High-Integrity Sequencing of Spike Gene for SARS-CoV-2 Variant Determination.

For tiling of the SARS-CoV-2 genome, the ARTIC Network provided a V4 protocol using 99 pairs of primers for amplicon production and is currently the widely used amplicon-based approach. However, this technique has regions of low sequence coverage and is labour-, time-, and cost-intensive. Moreover, it requires 14 pairs of primers in two separate PCRs to obtain spike gene sequences. To overcome these disadvantages, we proposed a single PCR to efficiently detect spike gene mutations. We proposed a bioinformatic protocol that can process FASTQ reads into spike gene consensus sequences to accurately call spike protein variants from sequenced samples or to fairly express the cases of missing amplicons. We evaluated the in silico detection rate of primer sets that yield amplicon sizes of 400, 1200, and 2500 bp for spike gene sequencing of SARS-CoV-2 to be 59.49, 76.19, and 92.20%, respectively. The in silico detection rate of our proposed single PCR primers was 97.07%. We demonstrated the robustness of our analytical protocol against 3000 Oxford Nanopore sequencing runs of distinct datasets, thus ensuring high-integrity sequencing of spike genes for variant SARS-CoV-2 determination. Our protocol works well with the data yielded from versatile primer designs, making it easy to determine spike protein variants.

Susceptibility of Elizabethkingia spp. to commonly tested and novel antibiotics and concordance between broth microdilution and automated testing methods.

OBJECTIVES: We aimed to determine susceptibilities of Elizabethkingia spp. to 25 commonly tested and 8 novel antibiotics, and to compare the performance of different susceptibility testing methods. METHODS: Clinical isolates of Elizabethkingia spp., Chryseobacterium spp. and Flavobacterium spp. collected during 2002-18 (n = 210) in a nationwide surveillance programme in Taiwan were speciated by 16S rRNA sequencing. MICs were determined by broth microdilution. The broth microdilution results of 18 common antibiotics were compared with those obtained by the VITEK 2 automated system. RESULTS: Among the Elizabethkingia spp. identified (n = 108), Elizabethkingia anophelis was the most prevalent (n = 90), followed by Elizabethkingia meningoseptica (n = 7) and Elizabethkingia miricola cluster [E. miricola (n = 6), Elizabethkingia bruuniana (n = 3) and Elizabethkingia ursingii (n = 2)]. Most isolates were recovered from respiratory or blood specimens from hospitalized, elderly patients. PFGE showed two major and several minor E. anophelis clones. All isolates were resistant to nearly all the tested ß-lactams. Doxycycline, minocycline and trimethoprim/sulfamethoxazole inhibited >90% of Elizabethkingia spp. Rifampin inhibited E. meningoseptica (100%) and E. anophelis (81.1%). Fluoroquinolones and tigecycline were active against E. meningoseptica and E. miricola cluster isolates. Novel antibiotics, including imipenem/relebactam, meropenem/vaborbactam, ceftazidime/avibactam, cefepime/zidebactam, delafloxacin, eravacycline and omadacycline were ineffective but lascufloxacin inhibited half of Elizabethkingia spp. The very major discrepancy rates of VITEK 2 were >1.5% for ciprofloxacin, moxifloxacin and vancomycin. Major discrepancy rates were >3% for amikacin, tigecycline, piperacillin/tazobactam and trimethoprim/sulfamethoxazole. CONCLUSIONS: MDR, absence of standard interpretation criteria and poor intermethod concordance necessitate working guidelines to facilitate future research of emerging Elizabethkingia spp.

Activation of ATP citrate lyase by mTOR signal induces disturbed lipid metabolism in hepatitis B virus pre-S2 mutant tumorigenesis.

UNLABELLED: The development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) has been found to be associated with disturbed lipid metabolism. To elucidate the role of lipid metabolism in HBV tumorigenesis, we investigated the dynamic pattern of lipid metabolism in HBV pre-S2 mutant-induced tumorigenesis. Lipid and gene expression profiles were analyzed in an in vitro culture system and in transgenic mouse livers harboring HBV pre-S2 mutant. The pre-S2 mutant transgenic livers showed a biphasic pattern of lipid accumulation, starting from mild fatty change in early (1 month) transgenic livers, which subsided and then, remarkably, increased in HCC tissues. This biphasic pattern was synchronized with ATP citrate lyase (ACLY) activation. Further analyses revealed that the pre-S2 mutant initiated an endoplasmic reticulum (ER) stress-dependent mammalian target of rapamycin (mTOR) signalling cascade. The pre-S2 mutant-induced mTOR signal activated the sterol regulatory element binding transcription factor 1 (SREBF1) to upregulate ACLY, which then activated the fatty acid desaturase 2 (FADS2), mediated through ACLY-dependent histone acetylation. Such an ER stress-dependent mTOR signal cascade also is important for the proliferation of hepatocytes in vitro and is further validated in HBV-related HCC tissues. IMPORTANCE: Aberrations of lipid metabolism frequently occur in chronic HBV infection. Our results provide a potential mechanism of disturbed lipid metabolism in HBV pre-S2 mutant-induced tumorigenesis, which should be valuable for the design of HCC chemoprevention in high-risk HBV carriers.

The emerging role of hepatitis B virus pre-S2 deletion mutant proteins in HBV tumorigenesis.

Chronic hepatitis B virus (HBV) infection can cause hepatocellular carcinoma (HCC). Several hypotheses have been proposed to explain the mechanisms of HBV tumorigenesis, including inflammation and liver regeneration associated with cytotoxic immune injuries and transcriptional activators of mutant HBV gene products. The mutant viral oncoprotein-driven tumorigenesis is prevailed at the advanced stage or anti-HBe-positive phase of chronic HBV infection. Besides HBx, the pre-S2 (deletion) mutant protein represents a newly recognized oncoprotein that is accumulated in the endoplasmic reticulum (ER) and manifests as type II ground glass hepatocytes (GGH). The retention of pre-S2 mutant protein in ER can induce ER stress and initiate an ER stress-dependent VEGF/Akt/mTOR and NFκB/COX-2 signal pathway. Additionally, the pre-S2 mutant large surface protein can induce an ER stress-independent pathway to transactivate JAB-1/p27/RB/cyclin A,D pathway, leading to growth advantage of type II GGH. The pre-S2 mutant protein-induced ER stress can also cause DNA damage, centrosome overduplication, and genomic instability. In 5-10% of type II GGHs, there is co-expression of pre-S2 mutant protein and HBx antigen which exhibited enhanced oncogenic effects in transgenic mice. The mTOR signal cascade is consistently activated throughout the course of pre-S2 mutant transgenic livers and in human HCC tissues, leading to metabolic disorders and HCC tumorigenesis. Clinically, the presence of pre-S2 deletion mutants in sera frequently develop resistance to nucleoside analogues anti-virals and predict HCC development. The pre-S2 deletion mutants and type II GGHs therefore represent novel biomarkers of HBV-related HCCs. A versatile DNA array chip has been developed to detect pre-S2 mutants in serum. Overall, the presence of pre-S2 mutants in serum has implications for anti-viral treatment and can predict HCC development. Targeting at pre-S2 mutant protein-induced, ER stress-dependent, mTOR signal cascade and metabolic disorders may offer potential strategy for chemoprevention or therapy in high risk chronic HBV carriers.

Histone deacetylase inhibitor suberoylanilide hydroxamic acid suppresses the pro-oncogenic effects induced by hepatitis B virus pre-S2 mutant oncoprotein and represents a potential chemopreventive agent in high-risk chronic HBV patients.

Chronic hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC). The pre-S(2) mutant large HBV surface antigen (LHBS) in type II ground glass hepatocytes (GGHs) has been recognized as an emerging viral oncoprotein; it directly interacts with the c-Jun activation domain-binding protein 1 (JAB1) and subsequently causes hyperphosphorylation of the tumor-suppressor retinoblastoma and, consequently, leads to disturbed cell cycle progression. The interaction of the pre-S(2) mutant LHBS with JAB1 could provide a potential target for chemoprevention. In this study, we found that the preneoplastic type II GGHs showed a significant decrease of the cyclin-dependent kinase inhibitor p27(Kip1), which serves as a marker for pre-S(2) mutant-JAB1 complex formation. The histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) elevated expression of the tumor-suppressor thioredoxin-binding protein 2 (TBP2), which subsequently enhanced the JAB1-TBP2 interaction and abolished the pre-S(2) mutant LHBS-induced degradation of p27(Kip1), which, in turn, recovered the normal cell cycle checkpoint. The pre-S(2) mutant LHBS-induced pro-oncogenic effects: increased cell proliferation, nuclear/cytoplasmic ratio and proliferating cell nuclear antigen expression, were all greatly ameliorated after SAHA treatments, which suggested SAHA as a promising chemopreventive agent for the pre-S(2) mutant oncoprotein-induced HCC. In conclusion, this study provides the mechanism of histone deacetylase (HDAC) inhibitor in preventing the pre-S(2) mutant-induced oncogenic phenotype. The HDAC inhibitor SAHA is therefore a potential chemopreventive agent for high-risk chronic HBV patients who may develop HCC.

Milrinone therapy for enterovirus 71-induced pulmonary edema and/or neurogenic shock in children: a randomized controlled trial.

OBJECTIVE: Enterovirus 71-induced brainstem encephalitis with pulmonary edema and/or neurogenic shock (stage 3B) is associated with rapid mortality in children. In a small pilot study, we found that milrinone reduced early mortality compared with historical controls. This prospective, randomized control trial was designed to provide more definitive evidence of the ability of milrinone to reduce the 1-week mortality of stage 3B enterovirus 71 infections. DESIGN: Prospective, unicenter, open-label, randomized, controlled study. SETTING: Inpatient ward of a large tertiary teaching hospital in Ho Chi Minh City, Vietnam. PATIENTS: Children (≤ 18 yr old) admitted with proven enterovirus 71-induced pulmonary edema and/or neurogenic shock. INTERVENTIONS: Patients were randomly assigned to receive intravenous milrinone (0.5 µg/kg/min) (n = 22) or conventional management (n = 19). Both groups received dopamine or dobutamine and intravenous immunoglobulin. MEASUREMENTS AND MAIN RESULTS: The primary endpoint was 1-week mortality. The secondary endpoints included length of ventilator dependence and hospital stay and adverse events. The median age was 2 years with a predominance of boys in both groups. The 1-week mortality was significantly lower, 18.2% (4/22) in the milrinone compared with 57.9% (11/19) in the conventional management group (relative risk = 0.314 [95% CI, 0.12-0.83], p = 0.01). The median duration of ventilator-free days was longer in the milrinone treatment group (p = 0.01). There was no apparent neurologic sequela in the survivors in either group, and no drug-related adverse events were documented. CONCLUSIONS: Milrinone significantly reduced the 1-week mortality of enterovirus 71-induced pulmonary edema and/or neurogenic shock without adverse effects. Further studies are needed to determine whether milrinone might be useful to prevent progression of earlier stages of brainstem encephalitis.

Cost-effective complete genome sequencing using the MinION platform for identification of recombinant enteroviruses.

IMPORTANCE: By employing a cost-effective approach for complete genome sequencing, the study has enabled the identification of novel enterovirus strains and shed light on the genetic exchange events during outbreaks. The success rate of genome sequencing and the scalability of the protocol demonstrate its practical utility for routine enterovirus surveillance. Moreover, the study's findings of recombinant strains of EVA71 and CVA2 contributing to epidemics in Malaysia and Taiwan emphasize the need for accurate detection and characterization of enteroviruses. The investigation of the whole genome and upstream ORF sequences has provided insights into the evolution and spread of enterovirus subgenogroups. These findings have important implications for the prevention, control, and surveillance of enteroviruses, ultimately contributing to the understanding and management of enterovirus-related illnesses.

Novel feedback inhibition of surface antigen synthesis by mammalian target of rapamycin (mTOR) signal and its implication for hepatitis B virus tumorigenesis and therapy.

UNLABELLED: Ground glass hepatocytes (GGHs) harboring hepatitis B virus (HBV) pre-S mutants have been recognized as precursor lesions of hepatocellular carcinoma (HCC). Previously, we observed the activation of mammalian target of rapamycin (mTOR) in GGHs and HCCs, together with a decreased expression of HBV surface antigen (HBsAg) in HCC tissues. It is, therefore, hypothesized that the activation of mTOR during HBV tumorigenesis may potentially down-regulate HBsAg expression. In this study, we verified an inverse relationship between the expression of HBsAg and phosphorylated mTOR (p-mTOR) in 13 of 20 paired nontumorous liver and HCC tissues. In vitro, wild-type or mutant pre-S proteins could activate mTOR in the HuH-7 cell line. Interestingly, the up-regulated mTOR, in turn, suppressed HBsAg synthesis at the transcriptional level via the transcription factor, Yin Yang 1 (YY1), which bound to nucleotide 2812-2816 of the pre-S1 promoter. This inhibitory effect by the mTOR signal could be abolished by the knockdown of histone deacetylase 1 (HDAC1). Furthermore, YY1 was physically associated with HDAC1 in a manner dependent on mTOR activation. Collectively, pre-S protein-induced mTOR activation may recruit the YY1-HDAC1 complex to feedback suppress transcription from the pre-S1 promoter. CONCLUSION: The activation of mTOR signal in GGHs may feedback suppress HBsAg synthesis during HBV tumorigenesis and explain the observed decrease or absence of HBsAg in HCC tissues. Therapy using mTOR inhibitors for HCCs may potentially activate HBV replication in patients with chronic HBV infection.

Rapid and Routine Molecular Typing Using Multiplex Polymerase Chain Reaction and MinION Sequencer.

Molecular typing is an essential tool that has been extensively applied in laboratories as well as in clinical settings. Next-generation sequencing technologies promise high-throughput and cost-effective molecular applications; however, the accessibility of these technologies is limited due to the high capital cost. Oxford Nanopore Technologies (ONT) offers a MinION device with the advantages of real-time data analysis, rapid library preparation, and low cost per test. However, the advantages of the MinION device are often overshadowed by its lower raw accuracy. Herein, we present a concise multilocus sequence typing protocol of Staphylococcus aureus using multiplex polymerase chain reaction and Rapid Barcoding Kit for barcoding and MinION device for sequencing. Moreover, to clarify the effects of carryover DNA on tasks that require high sequence accuracy, we used the MinION flow cell in successive runs of washing and reusing. Our results revealed that the MinION flow cell could achieve accurate typing of a total of 467 samples with 3,269 kilobase-long genes within a total of 5 runs. This thus demonstrates the effectiveness of a portable nanopore MinION sequencer in providing accurate, rapid, and routine molecular typing.

In vitro and in vivo efficacy of minocycline-based therapy for Elizabethkingia anophelis and the impact of reduced minocycline susceptibility.

OBJECTIVES: Elizabethkingia anophelis is inherently resistant to multiple antibiotics, except minocycline. This study aimed to determine the in vitro and in vivo efficacy of minocycline monotherapy and combination therapy against susceptible strains and the impact of reduced minocycline susceptibility. METHODS: Three clinical isolates and one laboratory-induced mutant with reduced minocycline susceptibility were included. Time-kill and checkerboard assays were used to assess in vitro efficacy and synergy, respectively. Galleria mellonella infection and mouse pneumonia models were used to assess in vivo efficacy, and a mouse thigh infection model was used to determine the bacterial load. RESULTS: Minocycline monotherapy exerted a modest inhibitory effect on three clinical minocycline-susceptible E. anophelis isolates in vitro, but delayed G. mellonella death and improved infected mouse survival; it also significantly reduced the in vivo bacterial load. Minocycline had decreased efficacy on G. mellonella and mice infected by the mutant with reduced minocycline susceptibility. Genome comparison revealed several spontaneous mutations associated with reduced minocycline susceptibility. Among eight antibiotics tested in combination with minocycline, rifampin consistently showed in vitro synergy. The addition of rifampin (1 mg/L) reduced the mutant prevention concentration of minocycline from 2-4 mg/L to < 0.5 mg/L. However, compared with monotherapy, the combination of rifampin and minocycline did not further reduce the bacterial load or improve the survival of G. mellonella or mice. CONCLUSION: Minocycline monotherapy was in vivo effective against susceptible E. anophelis. Reduced minocycline susceptibility due to spontaneous mutation decreased its therapeutic efficacy. In combination with rifampin, it prevented the in vitro emergence of reduced susceptibility but did not provide additional in vivo survival benefit.

Distribution and Genomic Characterization of Third-Generation Cephalosporin-Resistant Escherichia coli Isolated from a Single Family and Home Environment: A 2-Year Longitudinal Study.

Third-generation cephalosporin-resistant Escherichia coli (CREC), particularly strains producing extended-spectrum ß-lactamases (ESBLs), are a global concern. Our study aims to longitudinally assemble the genomic characteristics of CREC isolates from fecal samples from an index patient with recurrent CREC-related urinary tract infections and his family and swabs from his home environment 12 times between 2019 and 2021 to investigate the distribution of antibiotic resistance genes. CREC identified using the VITEK 2 were subjected to nanopore whole-genome sequencing (WGS). The WGS of 27 CREC isolates discovered in 137 specimens (1 urine, 123 feces, and 13 environmental) revealed the predominance of ST101 and ST131. Among these sequence types, blaCTX-M (44.4%, n = 12) was the predominant ESBL gene family, with blaCTX-M-14 (n = 6) being the most common. The remaining 15 (55.6%) isolates harbored blaCMY-2 genes and were clonally diverse. All E. coli isolated from the index patient's initial urine and fecal samples belonged to O25b:H4-B2-ST131 and carried blaCTX-M-14. The results of sequence analysis indicate plasmid-mediated household transmission of blaCMY-2 or blaCTX-M-55. A strong genomic similarity was discovered between fecal ESBL-producing E. coli and uropathogenic strains. Furthermore, blaCMY-2 genes were widely distributed among the CREC isolated from family members and their home environment.

Furin and TMPRSS2 Resistant Spike Induces Robust Humoral and Cellular Immunity Against SARS-CoV-2 Lethal Infection.

An effective COVID-19 vaccine against broad SARS-CoV-2 variants is still an unmet need. In the study, the vesicular stomatitis virus (VSV)-based vector was used to express the SARS-CoV-2 Spike protein to identify better vaccine designs. The replication-competent of the recombinant VSV-spike virus with C-terminal 19 amino acid truncation (SΔ19 Rep) was generated. A single dose of SΔ19 Rep intranasal vaccination is sufficient to induce protective immunity against SARS-CoV-2 infection in hamsters. All the clones isolated from the SΔ19 Rep virus contained R682G mutation located at the Furin cleavage site. An additional S813Y mutation close to the TMPRSS2 cleavage site was identified in some clones. The enzymatic processing of S protein was blocked by these mutations. The vaccination of the R682G-S813Y virus produced a high antibody response against S protein and a robust S protein-specific CD8+ T cell response. The vaccinated animals were protected from the lethal SARS-CoV-2 (delta variant) challenge. The S antigen with resistance to enzymatic processes by Furin and TMPRSS2 will provide better immunogenicity for vaccine design.

A clustered ground-glass hepatocyte pattern represents a new prognostic marker for the recurrence of hepatocellular carcinoma after surgery.

BACKGROUND: The recurrence of hepatocellular carcinoma (HCC) after hepatectomy is a serious event. It has been demonstrated that different ground-glass hepatocyte (GGH) patterns harbor specific hepatitis B virus (HBV) pre-S deletion mutants and represent preneoplastic lesions in chronic HBV infection. In the current study, the authors investigated whether a specific GGH pattern in nontumorous liver tissues was associated with the recurrence of HBV-related HCC after surgery. METHODS: Clinicopathologic data from 82 patients with HBV-related HCC were reviewed. GGH patterns were assessed on hematoxylin and eosin-stained sections. Tissue hepatitis B surface antigen (HBsAg) expression was evaluated by immunohistochemical staining. Serum profiles of pre-S status, viral load, and HBV genotype were determined and correlated with clinical recurrence and survival after surgery. RESULTS: The results indicated that the clustered pattern of GGHs or HBsAg expression was associated significantly with decreased local recurrence-free survival (LRFS) during a mean follow-up of 46.4 months (P<.001). This biomarker was comparable to or better than the prognostic value of other parameters, such as multifocal tumors (P = .022), satellite nodules (P = .005), small cell dysplasia (P = .045), or elevated viral load (P = .027), to predict recurrent HCC. Multivariate analysis also revealed that type II GGHs, which expressed marginal HBsAg and consistently clustered in nodules, were independent variables associated with LRFS (P<.001) and overall survival (P = .003). CONCLUSIONS: The current results indicated that the assessment of GGH patterns or HBsAg expression in nontumorous liver tissues provides an easily recognized, new risk marker for the recurrence of HBV-related HCC after hepatic resection.

Increased infiltration of regulatory T cells in hepatocellular carcinoma of patients with hepatitis B virus pre-S2 mutant.

Hepatocellular carcinoma (HCC) is a frequent and deadly human cancer worldwide that is intimately associated with chronic hepatitis B virus (HBV) infection. Pre-S2 mutant is a HBV oncoprotein that plays important roles in HCC development and is linked to poor prognosis in HCC patients. However, the profiles of tumor-infiltrating lymphocytes in HCC tissues of pre-S2 mutant-positive patients remain unknown. In this study, we performed fluorescent immunohistochemistry staining to detect the infiltration of 'anti-tumor' cytotoxic T lymphocytes (CTLs) and 'pro-tumor' regulatory T cells (Tregs) in pre-S2 mutant-positive and -negative HCC patients. We showed that pre-S2 mutant-positive patients had a significantly higher infiltration of CD4+CD25+ cells and forkhead box P3 (Foxp3)-expressing cells but similar CTLs and lower granzyme B-expressing cells in HCC tissues compared with pre-S2 mutant-negative patients. Moreover, the percentage of pre-S2 plus pre-S1 + pre-S2 deletion (pre-S2 mutant) was positively correlated with the density of CD4+CD25+ cells and Foxp3-expressing cells but negatively with granzyme B-expressing cells in HCC tissues. Considering that increased intratumoral Tregs have been shown to promote tumor immune evasion, our data may provide new insights into the pathogenesis of HBV pre-S2 mutant-induced HCC and suggest that therapeutics targeting Tregs may be a promising strategy for treating pre-S2 mutant-positive high-risk patient population.

Enhanced expression of vascular endothelial growth factor-A in ground glass hepatocytes and its implication in hepatitis B virus hepatocarcinogenesis.

UNLABELLED: Ground glass hepatocytes (GGH) in chronic hepatitis B virus (HBV) infection harbor HBV pre-S deletion mutants in endoplasmic reticulum (ER) and exhibit complex biologic features such as ER stress, DNA damage, and growth advantage. The presence of pre-S mutants in serum has been shown to predict the development of hepatocellular carcinoma (HCC) in HBV carriers. GGHs hence represent a potentially preneoplastic lesion. Whether a specific growth factor is overexpressed and activated in GGHs remains to be clarified. In this study, growth factor(s) up-regulated by pre-S mutants was identified using a growth factor array in HuH-7 cells. Immunohistochemistry, reverse-transcriptase polymerase chain reaction, and Western blot analysis were performed to study the participation of these genes and their signal pathways in HuH-7 cells and liver tissues. We demonstrate that vascular endothelial growth factor-A (VEGF-A) was up-regulated by pre-S mutants in HuH-7 cells and further confirmed in GGHs by immunostaining. The VEGF-A up-regulation by pre-S mutants could be suppressed by vomitoxin, an ER stress inhibitor. Furthermore, pre-S mutants-expressed HuH-7 cells exhibited activation of Akt/mTOR (mammalian target of rapamycin) signaling and increased growth advantage, which could be inhibited by VEGF-A neutralization. Consistent with this notion, enhanced expression of VEGF-A and activation of Akt/mTOR signaling, comparable to the levels of paired HCC tissues, were also detected in HBV-related nontumorous livers. CONCLUSION: The enhanced expression of VEGF-A in GGHs provides potential mechanism to explain the progression from preneoplastic GGHs to HCC in chronic HBV infection.

Hepatitis B Virus Pre-S Mutants as Biomarkers and Targets for the Development and Recurrence of Hepatocellular Carcinoma.

Chronic hepatitis B virus (HBV) infection is a major risk factor for the development of hepatocellular carcinoma (HCC), the leading cause of cancer-related death worldwide. Despite progress in the prevention and therapy of HCC, high incidence and recurrence rates of HCC remain big threats, resulting in poor patient survival. Effective biomarkers and targets of HCC are therefore urgently needed for better management and to improve patient outcomes. Pre-S mutants have been well demonstrated as HBV oncoproteins that play important roles in HCC development through activation of multiple oncogenic signal pathways in hepatocytes, in vitro and in vivo. The presence of pre-S mutants in patients with chronic HBV infection and HBV-related HCC has been associated with a significantly higher risk of HCC development and recurrence after curative surgical resection, respectively. In this review, we summarize the roles of pre-S mutants as biomarkers for predicting HBV-related HCC development and recurrence, and highlight the pre-S mutants-activated oncogenic signal pathways as potential targets for preventing HBV-related HCC development.

nanoMLST: accurate multilocus sequence typing using Oxford Nanopore Technologies MinION with a dual-barcode approach to multiplex large numbers of samples.

Multilocus sequence typing (MLST) is one of the most commonly used methods for studying microbial lineage worldwide. However, the traditional MLST process using Sanger sequencing is time-consuming and expensive. We have designed a workflow that simultaneously sequenced seven full-length housekeeping genes of 96 meticillin-resistant Staphylococcus aureus isolates with dual-barcode multiplexing using just a single flow cell of an Oxford Nanopore Technologies MinION system, and then we performed bioinformatic analysis for strain typing. Fifty-one of the isolates comprising 34 sequence types had been characterized using Sanger sequencing. We demonstrate that the allele assignments obtained by our nanopore workflow (nanoMLST, available at https://github.com/jade-nhri/nanoMLST) were identical to those obtained by Sanger sequencing (359/359, with 100 % agreement rate). In addition, we estimate that our multiplex system is able to perform MLST for up to 1000 samples simultaneously; thus, providing a rapid and cost-effective solution for molecular typing.

Next-Generation Sequencing-Based Quantitative Detection of Hepatitis B Virus Pre-S Mutants in Plasma Predicts Hepatocellular Carcinoma Recurrence.

Hepatocellular carcinoma (HCC) is among the most common and lethal human cancers worldwide. Despite curative resection, high recurrence of HCC remains a big threat, leading to poor patient outcomes. Hepatitis B virus (HBV) pre-S mutants, which harbor deletions over pre-S1 and pre-S2 gene segments of large surface proteins, have been implicated in HCC recurrence. Therefore, a reliable approach for detection of pre-S mutants is urgently needed for predicting HCC recurrence to improve patient survival. In this study, we used a next-generation sequencing (NGS)-based platform for quantitative detection of pre-S mutants in the plasma of HBV-related HCC patients and evaluated their prognostic values in HCC recurrence. We demonstrated that the presence of deletions spanning the pre-S2 gene segment and the high percentage of pre-S2 plus pre-S1 + pre-S2 deletions, either alone or in combination, was significantly and independently associated with poor recurrence-free survival and had greater prognostic performance than other clinicopathological and viral factors in predicting HCC recurrence. Our data suggest that the NGS-based quantitative detection of pre-S mutants in plasma represents a promising approach for identifying patients at high risk for HBV-related HCC recurrence after surgical resection in a noninvasive manner.

Increased Expression of Programmed Death Ligand 1 in Hepatocellular Carcinoma of Patients with Hepatitis B Virus Pre-S2 Mutant.

PURPOSE: Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), a leading cause of cancer-related death worldwide. The HCC patients who harbor HBV pre-S2 mutant, an oncoprotein that plays key roles in HCC development, have been closely associated with a worse prognosis after curative surgical resection, suggesting an urgent need for alternative therapeutic options to improve their survival. In this study, we aimed to evaluate the expression profiles of programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1), two of the most well-studied immune checkpoint molecules that promote tumor immune evasion, in tumor of the pre-S2 mutant-positive/high HCC patients. METHODS: We classified 40 HBV-related HCC patients into the pre-S2-positive/high and -negative/low groups by a next-generation sequencing-based approach. The fluorescent immunohistochemistry staining was performed to detect the expression of PD-1 and PD-L1 in HCC tissues of patients. RESULTS: We showed that patients with either deletion spanning pre-S2 gene segment or high percentage of pre-S2 plus pre-S1+pre-S2 deletion (the pre-S2 mutant-positive/high group) exhibited a significantly higher density of PD-L1-positive cells in HCC tissues than those without. Moreover, the percentage of pre-S2 plus pre-S1+pre-S2 deletion displayed a high positive correlation with the density of PD-L1-positive cells in HCC tissues. CONCLUSION: The increased expression of PD-L1 in tumor tissues of the pre-S2 mutant-positive HCC patients suggest that pre-S2 mutant may play a potential role in dysregulation of tumor immune microenvironment in the progression of HBV-related HCC, implicating for the development of future therapeutic strategies.

Hepatitis B virus pre-S2 deletion (nucleotide 1 to 54) in plasma predicts recurrence of hepatocellular carcinoma after curative surgical resection.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Despite curative surgical resection, high recurrence of HCC after surgery results in poor patient survival. To develop prognostic markers is therefore important for better prevention and therapy of recurrent HCC to improve patient outcomes. Deletion mutations over the pre-S1 and pre-S2 gene segments of hepatitis B virus (HBV) have been closely associated with recurrence of HCC after curative surgical resection. In this study, we applied a next-generation sequencing-based approach to further evaluate the association of pre-S deletion regions with HCC recurrence. We demonstrated that the pre-S2 deletion (nucleotide 1 to 54) was the most predominant deletion regions of pre-S gene in plasma of HBV-related HCC patients. Moreover, patients with the pre-S2 deletion (nucleotide 1 to 54) exhibited a significantly higher risk of HCC recurrence after curative surgical resection than those without. The pre-S2 deletion (nucleotide 1 to 54) in plasma represented a prognostic factor that independently predicted HCC recurrence with greater performance than other clinicopathological and viral factors. Our data suggest that detection of the pre-S2 deletion (nucleotide 1 to 54) in plasma may be a promising noninvasive strategy for identifying patients at high risk for HCC recurrence after curative surgical resection.