Oncolytic virotherapy is a novel and intriguing treatment strategy for cancer therapy. However, the clinical potential of oncolytic virus as single agent is limited. M1 virus is a promising oncolytic virus that has been tested in preclinical studies. In this study, we investigated the effect of the combination use of M1 virus and Bcl-2 family inhibitors. A chemical compounds screening including ten Bcl-2 family inhibitors demonstrated that pan-Bcl-2 inhibitors selectively augmented M1 virus oncolysis in cancer cells at very low doses. The mechanism of the enhanced antitumor effect of pan-Bcl-2 inhibitors with M1 virus is mainly due to the inhibition of Bcl-xL, which synergizes with M1-induced upregulation of Bak to trigger apoptosis. In xenograft mouse models and patient-derived tumor tissues, the combination of M1 and pan-Bcl-2 inhibitors significantly inhibited tumor growth and prolonged survival, suggesting the potential therapeutic value of this strategy. These findings offer insights into the synergy between Bcl-xL inhibition and oncolytic virus M1 as a combination anticancer treatment modality.

Background/Aims: Hainan Island has been inhabited by the “Li” aboriginal minority for centuries where the HCV genotype distribution patterns maybe remarkably different from other parts of China. We aimed to provide a better understanding of the infection with HCV genotype 6 among “Li” aboriginals on Hainan Island. Methods: Firstly, using RT-PCR and DNA sequencing to determined 517 partial HCV Core-E1(115 from Li Ethnic, 402 from Han Ethnic) and 8 full-length genomes from Li ethnic in Hainan Island successfully, and then using the phylogenetic tree to determine the HCV genotype distribution and analyze the evolution of them. Results: Phylogenetic tree analysis showed that the distribution pattern of HCV genotypes among the Han and Li ethnic population exhibits significant diferences: 6a was the most prevalent subtype in Han ethnic of Hainan Island followed by 1b, 3b, 2a, 3a, and 1a. All genomes from Li ethnic were classified into genotype 6, while 84 out of 115 (73%) could not be classified. Nine sequences (HN1350 et al.) from Li ethnic might be assigned to a new subtype 6xh as their p-distances ranged from 5.9∼9.7%. Furthermore, we sequenced and characterized full-length genomes for eight HCV-6 isolates which were all from Li ethnic in Hainan Island. Among these isolates, the HN1350 was classified as a new subtype: 6xh. Conclusion: Overall, we firstly defined a new subtype of genotype 6xh through partial and new full length genome. And we found a unique distribution pattern of HCV 6 in the Li tribe, which might provide a better way to understand the genetic diversity of HCV-6 and to investigate the phylogeny of HCV strains from Li tribe.

We characterized full-length genomes for 15 HCV-6 isolates, all from Asian immigrants living in North America. Among these isolates, nine were novel variants showing >15% nucleotide differences from their nearest relatives, representing lineages distinct from known subtypes. The other six were classified into subtypes 6c, 6h, 6q, 6r, and 6s. The partial sequences were also determined for five additional HCV-6 isolates, three from the US and two from Canada. The latter two were assigned to new subtype 6xf as they were found to classify with two other isolates for which we recently reported their full-length genomes. We further analyzed partial Core-E1 sequences of 100 HCV-6 isolates sampled in North America, seven from the US and 93 from Canada and all from Asian immigrants except for four from Caucasians. These 100 isolates belonged to 20 assigned subtypes and 16 unclassified lineages showing great genetic diversity and enhanced significance to public health.

Human T-cell leukemia virus type 1 (HTLV-1)-associated adult T-cell leukemia and T-cell lymphoma (ATL) are aggressive diseases with poor prognoses, limited therapeutic options, and no curative treatment. In this study, we used a mouse model of ATL and restored expression of the microRNA, miR-124a, to identify in vivo downstream effectors responsible for its tumor-suppressive functions in ATL cells. Our results revealed that STAT3, a direct target of miR-124a, is constitutively activated in HTLV-I-transformed cells and ATL cells, and activating STAT3 mutations were detected in 25.5% of primary ATL patients. Interestingly, we found that the STAT3 downstream kinase effector, Pim1, is constitutively activated in ATL cells. The dependence of ATL cells to Pim1 activity was demonstrated using 2 Pim1 small inhibitors, SMI-4a and AZD1208. These studies indicated that HTLV-I-transformed and ATL cells, but not normal peripheral blood mononuclear cells, are highly sensitive to AZD1208, and the inhibition of Pim1 signaling triggers an apoptotic signal in leukemic cells. Finally, preclinical testing of AZD1208 in a mouse model of ATL resulted in significant prevention of tumor growth in vivo. In conclusion, our studies suggest that constitutive activation of the STAT3-Pim1 pathway represents a novel therapeutic target for the treatment of ATL.

Cancers figure among the leading causes of morbidity and mortality worldwide. The number of new cases is expected to rise by about 70% over the next 2 decades. Development of novel therapeutic agents is urgently needed for clinical cancer therapy. Alphavirus M1 is a Getah-like virus isolated from China with a genome of positive single-strand RNA. We have previously identified that alphavirus M1 is a naturally existing oncolytic virus with significant anticancer activity against different kinds of cancer (e.g., liver cancer, bladder cancer, and colon cancer). To support the incoming clinical trial of intravenous administration of alphavirus M1 to cancer patients, we assessed the safety of M1 in adult nonhuman primates. We previously presented the genome sequencing data of the cynomolgus macaques (Macaca fascicularis), which was demonstrated as an ideal animal species for virus infection study. Therefore, we chose cynomolgus macaques of either sex for the present safety study of oncolytic virus M1. In the first round of administration, five experimental macaques were intravenously injected with six times of oncolytic virus M1 (1 x 10(9) pfu/dose) in 1 week, compared with five vehicle-injected control animals. The last two rounds of injections were further completed in the following months in the same way as the first round. Body weight, temperature, complete blood count, clinical biochemistries, cytokine profiles, lymphocytes subsets, neutralizing antibody, and clinical symptoms were closely monitored at different time points. Magnetic resonance imaging was also performed to assess the possibility of encephalitis or arthritis. As a result, no clinical, biochemical, immunological, or medical imaging or other pathological evidence of toxicity was found during the whole process of the study. Our results in cynomolgus macaques suggested the safety of intravenous administration of oncolytic virus M1 in cancer patients in the future.

Background/Aims: Different genotypes of HCV may differ in both disease progression and response to antiviral therapies. Hainan Island has been inhabited by the "Li" aboriginal minority for centuries. We aimed to provide a better understanding of HCV infection on Hainan Island, so that the information would help improve strategies for HCV prevention and control on the island and in the wider country. Methods: Using RT-PCR and DNA sequencing, we determined HCV sequences from 100 patients living on Hainan Island. Results: Phylogenetic analysis classified these sequences into six subtypes: 6a (n=35), 1b (n=31), 3b (n=16), 2a (n=8), 3a (n=6), and 1a (n=4). By including reference sequences reported from elsewhere in China, phylogeographic trees were reconstructed to indicate their migration patterns. While the predominant 6a isolates were estimated to have origins in Guangdong and Guangxi provinces, the increase in 3b strains must have resulted from IDU network transmission from the southwest. A Bayesian Skyline Plot for subtype 1a, which is rare in China, showed a rapid population growth since 1998. Although slowed in rate around 2005, this growth continued to the present. Not found for any other HCV lineage. Conclusions: Overall, a delayed growth pattern may indicate the unique history of 1a dissemination in China and its recently increasing prevalence, despite measures taken to improve HCV prevention. (C) 2016 T he Author(s) Published by S. Karger AG, Basel

The optimal treatment of hepatitis C virus (HCV) genotype 6 is unclear owing to its limited geographic distribution. Because of a high predictive value of rapid virological response (RVR) for sustained virological response (SVR), we conducted an open-label randomized controlled trial to compare 24- and 48-week peginterferon/ribavirin combination therapy for patients with HCV genotype 6 in Southern China who achieved an RVR.Treatment-naive, non-cirrhotic patients with chronic hepatitis C genotype 6 were treated with pegylated interferon α-2a (180 μg/week) and ribavirin (800-1,200 mg, according to weight) for 4 weeks. Patients who achieved an RVR, which was defined as HCV RNA negativity at week 4 (<50 IU), were randomized to receive either an additional 20 or 44 weeks of treatment (24- and 48-week treatment groups, respectively). The primary outcome measure was SVR. From January 2011 to June 2014, 152(152/210, 72.4%) patients with HCV genotype 6a and RVR were randomized 1:1 to the 24- or 48-week treatment group. The SVR rates in the 24- and 48-week groups in the intention-to-treat analysis were 90.8% (69/76) and 88.2% (67/76), respectively; those in the per-protocol analysis were 95.7% (67/70) and 97.0% (64/66), respectively. More patients in the 48-week group had anemia (46.1% vs. 28.9%, P = 0.03), but other adverse events were comparable between the groups. The limitation of the present study was that only patients from Southern China were enrolled which may inhibit the extensive application of the findings.Twenty-four weeks of peginterferon/ribavirin combination therapy was non-inferior to 48 weeks in patients with HCV genotype 6a in Southern China who achieved an RVR.ClinicalTrials.gov NCT01263860.

An HCV outbreak occurred in 2012 in China, affecting hundreds of patients. We characterized HCV subtype 2a and 6a sequences from 60 and 102 patients, respectively, and co-analyzed them with 82 local controls and 103 calibrating references. The close grouping of the patients׳ sequences contrasted sharply with the diversity of local controls. Scaled by the calibrating references, the emergence of patients׳ isolates was estimated at 2–5 years before sampling. In contrast, the controls intermingled with the calibrating references that were much older. For both subtypes, the major and minor clusters could be defined, with the closeness to indicate linked transmission. Conclusion: HCV sequences from the study patients grouped into three subtype 2a and two subtype 6a clusters, in addition to three 6a solitary branches, representing descendants of eight earlier strains that were distinct and otherwise sporadic. Due to iatrogenic transmission through reusing needles, five strains were highly selected and preferentially spread.

Background: Although human leukocyte antigens (HLA) have been shown in association with the outcomes of hepatitis C virus (HCV) infection among different ethnic groups, such studies remain absent in China, where the HCV prevalence is higher than the global average. Methods: In this study, 426 HCV-infected and 709 uninfected blood donors were analyzed, among whom the HLA alleles were sequenced using a high-resolution genotyping method. Results: At the 2-digit level, none of the alleles showed a statistical difference between the HCV-infected and uninfected groups. However, at the 4-digit level, the HLA-B alleles B*15:01 and B*15:02 showed an opposite association with HCV infection, i.e. B*15:01 was significantly higher in the HCV-infected group (odds ratio, OR = 1.561, p = 0.010), while B*15:02 was significantly higher in the uninfected group (OR = 0.778, p = 0.016). We also identified a higher frequency of B*13:02 in the HCV-infected group (OR = 1.515, p = 0.009) and a higher frequency of B*07:05 in the uninfected group (OR = 0.299, p = 0.001). Conclusions: The frequencies of four HLA alleles, B*07:05, B*13:02, B*15:01, and B*15:02, were found to be significantly different between the HCV-infected and uninfected blood donors in China, revealing an inverse relation of B*15:01 and B*15:02 with HCV infection. This finding suggests that the ethnic genetic variations of HLA may greatly affect the host immune responses against HCV.

We characterized the full-length genomes of 22 hepatitis C virus genotype 6 (HCV-6) isolates: 10 from Vietnam (classified into subtypes 6e, 6h, 6p, 6r, 6s, and 6u), one from China (confirmed as a new subtype 6xd), and 11 from the Lao PDR (representing a new subtype 6xe plus eight novel variants). With these 22 new genomes, HCV-6 now has a diverse and extended taxonomic structure, comprised of 28 assigned subtypes (denoted 6a-6xe) and 27 unassigned lineages, all of which have been represented by full-length genomes. Our phylogenetic analyses also included many partially-sequenced novel variants of HCV-6 from Lao PDR. This revealed that Lao HCV isolates are genetically very diverse and are phylogenetically distributed in multiple lineages within genotype 6. Our results suggest that HCV-6 has been maintained in Laos, a landlocked country, since the common ancestor of genotype 6 and indicates historical dispersal of HCV-6 across Southeast Asia.

We characterized the full-length genomes for nine novel variants of HCV genotype 4 (HCV-4), representing a new subtype 4s and eight unclassified lineages. They were obtained from patients who resided in Canada but all had origins in Africa. An extended maximum clade credibility (MCC) tree was reconstructed after the inclusion of 30 reference sequences. It differentiated 18 assigned subtypes and 10 unclassified lineages within HCV-4. Similar analysis of 102 partial NS5B sequences resulted in another MCC tree that revealed 22 assigned subtypes (4a-4t, 4w, and 4v) and 30 unclassified lineages at the subtype level. Our study shows that HCV-4 is taxonomically complex and it displays high genetic diversity to support an African origin.

ABSTRACT We investigated the migration patterns of hepatitis C virus (HCV) in China. Partial E1 and/or NS5B sequences from 411 volunteer blood donors sampled in 17 provinces and municipalities located in five large regions, the north-northeast, northwest, southwest, central south, and southeast, were characterized. The sequences were classified into eight subtypes (1a, n = 3; 1b, n = 183; 2a, n = 83; 3a, n = 30; 3b, n = 44; 6a, n = 55; 6n, n = 10; 6v, n = 1) and a new subtype candidate. Bayesian evolutionary analysis by sampling trees of the E1 sequences of the five major subtypes revealed distinct migration patterns. Subtype 1b showed four groups: one is prevalent nationwide with possible origins in the north-northeast; two are locally epidemic in the central south and northwest, respectively, and have spread sporadically to other regions; and the fourth one is likely linked to the long-distance dispersion among intravenous drug users from the northwest. Subtype 2a showed two groups: the larger one was mainly restricted to the northwest and seemed to show a trend toward migration via the Silk Road; the smaller one was geographically mixed and may represent descendants of those that spread widely during the contaminated plasma campaign in the 1990s. Subtype 3a exhibited three well-separated geographic groups that may be epidemically unrelated: one showed origins in the northwest, one showed origins in the southwest, and the other showed origins in the central south. In contrast, subtype 3b had a mixture of geographic origins, suggesting migrations from the southwest to the northwest and sporadically to other regions. Structurally resembling the tree for subtype 3a, the tree for subtype 6a showed four groups that may indicate migrations from the central south to southeast, southwest, and northwest. Strikingly, no subtype 6a strain was identified in the north-northeast. IMPORTANCE With a population of greater than 1.3 billion and a territory of >9.6 million square kilometers, China has a total of 34 provinces and municipalities. In such a vast country, the epidemic history and migration trends of HCV are thought to be unique and complex but variable among regions and are unlikely to be represented by those observed in only one or at best a few provinces and municipalities. However, due to the difficulties in recruiting patients, all previous studies for this purpose have been based only on data from limited regions, and therefore, geographical biases were unavoidable. In this study, such biases were greatly reduced because we utilized samples collected from volunteer blood donors in 17 provinces and municipalities. To our knowledge, this is the first study in which the HCV isolates represented such a large portion of the country, and thus, the results should shed light on the current understanding of HCV molecular epidemiology.

We completely sequenced nine HCV-6 variants from Vietnam. They are grouped into six lineages beyond the 24 assigned subtypes, 6a-6xa, and 14 unclassified lineages that have been recently described with full-length genomes. Co-analysis with reference sequences in the NS5B region identified additional 22 such lineages, which made the total taxonomic number of HCV-6 increased to 66 that might be recognized at the subtype level. Because two of these six lineages revealed in this study each had >3 epidemiologically unlinked isolates identified, we proposed to assign them new subtypes 6xb and 6xc in following the eXtended format recently recommended in the expanded HCV nomenclature.

We characterized the full-length genomes of nine hepatitis C virus genotype 3 (HCV-3) isolates: QC7, QC8, QC9, QC10, QC34, QC88, NE145, NE274 and 811. To the best of our knowledge, NE274 and NE145 were the first full-length genomes for confirming the provisionally assigned subtypes 3d and 3e, respectively, whereas 811 represented the first HCV-3 isolate that had its extreme 3' UTR terminus sequenced. Based on these full-length genomes, together with 42 references representing eight assigned subtypes and an unclassified variant of HCV-3, and 10 sequences of six other genotypes, a timescaled phylogenetic tree was reconstructed after an evolutionary analysis using a coalescent Bayesian procedure. The results indicated that subtypes 3a, 3d and 3e formed a subset with a common ancestor dated to ~202.89 [95% highest posterior density (HPD): 160.11, 264.6] years ago. The analysis of all of the HCV-3 sequences as a single lineage resulted in the dating of the divergence time to ~457.81 (95% HPD: 350.62, 587.53) years ago, whereas the common ancestor of all of the seven HCV genotypes dated to ~780.86 (95% HPD: 592.15, 1021.34) years ago. As subtype 3h and the unclassified variant were relatives, and represented the oldest HCV-3 lineages with origins in Africa and the Middle East, these findings may indicate the ancestral origin of HCV-3 in Africa. We speculate that the ancestral HCV-3 strains may have been brought to South Asia from Africa by land and/or across the sea to result in its indigenous circulation in that region. The spread was estimated to have occurred in the era after Vasco da Gama had completed his expeditions by sailing along the eastern coast of Africa to India. However, before this era, Arabians had practised slave trading from Africa to the Middle East and South Asia for centuries, which may have mediated the earliest spread of HCV-3.

UNLABELLED:We characterized a novel group of HCV variants that are genetically related but distinct from each other belonging to genotype 6 (HCV-6). From 26 infected Austronesian-descended aborigines on Hainan Island, China, HCV sequences were determined followed by genetic analyses. Six nearly full-length genomes and 20 E1 sequences of HCV were obtained, which differ from each other and from all known HCV lineages by nucleotides above the intra-subtype level of 13%. Together with subtypes 6g and 6w, they constitute a phylogenetic group sharing a common ancestor dating from the end of the 12th century. CONCLUSION:Our data indicate the maintenance of an isolated HCV-6 indigenous circulation on Hainan Island at least for six centuries.

We characterized the full-length genomes of 16 distinct hepatitis C virus genotype 1 (HCV-1) isolates. Among them, four represented the first full-length genomes for subtypes 1d (QC103), 1i (QC181), 1j (QC329) and 1k (QC82), and another four corresponded to subtypes 1c (QC165), 1g (QC78), 1h (QC156) and 1e (QC172). Both QC196 and QC87 were assigned into a new subtype 1m, and QC113 and QC74 into another new subtype 1n. The remaining four (QC60, QC316, QC152 and QC180) did not classify among the established subtypes and corresponded to four new lineages. Subtypes 1j, 1k, 1m, 1n and the unclassified isolate QC60 were identified in Haitian immigrants. In the updated HCV nomenclature of 2005, a total of 12 subtypes of HCV-1 were designated. Including the data from the present study, all but subtype 1f now have their full-length genomes defined. Further analysis of partial NS5B sequences available in GenBank denoted a total of 21 unclassified lineages, indicating the taxonomic complexity of HCV-1. Among them, six have had their full-length genomes characterized. Based on the available full-length genome sequences, a timescale phylogenetic tree was reconstructed which estimated important time points in the evolution of HCV-1. It revealed that subtype 1a diverged from its nearest relatives 135 years ago and subtype 1b diverged from its nearest relatives 112 years ago. When subtypes 1a, 1j, 1k, 1m, 1n and six close relatives (all but one from Haitian immigrants) were considered as a whole, the divergence time was 176 years ago. This diversification was concurrent with the time period when the transatlantic slave trade was active. When taking all the HCV-1 isolates as a single lineage, the divergence time was 326 years ago. This analysis suggested the existence of a recent common ancestor for subtype 1a and the Haitian variants; a co-origin for subtypes 1b, 1i and 1d was also implied.

Vietnam has a unique history in association with foreign countries, which may have resulted in multiple introductions of the alien HCV strains to mix with those indigenous ones. In this study, we characterized the HCV sequences in Core-E1 and NS5B regions from 236 Vietnamese individuals. We identified multiple HCV lineages; 6a, 6 e, 6h, 6k, 6l, 6 o, 6p, and two novel variants may represent the indigenous strains; 1a was probably introduced from the US; 1b and 2a possibly originated in East Asia; while 2i, 2j, and 2m were likely brought by French explorers. We inferred the evolutionary history for four major subtypes: 1a, 1b, 6a, and 6 e. The obtained Bayesian Skyline Plots (BSPs) consistently showed the rapid HCV population growth from 1955 to 1963 until 1984 or after, corresponding to the era of the Vietnam War. We also estimated HCV growth rates and reconstructed phylogeographic trees for comparing subtypes 1a, 1b, and HCV-2.

In this study, we characterized the full-length genomes of 16 HCV isolates obtained from patients in a single hospital in China using overlapping PCR followed by DNA sequencing. The obtained genomes are 9414-9628 nucleotides in length, and each genome contains a single ORF of 9021-9102 nucleotides. Nine genomes represent the common subtypes 1a, 1b, 2a, 2b, 3a, 3b, and 6a, while seven represent the infrequent lineages 1c, 2f, 4d, and 5a, and two novel genotype 6 variants. GZ51969 and GZ52540 are subtype 1b isolates belonging to two unique clusters designated A and B, which account for 29.5% and 59.5% of the 1b infections in China, respectively. ZS542 and GZ98799 represent the first two complete genomes of the provisionally assigned subtype 2f. ZS96 and ZS202 are novel genotype 6 variants that may qualify for two new subtypes. ZS17, ZS537, and ZS631 represent three alien subtypes, namely, 1c, 4d, and 5a, which were detected in China for the first time in this study and may have been recently introduced as a result of globalization. Taken together, these results confirmed a large variety of HCV taxonomic lineages in China through the sequencing of their full-length genomes. These lineages represent six genotypes, 11 subtypes, and two novel variants. They were characterized for achieving a better understanding of the HCV genetic variation patterns and for possible future research applications.