fever. Chest radiography and computed tomography revealed a right-sided, thick-walled cavity with diseased lung parenchyma, and sputum microscopy confirmed fungal forms of aspergillus. He subsequently developed a left-sided pneumonia caused by aspiration of the right-sided abscess. Because lung resection was considered too invasive, we performed simplified cavernostomy using the Alexis Wound Protector (XXS) using local anesthesia. In addition to the excellent drainage outcomes, this procedure was both simple and efficient. Moreover, the procedure was associated with less pain and physical limitation for the patient., (Copyright © 2014 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

We report the case of a 28-year-old woman with primary intercostal leiomyoma who presented with a complaint of right chest pain. Contrast-enhanced computed tomography (CT) demonstrated a slightly enhanced solid mass in the right anterior fifth intercostal space. Percutaneous needle biopsy revealed spindle cells without evidence of malignancy. Wide en bloc excision of the chest wall, including the anterior fifth and sixth ribs and the upper costal arch, was performed. This way, the mass was completely resected without exposure, and the chest wall defect was reconstructed using a Gore-Tex(®) dual mesh. Histopathological analysis confirmed localized primary intercostal leiomyoma. The patient has been disease-free for more than 2 months since surgery.Primary leiomyomas of the chest wall are extremely rare. To the best of our knowledge, 9 cases of leiomyoma of the pleura have been reported till date, but this is the first case report of an intercostal leiomyoma of the chest wall. This case report describes the clinical course of this case and presents a review of the relevant literature.

We report a case of retroesophageal parathyroid adenoma coexisting with an aberrant right subclavian artery (ARSA). A 63-year-old female presented with elevated serum calcium and intact parathyroid hormone levels. 99mTc-methoxyisobutylisonitrile scintigram and enhanced chest computed tomography (CT) revealed a mediastinal tumor measuring 25×15×7 mm located posterior to the esophagus. Three-dimensional CT provided accurate anatomical location of the tumor and the ARSA. We safely resected the ectopic parathyroid adenoma using video-assisted thoracic surgery owing to the accurate location of the adenoma and its relationship to the surrounding anatomical structures including the ARSA.

Thoracoscopic pulmonary segmentectomy of the lung is sometime adopted for the lung cancer, but a problem with segmentectomy is variable anatomy. Recently, we are exploring the impact of three-dimensional models using rapid-prototyping technique. It is useful for decision making, surgical planning, and intraoperative orientation for surgical treatment in patient with lung cancer who underwent pulmonary segmentectomy. These newly created models allow us to clearly identify the surgical margin and the intersegmental plane, vessels, and bronchi related to the cancer in the posterior segment. To the best of our knowledge, there are few reports describing a pulmonary model so far.

Lung surgeries in patients with bronchial variations have rarely been reported. Here, we describe the case of a patient along with lung cancer with variant anatomy of the right upper lobe bronchus. This variation was evaluated by three-dimensional multi-detector computed tomography angiography with bronchography and a three-dimensional printing model using rapid prototyping. The variant anterior segment bronchus (S3) of the right upper lobe arising from the middle lobe bronchus was confirmed before surgery using the printing model, which helped to determine the extent of resection required and facilitated the understanding of the patient's anatomy during surgery. A thoracoscopic anterior segmentectomy and middle lobectomy were performed. The printing model was useful for detecting and evaluating the variant bronchi.

Lung segmentectomy with bronchial variation has rarely been reported. We report the case of a lung cancer patient with variant anatomy of the right upper lobe bronchus.Thoracoscopic posterior segmentectomy of the right upper lobe was performed. Variant bronchus and related blood vessels were confirmed preoperatively by three-dimensional multidetector computed tomography (3D-MDCT), which facilitated visualization of the patient's anatomy during surgery.

Background: The right top pulmonary vein (PV) has been defined as an anomalous branch of the right superior PV draining into the left atrium (LA); however, various PV anomalies and terminologies have been reported. To clarify the concept of the right top PV, we reviewed the literature and our cases., Methods: We reviewed the literature on the right top PV, right PV passing behind the intermediate bronchus (BIB), and related PV anomalies. We also reviewed our anomalous PV cases, which were analyzed using 3-dimensional computed tomography (3D-CT)., Results: Authors of the previous reports were radiologists, surgeons, and cardiologists. The terminologies used in the literature included PV branch crossing BIB, right upper lobe vein posterior to the bronchus intermedius, and right isolated superior posterior branch. The frequency of the anomaly in the literature ranged from 0.3% to 9.3%. Anomalous PVs originated from either the right upper lobe or the lower lobe. We found the following among 303 patients with chest disorders at our hospital: 10 (3.3%) of these PV anomalies were observed-4 drained directly into the LA and the other 4 drained into the right superior PV. Among 9 patients who were analyzed and had complete interlobar fissures between the upper and lower lobes, 4 patients had drainage from both the right upper and the lower lobes. Eight PVs passed BIB, 1 passed behind the main bronchus (BMB), and the other passed both BIB and BMB., Conclusions: We propose that the term right top PV should be used in a broad sense, being defined as "an anomalous branch of PV draining directly into the left atrium (LA)," and that it be classified into 6 types., (Copyright © 2013 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

To "mark" the nitrogen atoms in phenyl-(2-pyridyl)-(3-pyridyl)-(4-pyridyl)methane (1), we have synthesized the corresponding tris(pyridine N-oxide) 2 by oxidation of 1 with m-chloroperbenzoic acid. The nitrogen atoms of 2 are unequivocally determined by the X-ray crystal analysis of a single crystal of rac-2 whereas the nitrogen atoms cannot be assigned at all in the case of rac-1. N-Oxide 2 can be resolved by chiral high-performance liquid chromatography under similar conditions to those used for the resolution of 1. The calculated circular dichroism (CD) curve for (R)-2 on the basis of time-dependent density functional theory reproduces the experimental spectra very well to suggest that the second-eluted fraction ([CD(+)283]-2) is the R isomer, namely (R)-[CD(+)283]-2. The independent absolute configuration determinations for 1 and 2 are in keeping with the chemical correlation between the two compounds by oxidation of (R)-1 into (R)-2., (Copyright © 2011 Wiley-Liss, Inc.)

A 22-year-old man was referred for hemoptysis and general fatigue after exercise. Arteriography demonstrated an anomalous artery arising from the descending aorta supplying the lingular and all of the basal segments of the left lung. The feeding areas of the pulmonary and anomalous arteries were mutually exclusive. He underwent division of the anomalous artery and combined resection of the diseased segments. The upper division of the upper lobe and the superior segment of the lower lobe were spared. His symptoms were greatly improved postoperatively. The preoperative anatomic evaluation of anomalous vessels is crucial in surgical management.

Tetrakis(bithienyl)methane and tetrakis(terthienyl)methane have been synthesized from tetrakis(2-thienyl)methane by use of Suzuki-Miyaura coupling as a key reaction. Their trimethylsilyl (TMS) derivatives are also synthesized. X-ray analysis reveals that each oligothiophene moiety tends to adopt anti-conformations and show relatively small torsion angles between the adjacent thiophene rings. While the longest absorption maxima of these tetrakis(oligothienyl)methanes exhibit only a slight bathochromic shift compared to the corresponding linear oligothiophene derivative, tetrakis(bithienyl)methane and its TMS derivative exhibit an appreciable red-shift in their fluorescence spectra. The intramolecular interaction between thienyl groups of tetrakis(2-thienyl)methane is supported by DFT calculation.

Objective: Ground-glass opacity (GGO) of the lung is being frequently detected by thin section computed tomography scan. However, the long term management of detected GGO is still unclear. To establish follow-up plans, we performed the clinical and radiological review to identify the factors that are closely associated with GGO growth., Methods: We retrospectively analyzed computed tomography images of 125 GGOs that were stable for 3 months between 1999 and 2006 at the Cancer Institute Hospital, Tokyo. To identify factors that affect the roentgenological growth, the time to GGO growth curve by Kaplan-Meyer method was evaluated in terms of gender, age, smoking, initial size, existence of a solid part, GGO density, location, multiplicity, and lung cancer history by univariate and multivariate analyses., Results: The median observation period was 1048 days (177-3269) and 26 of 125 GGOs (21%) grew. The estimated growth population for 5 years was 30%. The growth was more frequently seen in the elderly (p = 0.017), in part-solid GGO (p < 0.01) and in GGO of larger than 10 mm (p < 0.01, logrank test). By multivariate analysis, initial size (p < 0.01, Cox's model) and history of lung cancer (p = 0.017, logistic model) were independent factors that were significantly associated with GGO growth. Fifty GGOs that were 10 mm or smaller and without a lung cancer history did not grow within 3.5 years., Conclusions: After initial management and 3 month follow-up, larger size (more than 10 mm) and a history of lung cancer are risk factors for GGO growth, and therefore should be considered when making a follow-up plan.

We investigated the necessity of preparation for blood transfusion in gastric cancer surgery to save costs for blood typing, antibody screening, cross-matching, and disposal of the blood product. The subjects of the study were 52 patients who underwent gastric cancer surgery at our department between 2000 and 2004. The requirement for blood transfusion during surgery was investigated in terms of patient characteristics, hemoglobin before surgery, and performance status as well as treatment regimen. Furthermore, economic effects were investigated when typing and screening (T&S) were performed instead of typing and cross-matching (T&X). Of 9 patients who received blood transfusion, 8 had gastric cancer of stage IIIB or higher, or underwent combined resection. Blood transfusion was not used in surgery for patients with early gastric cancer. The volumes of blood prepared, lost, and disposed of in 28 patients who underwent T&X were 831.3+/-249.4, 219.3+/-228.5 and 600+/-333.1 ml, respectively, whereas the blood loss in 24 patients who underwent T&S was 161.1+/-95.6 ml; this difference had a major economic effect. The practice of T&S for patients undergoing gastric surgery in the absence of combined resection for early gastric cancer seems to be a safe and cost-effective practice that abrogates disposal of blood in hospital management.

The title compound, a prototypical chiral molecule based on a tetraarylmethane framework, has been synthesized in five steps from (2-pyridyl)-(3-pyridyl)ketone. X-ray crystallographic analysis revealed the tetraarylmethane framework of the molecule but did not determine the positions of the nitrogen atoms because the crystal is a racemic compound and the aryl groups are disordered in the crystal. The optical resolution of the title compound was achieved by chiral HPLC with a Chiralcel OD column. The CD spectra of the two fractions in acetonitrile exhibited opposite signs as expected for a pair of enantiomers. Their CD spectra are changed in 2 M HCl due to protonation. The calculated CD curve for the target molecule based on time-dependent density functional theory (TDDFT) reproduces the experimental result very well, thus suggesting that the first eluted fraction is the R isomer in terms of absolute configuration.

Background: We have developed a new technique for treatment of intramucosal carcinoma which exceeds the standard indication for endoscopic mucosal resection and carcinoma invading the submucosa without lymph node metastasis that are located in the posterior wall of the stomach, which we refer to as laparoscopic intragastric full-thickness excision (LIFE) under flexible endoscopic control., Surgical Technique: Three pigs were used for the study. Three trocars were used. The first trocar (trocar # 1) was placed in the subumbilical region to introduce the videoscope, whereas the second and third trocars (trocar # 2 and trocar # 3) were punctured percutaneously into the abdominal cavity. A straight needle with 3-0 silk suture was attached to a T-bar on the wire side and inserted into the abdominal cavity. An area adjacent to the lesion in the posterior wall of the stomach was pierced by the straight needle, which was then pulled into the stomach using the forceps of the endoscope. The T-bar, after being passed through the abdominal wall, was fixed outside the gastric wall, and trocar # 3 was repositioned in the stomach by the percutaneous transgastric route. The posterior wall of the stomach was pulled inward by the T-bar, and the lesion was removed by several excisions with laparoscopic stapling devices inserted through trocar # 3; extraction of the specimen was achieved through trocar # 3. The gastrotomy site was suture-closed using instruments positioned through trocar # 2 and trocar # 3 under laparoscopy., Conclusions: Based on a feasibility study in pigs, the LIFE procedure can be performed for lesions of the posterior wall of the stomach.