Molecular detection of Babesia and Hepatozoon species and morphological characteristics of Babesia species in Japanese wild boars.

We investigated intraerythrocytic Babesia parasites in 21 Japanese wild boars, Sus scrofa leucomystax, captured in Wakayama Prefecture on the mainland from 2008 to 2009 and in 31 Japanese wild boars from 2011 to 2013 in Kochi Prefecture on Shikoku Island, Japan. We detected small subunit ribosomal RNA (18S rRNA) gene (SSUrDNA) fragments of a Babesia species in 17 boars from Wakayama and 18 boars from Kochi. The nearly full SSUrDNA sequence (1669 bps) of this species was determined. A FASTA search revealed that the SSUrDNA sequence of the Babesia sp. in Japanese wild boars was the most homologous to those of several Babesia isolates reported as Babesia gibsoni. Phylogenetic analysis showed that the Babesia sp. found in Japanese wild boars was the closest relative to B. gibsoni but made a different clade from B. gibsoni. The Babesia sp. in Japanese wild boars was completely different from Babesia sp. Suis found in a European domestic pig, Sus scrofa domesticus. By microscopic examination, ring-shaped, oval and pear-shaped small sized intraerythrocytic parasites were observed on blood smears of 12 of 18 Japanese wild boars whose blood smears could be examined in Wakayama. We also detected SSUrDNA fragments of a Hepatozoon species in 6 of the 21 wild boars from Wakayama. The nearly full SSUrDNA sequence (1774 bps) of the Hepatozoon sp. was shown to be identical to that of Hepatozoon apri.

Double-filtration plasmapheresis plus IFN for HCV-1b patients with non-sustained virological response to previous combination therapy: early viral dynamics.

Double-filtration plasmapheresis (DFPP) was approved in Japan in April 2008 for the retreatment of chronic hepatitis C patients with genotype 1b and high viral loads, whose hepatitis C virus was not eradicated by earlier IFN therapy or by pegylated IFN plus ribavirin (PEG-IFN/RBV) combination therapy. In this study, we assessed the early viral dynamics of 9 patients with non-sustained virological response to the combination therapy. The overall viral dynamics of DFPP plus IFN treatment with or without RBV for 4 weeks showed a reduction of > or =1 log in the viral load in 22% (2 of 9 patients), 55.6% (5/9), 77.8% (7/9) and 77.8% (7/9) at 24 h, 1, 2 and 4 weeks after the start of treatment. By contrast, DFPP plus consecutive intravenous IFN-beta for 4 weeks reduced the viral load by > or = 1 log in 33% (2/6), 50% (3/6), 83.3% (5/6) and 83.3% (5/6) at 24 h, 1, 2 and 4 weeks. The viral load declined by > or = 2 log in 50% (3/6) at 4 weeks after the start of treatment. DFPP plus consecutive intravenous IFN-beta for 4 weeks is a promising treatment for non-sustained virological response patients.

HCV replication suppresses cellular glucose uptake through down-regulation of cell surface expression of glucose transporters.

BACKGROUND/AIMS: Persistent infection with hepatitis C virus (HCV) causes extrahepatic diseases, including diabetes. We investigated the possible effect(s) of HCV replication on cellular glucose uptake and expression of the facilitative glucose transporter (GLUT) 2 and 1. METHODS: We used Huh-7.5 cells harboring either an HCV subgenomic RNA replicon (SGR) or an HCV full-genomic RNA replicon (FGR), HCV-infected cells, and the respective cells treated with interferon (IFN). We also used liver tissue samples obtained from patients with or without HCV infection. RESULTS: Glucose uptake and surface expression of GLUT2 and GLUT1 were suppressed in SGR, FGR and HCV-infected cells compared to the control cells. Expression levels of GLUT2 mRNA, but not GLUT1 mRNA, were lower in SGR, FGR and HCV-infected cells than in the control. Luciferase reporter assay demonstrated decreased GLUT2 promoter activities in SGR, FGR and HCV-infected cells. IFN treatment restored glucose uptake, GLUT2 surface expression, GLUT2 mRNA expression and GLUT2 promoter activities. Also, GLUT2 expression was reduced in hepatocytes of liver tissues obtained from HCV-infected patients. CONCLUSIONS: HCV replication down-regulates cell surface expression of GLUT2 partly at the transcriptional level, and possibly at the intracellular trafficking level as suggested for GLUT1, thereby lowering glucose uptake by hepatocytes.

Sequence variation in hepatitis C virus nonstructural protein 5A predicts clinical outcome of pegylated interferon/ribavirin combination therapy.

UNLABELLED: A substantial proportion of hepatitis C virus (HCV)-1b-infected patients still do not respond to interferon-based therapy. This study aims to explore a predictive marker for the ultimate virological response of HCV-1b-infected patients treated with pegylated interferon/ribavirin (PEG-IFN/RBV) combination therapy. Nonstructural protein 5A (NS5A) sequences of HCV in the pretreated sera of 45 patients infected with HCV-1b were analyzed. The mean number of mutations in the variable region 3 (V3) plus its upstream flanking region of NS5A (amino acid 2334-2379), referred to as IFN/RBV resistance-determining region (IRRDR), was significantly higher for HCV isolates obtained from patients who later achieved sustained virological response (SVR) by PEG-IFN/RBV than for those in patients undergoing non-SVR. The receiver operating characteristic curve analysis estimated six mutations in IRRDR as the optimal threshold for SVR prediction. Indeed, 16 (76%) of 21 SVR, but only 2 (8%) of 24 non-SVR, had HCV with six or more mutations in IRRDR (IRRDR > or = 6) (P < 0.0001). All of 18 patients infected with HCV of IRRDR of 6 or greater examined showed a significant (> or =1 log) reduction or disappearance of serum HCV core antigen titers within 24 hours after initial dose of PEG-IFN/RBV, whereas 10 (37%) of 27 patients with HCV of IRRDR of 5 or less did (P < 0.0001). The positive predictive value of IRRDR of 6 or greater for SVR was 89% (16/18; P = 0.0007), with its negative predictive value for non-SVR being 81% (22/27; P = 0.0008). CONCLUSION: A high degree (> or =6) of sequence variation in IRRDR would be a useful marker for predicting SVR, whereas a less diverse (< or =5) IRRDR sequence predicts non-SVR.

[Hepatitis C virus NS3/4A with different secondary structures at amino-terminus has different serine protease activities and inhibitory activities on host cell].

OBJECTIVE: To construct the point mutation plasmids expressing NS3/4A with different secondary structure of the amino-terminal 120 residues of NS3, and to investigate the differences in serine protease and inhibitory effects on host cells between each subgroup. METHODS: The point mutation plasmids were constructed, which expressed NS3/4A with the corresponding secondary structures of subgroup, and were named as A1-2, A2-1, A2-2, B1-1, B1-2, B2-1, and B2-2, with the backbone of M-H05-5 (A1-1). Western blot was performed to detect the expression of NS3/4A and the difference in in cis and in trans NS3 serine protease activity between each subgroup. The inhibitory effects of HCV NS3/4A with different amino-terminal secondary structures on IFN-beta production and p53-dependent transcriptional activation were revealed by Luciferase reporter assay. RESULTS: Western blot revealed the successful expression of the constructs and the incomplete cleavage of NS3/4A in subgroup A2-1 and B2-1, indicating that the in cis NS3 serine protease activities of subgroup A2-1 and B2-1 were weaker compared with that of the other subgroups. By using NS5A/5BDeltaC as a substrate for NS3/4A serine protease, it was also found that the in trans NS3 serine protease activities of subgroup A2-1 and B2-1 were also weaker compared with that of the other subgroups. Differences in inhibitory effects of HCV NS3 on IFN-beta promoter activity and on p53-dependent luciferase gene transcriptional activation were also observed between subgroup A2-1, B2-1 and the other subgroups. CONCLUSION: HCV NS3/4A with different secondary structures at amino-terminus has different serine protease activities and inhibitory activities on host cell functions.

Novel subgenotypes of hepatitis B virus genotypes C and D in Papua, Indonesia.

Eight genotypes (A to H) and nine subtypes (adw2, adw4, ayw1, ayw2, ayw3, ayw4, adrq+, adrq-, and ayr) of hepatitis B virus (HBV) have been identified worldwide. They appear to be associated with geographical distribution, virological characteristics, and possibly clinical outcomes. We performed sequence analysis of part of the S gene and the entire precore/core gene of HBV isolates obtained from HBsAg-positive blood donors in Papua Province, Indonesia. Phylogenetic analysis of the S gene sequences revealed that 23 (85.2%) of the 27 HBV isolates tested belonged to genotype C (HBV/C) and 2 (7.4%) each to HBV/B and HBV/D. Interestingly, 19 (82.6%) of the 23 isolates of HBV/C clustered in a branch that was distinct from the previously reported subgenotypes C1 to C5 (HBV/C1 to HBV/C5). Similarly, two isolates of HBV/D clustered in a branch distinct from the reported subgenotypes HBV/D1 to HBV/D5. Phylogenetic analysis of the entire precore/core gene confirmed the consistent presence of the distinct branches in HBV/C and HBV/D. We therefore propose novel subgenotypes designated HBV/C6 and HBV/D6. The majority of HBV/C6 isolates in Papua had alanine at positions 159 and 177 (A159/A177) in the HBsAg. A159/A177 is different from the determinants for adrq+ (A159/V177), found throughout Asia, and adrq- (V159/A177), found in New Caledonia and Polynesia, possibly representing a unique antigenic group (provisionally referred to as adrq indeterminate). In conclusion, we have identified two novel HBV subgenotypes, HBV/C6 and HBV/D6, the first of which is the most prevalent subgenotype of HBV in Papua, Indonesia.

[SSPE virus and pathogenesis].

Subacute sclerosing panencephalitis (SSPE) is caused by particular mutants of measles virus, which are often referred to as SSPE virus. SSPE virus is characterized by (i) the inability to produce infectious viral particles, (ii) the neuropathogenicity in animal models as well as in humans, and (iii) the prolonged persistence in vivo over many years. The viral genome exhibits particular mutations, called biased hypermutation, most notably in the M gene, followed by the F and H genes. Consequently, the M, F and H proteins are mutated, which is thought to account for the characteristic features of SSPE virus. The possible mechanism of long-term persistence of the virus after the recovery of measles is also discussed.

Inhibition of measles virus and subacute sclerosing panencephalitis virus by RNA interference.

Subacute sclerosing panencephalitis (SSPE) is a rare, but fatal outcome of measles virus (MeV) infection. SSPE develops after prolonged persistence of mutated MeV called SSPE virus. Although a combination therapy using interferon and inosiplex or ribavirin appears to prolong survival time to some extent, there is currently no effective treatment to completely cure SSPE and a new treatment strategy is greatly needed. In this study, we adopted RNA interference (RNAi) strategy and examined whether small interfering RNAs (siRNAs) can be used to inhibit replication of MeV and SSPE virus. We report here that siRNAs targeted against L mRNA of MeV, either synthetic siRNAs or those generated by pcPUR+U6i-based expression plasmids, effectively and specifically inhibited replication of both MeV and SSPE virus without exhibiting any cytotoxic effect. The L protein of MeV is a major component of RNA-dependent RNA polymerase that is essential for viral RNA replication, and yet it is least abundant among all the MeV proteins expressed. Therefore, mRNA encoding the L protein would be a good target for RNAi strategy. The present results imply the possibility that our siRNAs against MeV L mRNA are among the potential candidates to be used to treat patients with SSPE.

Development of absolute quantification method for genotype-specific Babesia microti using real-time PCR and practical experimental tips of real-time PCR.

Babesia microti, a rodent babesia, is known as a pathogen of zoonosis, human babesiosis, is composed of several genotypes of small subunit ribosomal RNA gene (SSUrDNA) and different genotypes have been suggested to have different infectivity and pathogenicity to humans. We established a real-time PCR assay using SYBR Green I, which allows specific detection and absolute quantification for each SSUrDNA-type-B. microti of four SSUrDNA-types found in Japanese rodents even in mixed infection. In this assay, four genotype-specific primer pairs targeted on internal transcribed spacer 1 or 2 sequences were used. Primer pairs have the characteristics for a high specificity for homologous genotype DNA. The calibration curves of cycle threshold (Ct) values versus log concentrations of DNA for all four genotypes were linear over 107 fold range of DNA concentrations with correlation coefficient from 0.95 to 1 and sufficient amplification efficiency from 90% to 110%. The standard curves for all four genotypes were not changed even in the presence of heterologous DNA. In this paper, we introduce how to establish and perform the genotype-specific real-time PCR and our practical experimental tips to be recommended.

Continuous in vivo culture and indirect fluorescent antibody test for zoonotic protozoa of Babesia microti.

Babesiosis has attracted attention as a zoonotic disease. The disease is caused in immunocompetent individuals almost solely by Babesia microti, a rodent babesia. Most cases of human babesiosis by B. microti have been reported in the endemic foci of the Northeastern coastal areas and upper Midwest regions of the United States, while some sporadic cases have recently been reported in several Asian countries including Japan. Our previous surveys identified that four small subunit ribosomal RNA gene (SSUrDNA) types of B. microti parasitize Japanese rodents. Indirect fluorescent antibody test (IFAT) is often performed for the diagnosis of babesiosis together with microscopical examination of thin blood smears and PCR. We established IFAT against four SSUrDNA-types of B. microti using erythrocytes of SCID mice or Syrian hamsters infected with each SSUrDNA-type B. microti. The results of IFAT for sera of ICR mice or Syrian hamsters infected with each SSUrDNA-type B. microti demonstrated that the four SSUrDNA-types of B. microti have different serotypes. Here, we report technical or practical procedures of IFAT, which gains sufficiently stable results, including procedures of continuous in vivo culture of B. microti.

Inhibition of protein synthesis by the nonstructural proteins NS4A and NS4B of hepatitis C virus.

Possible inhibitory effects of hepatitis C virus (HCV) proteins on cellular protein synthesis were analyzed using transient expression system. The core protein, the nonstructural protein 4A (NS4A) and NS4B, but not NS3, NS5A or NS5B, inhibited p21/Waf1 expression post-transcriptionally. Further analysis revealed that the inhibition by NS4A and NS4B was mediated at least partly, if not entirely, at the translation level. NS4A-mediated translational inhibition was counteracted to some extent by NS3 co-expressed either in trans or cis. Co-expression of NS4A and NS4B exerted an additive effect on the translational inhibition. The N-terminal two-thirds of NS4A (amino acids 1-40) was shown to be involved in the translational inhibition. We also tested possible inhibitory effects of NS4A and NS4B on synthesis of other cellular proteins in parallel with p21/Waf1. NS4A and NS4B inhibited p21/Waf1 most strongly, followed by RNase L, p53, a C-terminally truncated form of CREB-RP and 2'-5' oligoadenylate synthetase. p21/Waf1, RNase L and p53 are known to have the PEST (proline-glutamic acid-serine-threonine) motif with relatively high scores in their sequences and considered to be sensitive to intracellular degradation. Taken together, our results suggest that NS4A and NS4B each mediate translational inhibition and, probably, increased degradation of certain cellular proteins.

Cleavage of the hepatitis C virus NS5A protein by caspase-3 in the interferon sensitivity-determining region in a sequence-dependent manner.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) has versatile functions and has been implicated in viral pathogenesis, including interferon (IFN) resistance and hepatocarcinogenesis. It has been reported that NS5A is cleaved into a few fragments by a caspase(s) or caspase-like enzyme(s) under certain conditions. Two cleavage sites have been mapped to the Asp residues at positions 154 and 398 (D154 and D398). However, other possible cleavage sites were not determined yet so far. In this study, we demonstrated caspase-3-mediated NS5A cleavage upon apoptotic stimuli and identified a new site as the third cleavage target. This site was mapped to D251, which lies within IFN sensitivity-determining region (ISDR). Although D251 was conserved among all HCV subtype 1b (HCV-1b) strains tested, the consensus caspase-3 recognition sequence (D248-X-X-D251) was not conserved due to the sequence variation at position 248 in ISDR. Furthermore, A252 was found to be necessary for efficient cleavage of NS5A. The virological significance of the HCV strain-dependent NS5A cleavage at this site awaits further investigation.

A transgenic mouse line with a 58-kb fragment deletion in chromosome 11E1 that encompasses part of the Fam20a gene and its upstream region shows growth disorder.

Growth disorder is an umbrella term for a range of abnormal growth patterns, such as unusually fast or slow growth in infants or children. The causes of growth disorder include hormonal irregularities, chronic disease, complications during pregnancy or genetic conditions. A complex trait such as body size is influenced by multiple genes as well as environmental factors, giving rise to a continuous spectrum of phenotypes. This causal complexity makes discovery of the genetic determinants of growth disorder rather difficult. We here report our discovery of a transgenic mouse line exhibiting growth disorder, which we happened to discover in the course of generating transgenic mice expressing a viral gene. Although these mice did not express any corresponding viral mRNA or protein due to a deletion in the transgene, they showed slow growth in the 5 weeks after birth and ceased growing thereafter, while maintaining a weight equivalent to that of 3-week-old normal mice. Histopathological analysis of the organs of these mice revealed that malnutrition and metabolic disorder occurred at 5 weeks after birth in the liver. Genetic analysis has revealed that the growth disorder is associated with a 58-kb fragment deletion in chromosome 11E1 that encompasses part of the Fam20a gene and part of its upstream region. The present study thus points out for the first time the possible link between Fam20a mutation and growth disorder.

Autoimmune thrombocytopenic purpura during pegylated interferon α treatment for chronic hepatitis C.

We describe a 72-year-old woman with chronic hepatitis C and autoimmune thrombocytopenic purpura (AITP) during pegylated interferon (PEG-IFN) alpha. Immunoglobulin G and antinuclear antibody were 2,113 mg/dL and 1,280 at the start, respectively. A liver biopsy negated autoimmune hepatitis. After a 48-week combination therapy with ribavirin, PEG-IFN alpha-2a was administered. At the 30th month, the platelet count was decreased to 1.1 x 10(4)/microL. Bone marrow biopsy disclosed normocellular marrow compatible with AITP. The platelet-associated IgG (PAIgG) titer rose to 500 ng/10(7) cells. Corticosteroid therapy was successful, and the platelet count and PAIgG titer reverted to 6.4 x 10(4)/microL and 57.3 ng/10(7) cells, respectively.

[Construction of pSG5/NS5A5BdeltaC and its expression in Huh7 cells].

AIM: To construct the plasmid pSG5/NS5A5BdeltaC, and to investigate its expression in Huh7 cells. METHODS: The plasmidpTM1-NS2NS5A5BdeltaC was taken as the template. The primers were designed according to the characteristics of the sequence and endoenzyme cleavage sites in HCV NS5A5BdeltaC and vector pTM1, pSG5. The HCV NS5A5BdeltaC gene fragment was amplified by PCR from pTM1-NS2NS5A5BdeltaC and inserted into vector pSG5. The positive clones were screened by Ampicillin and identified by the restriction endoenzyme Sac I and Bgl II digestion and agarose gel electrophoresis. The constructs were transfected into Huh7 cells with FuGene 6 reagents. Immunofluorescence and Western blot were performed to detect the expression of the constructs in Huh7 cells. RESULTS: Endoenzyme digestion analysis showed that the size and the inserting orientation of the fragment met the design expectation. Immunofluorescence staining displayed the expression of HCV NS5A5BdeltaC protein, which was located in the cytoplasm, and the expression rate reached as high as 40%. SDS-PAGE analysis showed that the relative molecular mass of the expressed product by pSG5/NS5A5BdeltaC was about 82 ku, which was consistent with the theoretical value. CONCLUSION: pSG5/NS5A5BdeltaC is successfully constructed, and it can be expressed transiently in Huh7 cells, which would lay a foundation for the further study on function of HCV polyprotein.

Single-point mutations of the M protein of a measles virus variant obtained from a patient with subacute sclerosing panencephalitis critically affect solubility and subcellular localization of the M protein and cell-free virus production.

Subacute sclerosing panencephalitis (SSPE) is caused by variants of wild-type measles virus (MV). Such MV variants lack almost completely the ability to produce cell-free progeny virus. We recently isolated an MV variant that has only three amino acid mutations (L165P,L250P and Y282H) in the M protein compared with MV field isolates of the same genotype. In the present study, we analyzed the significance of these mutations with regard to the characteristics of the M protein and progeny virus production. We found that each of the three mutations rendered the M protein insoluble in 0.5% Triton X-100 and altered its subcellular localization, either when ectopically expressed alone using a plasmid-based expression system or when expressed in the context of viral replication. Moreover, each of the three mutations markedly, but not completely, impaired the ability of MV to produce cell-free progeny virus, with the degree of impairment being the same as for all three mutations together. These results suggest the possibility that the changes in the solubility and subcellular localization of the M protein determine the ability to produce cell-free progeny virus, at least to some extent, and play a role in the pathogenicity of variants causing SSPE.

Efficient production of infectious hepatitis C virus with adaptive mutations in cultured hepatoma cells.

Robust production of infectious hepatitis C virus (HCV) in cell culture was realized by using the JFH1 strain and the homologous chimeric J6/JFH1 strain in Huh-7.5 cells, a highly HCV-permissive subclone of Huh-7 cells. In this study, we aimed to establish a more efficient HCV-production system and to gain some insight into the adaptation mechanisms of efficient HCV production. By serial passaging of J6/JFH1-infected Huh-7.5 cells, we obtained culture-adapted J6/JFH1 variants, designated P-27, P-38 and P-47. Sequence analyses revealed that the adaptive mutant viruses P-27, P-38 and P-47 possessed eight mutations [four in E2, two in NS2, one in NS5A and one in NS5B), 10 mutations [two additional mutations in the 5'-untranslated region (5'-UTR) and core] and 11 mutations (three additional mutations in 5'-UTR, core and NS5B), respectively. We introduced amino acid substitutions into the wild-type J6/JFH1 clone, generated recombinant viruses with adaptive mutations and analysed their infectivity and ability to produce infectious viruses. The viruses with the adaptive mutations exhibited higher expression of HCV proteins than did the wild type in Huh-7.5 cells. Moreover, we provide evidence suggesting that the mutation N534H in the E2 glycoprotein of the mutant viruses conferred an advantage at the entry level. We thus demonstrate that an efficient HCV-production system could be obtained by introducing adaptive mutations into the J6/JFH1 genome. The J6/JFH1-derived mutant viruses presented here would be a good tool for producing HCV particles with enhanced infectivity and for studying the molecular mechanism of HCV entry.

Hepatitis C virus NS5A protein interacts with and negatively regulates the non-receptor protein tyrosine kinase Syk.

Hepatitis C virus (HCV) is the major causative agent of hepatocellular carcinoma. However, the precise mechanism underlying the carcinogenesis is yet to be elucidated. It has recently been reported that Syk, a non-receptor protein tyrosine kinase, functions as a potent tumour suppressor in human breast carcinoma. This study first examined the possible effect of HCV infection on expression of Syk in vivo. Immunohistochemical analysis revealed that endogenous Syk, which otherwise was expressed diffusely in the cytoplasm of normal hepatocytes, was localized near the cell membrane with a patchy pattern in HCV-infected hepatocytes. The possible interaction between HCV proteins and Syk in human hepatoma-derived Huh-7 cells was then examined. Immunoprecipitation analysis revealed that NS5A interacted strongly with Syk. Deletion-mutation analysis revealed that an N-terminal portion of NS5A (aa 1-175) was involved in the physical interaction with Syk. An in vitro kinase assay demonstrated that NS5A inhibited the enzymic activity of Syk and that, in addition to the N-terminal 175 residues, a central portion of NS5A (aa 237-302) was required for inhibition of Syk. Moreover, Syk-mediated phosphorylation of phospholipase C-gamma1 was downregulated by NS5A. An interaction of NS5A with Syk was also detected in Huh-7.5 cells harbouring an HCV RNA replicon or infected with HCV. In conclusion, these results demonstrated that NS5A interacts with Syk resulting in negative regulation of its kinase activity. The results indicate that NS5A may be involved in the carcinogenesis of hepatocytes through the suppression of Syk kinase activities.

Generation of recombinant adenovirus expressing siRNA against the L mRNA of measles virus and subacute sclerosing panencephalitis virus.

Subacute sclerosing panencephalitis (SSPE) is a fatal neurodegenerative disease caused by prolonged persistent infection of the central nervous system with a measles virus (MV) mutant called SSPE virus. At present, there is no effective treatment to completely cure SSPE and development of a new therapeutic measure(s) against this fatal slow virus infection is needed. We previously reported that replication of MV and SSPE virus was effectively inhibited by small interfering RNA (siRNA), either chemically synthetic or plasmid-driven ones, that were targeted against different sequences of the mRNA for the L protein of MV. In this study, we have generated recombinant adenovirus expressing the siRNAs (rAd-siRNA-MV-L2, -L4 and -L5) and demonstrated that these rAd-siRNAs efficiently inhibited replication of MV and SSPE virus in a dose-dependent manner. Due to their high capacity for gene delivery to nerve cells and the potential to inhibit SSPE virus replication, the rAd-siRNAs could be a good candidate for a novel therapeutic measure against SSPE.

Prediction of efficient virological response to pegylated interferon/ribavirin combination therapy by NS5A sequences of hepatitis C virus and anti-NS5A antibodies in pre-treatment sera.

A considerable number of patients infected with Hepatitis C virus subtype 1b (HCV-1b) do not respond to pegylated interferon/ribavirin combination therapy. In this study we explored a useful factor(s) to predict treatment outcome. A total of 47 HCV-1b-infected patients were treated with pegylated interferon/ ribavirin for 48 weeks. Sera of the patients were examined for the entire NS5A sequence of the HCV genome, HCV RNA titers and anti-NS5A antibodies. According to their responses, the patients were divided into two groups, early viral responders who cleared the virus by week 16 (EVR[16w]) and those who did not (Non-EVR[16w]). The mean number of mutations in the V3 region (aa 2356 to 2379) or that in the V3 region plus its N-terminally flanking region, which we refer to as interferon/ribavirin resistancedetermining region (IRRDR; aa 2334 to 2379), of NS5A obtained from the pretreatment sera was signifi-cantly larger for EVR(16w) compared with Non-EVR(16w). Moreover, HCV-1b isolates with > or =5 mutations in V3 or those with > or =6 mutations in IRRDR were almost exclusively found in EVR(16w). Also, the presence of detectable levels of anti-NS5A antibodies in the pretreatment sera was closely associated with EVR(16w). In conclusion, a high degree of sequence variation in V3 (> or =5) or IRRDR (> or =6) and the presence of detectable levels of anti-NS5A antibodies in the pretreatment sera would be useful factors to predict EVR(16w). On the other hand, a less diverse sequence in V3 (< or =4) or IRRDR (< or =5) together with the absence of detectable anti-NS5A antibodies could be a predictive factor for Non-EVR(16w).