Structural insights into the multifunctionality of rabies virus P3 protein.

Viruses form extensive interfaces with host proteins to modulate the biology of the infected cell, frequently via multifunctional viral proteins. These proteins are conventionally considered as assemblies of independent functional modules, where the presence or absence of modules determines the overall composite phenotype. However, this model cannot account for functions observed in specific viral proteins. For example, rabies virus (RABV) P3 protein is a truncated form of the pathogenicity factor P protein, but displays a unique phenotype with functions not seen in longer isoforms, indicating that changes beyond the simple complement of functional modules define the functions of P3. Here, we report structural and cellular analyses of P3 derived from the pathogenic RABV strain Nishigahara (Nish) and an attenuated derivative strain (Ni-CE). We identify a network of intraprotomer interactions involving the globular C-terminal domain and intrinsically disordered regions (IDRs) of the N-terminal region that characterize the fully functional Nish P3 to fluctuate between open and closed states, whereas the defective Ni-CE P3 is predominantly open. This conformational difference appears to be due to the single mutation N226H in Ni-CE P3. We find that Nish P3, but not Ni-CE or N226H P3, undergoes liquid-liquid phase separation and this property correlates with the capacity of P3 to interact with different cellular membrane-less organelles, including those associated with immune evasion and pathogenesis. Our analyses propose that discrete functions of a critical multifunctional viral protein depend on the conformational arrangements of distant individual domains and IDRs, in addition to their independent functions.

Enterovirus 3A protein disrupts endoplasmic reticulum homeostasis through interaction with GBF1.

Enteroviruses are single-stranded, positive-sense RNA viruses causing endoplasmic reticulum (ER) stress to induce or modulate downstream signaling pathways known as the unfolded protein responses (UPR). However, viral and host factors involved in the UPR related to viral pathogenesis remain unclear. In the present study, we aimed to identify the major regulator of enterovirus-induced UPR and elucidate the underlying molecular mechanisms. We showed that host Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1), which supports enteroviruses replication, was a major regulator of the UPR caused by infection with enteroviruses. In addition, we found that severe UPR was induced by the expression of 3A proteins encoded in human pathogenic enteroviruses, such as enterovirus A71, coxsackievirus B3, poliovirus, and enterovirus D68. The N-terminal-conserved residues of 3A protein interact with the GBF1 and induce UPR through inhibition of ADP-ribosylation factor 1 (ARF1) activation via GBF1 sequestration. Remodeling and expansion of ER and accumulation of ER-resident proteins were observed in cells infected with enteroviruses. Finally, 3A induced apoptosis in cells infected with enteroviruses via activation of the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/C/EBP homologous protein (CHOP) pathway of UPR. Pharmaceutical inhibition of PERK suppressed the cell death caused by infection with enteroviruses, suggesting the UPR pathway is a therapeutic target for treating diseases caused by infection with enteroviruses.IMPORTANCEInfection caused by several plus-stranded RNA viruses leads to dysregulated ER homeostasis in the host cells. The mechanisms underlying the disruption and impairment of ER homeostasis and its significance in pathogenesis upon enteroviral infection remain unclear. Our findings suggested that the 3A protein encoded in human pathogenic enteroviruses disrupts ER homeostasis by interacting with GBF1, a major regulator of UPR. Enterovirus-mediated infections drive ER into pathogenic conditions, where ER-resident proteins are accumulated. Furthermore, in such scenarios, the PERK/CHOP signaling pathway induced by an unresolved imbalance of ER homeostasis essentially drives apoptosis. Therefore, elucidating the mechanisms underlying the virus-induced disruption of ER homeostasis might be a potential target to mitigate the pathogenesis of enteroviruses.

Chemical screening of inhibitors specific for MdDOX-Co that cause an apple columnar tree-shape.

MdDOX-Co, the ectopic expression of which is considered to cause the apple columnar tree shape, belongs to the 2-oxoglutarate-dependent dioxygenase (2ODD) family. It adds a hydroxyl group to position 12 of gibberellins (GAs). However, the 2ODD enzymes related to GA biosynthesis and catabolism are phylogenetically distinct from MdDOX-Co. Thus, it is possible that substrates other than GAs exist in MdDOX-Co. To identify the previously unidentified substrate(s) of MdDOX-Co, we searched for MdDOX-Co-specific inhibitors. Chemical screening using gas chromatography-mass spectrometry was performed to investigate the effects of 2400 compounds that inhibited the catalytic reaction of MdDOX-Co, but not the catabolic reaction of GA 2-oxidase, an enzyme involved in GA catabolism. By applying two positive compounds in Arabidopsis, a chemical 3-((2-chloro-6-fluorobenzyl)thio)-5,7-dimethyl-5H-pyrazolo[3,4-e][1,4,2]dithiazine-1,1-dioxide designated as TPDD that did not inhibit GA biosynthesis was selected. The structure-activity relationships among the TPDD analogs were also obtained.

The apple gene responsible for columnar tree shape reduces the abundance of biologically active gibberellin.

Ectopic expression of the apple 2-oxoglutarate-dependent dioxygenase (DOX, 2ODD) gene, designated MdDOX-Co, is thought to cause the columnar shape of apple trees. However, the mechanism underlying the formation of such a unique tree shape remains unclear. To solve this problem, we demonstrated that Arabidopsis thaliana overexpressing MdDOX-Co contained reduced levels of biologically active gibberellin (GA) compared with wild type. In summary: (i) with biochemical approaches, the gene product MdDOX-Co was shown to metabolize active GA A4 (GA4 ) to GA58 (12-OH-GA4 ) in vitro. MdDOX-Co also metabolized its precursors GA12 and GA9 to GA111 (12-OH-GA12 ) and GA70 (12-OH-GA9 ), respectively; (ii) Of the three 12-OH-GAs, GA58 was still active physiologically, but not GA70 or GA111 ; (iii) Arabidopsis MdDOX-Co OE transformants converted exogenously applied deuterium-labeled (d2 )-GA12 to d2 -GA111 but not to d2 -GA58 , whereas transformants converted applied d2 -GA9 to d2 -GA58 ; (iv) GA111 is converted poorly to GA70 by GA 20-oxidases in vitro when GA12 is efficiently metabolized to GA9 ; (v) no GA58 was detected endogenously in MdDOX-Co OE transformants. Overall, we conclude that 12-hydroxylation of GA12 by MdDOX-Co prevents the biosynthesis of biologically active GAs in planta, resulting in columnar phenotypes.

Dengue Virus Type 1 Infection in Traveler Returning from Tanzania to Japan, 2019.

The largest outbreak of dengue fever in Tanzania is ongoing. Dengue virus type 1 was diagnosed in a traveler who returned from Tanzania to Japan. In phylogenetic analysis, the detected strain was close to the Singapore 2015 strain, providing a valuable clue for investigating the dengue outbreak in Tanzania.

Identification of candidate genes responsible for the susceptibility of apple (Malus × domestica Borkh.) to Alternaria blotch.

BACKGROUND: The mechanism underlying the interaction between host plant and host-selective toxin (HST)-producing Alternaria alternata during infection is of particular interest for sustainable crop production. Alternaria blotch of apple (Malus × domestica Borkh.) caused by A. alternata apple pathotype is a major disease particularly in East Asia, which is the largest producer of apples globally. A single dominant gene, Alt, controls the susceptibility of the apple cultivar 'Delicious' to Alternaria blotch. In this study, we fine mapped the Alt locus and characterized three potential candidate genes. RESULTS: We used 797 F1 individuals derived from 15 crosses between apple accessions susceptible (Alt/alt) and resistant (alt/alt) to Alternaria blotch to construct physical and genetic maps of the Alt locus located on the top of chromosome 11. Susceptible accessions were derived from 'Delicious.' To fine map the Alt locus, we constructed a BAC library of 'Starking Delicious,' a sport of 'Delicious,' and used graphical genotyping to delimit the Alt locus to a region of 43 kb. Three genes predicted within the candidate Alt region were potentially involved in plant defense response, among which the gene encoding a coiled coil-nucleotide binding-leucine rich repeat (CC-NB-LRR) type disease resistance protein was the most promising. Moreover, a 12-bp insertion was uniquely identified in the 5' untranslated region of the Alt-associated allele of this gene, the presence or absence of which co-segregated with the susceptibility or resistance to A. alternata apple pathotype, respectively, among 43 tested cultivars including old ones and founders of modern apple breeding. CONCLUSION: A disease resistance protein has been suggested as a determinant of susceptibility/resistance to HST-producing A. alternata for the first time. Our finding provides new insight into the mechanism of HST-mediated disease control used by A. alternata against host plants.

Molecular Function Analysis of Rabies Virus RNA Polymerase L Protein by Using an L Gene-Deficient Virus.

While the RNA-dependent RNA polymerase L protein of rabies virus (RABV), a member of the genus Lyssavirus of the family Rhabdoviridae, has potential to be a therapeutic target for rabies, the molecular functions of this protein have remained largely unknown. In this study, to obtain a novel experimental tool for molecular function analysis of the RABV L protein, we established by using a reverse genetics approach an L gene-deficient RABV (Nishi-ΔL/Nluc), which infects, propagates, and correspondingly produces NanoLuc luciferase in cultured neuroblastoma cells transfected to express the L protein. trans-Complementation with wild-type L protein, but not that with a functionally defective L protein mutant, efficiently supported luciferase production by Nishi-ΔL/Nluc, confirming its potential for function analysis of the L protein. Based on the findings obtained from comprehensive genetic analyses of L genes from various RABV and other lyssavirus species, we examined the functional importance of a highly conserved L protein region at positions 1914 to 1933 by a trans-complementation assay with Nishi-ΔL/Nluc and a series of L protein mutants. The results revealed that the amino acid sequence at positions 1929 to 1933 (NPYNE) is functionally important, and this was supported by other findings that this sequence is critical for binding of the L protein with its essential cofactor, P protein, and thus also for L protein's RNA polymerase activity. Our findings provide useful information for the development of an anti-RABV drug targeting the L-P protein interaction.IMPORTANCE To the best of our knowledge, this is the first report on the establishment of an L gene-deficient, reporter gene-expressing virus in all species of the order Mononegavirales, also highlighting its applicability to a trans-complementation assay, which is useful for molecular function analyses of their L proteins. Moreover, this study revealed for the first time that the NPYNE sequence at positions 1929 to 1933 in the RABV L protein is important for L protein's interaction with the P protein, consistent with and extending the results of a previous study showing that the P protein-binding domain in the L protein is located in its C-terminal region, at positions 1562 to 2127. This study indicates that the NPYNE sequence is a promising target for the development of an inhibitor of viral RNA synthesis, which has high potential as a therapeutic drug for rabies.

Roles of the Rabies Virus Phosphoprotein Isoforms in Pathogenesis.

UNLABELLED: Rabies virus (RABV) P gene mRNA encodes five in-frame start codons, resulting in expression of full-length P protein (P1) and N-terminally truncated P proteins (tPs), designated P2, P3, P4, and P5. Despite the fact that some tPs are known as interferon (IFN) antagonists, the importance of tPs in the pathogenesis of RABV is still unclear. In this study, to examine whether tPs contribute to pathogenesis, we exploited a reverse genetics approach to generate CE(NiP)ΔP2-5, a mutant of pathogenic CE(NiP) in which the P gene was mutated by replacing all of the start codons (AUG) for tPs with AUA. We confirmed that while CE(NiP) expresses detectable levels of P2 and P3, CE(NiP)ΔP2-5 has an impaired ability to express these tPs. After intramuscular inoculation, CE(NiP)ΔP2-5 caused significantly lower morbidity and mortality rates in mice than did CE(NiP), indicating that tPs play a critical role in RABV neuroinvasiveness. Further examinations revealed that this less neuroinvasive phenotype of CE(NiP)ΔP2-5 correlates with its impaired ability to replicate in muscle cells, indicative of the importance of tPs in viral replication in muscle cells. We also demonstrated that CE(NiP)ΔP2-5 infection induced a higher level of Ifn-ß gene expression in muscle cells than did CE(NiP) infection, consistent with the results of an IFN-ß promoter reporter assay suggesting that all tPs function to antagonize IFN induction in muscle cells. Taken together, our findings strongly suggest that tPs promote viral replication in muscle cells through their IFN antagonist activities and thereby support infection of peripheral nerves. IMPORTANCE: Despite the fact that previous studies have demonstrated that P2 and P3 of RABV have IFN antagonist activities, the actual importance of tPs in pathogenesis has remained unclear. Here, we provide the first evidence that tPs contribute to the pathogenesis of RABV, especially its neuroinvasiveness. Our results also show the mechanism underlying the neuroinvasiveness driven by tPs, highlighting the importance of their IFN antagonist activities, which support viral replication in muscle cells.

Isolation and characterization of a novel type of rotavirus species A in sugar gliders (Petaurus breviceps).

To estimate the risk of interspecies transmission of rotavirus species A (RVA) from exotic pets to other mammalian species, the prevalence of RVA in sugar gliders (Petaurus breviceps) was investigated. RVAs were detected in 10 of 44 sugar gliders by reverse transcription (RT)-semi-nested PCR. These viruses were classified as G27P[3] and G27P[36] genotypes, with G27 and P[36] being new genotypes as assigned by the Rotavirus Classification Working Group. To characterize sugar glider RVA in detail, one strain, RVA/SugarGlider-tc/JPN/SG385/2012/G27P[36] (SG385-tc), was isolated. All of the genes of the strain were classified as new genotypes (G27-P[36]-I19-R10-C10-M9-A20-N11-T13-E17-H12). The enterotoxin domain in NSP4, which is important for the induction of diarrhoea, was conserved between SG385-tc and previously reported mammalian strains, suggesting the potential of sugar glider RVA to cause diarrhoea in mammalian species. In fact, seven out of nine suckling mice inoculated orally with 3.9 × 104 f.f.u. of strain SG385-tc had diarrhoea and the 50 % diarrhoea-inducing dose (DD50) of strain SG385-tc in suckling mice was 1.2 × 104 f.f.u. Our findings suggest that sugar glider RVA is infective to and possibly pathogenic in other mammalian species.

Isolation of a sp. nov. Ljungan virus from wild birds in Japan.

Ljungan virus (LV) has been isolated/detected from rodents in a limited area including European countries and the USA. In this study, we isolated an LV strain from faecal samples of wild birds that had been collected in Japan, and determined the nearly complete sequence of the genome. Sequence analyses showed that the isolate possesses an LV-like genomic organization: 5UTR-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3UTR. Phylogenetic and similarity analyses based on the VP1 region indicated that the strain constitutes a novel genotype within LV. In addition, we identified species origin of the faeces as gull species by using the DNA barcoding technique. These data suggested that the novel LV strain infected a gull species, in which the virus had not been identified. Taken together, this study has provided the first evidence of the presence of a novel LV in Japan, highlighting the possibility of LV infection in birds.

Purification, gene cloning, and characterization of γ-butyrobetainyl CoA synthetase from Agrobacterium sp. 525a.

The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70-95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent Km values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM.

Expression of a putative dioxygenase gene adjacent to an insertion mutation is involved in the short internodes of columnar apples (Malus × domestica).

Determining the molecular mechanism of fruit tree architecture is important for tree management and fruit production. An apple mutant 'McIntosh Wijcik', which was discovered as a bud mutation from 'McIntosh', exhibits a columnar growth phenotype that is controlled by a single dominant gene, Co. In this study, the mutation and the Co gene were analyzed. Fine mapping narrowed the Co region to a 101 kb region. Sequence analysis of the Co region and the original wild-type co region identified an insertion mutation of an 8202 bp long terminal repeat (LTR) retroposon in the Co region. Segregation analysis using a DNA marker based on the insertion polymorphism showed that the LTR retroposon was closely associated with the columnar growth phenotype. RNA-seq and RT-PCR analysis identified a promising Co candidate gene (91071-gene) within the Co region that is specifically expressed in 'McIntosh Wijcik' but not in 'McIntosh'. The 91071-gene was located approximately 16 kb downstream of the insertion mutation and is predicted to encode a 2-oxoglutarate-dependent dioxygenase involved in an unknown reaction. Overexpression of the 91071-gene in transgenic tobaccos and apples resulted in phenotypes with short internodes, like columnar apples. These data suggested that the 8202 bp retroposon insertion in 'McIntosh Wijcik' is associated with the short internodes of the columnar growth phenotype via upregulated expression of the adjacent 91071-gene. Furthermore, the DNA marker based on the insertion polymorphism could be useful for the marker-assisted selection of columnar apples.

Genomic dissection of a 'Fuji' apple cultivar: re-sequencing, SNP marker development, definition of haplotypes, and QTL detection.

'Fuji' is one of the most popular and highly-produced apple cultivars worldwide, and has been frequently used in breeding programs. The development of genotypic markers for the preferable phenotypes of 'Fuji' is required. Here, we aimed to define the haplotypes of 'Fuji' and find associations between haplotypes and phenotypes of five traits (harvest day, fruit weight, acidity, degree of watercore, and flesh mealiness) by using 115 accessions related to 'Fuji'. Through the re-sequencing of 'Fuji' genome, total of 2,820,759 variants, including single nucleotide polymorphisms (SNPs) and insertions or deletions (indels) were detected between 'Fuji' and 'Golden Delicious' reference genome. We selected mapping-validated 1,014 SNPs, most of which were heterozygous in 'Fuji' and capable of distinguishing alleles inherited from the parents of 'Fuji' (i.e., 'Ralls Janet' and 'Delicious'). We used these SNPs to define the haplotypes of 'Fuji' and trace their inheritance in relatives, which were shown to have an average of 27% of 'Fuji' genome. Analysis of variance (ANOVA) based on 'Fuji' haplotypes identified one quantitative trait loci (QTL) each for harvest time, acidity, degree of watercore, and mealiness. A haplotype from 'Delicious' chr14 was considered to dominantly cause watercore, and one from 'Ralls Janet' chr1 was related to low-mealiness.

Persistence of the rotavirus A genome in mesenteric lymph nodes of cattle raised on farms.

Previous studies revealed that rotavirus A (RVA) is present in not only the small intestine but also various organs. It was reported that RVA persisted in mesenteric lymph nodes (MLNs) in experimental models. However, there have been no reports focused on RVA in MLNs of animals under natural conditions. In this study, in order to investigate the persistence of the RVA genome in MLNs in cattle under natural conditions, reverse transcription-semi-nested PCR was carried out to detect RVA genomes in the MLNs from 17 calves that had been subjected to autopsy examinations. RVA genomes were detected in MLNs from 10 (˜60  %) of the 17 autopsied calves. MLNs from 170 healthy adult cattle that had been slaughtered were also examined; 15 (∼10  %) of the 170 cattle had RVA genomes in their MLNs, indicating that RNA genomes are found frequently in MLNs of cattle under natural conditions. Genetic analyses revealed that RVAs in MLNs were classified as G and/or P genotypes generally prevalent in bovines. Basically, the strains in intestinal contents were genetically identical to those in MLNs from individual cattle, suggesting that bovine RVAs have the ability to spread from the intestine to MLNs. Furthermore, amongst RVA-positive cattle, six of 10 autopsied calves and 12 of 15 healthy adult cattle were negative for the virus in the intestinal contents, indicating that bovine RVA genomes can persist in MLNs after viral clearance in the digestive tract.

Interaction of rabies virus P-protein with STAT proteins is critical to lethal rabies disease.

BACKGROUND: Rabies virus (RABV) causes rabies disease resulting in >55,000 human deaths/year. The multifunctional RABV P-protein has essential roles in genome replication, and forms interactions with cellular STAT proteins that are thought to underlie viral antagonism of interferon-dependent immunity. However, the molecular details of P-protein-STAT interaction, and its importance to disease are unresolved. METHODS: Studies were performed using sequence/structure analysis, mutagenesis, immunoprecipitation, luciferase and qRT-PCR-based signaling assays, confocal microscopy and reverse genetics/in vivo infection. RESULTS: We identified a hydrophobic pocket of the P-protein C-terminal domain as critical to STAT-binding/antagonism. This interface was found to be functionally and spatially independent of the region responsible for N-protein interaction, which is critical to genome replication. Based on these findings, we generated the first mutant RABV lacking STAT-association. Growth of the virus in vitro was unimpaired, but it lacked STAT-antagonist function and was highly sensitive to interferon. Importantly, growth of the virus was strongly attenuated in brains of infected mice, producing no major neurological symptoms, compared with the invariably lethal wild-type virus. CONCLUSIONS: These data represent direct evidence that P-protein-STAT interaction is critical to rabies, and provide novel insights into the mechanism by which RABV coordinates distinct functions in interferon antagonism and replication.

Identification of a novel aminopropyltransferase involved in the synthesis of branched-chain polyamines in hyperthermophiles.

Longer- and/or branched-chain polyamines are unique polycations found in thermophiles. N(4)-aminopropylspermine is considered a major polyamine in Thermococcus kodakarensis. To determine whether a quaternary branched penta-amine, N(4)-bis(aminopropyl)spermidine, an isomer of N(4)-aminopropylspermine, was also present, acid-extracted cytoplasmic polyamines were analyzed by high-pressure liquid chromatography, gas chromatography (HPLC), and gas chromatography-mass spectrometry. N(4)-bis(aminopropyl)spermidine was an abundant cytoplasmic polyamine in this species. To identify the enzyme that catalyzes N(4)-bis(aminopropyl)spermidine synthesis, the active fraction was concentrated from the cytoplasm and analyzed by linear ion trap-time of flight mass spectrometry with an electrospray ionization instrument after analysis by the MASCOT database. TK0545, TK0548, TK0967, and TK1691 were identified as candidate enzymes, and the corresponding genes were individually cloned and expressed in Escherichia coli. Recombinant forms were purified, and their N(4)-bis(aminopropyl)spermidine synthesis activity was measured. Of the four candidates, TK1691 (BpsA) was found to synthesize N(4)-bis(aminopropyl)spermidine from spermidine via N(4)-aminopropylspermidine. Compared to the wild type, the bpsA-disrupted strain DBP1 grew at 85°C with a slightly longer lag phase but was unable to grow at 93°C. HPLC analysis showed that both N(4)-aminopropylspermidine and N(4)-bis(aminopropyl)spermidine were absent from the DBP1 strain grown at 85°C, demonstrating that the branched-chain polyamine synthesized by BpsA is important for cell growth at 93°C. Sequence comparison to orthologs from various microorganisms indicated that BpsA differed from other known aminopropyltransferases that produce spermidine and spermine. BpsA orthologs were found only in thermophiles, both in archaea and bacteria, but were absent from mesophiles. These findings indicate that BpsA is a novel aminopropyltransferase essential for the synthesis of branched-chain polyamines, enabling thermophiles to grow in high-temperature environments.

Involvement of the rabies virus phosphoprotein gene in neuroinvasiveness.

Rabies virus (RABV), which is transmitted via a bite wound caused by a rabid animal, infects peripheral nerves and then spreads to the central nervous system (CNS) before causing severe neurological symptoms and death in the infected individual. Despite the importance of this ability of the virus to spread from a peripheral site to the CNS (neuroinvasiveness) in the pathogenesis of rabies, little is known about the mechanism underlying the neuroinvasiveness of RABV. In this study, to obtain insights into the mechanism, we conducted comparative analysis of two fixed RABV strains, Nishigahara and the derivative strain Ni-CE, which cause lethal and asymptomatic infections, respectively, in mice after intramuscular inoculation. Examination of a series of chimeric viruses harboring the respective genes from Nishigahara in the genetic background of Ni-CE revealed that the Nishigahara phosphoprotein (P) gene plays a major role in the neuroinvasiveness by mediating infection of peripheral nerves. The results obtained from both in vivo and in vitro experiments strongly suggested that the Nishigahara P gene, but not the Ni-CE P gene, is important for stable viral replication in muscle cells. Further investigation based on the previous finding that RABV phosphoprotein counteracts the host interferon (IFN) system demonstrated that the Nishigahara P gene, but not the Ni-CE P gene, functions to suppress expression of the beta interferon (IFN-ß) gene (Ifn-ß) and IFN-stimulated genes in muscle cells. In conclusion, we provide the first data strongly suggesting that RABV phosphoprotein assists viral replication in muscle cells by counteracting the host IFN system and, consequently, enhances infection of peripheral nerves.

Identification of QTLs for fruit quality traits in Japanese apples: QTLs for early ripening are tightly related to preharvest fruit drop.

Many important apple (Malus × domestica Borkh.) fruit quality traits are regulated by multiple genes, and more information about quantitative trait loci (QTLs) for these traits is required for marker-assisted selection. In this study, we constructed genetic linkage maps of the Japanese apple cultivars 'Orin' and 'Akane' using F1 seedlings derived from a cross between these cultivars. The 'Orin' map consisted of 251 loci covering 17 linkage groups (LGs; total length 1095.3 cM), and the 'Akane' map consisted of 291 loci covering 18 LGs (total length 1098.2 cM). We performed QTL analysis for 16 important traits, and found that four QTLs related to harvest time explained about 70% of genetic variation, and these will be useful for marker-assisted selection. The QTL for early harvest time in LG15 was located very close to the QTL for preharvest fruit drop. The QTL for skin color depth was located around the position of MYB1 in LG9, which suggested that alleles harbored by 'Akane' are regulating red color depth with different degrees of effect. We also analyzed soluble solids and sugar component contents, and found that a QTL for soluble solids content in LG16 could be explained by the amount of sorbitol and fructose.

Polypoid Malignant Melanoma with Spontaneous Decapitation Showing Favorable Prognosis: A Case Report.

Introduction: Regression of malignant melanoma (MM) is a commonly observed phenomenon, which refers to disappearance or loss of all or part of MM. It can be identified both clinically and histologically, and high probability of histological regression of MM (10-58%) has been reported. However, the decapitation of skin tumor has rarely been described in the English literature, and decapitation in MM has not been reported. Here, we report the case of polypoid MM with spontaneous decapitation. Case Presentation: An 81-year-old Japanese woman was referred to our hospital due to a polypoid nodule on her cheek. She was diagnosed with MM at stage IIC (T4bN0M0) by histological examinations. Three weeks after the biopsy, the nodule decapitated spontaneously without bleeding, and an ulcer developed on the base of the polypoid nodule. The histological examination of the additionally resected ulcerative lesion under the nodule revealed infiltration of T cells mainly composed of CD8⁺ natural killer T cells. No recurrence or metastasis has been observed for 4 years. Conclusion: This is the first case report of polypoid MM with spontaneous decapitation, which may be attributed to natural killer T cells. Moreover, this case shows favorable prognosis, while it is said that the regression in thick MM does not have prognostic advantage and polypoid topography has been reported to be related to extremely poor prognosis. Further investigations are needed to evaluate the prognostic advantage of decapitation in MM and other skin tumors.

Shedding of rubella virus in postsymptomatic individuals; viral RNA load is a potential indicator to estimate candidate patients excreting infectious rubella virus.

BACKGROUND: Since the first isolation of rubella virus (RuV) in 1962, comprehensive data regarding the quantitative evaluation of RuV shedding remain unavailable. In this study, we evaluated the shedding of viral RNA and infectious virus in patients with acute RuV infection. STUDY DESIGN: We analyzed 767 specimens, including serum/plasma, peripheral blood mononuclear cells (PBMCs), throat swabs, and urine, obtained from 251 patients with rubella. The viral RNA load and the presence of infectious RuV were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and virus isolation. RESULTS: Virus excretion peaked 0-2 days after rash onset and decreased over time. The median viral RNA load dropped to an undetectable level on day 3 after rash onset in serum/plasma, day 2 in PBMCs, days 10-13 in throat swabs, and days 6-7 in urine. Infectious virus could be isolated for up to day 2 after rash onset in serum/plasma, day 1 in PBMCs, days 8-9 in throat swabs, and days 4-5 in urine. The minimum viral RNA load that allowed virus isolation was 961 copies/mL in serum/plasma, 784 copies/mL in PBMCs, 650 copies/mL in throat swabs, and 304 copies/mL in urine. A higher viral RNA load indicated a higher likelihood of the presence of infectious virus. CONCLUSION: These findings would contribute to improve algorithms for rubella surveillance and diagnosis. In addition, this study indicates that the results of RT-qPCR enable efficient rubella control by estimating candidate patients excreting infectious virus, which could help prevent viral transmission at an early stage and eliminate rubella ultimately.