Rapid quantitative detection system for measles virus-neutralizing antibodies using HiBiT-tagged virus-like particles.

Immunological testing to detect neutralizing antibodies (NAbs) is important in measles (MV) infection control. Currently, the plaque reduction neutralization test is the only credible method for measuring actual virus NAbs; however, its feasibility is hampered by drawbacks, such as long turnaround times, low throughput, and the need for laboratory biosafety equipment. To solve these problems, we developed a simple and rapid MV-NAb detection system using lentivirus-based virus-like particles incorporated with the NanoLuc fragment peptide HiBiT comprising the MV fusion protein and hemagglutinin on their exterior surface. Overall, this simple, safe, and rapid method could be used to detect MV NAbs.

Generation of a photocontrollable recombinant bovine parainfluenza virus type 3.

Bovine parainfluenza virus type 3 (BPIV3) is a promising vaccine vector against various respiratory virus infections, including the human PIV3, respiratory syncytial virus, and severe acute respiratory syndrome-coronavirus 2 infections. In this study, we combined the Magnet system and reverse genetic approach to generate photocontrollable BPIV3. An optically controllable Magnet gene was inserted into the H2 region of the BPIV3 large protein gene, which encodes an RNA-dependent RNA polymerase. The generated photocontrollable BPIV3 grew in specific regions of the cell sheet only when illuminated with blue light, suggesting that spatiotemporal control can aid in safe clinical applications of BPIV3.

Establishment of measles virus receptor-expressing Vero cells lacking functional poliovirus receptors.

Global efforts are underway to eliminate measles and rubella, and active viral surveillance is the key to achieving this goal. In addition, the World Health Organization announced guidelines for handling materials potentially infectious for poliovirus (PV) to minimize the risk of PV reintroduction and to achieve PV eradication. To support global efforts, we established new PV-non-susceptible cell lines that are useful for the isolation of measles virus (MeV) and rubella virus (RuV) (Vero ΔPVR1/2 hSLAM+). In the cell lines, MeV and RuV replicated efficiently, with no concern regarding PV replication.

Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry.

The lipid composition of the host cell membrane is one of the key determinants of the entry of enveloped viruses into cells. To elucidate the detailed mechanisms behind the cell entry of rubella virus (RuV), one of the enveloped viruses, we searched for host factors involved in such entry by using CRISPR/Cas9 genome-wide knockout screening, and we found sphingomyelin synthase 1 (SMS1), encoded by the SGMS1 gene, as a candidate. RuV growth was strictly suppressed in SGMS1-knockout cells and was completely recovered by the overexpression of enzymatically active SMS1 and partially recovered by that of SMS2, another member of the SMS family, but not by that of enzymatically inactive SMS1. An entry assay using pseudotyped vesicular stomatitis virus possessing RuV envelope proteins revealed that sphingomyelin generated by SMSs is crucial for at least RuV entry. In SGMS1-knockout cells, lipid mixing between the RuV envelope membrane and the membrane of host cells occurred, but entry of the RuV genome from the viral particles into the cytoplasm was strongly inhibited. This indicates that sphingomyelin produced by SMSs is essential for the formation of membrane pores after hemifusion occurs during RuV entry. IMPORTANCE Infection with rubella virus during pregnancy causes congenital rubella syndrome in infants. Despite its importance in public health, the detailed mechanisms of rubella virus cell entry have only recently become somewhat clearer. The E1 protein of rubella virus is classified as a class II fusion protein based on its structural similarity, but it has the unique feature that its activity is dependent on calcium ion binding in the fusion loops. In this study, we found another unique feature, as cellular sphingomyelin plays a critical role in the penetration of the nucleocapsid into the cytoplasm after hemifusion by rubella virus. This provides important insight into the entry mechanism of rubella virus. This study also presents a model of hemifusion arrest during cell entry by an intact virus, providing a useful tool for analyzing membrane fusion, a biologically important phenomenon.

Heat Shock Protein 90 Ensures the Integrity of Rubella Virus p150 Protein and Supports Viral Replication.

Two viral nonstructural proteins, p150 and p90, are expressed in rubella virus (RUBV)-infected cells and mediate viral genome replication, presumably using various host machineries. Molecular chaperones are critical host factors for the maintenance of cellular proteostasis, and certain viral proteins use this chaperone system. The RUBV p150 and p90 proteins are generated from a precursor polyprotein, p200, via processing by the protease activity of its p150 region. This processing is essential for RUBV genome replication. Here we show that heat shock protein 90 (HSP90), a molecular chaperone, is an important host factor for RUBV genome replication. The treatment of RUBV-infected cells with the HSP90 inhibitors 17-allylamino-17-desmethoxygeldanamycin (17-AAG) and ganetespib suppressed RUBV genome replication. HSP90α physically interacted with p150, but not p90. Further analyses into the mechanism of action of the HSP90 inhibitors revealed that HSP90 activity contributes to p150 functional integrity and promotes p200 processing. Collectively, our data demonstrate that RUBV p150 is a client of the HSP90 molecular chaperone and that HSP90 functions as a key host factor for RUBV replication.IMPORTANCE Accumulating evidence indicates that RNA viruses use numerous host factors during replication of their genomes. However, the host factors involved in rubella virus (RUBV) genome replication are largely unknown. In this study, we demonstrate that the HSP90 molecular chaperone is needed for the efficient replication of the RUBV genome. Further, we reveal that HSP90 interacts with RUBV nonstructural protein p150 and its precursor polyprotein, p200. HSP90 contributes to the stability of p150 and the processing of p200 via its protease domain in the p150 region. We conclude that the cellular molecular chaperone HSP90 is a key host factor for functional maturation of nonstructural proteins for RUBV genome replication. These findings provide novel insight into this host-virus interaction.

Nationwide Molecular Epidemiology of Measles Virus in Japan Between 2008 and 2017.

Genotyping evidence that supports the interruption of endemic measles virus (MV) transmission is one of the essential criteria to be verified in achieving measles elimination. In Japan since 2014, MV genotype analyses have been performed for most of the measles cases in prefectural public health institutes nationwide. With this strong molecular epidemiological data, Japan was verified to have eliminated measles in March, 2015. However, even in the postelimination era, sporadic cases and small outbreaks of measles have been detected repeatedly in Japan. This study investigated the nationwide molecular epidemiology of MV between 2008 and 2017. The 891 strains in the total period between 2008 and 2017 belonged to seven genotypes (D5, D4, D9, H1, G3, B3, and D8) and 124 different MV sequence variants, based on the 450-nucleotide sequence region of the N gene (N450). The 311 MV strains in the postelimination era between 2015 and 2017 were classified into 1, 7, 8, and 32 different N450 sequence variants in D9, H1, B3, and D8 genotypes, respectively. Analysis of the detection period of the individual N450 sequence variants showed that the majority of MV strains were detected only for a short period. However, MV strains, MVs/Osaka.JPN/29.15/ [D8] and MVi/Hulu Langat.MYS/26.11/ [D8], which are named strains designated by World Health Organization (WHO), have been detected in many cases over 2 or 3 years between 2015 and 2017. The WHO-named strains have circulated worldwide, causing outbreaks in many countries. Epidemiological investigation revealed repeated importation of these WHO-named strains into Japan. To demonstrate the elimination status (interruption of endemic transmission) in situations with repeated importation of the same strains is challenging. Nevertheless, the detailed sequence analysis of individual MV strains and chronological analysis of these strains provided sufficient evidence to show that Japan has still maintained its measles elimination status in 2017.

Liposome Flotation Assay for Studying Interactions Between Rubella Virus Particles and Lipid Membranes.

Rubella virus (RuV) is an enveloped, positive-sense single-stranded RNA virus that is pathogenic to humans. RuV binds to the target cell via the viral envelope protein E1, but the specific receptor molecules on the target cell are yet to be fully elucidated. Here, we describe a protocol for liposome flotation assay to study direct interactions between RuV particles and lipid membranes in a qualitative manner. Interactions are examined by a Nycodenz density gradient fractionation using UV-inactivated RuV particles and fluorescent-labeled liposomes consisting of pure lipids. Fractionated RuV particles are detected using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis for viral proteins. On the Nycodenz gradient, RuV particles bound to liposomes shift to lower density fractions than unbound RuV particles. Using this protocol, we provide compelling evidence that, at neutral pH in a calcium-dependent manner, RuV particles bind to lipid membranes containing both sphingomyelin (SM) and cholesterol in certain cell types.

Analysis of the effect of Sphingomyelinase on rubellavirus infectivity in two cell lines.

Rubella is a mildly contagious disease characterized by low-grade fever and a morbilliform rash caused by the rubella virus (RuV). Viruses often use cellular phospholipids for infection. We studied the roles of cellular sphingomyelin in RuV infection. Treatment of cells with sphingomyelinase (SMase) inhibited RuV infection in rabbit kidney-derived RK13 cells and African green monkey (Cercopithecus aethiops) kidney-derived Vero cells. Our data further demonstrated that RuV used cellular sphingomyelin and cholesterol for its binding to cells and membrane fusion at the step of virus entry. Detailed protocols of our assays, which assess the effects of SMase treatment on RuV infectivity in RK13 and Vero cells, are described.

Both Sphingomyelin and Cholesterol in the Host Cell Membrane Are Essential for Rubella Virus Entry.

Rubella virus (RuV) causes a systemic infection, and transplacental fetal infection causes congenital rubella syndrome. In this study, we showed that treatment of cells with sphingomyelinase inhibited RuV infection. Assays using inhibitors of serine palmitoyl transferase and ceramide transport protein demonstrated the contribution of sphingomyelin (SM) to RuV infection. Compelling evidence for direct binding of RuV to lipid membranes at neutral pH was obtained using liposome coflotation assays. The absence of either SM or cholesterol (Chol) abrogated the RuV-liposome interaction. SM and Chol (SM/Chol) were also critical for RuV binding to erythrocytes and lymphoid cells. Removal of Ca2+ from the assay buffer or mutation of RuV envelope E1 protein Ca2+-binding sites abrogated RuV binding to liposomes, erythrocytes, and lymphoid cells. However, RuV bound to various nonlymphoid adherent cell lines independently of extracellular Ca2+ or SM/Chol. Even in these adherent cell lines, both the E1 protein Ca2+-binding sites and cellular SM/Chol were essential for the early stage of RuV infection, possibly affecting envelope-membrane fusion in acidic compartments. Myelin oligodendrocyte glycoprotein (MOG) has recently been identified as a cellular receptor for RuV. However, RuV bound to MOG-negative cells in a Ca2+-independent manner. Collectively, our data demonstrate that RuV has two distinct binding mechanisms: one is Ca2+ dependent and the other is Ca2+ independent. Ca2+-dependent binding observed in lymphoid cells occurs by the direct interaction between E1 protein fusion loops and SM/Chol-enriched membranes. Clarification of the mechanism of Ca2+-independent RuV binding is an important next step in understanding the pathology of RuV infection.IMPORTANCE Rubella has a significant impact on public health as infection during early pregnancy can result in babies being born with congenital rubella syndrome. Even though effective rubella vaccines are available, rubella outbreaks still occur in many countries. We studied the entry mechanism of rubella virus (RuV) and found that RuV binds directly to the host plasma membrane in the presence of Ca2+ at neutral pH. This Ca2+-dependent binding is specifically directed to membranes enriched in sphingomyelin and cholesterol and is critical for RuV infection. Importantly, RuV also binds to many cell lines in a Ca2+-independent manner. An unidentified RuV receptor(s) is involved in this Ca2+-independent binding. We believe that the data presented here may aid the development of the first anti-RuV drug.

Analysis of VSV pseudotype virus infection mediated by rubella virus envelope proteins.

Rubella virus (RV) generally causes a systemic infection in humans. Viral cell tropism is a key determinant of viral pathogenesis, but the tropism of RV is currently poorly understood. We analyzed various human cell lines and determined that RV only establishes an infection efficiently in particular non-immune cell lines. To establish an infection the host cells must be susceptible and permissible. To assess the susceptibility of individual cell lines, we generated a pseudotype vesicular stomatitis virus bearing RV envelope proteins (VSV-RV/CE2E1). VSV-RV/CE2E1 entered cells in an RV envelope protein-dependent manner, and thus the infection was neutralized completely by an RV-specific antibody. The infection was Ca2+-dependent and inhibited by endosomal acidification inhibitors, further confirming the dependency on RV envelope proteins for the VSV-RV/CE2E1 infection. Human non-immune cell lines were mostly susceptible to VSV-RV/CE2E1, while immune cell lines were much less susceptible than non-immune cell lines. However, susceptibility of immune cells to VSV-RV/CE2E1 was increased upon stimulation of these cells. Our data therefore suggest that immune cells are generally less susceptible to RV infection than non-immune cells, but the susceptibility of immune cells is enhanced upon stimulation.

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

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

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

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

Trichloroethylene exposure aggravates behavioral abnormalities in mice that are deficient in superoxide dismutase.

Trichloroethylene (TCE) has been implicated as a causative agent for Parkinson's disease (PD). The administration of TCE to rodents induces neurotoxicity associated with dopaminergic neuron death, and evidence suggests that oxidative stress as a major player in the progression of PD. Here we report on TCE-induced behavioral abnormality in mice that are deficient in superoxide dismutase 1 (SOD1). Wild-type (WT) and SOD1-deficient (Sod1(-/-)) mice were intraperitoneally administered TCE (500 mg/kg) over a period of 4 weeks. Although the TCE-administrated Sod1(-/-) mice showed marked abnormal motor behavior, no significant differences were observed among the experimental groups by biochemical and histopathological analyses. However, treating mouse neuroblastoma-derived NB2a cells with TCE resulted in the down regulation of the SOD1 protein and elevated oxidative stress under conditions where SOD1 production was suppressed. Taken together, these data indicate that SOD1 plays a pivotal role in protecting motor neuron function against TCE toxicity.

The SOD1 transgene expressed in erythroid cells alleviates fatal phenotype in congenic NZB/NZW-F1 mice.

Oxidative stress due to a superoxide dismutase 1 (SOD1) deficiency causes anemia and autoimmune responses, which are phenotypically similar to autoimmune hemolytic anemia (AIHA) and systemic lupus erythematosus (SLE) in C57BL/6 mice and aggravates AIHA pathogenesis in New Zealand black (NZB) mice. We report herein on an evaluation of the role of reactive oxygen species (ROS) in a model mouse with inherited SLE, that is, F1 mice of the NZB × New Zealand white (NZW) strain. The ROS levels within red blood cells (RBCs) of the F1 mice were similar to the NZW mice but lower compared to the NZB mice throughout adult period. Regarding SLE pathogenesis, we examined the effects of an SOD1 deficiency or the overexpression of human SOD1 in erythroid cells by establishing corresponding congenic F1 mice. A SOD1 deficiency caused an elevation in ROS production, methemoglobin content, and hyperoxidation of peroxiredoxin in RBC of the F1 mice, which were all consistent with elevated oxidative stress. However, while the overexpression of human SOD1 in erythroid cells extended the life span of the congenic F1 mice, the SOD1 deficiency had no effect on life span compared to wild-type F1 mice. It is generally recognized that NZW mice possess a larval defect in the immune system and that NZB mice trigger an autoimmune reaction in the F1 mice. Our results suggest that the oxidative insult originated from the NZB mouse background has a functional role in triggering an aberrant immune reaction, leading to fatal responses in F1 mice.

Analysis of the temperature sensitivity of Japanese rubella vaccine strain TO-336.vac and its effect on immunogenicity in the guinea pig.

The marker of Japanese domestic rubella vaccines is their lack of immunogenicity in guinea pigs. This has long been thought to be related to the temperature sensitivity of the viruses, but supporting evidence has not been described. In this study, we generated infectious clones of TO-336.vac, a Japanese domestic vaccine, TO-336.GMK5, the parental virus of TO-336.vac, and their mutants, and determined the molecular bases of their temperature sensitivity and immunogenicity in guinea pigs. The results revealed that Ser(1159) in the non-structural protein-coding region was responsible for the temperature sensitivity of TO-336.vac dominantly, while the structural protein-coding region affected the temperature sensitivity subordinately. The findings further suggested that the temperature sensitivity of TO-336.vac affected the antibody induction in guinea pigs after subcutaneous inoculation.

SOD1 deficiency induces the systemic hyperoxidation of peroxiredoxin in the mouse.

A deficiency of superoxide dismutase 1 (SOD1) or peroxiredoxin (Prx) 2 causes anemia in mice due to elevated oxidative stress. In the current study, we investigated whether intrinsic oxidative stress caused by a SOD1 deficiency affected the redox status of Prx2 and other isoforms in red blood cells (RBCs) and several organs of mice. We observed a marked elevation in hyperoxidized Prx2 levels in RBCs from SOD1-deficient mice. Hyperoxidized Prx2 reportedly undergoes a rhythmic change in isolated RBCs under culture conditions. We confirmed such changes in RBCs from wild-type mice but observed no evident changes in SOD1-deficient RBCs. In addition, an elevation in hyperoxidized Prxs, notably Prx2 and Prx3, was observed in several organs from SOD1-deficient mice. However, a SOD1 deficiency had no impact on the wheel-running activity of the mice. Thus, although the redox status of some Prxs is systemically shifted to a more oxidized state as the result of a SOD1 deficiency, which is associated with anemia and some diseases, a redox imbalance appears to have no detectable effect on the circadian activity of mice.

Short self-interacting N-terminal region of rubella virus capsid protein is essential for cooperative actions of capsid and nonstructural p150 proteins.

UNLABELLED: Nucleocapsid formation is a primary function of the rubella virus capsid protein, which also promotes viral RNA synthesis via an unknown mechanism. The present study demonstrates that in infected cells, the capsid protein is associated with the nonstructural p150 protein via the short self-interacting N-terminal region of the capsid protein. Mutational analyses indicated that hydrophobic amino acids in this N-terminal region are essential for its N-terminal self-interaction, which is critical for the capsid-p150 association. An analysis based on a subgenomic replicon system demonstrated that the self-interacting N-terminal region of the capsid protein plays a key role in promoting viral gene expression. Analyses using a virus-like particle (VLP) system also showed that the self-interacting N-terminal region of the capsid protein is not essential for VLP production but is critical for VLP infectivity. These results demonstrate that the close cooperative actions of the capsid protein and p150 require the short self-interacting N-terminal region of the capsid protein during the life cycle of the rubella virus. IMPORTANCE: The capsid protein of rubella virus promotes viral RNA replication via an unknown mechanism. This protein interacts with the nonstructural protein p150, but the importance of this interaction is unclear. In this study, we demonstrate that the short N-terminal region of the capsid protein forms a homo-oligomer that is critical for the capsid-p150 interaction. These interactions are required for the viral-gene-expression-promoting activity of the capsid protein, allowing efficient viral growth. These findings provide information about the mechanisms underlying the regulation of rubella virus RNA replication via the cooperative actions of the capsid protein and p150.

Functionally distinct effects of the C-terminal regions of IKKε and TBK1 on type I IFN production.

Inhibitor of κB kinase ε (IKKε) and TANK binding kinase 1 (TBK1), so-called non-canonical IKKs or IKK-related kinases, are involved in the cellular innate immunity by inducing type I IFNs. Two kinases commonly phosphorylate transcription factors IRF3 and IRF7 in type I IFN production pathway. In contrast to TBK1, underlying mechanisms of IKKε activation and regions required for activation of downstream molecules are poorly understood. In this study, we investigated regions of IKKε required for the activation of type I IFN promoter specially, by focusing on the C-terminal region. To show the functional significance of the IKKε C-terminal region on type I IFN production, we employed various mutant forms of IKKε and compared to corresponding region of TBK1. We identified the specific regions and residues of IKKε involved in the activation of downstream signaling. Interestingly, corresponding region and residues are not required for activation of downstream signaling by TBK1. The results highlight the importance of the C-terminal region in the functional activity of IKKε in innate immune response and also the difference in activation mechanisms between IKKε and the closely related TBK1.

Ascorbic acid reverses the prolonged anesthetic action of pentobarbital in Akr1a-knockout mice.

AIMS: Aldehyde reductase (AKR1A), a member of the aldo-keto reductase superfamily, is highly expressed in the liver and is involved in both the detoxification of carbonyl compounds and ascorbic acid biosynthesis. By comparison with wild-type mice, Akr1a-knockout (Akr1a(-/-)) mice and human Akrla-transgenic (Akr1a(tg/+)) mice experience different anesthetic actions from pentobarbital-prolonged in Akr1a-knockout (Akr1a(-/-)) mice and shortened in human Akrla-transgenic (Akr1a(tg/+)) mice. MAIN METHODS: We investigated this alteration in the anesthetic efficacy of pentobarbital in Akr1a genetically modified mice. KEY FINDINGS: Neither the cytosolic protein of wild-type mouse liver nor purified rat AKR1A directly reduced pentobarbital. Ascorbic acid administration neutralized the prolonged duration of the loss of the righting reflex (LORR) in Akr1a(-/-) mice, but preincubation of pentobarbital with ascorbic acid prior to administration did not change the anesthetic effect. Those results indicated that ascorbic acid does not directly reduce pentobarbital. Enzymatic activities and levels of the proteins of some cytochrome P450s that make up a potent detoxification system for pentobarbital showed no changes in the genetically modified mice examined. Thus, ascorbic acid also had no effect on the detoxification system in the liver. The prolonged duration of LORR in the Akr1a(-/-) mice caused by pentobarbital and the neutralization of the anesthetic effect by ascorbic acid together with other results imply that ascorbic acid alters the responses of the neuronal system to anesthetics. SIGNIFICANCE: Pentobarbital action is increased under conditions of ascorbic acid deficiency, and this may have to be taken into account when anesthetizing malnourished patients.