Pathological and virological findings of type I interferon receptor knockout mice upon experimental infection with Heartland virus.

Heartland virus (HRTV) causes generalized symptoms, severe shock, and multiple organ failure. We previously reported that interferon-α/ß receptor knockout (IFNAR-/-) mice infected intraperitoneally with 1 × 107 tissue culture-infective dose (TCID50) of HRTV died, while those subcutaneously infected with the same dose of HRTV did not. The pathophysiology of IFNAR-/- mice infected with HRTV and the mechanism underlying the difference in disease severity, which depends on HRTV infection route, were analyzed in this study. The liver, spleen, mesenteric and axillary lymph nodes, and gastrointestinal tract of intraperitoneally (I.P.) infected mice had pathological changes; however, subcutaneously (S.C.) infected mice only had pathological changes in the axillary lymph node and gastrointestinal tract. HRTV RNA levels in the mesenteric lymph node, lung, liver, spleen, kidney, stomach, intestine, and blood were significantly higher in I.P. infected mice than those in S.C. infected mice. Chemokine ligand-1 (CXCL-1), tumor necrosis factor (TNF)-α, interleukin (IL)-12, interferon (IFN)-γ, and IL-10 levels in plasma of I.P. infected mice were higher than those of S.C. infected mice. These results indicated that high levels of viral RNA and the induction of inflammatory responses in HRTV-infected IFNAR-/- mice may be associated with disease severity.

Dengue virus infection induces selective expansion of Vγ4 and Vγ6TCR γδ T cells in the small intestine and a cytokine storm driving vascular leakage in mice.

Dengue is a major health problem in tropical and subtropical regions. Some patients develop a severe form of dengue, called dengue hemorrhagic fever, which can be fatal. Severe dengue is associated with a transient increase in vascular permeability. A cytokine storm is thought to be the cause of the vascular leakage. Although there are various research reports on the pathogenic mechanism, the complete pathological process remains poorly understood. We previously reported that dengue virus (DENV) type 3 P12/08 strain caused a lethal systemic infection and severe vascular leakage in interferon (IFN)-α/ß and γ receptor knockout mice (IFN-α/ß/γRKO mice), and that blockade of TNF-α signaling protected mice. Here, we performed transcriptome analysis of liver and small intestine samples collected chronologically from P12/08-infected IFN-α/ß/γRKO mice in the presence/absence of blockade of TNF-α signaling and evaluated the cytokine and effector-level events. Blockade of TNF-α signaling mainly protected the small intestine but not the liver. Infection induced the selective expansion of IL-17A-producing Vγ4 and Vγ6 T cell receptor (TCR) γδ T cells in the small intestine, and IL-17A, together with TNF-α, played a critical role in the transition to severe disease via the induction of inflammatory cytokines such as TNF-α, IL-1ß, and particularly the excess production of IL-6. Infection also induced the infiltration of neutrophils, as well as neutrophil collagenase/matrix metalloprotease 8 production. Blockade of IL-17A signaling reduced mortality and suppressed the expression of most of these cytokines, including TNF-α, indicating that IL-17A and TNF-α synergistically enhance cytokine expression. Blockade of IL-17A prevented nuclear translocation of NF-κB p65 in stroma-like cells and epithelial cells in the small intestine but only partially prevented recruitment of immune cells to the small intestine. This study provides an overall picture of the pathogenesis of infection in individual mice at the cytokine and effector levels.

Long-term survival from progressive multifocal leukoencephalopathy in living-donor liver transplant recipient with preformed donor-specific antibody.

Intensive immunosuppression has enabled liver transplantation even in recipients with preformed donor-specific antibodies (DSA), an independent risk factor for graft rejection. However, these recipients may also be at high risk of progressive multifocal encephalopathy (PML) due to the comorbid immunosuppressed status. A 58-year-old woman presented with self-limited focal-to-bilateral tonic-clonic seizures 9 months after liver transplantation. She was desensitized using rituximab and plasma exchange before transplantation and was subsequently treated with steroids, tacrolimus, and everolimus after transplantation for her preformed DSA. Neurological examination revealed mild acalculia and agraphia. Cranial MRI showed asymmetric, cortex-sparing white matter lesions that increased over a week in the left frontal, left parietal, and right parieto-occipital lobes. Polymerase chain reaction (PCR) of the cerebrospinal fluid for the JC supported the diagnosis of PML. Immune reconstitution by reducing the immunosuppressant dose stopped lesion expansion, and PCR of the cerebrospinal fluid for the JC virus became negative. Graft rejection occurred 2 months after immune reconstitution, requiring readjustment of immunosuppressants. Forty-eight months after PML onset, the patient lived at home without disabling deficits. Intensive immunosuppression may predispose recipients to PML after liver transplantation with preformed DSA. Early immune reconstitution and careful monitoring of graft rejection may help improve outcomes.

Nationwide Laboratory Surveillance of Progressive Multifocal Leukoencephalopathy in Japan: Fiscal Years 2011-2020.

Progressive multifocal leukoencephalopathy (PML) is a devastating demyelinating disease caused by JC virus (JCV), predominantly affecting patients with impaired cellular immunity. PML is a non-reportable disease with a few exceptions, making national surveillance difficult. In Japan, polymerase chain reaction (PCR) testing for JCV in the cerebrospinal fluid (CSF) is performed at the National Institute of Infectious Diseases to support PML diagnosis. To clarify the overall profile of PML in Japan, patient data provided at the time of CSF-JCV testing over 10 years (FY2011-2020) were analyzed. PCR testing for 1537 new suspected PML cases was conducted, and 288 (18.7%) patients tested positive for CSF-JCV. An analysis of the clinical information on all individuals tested revealed characteristics of PML cases, including the geographic distribution, age and sex patterns, and CSF-JCV-positivity rates among the study subjects for each type of underlying condition. During the last five years of the study period, a surveillance system utilizing ultrasensitive PCR testing and widespread clinical attention to PML led to the detection of CSF-JCV in the earlier stages of the disease. The results of this study will provide valuable information not only for PML diagnosis, but also for the treatment of PML-predisposing conditions.

Japanese Encephalitis Virus Genotypes 1 and 3 Isolation in Kochi, Japan.

Japanese encephalitis virus (JEV) is a mosquito-borne virus belonging to the JEV serocomplex within the genus Flavivirus, family Flaviviridae. It has 5 genotypes, G1-G5, based on the envelope (E) protein nucleotide sequence. JEV G3 circulated in Japan until the early 1990s when it was replaced by G1. JEV G3 was isolated from swine serum samples (sw/Kochi/1/2004) in the Kochi Prefecture, western Japan, in 2004. In addition, the 2018 isolates from pigs and cows (sw/Kochi/492/2018 and bo/Kochi/211/2018) in the same prefecture were identified as G3. The nucleotide sequencing results of the sw/Kochi/492/2018 and bo/Kochi/211/2018 polyprotein region differed from those of the sw/Kochi/1/2004 strain described in our previous report. Seven JEV isolates were identified as G1 in the same geographical area as that in this study. This result indicates that both JEV G1 and G3 are present in the Kochi area.

Neutralizing mAbs against SFTS Virus Gn Protein Show Strong Therapeutic Effects in an SFTS Animal Model.

Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease with a high case fatality rate caused by the SFTS virus, and currently there are no approved specific treatments. Neutralizing monoclonal antibodies (mAbs) against the virus could be a therapeutic agent in SFTS treatment, but their development has not sufficiently been carried out. In the present study, mouse and human mAbs exposed to the viral envelope proteins Gn and Gc (16 clones each) were characterized in vitro and in vivo by using recombinant proteins, cell culture with viruses, and an SFTS animal model with IFNAR-/- mice. Neutralization activities against the recombinant vesicular stomatitis virus bearing SFTS virus Gn/Gc as envelope proteins were observed with three anti-Gn and six anti-Gc mAbs. Therapeutic activities were observed among anti-Gn, but not anti-Gc mAbs with neutralizing activities. These results propose an effective strategy to obtain promising therapeutic mAb candidates for SFTS treatment, and a necessity to reveal precise roles of the SFTS virus Gn/Gc proteins.

Characterization of Keterah orthonairovirus and evaluation of therapeutic candidates against Keterah orthonairovirus infectious disease.

The species Keterah orthonairovirus is a member of the genus Orthonairovirus. Few studies have focused on this species, and there remains no treatment for Issyk-Kul fever, an infectious disease caused by a Keterah orthonairovirus. This study was performed to characterize this species using two viruses, Issyk-Kul virus (ISKV) and Soft tick bunyavirus (STBV), in cell culture and type I interferon receptor knockout (IFNAR-/-) mice and to evaluate the efficacy of serum transfusion using a mouse model of ISKV infection. The two viruses replicated in many kinds of mammal- and tick-derived cell lines but showed few different characteristics in tropism and antigenicity against anti-viral sera in cell culture. Neither virus caused clinical signs in wild-type mice, but both caused lethal infection in IFNAR-/- mice. ISKV caused more acute death than STBV in IFNAR-/- mice. In both viral infections in IFNAR-/- mice, macroscopic abnormalities were prominent in the liver. Similar levels of viral genome between ISKV- and STBV-infected IFNAR-/- mice were observed in blood, liver, lymphoid tissues and adrenal gland at moribund stages. Hematologic abnormalities in IFNAR-/- mice infected with these viruses, including leukopenia and thrombocytopenia, and biochemical abnormalities indicating liver damage were prominent. In addition, blood levels of many kinds of cytokines and chemokines such as granulocyte colony-stimulating factor, interleukin-6, tumor necrosis factor-α, interferon gamma-induced protein 10 and monocyte chemoattractant protein-1 were elevated. ISKV-immunized serum transfusion after infection delayed the time to death of IFNAR-/- mice. Thus, the present study showed that the species Keterah orthonairovirus could proliferate in most mammal-derived cell lines and cause severe liver lesions and death in IFNAR-/- mice and that serum transfusion might be effective in treatment against Issyk-Kul fever.

Leu-to-Phe substitution at prM146 decreases the growth ability of Zika virus and partially reduces its pathogenicity in mice.

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.

Neuroinvasiveness of the MR766 strain of Zika virus in IFNAR-/- mice maps to prM residues conserved amongst African genotype viruses.

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/ß 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.

Therapeutic Experience of Progressive Multifocal Leukoencephalopathy Development during Ofatumumab Therapy for Chronic Lymphocytic Leukemia.

A 79-year-old man experienced cognitive impairment and visual field defects during ofatumumab therapy for chronic lymphocytic leukemia refractory to combination chemotherapy. Magnetic resonance imaging revealed T1-weighted low-intensity and T2-weighted high-intensity lesions with patchy gadolinium enhancement in the subcortical white matter. A diagnosis of progressive multifocal leukoencephalopathy was made after the detection of John Cunningham virus (JCV) DNA in his cerebrospinal fluid (CSF). Following plasma exchange and the administration of mirtazapine and mefloquine, the JCV DNA levels in the CSF decreased. However, the patient died 55 days after treatment was initiated. Ofatumumab treatment appears to be associated with the development of progressive multifocal leukoencephalopathy.

DNA mismatch repair deficiency and p53 abnormality are age-related events in mixed endometrial carcinoma with a clear cell component.

Mixed endometrial carcinoma (MEC) is defined as a tumor composed of two or more spatially distinct subtypes, at least one of which is serous or clear cell carcinoma. In this study, the clinicopathological features of 15 MEC cases containing a clear cell component (MEC-C) were investigated. The ages of patients ranged from 32 to 83 years (median, 61 years). The combinations of carcinoma components observed were endometrioid and clear cell in ten patients; endometrioid, clear cell and serous in three; and clear cell and serous in two. Immunohistochemically, nine had DNA mismatch repair (MMR) protein deficiency (MMR-d), nine had loss of ARID1A and three cases had aberrant p53 expression. MMR-d and loss of ARID1A showed a strong correlation. Only one case showed both MMR-d and aberrant p53 expression. The patients with MMR-d were younger than those without MMR-d (median; 58 years vs. 71 years). Loss of ARID1A also showed significant predilection for younger women than ARID1A intact cases. In conclusion, MMR-d was observed in 60 % of MEC-C, showed predilection for young women, and was associated with ARID1A loss. In contrast, non- MMR-d MEC-C occurred in elder women and some tumors may associate with TP53 mutation. These findings suggest that MEC-C develop via two different molecular mechanisms and they are age-related events.

Characterization of pseudotyped vesicular stomatitis virus bearing the heartland virus envelope glycoprotein.

The heartland virus (HRTV) is a novel phlebovirus that causes severe infections in the USA and closely related to the severe fever thrombocytopenia syndrome virus (SFTSV), a causative agent for SFTS in Asia. The entry mechanisms of HRTV remain unclear. Here, we developed the pseudotyped vesicular stomatitis virus bearing the HRTV glycoprotein (GP) (HRTVpv), and the antigenicity and the entry mechanisms of HRTV were analyzed. HRTVpv was neutralized by anti-SFTSV Gc antibody, but not the anti-SFTSV Gn antibodies. Entry of HRTVpv to cells was inhibited by bafilomycin A1 and dynasore, and but it was enhanced in cells overexpressed with C-type lectins. Production of infectious HRTVpv and SFTSVpv was reduced by Nn-DNJ, α-glucosidase inhibitor. The entry of HRTV occurs via pH- and dynamin-dependent endocytosis. Furthermore, Nn-DNJ may be a possible therapeutic agent against HRTV and SFTSV.

A highly attenuated vaccinia virus strain LC16m8-based vaccine for severe fever with thrombocytopenia syndrome.

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.

Several catechins and flavonols from green tea inhibit severe fever with thrombocytopenia syndrome virus infection in vitro.

INTRODUCTION: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne hemorrhagic fever caused by SFTS virus (SFTSV). The mortality rate of SFTS is pretty high, but no vaccines and antiviral drugs are currently available. METHODS: The antiviral effects of six green tea-related polyphenols, including four catechins and two flavonols, on SFTSV were evaluated to identify natural antiviral compounds. RESULTS: Pretreatment with all polyphenols inhibited SFTSV infection in a concentration-dependent manner. The half-maximal inhibitory concentrations of (-)-epigallocatechin gallate (EGCg) and (-)-epigallocatechin (EGC) were 1.7-1.9 and 11-39 µM, respectively. The selectivity indices of EGCg and EGC were larger than those of the other polyphenols. Furthermore, pretreatment with EGCg and EGC dose-dependently decreased viral attachment to the host cells. Additionally, the treatment of infected cells with EGCg and EGC inhibited infection more significantly at a lower multiplicity of infection (MOI) than at a higher MOI, and this effect was less effective than that of pretreatment. Pyrogallol, a trihydroxybenzene that is the structural backbone of both EGCg and EGC, also inhibited SFTSV infection, as did gallic acid. CONCLUSIONS: Our study revealed that green tea-related polyphenols, especially EGCg and EGC, are useful as candidate anti-SFTSV drugs. Furthermore, the structural basis of their antiviral activity was identified, which should enable investigations of more active drugs in the future.

Chimeric flavivirus enables evaluation of antibodies against dengue virus envelope protein in vitro and in vivo.

In a secondary dengue virus (DENV) infection, the presence of non-neutralizing antibodies (Abs), developed during a previous infection with a different DENV serotype, is thought to worsen clinical outcomes by enhancing viral production. This phenomenon is called antibody-dependent enhancement (ADE) of infection, and it has delayed the development of therapeutic Abs and vaccines against DENV, as they must be evaluated for the potential to induce ADE. Unfortunately, limited replication of DENV clinical isolates in vitro and in experimental animals hinders this evaluation process. We have, therefore, constructed a recombinant chimeric flavivirus (DV2ChimV), which carries premembrane (prM) and envelope (E) genes of type 2 DENV (DENV-2) R05-624 clinical (Thai) isolate in a backbone of Japanese encephalitis virus (Nakayama strain). DENV E-protein is the most important viral target, not only for neutralizing Abs, but also for infection-enhancing Abs. In contrast to DENV-2 R05-624, DV2ChimV replicated efficiently in cultured mammalian cells and was lethal in interferon-α/ß-γ-receptor double-knockout mice. With DV2ChimV, we were able to perform neutralization assays, in vitro and in vivo ADE assays, and in vivo protection assays. These results suggest that the chimeric virus is a powerful tool for evaluation of Abs against DENV.

A Prospective, Randomized, Open-Label Trial of Early versus Late Favipiravir Therapy in Hospitalized Patients with COVID-19.

Favipiravir is an oral broad-spectrum inhibitor of viral RNA-dependent RNA polymerase that is approved for treatment of influenza in Japan. We conducted a prospective, randomized, open-label, multicenter trial of favipiravir for the treatment of COVID-19 at 25 hospitals across Japan. Eligible patients were adolescents and adults admitted with COVID-19 who were asymptomatic or mildly ill and had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Patients were randomly assigned at a 1:1 ratio to early or late favipiravir therapy (in the latter case, the same regimen starting on day 6 instead of day 1). The primary endpoint was viral clearance by day 6. The secondary endpoint was change in viral load by day 6. Exploratory endpoints included time to defervescence and resolution of symptoms. Eighty-nine patients were enrolled, of whom 69 were virologically evaluable. Viral clearance occurred within 6 days in 66.7% and 56.1% of the early and late treatment groups (adjusted hazard ratio [aHR], 1.42; 95% confidence interval [95% CI], 0.76 to 2.62). Of 30 patients who had a fever (≥37.5°C) on day 1, times to defervescence were 2.1 days and 3.2 days in the early and late treatment groups (aHR, 1.88; 95% CI, 0.81 to 4.35). During therapy, 84.1% developed transient hyperuricemia. Favipiravir did not significantly improve viral clearance as measured by reverse transcription-PCR (RT-PCR) by day 6 but was associated with numerical reduction in time to defervescence. Neither disease progression nor death occurred in any of the patients in either treatment group during the 28-day participation. (This study has been registered with the Japan Registry of Clinical Trials under number jRCTs041190120.).

JC virus granule cell neuronopathy associated with Ruxolitinib: A case report and review of the literature.

•We report here a case of JC virus granule cell neuronopathy associated with Ruxolitinib•It is worthwhile considering the possibility of JCV-GCN in myelofibrosis patients receiving ruxiolitinib, who present with progressive cerebellar symptoms and cerebellar atrophy.•Combination therapy using mefloquine and mirtazapine may be an effective treatment.

Activation of platelet-derived growth factor receptor ß in the severe fever with thrombocytopenia syndrome virus infection.

Severe fever with thrombocytopenia syndrome (SFTS), an emerging viral infectious disease with a high case fatality rate, is caused by the SFTS virus (SFTSV). Although several cellular molecules involved in viral entry have been identified, the entry mechanisms of SFTSV remain unclear. In this study, we screened the protein kinase inhibitors in inhibitory effects on the infection of Vero cells with SFTSV using InhibitorSelect™ Protein Kinase Library Series (Merck & Co., Inc., Kenilworth, NJ, USA). Several types of inhibitors targeted to platelet-derived growth factor receptor ß (PDGFRß) inhibited the infection of Vero, Huh7, and NIH3T3 cells with SFTSV in a dose-dependent manner within the noncytotoxic range. In addition, these protein kinase inhibitors also inhibited the infection of the target cells with SFTSV glycoprotein (SFTSV-GP) pseudotyped virus (SFTSVpv). Activation of PDGFRß phosphorylation was detected in SFTSV-treated cells. The infectivities of SFTSVpv were specifically decreased not only in NIH3T3 cells treated with siRNA for PDGFRß but also in NIH3T3 cells treated with anti-PDGFRß neutralizing antibody in a dose-dependent manner. SFTSV growth and entry of SFTSVpv were also inhibited by Akt inhibitors. Activation of Akt phosphorylation was also detected in SFTSV-treated cells. These data indicate that PDGFRß is one of the important host factors in the entry steps of SFTSV.

Association of human cytomegalovirus (HCMV) neutralizing antibodies with antibodies to the HCMV glycoprotein complexes.

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.

Amino Acid at Position 166 of NS2A in Japanese Encephalitis Virus (JEV) is Associated with In Vitro Growth Characteristics of JEV.

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 (NS1-3Muar) exhibited a markedly diminished growth ability in murine neuroblastoma cells. Moreover, the growth rate of a Muar NS2A-bearing intertypic virus (NS2AMuar) 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 NS1-3Muar virus clones revealed that His-to-Tyr mutation at position 166 of NS2A (NS2A166) could rescue the low replication ability of NS1-3Muar in Neuro-2a cells. Notably, a virus harboring a Tyr-to-His substitution at NS2A166 (NS2AY166H) showed a decreased growth ability relative to that of the parental virus Mie/41/2002, whereas an NS2AMuar-based mutant virus, NS2AMuar-H166Y, showed a higher growth ability than NS2AMuar in Neuro-2a cells. Thus, these results indicate that the NS2A166 amino acid in JEV is critical for the growth and tissue tropism of JEV in vitro.