Development of quantified HIV-1 antigen panel for evaluating HIV Ag/Ab combination tests using the RT-qPCR method.

We established a human immunodeficiency virus type 1 (HIV-1) antigen (Ag) panel from culture supernatants of 27 HIV-1 isolates, including 11 HIV-1 subtypes, circulating recombinant forms (CRFs), and groups (HIV-1 types), to evaluate the HIV-1 Ag detection sensitivity and HIV-1 type specificity of three HIV-1 Ag/antibody (Ab) combination tests approved in Japan. The HIV-1 copy numbers were quantified by the reverse transcription quantitative polymerase chain reaction (RT-qPCR) method. They were diluted to four different copy numbers and used in this evaluation. Enzygnost HIV Integral IV gave HIV-positive results in nearly all samples, with the single exception being an HIV-negative result in a case with a value just below the cut-off in a CRF08_BC member (100,000 copies/mL). Genscreen HIV Ag-Ab ULT showed low sensitivity to HIV-1 group O members, but this is not an urgent problem as no HIV-1 group O infection cases have been reported in Japan. The detection sensitivity of Determine HIV Early Detect was lower than that of the aforementioned two tests by ten-to hundred-fold, indicating that the kit may have limited performance in the acute phase of HIV-1 infection. Our HIV-1 Ag panel is useful for evaluating the HIV-1 Ag sensitivity of HIV-1 Ag/Ab combination tests.

Engineered flavivirus vaccines control induction of crossreactive infection-enhancing and -neutralizing antibodies.

Flaviviruses are important human pathogens because of their global distribution and disease severity. The high structural similarity among flaviviruses induces cross-immunity, with individual flaviviruses exhibiting crossreactive infection-enhancing and/or -neutralizing activities against other flaviviruses. Unlike neutralizing antibodies, enhancing antibodies may increase the risk of disease severity. Vaccine-induced enhancement remains a concern in the development of flavivirus vaccines. Here, we immunized mice with DNA vaccine candidates (pcJEME, pcWNME or pcZIKME) against Japanese encephalitis virus (JEV), West Nile virus (WNV) or Zika virus (ZIKV), respectively, and investigated crossreactive neutralizing and enhancing antibody activities against seven flaviviruses. pcZIKME induced higher cross-neutralization against dengue viruses than against JEV and WNV. Moreover, pcZIKME with a single amino acid substitution (D87N) showed an increase in crossreactive neutralizing activity and a decrease in enhancing activities against other flaviviruses. A similar trend was observed in pcWNME. Engineered antigen might contribute to the development of safe and effective flavivirus vaccines.

Spread of genetically similar noroviruses in Bangkok, Thailand, through symptomatic and asymptomatic individuals.

Norovirus infection is a major cause of acute gastroenteritis, although some infected individuals are asymptomatic. GII.4 is the predominant genotype worldwide and, since 2000, has been the most prevalent in patients in Thailand with acute gastroenteritis. We screened stool samples for norovirus in 786 patients with acute gastroenteritis who were admitted to a hospital in Bangkok from 2017 to early 2019 and detected it in 136 specimens (17.3%). Eight and 124 specimens were positive for the GI and GII genogroups, respectively, and the remaining 4 specimens were double-positive. Nine genotypes (GI.3, GI.5, GII.2, GII.3, GII.4, GII.6, GII.8, GII.13, and GII.17) were identified from 140 strains, and 72 strains (51.4%) were GII.4. We had previously conducted a one-year survey of norovirus infection in residents of a community in Bangkok from May 2018 to April 2019 and found that a substantial portion of the residents were infected asymptomatically. The 9 genotypes identified in the patients were also commonly identified in the community residents. To investigate the relationship between noroviruses identified in the acute gastroenteritis patients and those identified in the community residents, phylogenetic tree analysis was conducted. Of the 9 genotypes, 8 showed similarities in both their genomic sequences and their deduced amino acid sequences. In addition, strain replacement of GI.3 was observed in both the patients and the community residents within the overlapping period. These results suggested that norovirus spreads efficiently to the community by simultaneously causing symptomatic and asymptomatic infections.

Nucleotide Sequence of HIV-1-Positive Specimen Reference Panel for Evaluation of HIV In Vitro Diagnostics in Japan.

HIV-1 subtype/circulating recombinant form (CRF) distribution of HIV-1-positive specimens for evaluating HIV in vitro diagnostics (IVDs) was examined and compared with the HIV-1 epidemic in Japan. The nucleotide sequences of the gag-pol region of 173 plasma specimens (84, provided in 2007, and 89 in 2013-2015) were determined. HIV-1 subtype/CRF classification was performed based on the phylogenetic analyses of the sequences. The subtype/CRF distribution resulting in this study was similar to that of a previous epidemiological report. Three CRF02_AG and one unique recombinant form, including subtype G and A regions, were observed in the 2013 and 2014 specimens, except in the 2007 specimens. The reference panel consisting of these specimens was practical for the evaluation of HIV IVDs in Japan.

Full genome-based characterization of G4P[6] rotavirus strains from diarrheic patients in Thailand: Evidence for independent porcine-to-human interspecies transmission events.

The exact evolutionary patterns of human G4P[6] rotavirus strains remain to be elucidated. Such strains possess unique and strain-specific genotype constellations, raising the question of whether G4P[6] strains are primarily transmitted via independent interspecies transmission or human-to-human transmission after interspecies transmission. Two G4P[6] rotavirus strains were identified in fecal specimens from hospitalized patients with severe diarrhea in Thailand, namely, DU2014-259 (RVA/Human-wt/THA/DU2014-259/2014/G4P[6]) and PK2015-1-0001 (RVA/Human-wt/THA/PK2015-1-0001/2015/G4P[6]). Here, we analyzed the full genomes of the two human G4P[6] strains, which provided the opportunity to study and confirm their evolutionary origin. On whole genome analysis, both strains exhibited a unique Wa-like genotype constellation of G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1. The NSP1 genotype A8 is commonly found in porcine rotavirus strains. Furthermore, on phylogenetic analysis, each of the 11 genes of strains DU2014-259 and PK2015-1-0001 appeared to be of porcine origin. On the other hand, the two study strains consistently formed distinct clusters for nine of the 11 gene segments (VP4, VP6, VP1-VP3, and NSP2-NSP5), strongly indicating the occurrence of independent porcine-to-human interspecies transmission events. Our observations provide important insights into the origin of zoonotic G4P[6] strains, and into the dynamic interaction between porcine and human rotavirus strains.

Identification of GII.14[P7] norovirus and its genomic mutations from a case of long-term infection in a post-symptomatic individual.

Norovirus is a leading cause of acute gastroenteritis worldwide. Norovirus shedding typically lasts one week to one month after the onset of diarrhea in immunocompetent hosts. The occurrence of mutations in the genome during infection has contributed to the evolution of norovirus. It has been suggested that genomic mutations in the P2-domain of capsid protein VP1, the major antigenic site for virus clearance, are involved in the evasion of host immunity and prolonged shedding of norovirus. In our previous study, we found a case of long-term shedding of GII.14 norovirus in a post-symptomatic immunocompetent individual that lasted about three months. In this study, we characterized the genomic sequence of the GII.14 strain to gain insight into the context of long-term shedding. By sequencing a 4.8 kb region of the genome corresponding to half of ORF1 and the entire ORF2 and ORF3, which encode several non-structural proteins and the structural proteins VP1 and VP2, the GII.14 strain was found to be classified as recombinant GII.14[P7]. Six point-mutations occurred during the three-month period of infection in a time-dependent manner in the genomic regions encoding RNA-dependent RNA polymerase, VP1, and VP2. Three of the six mutations were sense mutations, but no amino acid substitution was identified in the P2-domain of VP1. These results suggest that there is a mechanism by which long-term shedding of norovirus occurs in immunocompetent individuals independent of P2-domain mutations.

Anti-Chikungunya Virus Monoclonal Antibody That Inhibits Viral Fusion and Release.

Chikungunya fever, a mosquito-borne disease manifested by fever, rash, myalgia, and arthralgia, is caused by chikungunya virus (CHIKV), which belongs to the genus Alphavirus of the family Togaviridae Anti-CHIKV IgG from convalescent patients is known to directly neutralize CHIKV, and the state of immunity lasts throughout life. Here, we examined the epitope of a neutralizing mouse monoclonal antibody against CHIKV, CHE19, which inhibits viral fusion and release. In silico docking analysis showed that the epitope of CHE19 was localized in the viral E2 envelope and consisted of two separate segments, an N-linker and a ß-ribbon connector, and that its bound Fab fragment on E2 overlapped the position that the E3 glycoprotein originally occupied. We showed that CHIKV-E2 is lost during the viral internalization and that CHE19 inhibits the elimination of CHIKV-E2. These findings suggested that CHE19 stabilizes the E2-E1 heterodimer instead of E3 and inhibits the protrusion of the E1 fusion loop and subsequent membrane fusion. In addition, the antigen-bound Fab fragment configuration showed that CHE19 connects to the CHIKV spikes existing on the two individual virions, leading us to conclude that the CHE19-CHIKV complex was responsible for the large virus aggregations. In our subsequent filtration experiments, large viral aggregations by CHE19 were trapped by a 0.45-µm filter. This virion-connecting characteristic of CHE19 could explain the inhibition of viral release from infected cells by the tethering effect of the virion itself. These findings provide clues toward the development of effective prophylactic and therapeutic monoclonal antibodies against the Alphavirus infection.IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Neither a specific antiviral drug nor a commercial vaccine for CHIKV infection are available. Here, we show a detailed model of the docking between the envelope glycoprotein of CHIKV and our unique anti-CHIKV-neutralizing monoclonal antibody (CHE19), which inhibits CHIKV membrane fusion and virion release from CHIKV-infected cells. Homology modeling of the neutralizing antibody CHE19 and protein-protein docking analysis of the CHIKV envelope glycoprotein and CHE19 suggested that CHE19 inhibits the viral membrane fusion by stabilizing the E2-E1 heterodimer and inhibits virion release by facilitating the formation of virus aggregation due to the connecting virions, and these predictions were confirmed by experiments. Sequence information of CHE19 and the CHIKV envelope glycoprotein and their docking model will contribute to future development of an effective prophylactic and therapeutic agent.

Norovirus transmission mediated by asymptomatic family members in households.

The transmission of human norovirus excreted from infected persons occasionally causes sporadic infections and outbreaks. Both symptomatic patients and asymptomatic carriers have been reported to contribute to norovirus transmission, but little is known about the magnitude of the contribution of asymptomatic carriers. We carried out a 1-year survey of residents of a district of Bangkok, Thailand to determine the percentage of norovirus transmissions originating from asymptomatic individuals. We screened 38 individuals recruited from 16 families from May 2018 to April 2019 for GI and GII genotypes. Norovirus was detected every month, and 101 of 716 stool samples (14.1%) from individuals with no symptoms of acute gastroenteritis were norovirus-positive. The average infection frequency was 2.4 times per person per year. Fourteen genotypes were identified from the positive samples, with GII.4 being detected most frequently. Notably, 89.1% of the norovirus-positive samples were provided by individuals with no diarrhea episode. Similar to cases of symptomatic infections in Thailand, asymptomatic infections were observed most frequently in December. We detected 4 cases of NV infection caused by household transmission, and 3 of the 4 transmissions originated from asymptomatic individuals. We also identified a case in which norovirus derived from an asymptomatic individual caused diarrhea in a family member. These results suggest that asymptomatic individuals play a substantial role in both the maintenance and spreading of norovirus in a community through household transmission.

Full genome characterization of novel DS-1-like G9P[8] rotavirus strains that have emerged in Thailand.

The emergence and rapid spread of unusual DS-1-like intergenogroup reassortant rotaviruses having G1/3/8 genotypes have been recently reported from major parts of the world (Africa, Asia, Australia, Europe, and the Americas). During rotavirus surveillance in Thailand, three novel intergenogroup reassortant strains possessing the G9P[8] genotype (DBM2017-016, DBM2017-203, and DBM2018-291) were identified in three stool specimens from diarrheic children. In the present study, we determined and analyzed the full genomes of these three strains. On full-genomic analysis, all three strains were found to share a unique genotype constellation comprising both genogroup 1 and 2 genes: G9-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis demonstrated that each of the 11 genes of the three strains was closely related to that of emerging DS-1-like intergenogroup reassortant, human, and/or locally circulating human strains. Thus, the three strains were suggested to be multiple reassortants that had acquired the G9-VP7 genes from co-circulating Wa-like G9P[8] rotaviruses in the genetic background of DS-1-like intergenogroup reassortant (likely equine-like G3P[8]) strains. To our knowledge, this is the first description of emerging DS-1-like intergenogroup reassortant strains having the G9P[8] genotype. Our observations will add to the growing insights into the dynamic evolution of emerging DS-1-like intergenogroup reassortant rotaviruses through reassortment.

High prevalence of equine-like G3P[8] rotavirus in children and adults with acute gastroenteritis in Thailand.

Group A rotavirus (RVA) is a major cause of acute gastroenteritis in infants and young children worldwide. This study aims to clarify the distribution of G/P types and genetic characteristics of RVAs circulating in Thailand. Between January 2014 and September 2016, 1867 stool specimens were collected from children and adults with acute gastroenteritis in six provinces in Thailand. RVAs were detected in 514/1867 (27.5%) stool specimens. G1P[8] (44.7%) was the most predominant genotype, followed by G3P[8] (33.7%), G2P[4] (11.5%), G8P[8] (7.0%), and G9P[8] (1.3%). Unusual G3P[9] (0.8%), G3P[10] (0.4%), G4P[6] (0.4%), and G10P[14] (0.2%) were also detected at low frequencies. The predominant genotype, G1P[8] (64.4%), in 2014 decreased to 6.1% in 2016. In contrast, the frequency of G3P[8] markedly increased from 5.5% in 2014 to 65.3% in 2015 and 89.8% in 2016. On polyacrylamide gel electrophoresis, most (135/140; 96.4%) of the G3P[8] strains exhibited a short RNA profile. Successful determination of the nucleotide sequences of the VP7 genes of 98 G3P[8] strains with a short RNA profile showed that they are all equine-like G3P[8] strains. On phylogenetic analysis of genome segments of two representative Thai equine-like G3P[8] strains, it was noteworthy that they possessed distinct NSP4 genes, one bovine-like and the other human-like. Thus, we found that characteristic equine-like G3P[8] strains with a short RNA electropherotype are becoming highly prevalent in children and adults in Thailand.

The use of green fluorescent protein-tagged virus-like particles as a tracer in the early phase of chikungunya infection.

To visually examine the early phase of chikungunya virus (CHIKV) infection in target cells, we constructed a virus-like particle (VLP) in which the envelope protein E1 is fused with green fluorescent protein (GFP). This chikungunya VLP-GFP (CHIK-VLP-EGFP), purified by density gradient fractionation, was observed as 60-70 nm-dia. particles and was detected as tiny puncta of fluorescence in the cells. CHIK-VLP-EGFP showed binding properties similar to those of the wild-type viruses. Most of the fluorescence signals that had bound on Vero cells disappeared within 30 min at 37 °C, but not in the presence of anti-CHIKV neutralizing serum or an endosomal acidification inhibitor (bafilomycin A1), suggesting that the loss of fluorescence signals is due to the disassembly of the viral envelope following the internalization of CHIK-VLP-EGFP. In addition to these results, the fluorescence signals disappeared in highly susceptible Vero and U251MG cells but not in poorly susceptible A549 cells. Thus, CHIK-VLP-EGFP is a useful tool to examine the effects of the CHIKV neutralizing antibodies and antiviral compounds that are effective in the entry phase of CHIKV.

The dynamics of norovirus genotypes and genetic analysis of a novel recombinant GII.P12-GII.3 among infants and children in Bangkok, Thailand between 2014 and 2016.

Norovirus (NoV) is the leading cause of viral acute gastroenteritis among all age groups in the world. We performed a molecular epidemiological study of the NoVs prevalent in Bangkok between November 2014 and July 2016 to investigate the emergence of new NoV variants in Thailand. A total of 332 stool specimens were collected from hospitalized pediatric patients with acute gastroenteritis in Bangkok, Thailand. NoVs were detected by real-time PCR. The genome of the N-terminal/shell domain was amplified, the nucleotide sequence was determined, and phylogenetic analyses were performed. GII NoV was detected in 58 (17.5%) of the 332 specimens. GII.17, a genotype strain prevalent from 2014 to mid-2015, was hardly detected and replaced by the GII.3 genotype strain. Entire genome sequencing followed by phylogenetic analysis of the GII.3 genotype strains indicated that they are new recombinant viruses, because the genome encoding ORF1 is derived from a GII.12 genotype strain, whereas that encoding ORF2-3 is from a GII.3 genotype strain. The putative recombination breakpoints with the highest statistical significance were located around the border of 3Dpol and ORF2. The change in the prevalent strain of NoV seems to be linked to the emergence of new forms of recombinant viruses. These findings suggested that the swapping of the structural and non-structural proteins of NoV is a common mechanism by which new epidemic variants are generated in nature.

An Outbreak of Acute Hepatitis Caused by Genotype IB Hepatitis A Viruses Contaminating the Water Supply in Thailand.

BACKGROUND: In 2000, an outbreak of acute hepatitis A was reported in a province adjacent to Bangkok, Thailand. AIMS: To investigate the cause of the 2000 hepatitis A outbreaks in Thailand using molecular epidemiological analysis. METHODS: Serum and stool specimens were collected from patients who were clinically diagnosed with acute viral hepatitis. Water samples from drinking water and deep-drilled wells were also collected. These specimens were subjected to polymerase chain reaction (PCR) amplification and sequencing of the VP1/2A region of the hepatitis A virus (HAV) genome. The entire genome sequence of one of the fecal specimens was determined and phylogenetically analyzed with those of known HAV sequences. RESULTS AND CONCLUSIONS: Eleven of 24 fecal specimens collected from acute viral hepatitis patients were positive as determined by semi- nested reverse transcription PCR targeting the VP1/2A region of HAV. The nucleotide sequence of these samples had an identical genotype IB sequence, suggesting that the same causative agent was present. The complete nucleotide sequence derived from one of the samples indicated that the Thai genotype IB strain should be classified in a unique phylogenetic cluster. The analysis using an adjusted odds ratio showed that the consumption of groundwater was the most likely risk factor associated with the disease.

A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors.

Due to their high frequency of genomic mutations, human retroviruses often develop resistance to antiretroviral drugs. The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) is a significant obstacle to the effective long-term treatment of HIV infection. The development of a rapid and versatile drug-susceptibility assay would enable acquisition of phenotypic information and facilitate determination of the appropriate choice of antiretroviral agents. In this study, we developed a novel in vitro method, termed the Cell-free drug susceptibility assay (CFDSA), for monitoring phenotypic information regarding the drug resistance of HIV-1 protease (PR). The CFDSA utilizes a wheat germ cell-free protein production system to synthesize enzymatically active HIV-1 PRs directly from PCR products amplified from HIV-1 molecular clones or clinical isolates in a rapid one-step procedure. Enzymatic activity of PRs can be readily measured by AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) in the presence or absence of clinically used protease inhibitors (PIs). CFDSA measurement of drug resistance was based on the fold resistance to the half-maximal inhibitory concentration (IC50) of various PIs. The CFDSA could serve as a non-infectious, rapid, accessible, and reliable alternative to infectious cell-based phenotypic assays for evaluation of PI-resistant HIV-1.

Japanese external quality assessment program to standardize HIV-1 drug-resistance testing (JEQS2010 program) using in vitro transcribed RNA as reference material.

To design appropriate antiretroviral therapy regimens and avoid the emergence of human immunodeficiency virus (HIV)-1 variants with reduced susceptibility to antiretroviral drugs, genotypic drug-resistance testing (HIV genotyping) is strongly recommended. To monitor the quality of HIV genotyping in Japan, we performed an external quality assessment (EQA), named the Japanese external quality assessment program, to standardize HIV genotyping (JEQS). To accurately evaluate the quality of HIV genotyping, we employed as reference material (RM) a well-characterized sample, in vitro transcribed RNA (trRNA) that includes the HIV gag-pol sequence, and created a JEQS2010 panel consisting of three single variant and three mixed trRNA samples. All 11 participating laboratories showed high concordance rates (>96%) for the single variant samples. Eight laboratories also showed good rates of detecting minor variants, but three laboratories failed to detect the variants comprising one-half of the sample. These three laboratories used a common primer that had four internal mismatches to the minor trRNA clone. This program showed the usefulness of trRNA as RM, the high quality of HIV genotyping, and extensive interlaboratory variation in the ability to detect minor variants. These results suggest that improving the quality of HIV genotyping in Japan requires regularly implementing the EQA program and improving the HIV genotyping protocol in each laboratory.

DNA damage enhances integration of HIV-1 into macrophages by overcoming integrase inhibition.

BACKGROUND: The prevention of persistent human immunodeficiency virus type 1 (HIV-1) infection requires the clarification of the mode of viral transduction into resting macrophages. Recently, DNA double-strand breaks (DSBs) were shown to enhance infection by D64A virus, which has a defective integrase catalytic activity (IN-CA). However, the mechanism by which DSBs upregulate viral transduction was unclear. Here we analyzed the roles of DSBs during IN-CA-independent viral transduction into macrophages. RESULTS: We used cellular systems with rare-cutting endonucleases and found that D64A virus integrated efficiently into the sites of artificially induced DSBs. This IN-CA-independent viral transduction was blocked by an inhibitor of ataxia telangiectasia mutated protein (ATM) but was resistant to raltegravir (RAL), an inhibitor of integrase activity during strand transfer. Moreover, Vpr, an accessory gene product of HIV-1, induced DSBs in resting macrophages and significantly enhanced the rate of IN-CA-independent viral transduction into macrophages with concomitant production of secondary viruses. CONCLUSION: DSBs contribute to the IN-CA-independent viral infection of macrophages, which is resistant to RAL. Thus, the ATM-dependent cellular pathway and Vpr-induced DNA damage are novel targets for preventing persistent HIV-1 infection.

Lack of the trans-receptor mechanism of HIV-1 infection: CD4- and coreceptor-independent incorporation of HIV-1-resistant cells into syncytia induced by HIV-1.

Human immunodeficiency virus type 1 (HIV-1) infects cells through an interaction of HIV-1 envelope protein with CD4 and an appropriate coreceptor on target cells. This interaction often leads to cell fusion, and formation of syncytia. HIV-1-resistant cells expressing either CD4 or a coreceptor are often surrounding HIV-1-susceptible cells, expressing both CD4 and a compatible coreceptor, in vivo. It is therefore worthwhile to investigate whether these HIV-1-resistant cells could cooperate in HIV-1 infection or cell fusion leading to their incorporation into syncytia. When CD4-positive, coreceptor-negative cells were co-cultured with CD4-negative, coreceptor-positive cells and exposed to HIV-1, HIV-1 infection was not established, indicating that CD4 and the coreceptor expressed on different cell surfaces could not cooperate in HIV-1 entry. However, when HIV-1-resistant cells expressing CD4 or a coreceptor or lacking both were mixed with HIV-1-susceptible cells and inoculated with HIV-1, all these HIV-1-resistant cells were similarly incorporated into syncytia induced by HIV-1, indicating a CD4- and coreceptor-independent incorporation of HIV-1-resistant cells into syncytia. This incorporation was impaired by the transfection of these cells with siRNAs for adhesion molecules. Our study demonstrates that HIV-1-resistant cells can be incorporated into syncytia induced by HIV-1 and this incorporation may partially be mediated through adhesion molecules.

Elimination of hepatitis C virus from hepatocytes by a selective activation of therapeutic molecules.

To eliminate hepatitis C virus (HCV) from infected hepatocytes, we generated two therapeutic molecules specifically activated in cells infected with HCV. A dominant active mutant of interferon (IFN) regulatory factor 7 (IRF7) and a negative regulator of HCV replication, VAP-C (Vesicle-associated membrane protein-associated protein subtype C), were fused with the C-terminal region of IPS-1 (IFNß promoter stimulator-1), which includes an HCV protease cleavage site that was modified to be localized on the ER membrane, and designated cIRF7 and cVAP-C, respectively. In cells expressing the HCV protease, cIRF7 was cleaved and the processed fragment was migrated into the nucleus, where it activated various IFN promoters, including promoters of IFNα6, IFNß, and IFN stimulated response element. Activation of the IFN promoters and suppression of viral RNA replication were observed in the HCV replicon cells and in cells infected with the JFH1 strain of HCV (HCVcc) by expression of cIRF7. Suppression of viral RNA replication was observed even in the IFN-resistant replicon cells by the expression of cIRF7. Expression of the cVAP-C also resulted in suppression of HCV replication in both the replicon and HCVcc infected cells. These results suggest that delivery of the therapeutic molecules into the liver of hepatitis C patients, followed by selective activation of the molecules in HCV-infected hepatocytes, is a feasible method for eliminating HCV.

Differential anti-APOBEC3G activity of HIV-1 Vif proteins derived from different subtypes.

Antiretroviral cytidine deaminase APOBEC3G, which is abundantly expressed in peripheral blood lymphocytes and macrophages, strongly protects these cells against HIV-1 infection. The HIV-1 Vif protein overcomes this antiviral effect by enhancing proteasome-mediated APOBEC3G degradation and is key for maintaining viral infectivity. The 579-bp-long vif gene displays high genetic diversity among HIV-1 subtypes. Therefore, it is intriguing to address whether Vif proteins derived from different subtypes differ in their viral defense activity against APOBEC3G. Expression plasmids encoding Vif proteins derived from subtypes A, B, C, CRF01_AE, and CRF02_AG isolates were created, and their anti-APOBEC3G activities were compared. Viruses produced from cells expressing APOBEC3G and Vif proteins from different subtypes showed relatively different viral infectivities. Notably, subtype C-derived Vif proteins tested had the highest activity against APOBEC3G that was ascribed to its increased binding activity, for which the N-terminal domain of the Vif protein sequences was responsible. These results suggest that the biological differences of Vif proteins belonging to different subtypes might affect viral fitness and quasispecies in vivo.

Heparin identification test and purity test for OSCS in heparin sodium and heparin calcium by weak anion-exchange high-performance liquid chromatography.

Heparin sodium and heparin calcium, which are widely used as anti-coagulants, are known to potentially contain the natural impurity dermatan sulfate (DS). Recently serious adverse events occurred in patients receiving heparin sodium in the US, and a contaminant oversulfated chondroitin sulfate (OSCS) was found to be a cause of the events. To ensure the quality and safety of pharmaceutical heparins, there is need of a physicochemical identification test that can discriminate heparin from the heparin-related substances as well as a sensitive purity test for OSCS. Recently, HPLC with a strong-anion exchange column was proposed as the methods for identifying heparin and determination of OSCS in heparin sodium. Although this method is convenient and easy to perform, the only column suitable for this purpose is the Dionex IonPac AS11-HC column. In this study, we developed alternative identification test and test for OSCS in both heparin sodium and heparin calcium using a weak anion-exchange column. The identification test allowed for separation of heparin from the impurity DS and contaminant OSCS in a shorter time. The purity test provided enough sensitivity, specificity, linearity, recovery and repeatability for OSCS. We believe that our methods will be useful for quality control of pharmaceutical heparins.