Evaluation of a novel severe combined immunodeficiency mouse model for antiviral drug evaluation against Chandipura virus infection.

Chandipura virus (CHPV) is a negative-sense single-stranded RNA virus known to cause fatal encephalitis outbreaks in the Indian subcontinent. The virus displays tropism towards the pediatric population and holds significant public health concerns. Currently, there is no specific, effective therapy for CHPV encephalitis. In this study, we evaluated a novel C.B-17 severe combined immunodeficiency (SCID) mouse model which can be used for pre-clinical antiviral evaluation. Inoculation of CHPV developed a lethal infection in our model. Plaque assay and immunohistochemistry detected increased viral loads and antigens in various organs, including the brain, spinal cord, adrenal glands, and whole blood. We further conducted a proof-of-concept evaluation of favipiravir in the SCID mouse model. Favipiravir treatment improved survival with pre-symptomatic (days 5-14) and post-symptomatic (days 9-18) treatment. Reduced viral loads were observed in whole blood, kidney/adrenal gland, and brain tissue with favipiravir treatment. The findings in this study demonstrate the utility of SCID mouse for in vivo drug efficacy evaluation and the potential efficacy of favipiravir against CHPV infection.

Higher hematocrit level associated with higher 5-aminolevulinic acid-induced perioperative blood pressure change.

BACKGROUND: 5-aminolevulinic acid (5-ALA) is used for photodynamic diagnosis-assisted surgeries. Hypotension is among 5-ALA-related adverse effects. 5-ALA metabolism requires iron. The red cell life span is 120 days and heme iron is daily recycled. Higher hematocrit is likely to correlate with higher recycled iron. We previously reported 5-ALA-induced hemodynamics in urological surgery. This analysis aimed to determine the association between 5-ALA-induced perioperative systolic blood pressure (SBP) changes and the hematocrit. METHODS: This retrospective study enrolled consecutive patients who underwent transurethral resection of bladder tumor from August 2018 to December 2020. The patients were classified into the 5-ALA-pretreated patients (5-ALA group; n = 26) and non-pretreated patients (control group; n = 97). We evaluated the correlation between SBP change rates and hematocrit levels. The primary analyses included the difference in correlations between the two groups. Subsequently, the correlations were analyzed in the 5-ALA group and control group, respectively. RESULTS: The correlations significantly differed between the two groups preoperatively (P<0.001), during surgery (P = 0.014), postoperatively (P = 0.001), and on the following morning (P = 0.002). The correlations between SBP changes and the hematocrit in the 5-ALA group were significant before patients entered the operation room (Spearman's rank correlation coefficient [rS]=-0.449, P = 0.024), before anesthesia induction (rS=-0.584, P = 0.002), during surgery (rS=-0.401, P = 0.047), after operation (rS=-0.658, P<0.001), and on the following morning (rS=-0.547, P = 0.004). Those in the control group were not significant. CONCLUSIONS: The hematocrit levels were significantly correlated with perioperative 5-ALA-induced SBP changes. The association was again observed the next day. Higher hematocrit may be a factor for 5-ALA-induced hemodynamic changes.

Radical Resection for Second EGFR-mutated Primary Lung Cancer Following Immune Checkpoint Inhibitor Monotherapy for Stage IV Lung Adenocarcinoma.

A 78-year-old woman with multiple lung nodules, epithelial growth factor receptor (EGFR) exon 20 insertion mutations, and diagnosed with advanced lung adenocarcinoma (cT4N3M1a, stage IVA), was referred to our hospital. She received immune checkpoint inhibitor (ICI) therapy. The therapy showed remarkable antitumor effects; only a single nodule remained in the right upper lobe. The nodule was diagnosed as adenocarcinoma through a biopsy. We subsequently performed right upper lobectomy for multiple primary lung cancer (MPLC). The surgical specimen contained EGFR exon 19 deletion mutations and not exon 20 insertion mutations.

Differences in 5-Aminolevulinic Acid-Induced Hemodynamic Changes between Patients Undergoing Neurosurgery and Urological Surgery.

INTRODUCTION: Oral 5-aminolevulinic acid (5-ALA) is often used for photodynamic diagnosis-assisted glioma or bladder tumor surgery. 5-ALA affects blood pressure (BP). In fact, hypotension is a well-known adverse effect of 5-ALA in urology. However, information regarding 5-ALA-induced hemodynamic changes in neurosurgery remains limited. Furthermore, the duration of hypotension and how 5-ALA affects the heart rate (HR) are yet to be determined. Thus, in this study, we aimed to elucidate 5-ALA-induced perioperative hemodynamic changes in neurosurgery and urological surgery by examining real-world data. METHODS: Consecutive patients who underwent neurosurgery (neurosurgery patients; 5-ALA-pretreated vs. non-pretreated [17 vs. 16], from January 2014 to March 2021) and urological surgery (urological surgery patients; 5-ALA-pretreated vs. non-pretreated [26 vs. 101], from August 2018 to September 2020) were enrolled. Differences in hemodynamics were evaluated using the linear mixed model. BP and HR in 5-ALA-pretreated patients were compared with those in non-pretreated patients. Differences in 5-ALA-induced preoperative BP changes were compared between the neurosurgery patients and urological surgery patients. RESULTS: 5-ALA scarcely affected the hemodynamics in neurosurgery patients, whereas 5-ALA-induced hemodynamic changes were clearly observed in urological surgery patients. Hemodynamic parameters were found to be not significantly different between 5-ALA-pretreated and non-pretreated neurosurgery patients. The preoperative, intraoperative, and postoperative BP in 5-ALA-pretreated urological surgery patients were significantly lower than those in the non-pretreated patients. Preoperatively, two 5-ALA-pretreated urological surgery patients had severe postural hypotension (systolic BP <50 mmHg), and one of them did not continue with the surgery because of prolonged severe hypotension. The BP in 5-ALA-pretreated urological surgery patients tended to be persistently lower for 9 h after 5-ALA pretreatment. The preoperative and postoperative HR values were higher in 5-ALA-pretreated urological surgery patients. Cumulative incidences of BP reduction and HR elevation were significantly higher in 5-ALA-pretreated urological surgery patients. The preoperative BP reduction in 5-ALA-pretreated urological surgery patients was significantly larger than that in neurosurgery patients. CONCLUSIONS: 5-ALA-induced hemodynamics may differ between neurosurgery patients and urological surgery patients. 5-ALA may affect BP for at least 9 h.

Favipiravir treatment prolongs the survival in a lethal mouse model intracerebrally inoculated with Jamestown Canyon virus.

BACKGROUND: Jamestown Canyon virus (JCV) is a mosquito-borne orthobunyavirus that causes acute febrile illness, meningitis, and meningoencephalitis, primarily in North American adults. Currently, there are no available vaccines or specific treatments against JCV infections. METHODOLOGY/PRINCIPAL FINDINGS: The antiviral efficacy of favipiravir (FPV) against JCV infection was evaluated in vitro and in vivo in comparison with that of ribavirin (RBV) and 2'-fluoro-2'-deoxycytidine (2'-FdC). The in vitro inhibitory effect of these drugs on JCV replication was evaluated in Vero and Neuro-2a (N2A) cells. The efficacy of FPV in the treatment of JCV infection in vivo was evaluated in C57BL/6J mice inoculated intracerebrally with JCV, as per the survival, viral titers in the brain, and viral RNA load in the blood. The 90% inhibitory concentrations (IC90) of FPV, RBV, and 2'-FdC were 41.0, 61.8, and 13.6 µM in Vero cells and 20.7, 25.8, and 8.8 µM in N2A cells, respectively. All mice infected with 1.0×104 TCID50 died or were sacrificed within 10 days post-infection (dpi) without treatment. However, mice treated with FPV for 5 days [initiated either 2 days prior to infection (-2 dpi-2 dpi) or on the day of infection (0 dpi-4 dpi)] survived significantly longer than control mice, administered with PBS (p = 0.025 and 0.011, respectively). Moreover, at 1 and 3 dpi, the virus titers in the brain were significantly lower in FPV-treated mice (0 dpi-4 dpi) versus PBS-treated mice (p = 0.002 for both 1 and 3 dpi). CONCLUSIONS/SIGNIFICANCE: Although the intracerebral inoculation route is thought to be a challenging way to evaluate drug efficacy, FPV inhibits the in vitro replication of JCV and prolongs the survival of mice intracerebrally inoculated with JCV. These results will enable the development of a specific antiviral treatment against JCV infections and establishment of an effective animal model.

Development of an assay for detecting the residual viable virus in inactivated rabies vaccine by enzyme-linked immunosorbent assay.

Rabies is a zoonotic disease that can be prevented by vaccination. The confirmation of rabies virus inactivation is a critical step during the vaccine quality test; however, the current protocol conducted in Japan requires a large number of mice. The development and introduction of animal-free alternative assays are essential from the perspective of the 3Rs (reduction, refinement, and replacement) of animal testing. Here, we propose a novel inactivation assay for confirming the complete inactivation of the viable rabies virus using cultured Neuro-2a cells and an enzyme-linked immunosorbent assay (ELISA). The detection ability of ELISA was similar to that of a direct immunofluorescence assay, with the detection limit of ELISA being as low as 0.014 focus forming units/test. These results suggest that the assay could be used as a viral inactivation test. In comparison with a traditional in vivo assay, this assay has a higher detection ability, an objective interpretation, and would shorten the test duration from 25 days to 8 days.

Non-bacterial cystitis with increased expression of programmed death-ligand 1 in the urothelium: An unusual immune-related adverse event during treatment with pembrolizumab for lung adenocarcinoma.

INTRODUCTION: Immune checkpoint inhibitors are now a standard therapeutic option for lung adenocarcinoma. However, Immune checkpoint inhibitors often induce various immune-related adverse events. CASE PRESENTATION: The patient was a 78-year-old woman with lung adenocarcinoma who had a partial response to pembrolizumab. During treatment, she complained of pollakiuria and nocturia with painful micturition. Histological analysis revealed infiltration of CD8-positive and/or TIA-1 cytotoxic granule-associated RNA binding protein-positive lymphocytes and programmed death-ligand 1 expression in the urothelium. A diagnosis of immune-related adverse event cystitis was made based on these clinical and pathological findings. The patient's subjective symptoms and findings on cystoscopy improved dramatically after treatment with prednisolone. CONCLUSION: Immune checkpoint inhibitors-induced cystitis is extremely rare. This report is the first to include an immunohistochemical analysis of the urothelial epithelium in immune-related adverse event cystitis and describes an instructive case.

Seroprevalence of Jamestown Canyon virus in the Japanese general population.

BACKGROUND: Jamestown Canyon virus (JCV) is a mosquito-borne orthobunyavirus that causes acute febrile illness, meningitis, and meningoencephalitis, mainly among adults. JCV is widely distributed in North America and the number of JCV cases in the U.S. has increased in recent years. Therefore, the central nervous system disease caused by JCV can be considered a potentially re-emerging viral disease. However, the seroprevalence of JCV is unknown in Japan. The purpose of this study is to evaluate the seroprevalence of JCV in the Japanese population. METHODS: We used an IgG enzyme-linked immunosorbent assay (IgG-ELISA) with JCV-infected cell-lysates and/or a neutralizing (NT) antibody assay. The cut-off value of IgG-ELISA was determined using IgG-ELISA to analyze serum specimens from 37 healthy Japanese donors. IgG-ELISA was validated by assessing its sensitivity and specificity, using 38 human serum samples previously tested for the presence or absence of antibodies against JCV and snowshoe hare virus (SSHV), in an in-house NT antibody assay conducted by the Public Health Agency of Canada. The seroepidemiological study was performed using IgG-ELISA and NT antibody assay to analyze 246 human serum samples from the serum bank of the National Institute of Infectious Diseases (NIID) in Japan. RESULTS: The cut-off value of IgG-ELISA was determined at 0.20, based on the mean (- 0.075) and standard deviation (0.092) values using Japanese donors' sera. The sensitivity and the specificity of IgG-ELISA determined using 25 JCV-positive and 4 JCV-negative serum samples were 96 and 100%, respectively. Analysis of the 246 Japanese serum samples revealed that no specimen showed a higher value than the cut-off value of IgG-ELISA, and no sample tested positive by the NT antibody assay. CONCLUSIONS: Our results showed that JCV is not circulating significantly in Japan. To the best of our knowledge, this is the first report to demonstrate the seroprevalence of JCV in the general population in Japan.

High mobility group box 1 can be used to monitor perioperative course in patients with liver cancer.

OBJECTIVE: High mobility group box 1 (HMGB1) is produced by inflammation. Regarding liver injuries, HMGB1 is reportedly involved in liver regeneration. The present study investigated the use of HMGB1 as a postoperative marker of surgical course in patients with liver cancer. METHODS: Patients were enrolled if they had liver cancer, had undergone liver surgery, and did not develop postsurgical complications. Patients who received emergency surgery or patients with unresectable cancerous lesions were excluded. Blood samples were preoperatively obtained as well as at 1 day, 1 week, and 4 weeks following surgery; white blood cell count, serum C-reactive protein, serum albumin, and serum HMGB1 levels were measured. RESULTS: A total of 36 patients were included in this study. HMGB1 levels significantly changed over time, increasing from a median of 7.1 ng/ml (preoperatively) to 13.9 ng/ml at 1 week postoperatively, and then decreased to 6.3 ng/ml at 4 weeks postoperatively. Peak HMGB1 levels were delayed, and elevated HMGB1 levels persisted as compared with the changes in conventional markers. CONCLUSIONS: HMGB1 indicates a unique perioperative inflammatory state in patients with liver cancer. Serum HMGB1 may serve as a marker for monitoring surgical course in patients undergoing surgery for liver cancer.

Development of a recombinant replication-deficient rabies virus-based bivalent-vaccine against MERS-CoV and rabies virus and its humoral immunogenicity in mice.

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.

In vitro evaluation of minimum inhibitory concentration of several antibacterial agents against Rickettsia japonica using a plaque reduction assay system.

The study was conducted to determine the minimum inhibitory concentrations (MICs) of several antibacterial agents against Rickettsia japonica, which causes Japanese spotted fever. A plaque reduction assay as an in vitro culture method was conducted to determine the MICs of antibacterial agents (4 types of tetracyclines: tetracycline, doxycycline, minocycline, and tigecycline; 3 types of quinolones: ciprofloxacin, ofloxacin, and levofloxacin; and 2 types of macrolides: azithromycin and clarythromycin) against R. japonica. R. japonica was sensitive to the antibacterial agents tested with MICs similar to those against other spotted fever rickettsia determined in previously described plaque reduction assays.

Ribonucleotide reductase M2 promotes RNA replication of hepatitis C virus by protecting NS5B protein from hPLIC1-dependent proteasomal degradation.

Hepatitis C virus (HCV) establishes a chronic infection that can lead to cirrhosis and hepatocellular carcinoma. The HCV life cycle is closely associated with host factors that promote or restrict viral replication, the characterization of which could help to identify potential therapeutic targets. To this end, here we performed a genome-wide microarray analysis and identified ribonucleotide reductase M2 (RRM2) as a cellular factor essential for HCV replication. We found that RRM2 is up-regulated in response to HCV infection in quiescent hepatocytes from humanized chimeric mouse livers. To elucidate the molecular basis of RRM2 expression in HCV-infected cells, we used HCV-infected hepatocytes from chimeric mice and hepatoma cells infected with the HCV strain JFH1. Both models exhibited increased RRM2 mRNA and protein expression levels. Moreover, siRNA-mediated silencing of RRM2 suppressed HCV replication and infection. Of note, RRM2 and RNA polymerase nonstructural protein 5B (NS5B) partially co-localized in cells and co-immunoprecipitated, suggesting that they might interact. RRM2 knockdown reduced NS5B expression, which depended on the protein degradation pathway, as NS5B RNA levels did not decrease and NS5B protein stability correlated with RRM2 protein levels. We also found that RRM2 silencing decreased levels of hPLIC1 (human homolog 1 of protein linking integrin-associated protein and cytoskeleton), a ubiquitin-like protein that interacts with NS5B and promotes its degradation. This finding suggests that there is a dynamic interplay between RRM2 and the NS5B-hPLIC1 complex that has an important function in HCV replication. Together, these results identify a role of host RRM2 in viral RNA replication.

Spinal myoclonus following neuraxial anesthesia: a literature review.

Spinal myoclonus (SM) is a rare neurologic movement disorder following neuraxial anesthesia (NA). SM following NA (SM-NA) has insufficient clinical information and its pathogenesis remains to be elucidated. The aim of this review article was to summarize the past cases and consider SM-NA pathophysiology. Based on our PubMed search, it was revealed that SM-NA develops within several hours after neuraxial local anesthetic (LA) administration and resolves in a day without leaving neurologic compilations. It occurs primarily in the lower extremities, but can sometimes spread upward and affect the upper extremities and trunk. Although statistical adjustments are indispensable, analysis of the previous cases provided important facts that seem to be related with the mechanism of SM-NA. The frequently used LAs for spinal anesthesia were hyperbaric. SM-NA occurrence was more frequent in women. After initiation of spinal anesthesia, intrathecal hyperbaric LA distributes cephalad. In the LA elimination process, the large concentration differences in intrathecal LA may induce the partially functioning spinal neurons, resulting in myoclonus generation. The morphological features of the lumbar spine in women can predispose to a higher LA concentration difference. SM-NA is an unpredictable and rare neural complication following NA and should be confirmed by basic experiments and large-scale researches.

Preanesthetic severe postural hypotension following 5-aminolevulinic acid pretreatment in patients for photodynamic diagnosis-assisted urological surgery.

BACKGROUND: 5-Aminolevulinic acid (5-ALA) is utilized for photodynamic diagnosis-assisted (PDD) surgery. However, it has been associated with vasodilation, hence, occasional hypotension. CASE PRESENTATION: We encountered two patients who had severe postural hypotension following 5-ALA pretreatment prior to an operation. They were scheduled for urological PDD surgery, but upon standing to walk to the operation room, they felt sick because of severe hypotension. One of them underwent the surgery after recovery, but the other surgery was canceled due to a prolonged hypotension that lasted for more than a day. CONCLUSIONS: Severe postural hypotension may develop as a result of the high concentration of porphyrin precursors, which may affect the nervous system. Severe postural hypotension may be due to 5-ALA-induced autonomic dysfunction as well as vasodilative action of 5-ALA. These observations suggest that in addition to the careful monitoring of patients' vital signs, standing should be avoided following 5-ALA pretreatment.

Acyclovir Sensitivity and Neurovirulence of Herpes Simplex Virus Type 1 with Amino Acid Substitutions in the Viral Thymidine Kinase Gene, Which Were Detected in the Patients with Intractable Herpes Simplex Encephalitis Previously Reported.

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.

A case of spinal myoclonus in a patient with elective cesarean section.

BACKGROUND: Transient myoclonic involuntary movements, typically referred to as spinal myoclonus (SM), rarely develop in the extremities following neuraxial anesthesia (NA). NA indications in patients with history of SM following NA (SM-NA) are unknown. CASE PRESENTATION: A 33-year-old woman developed SM-NA after elective cesarean section (CS). Approximately 130 min after spinal anesthesia induction, she began exhibiting involuntary movements, which became most severe after approximately 3 h. The involuntary movements gradually decreased without treatments and disappeared after approximately 5 h. The patient underwent CS on three occasions. The first CS (age, 29 years) was under a combination of spinal and epidural anesthesia. The third CS (age, 35 years) was completed using only spinal anesthesia. There were no neurological events during the postoperative courses for the first and third CS. CONCLUSIONS: SM-NA can unexpectedly occur, and history of SM-NA may not be contraindicative for repeated NA.

Near-infrared spectroscopy might be a useful tool for predicting the risk of vascular complications after pediatric liver transplants: Two case reports.

In patients that have undergone liver transplants, a postoperative reduction in the blood flow of the liver graft represents a critical complication. We recently encountered an interesting phenomenon; that is, we found that the rSO2 level of the liver graft, as measured by NIRS, drops in patients that subsequently require an emergency liver biopsy. An 8-month-old female and an 8-month-old male underwent living donor liver transplants for biliary atresia. In both cases, a reduction in rSO2 was detected before an emergency liver biopsy was required. As a result of biopsy examinations, both patients were diagnosed with acute graft rejection. NIRS might be useful for graft management during the postoperative period in pediatric patients that undergo liver transplantation. After a liver transplant, a reduction in the rSO2 of the graft might be indicative of the onset of vascular complications.