Modeling and Analysis of HIV-1 Pol Polyprotein as a Case Study for Predicting Large Polyprotein Structures.

Acquired immunodeficiency syndrome (AIDS) is caused by human immunodeficiency virus (HIV). HIV protease, reverse transcriptase, and integrase are targets of current drugs to treat the disease. However, anti-viral drug-resistant strains have emerged quickly due to the high mutation rate of the virus, leading to the demand for the development of new drugs. One attractive target is Gag-Pol polyprotein, which plays a key role in the life cycle of HIV. Recently, we found that a combination of M50I and V151I mutations in HIV-1 integrase can suppress virus release and inhibit the initiation of Gag-Pol autoprocessing and maturation without interfering with the dimerization of Gag-Pol. Additional mutations in integrase or RNase H domain in reverse transcriptase can compensate for the defect. However, the molecular mechanism is unknown. There is no tertiary structure of the full-length HIV-1 Pol protein available for further study. Therefore, we developed a workflow to predict the tertiary structure of HIV-1 NL4.3 Pol polyprotein. The modeled structure has comparable quality compared with the recently published partial HIV-1 Pol structure (PDB ID: 7SJX). Our HIV-1 NL4.3 Pol dimer model is the first full-length Pol tertiary structure. It can provide a structural platform for studying the autoprocessing mechanism of HIV-1 Pol and for developing new potent drugs. Moreover, the workflow can be used to predict other large protein structures that cannot be resolved via conventional experimental methods.

Detection of Q129H Immune Escape Mutation in Apparently Healthy Hepatitis B Virus Carriers in Southwestern Nigeria.

As the global effort to eradicate hepatitis B continues, immune escape mutations (IEMs) and drug resistance mutations (DRMs) affecting its diagnosis, treatment, and prevention are compromising this goal. However, knowledge about the prevalence and circulation of these mutations in Nigeria is scarce. Serum samples (n = 199) from apparently healthy prospective blood donors, pregnant women, and individuals presenting with fever in southwestern Nigeria were analyzed for the presence of IEMs and DRMs by means of nested PCR in the HBV S (HBs) and HBV polymerase (Pol) genes, followed by phylogenetic and mutational analyses. In total, 25.1% (n = 50/199) of samples were positive for HBV, as measured by PCR. In 41 samples (20.6%), both fragments could be amplified, whereas the HBs gene and the Pol gene fragment alone were detected in 0.5% (n = 1/199) and 4% (n = 8/199) of samples, respectively. Sequences were successfully obtained for all 42 HBs gene fragments but for only 31/49 Pol gene fragments (totaling 73 sequences from 44 individuals). All sequences were identified as HBV genotype E. IEMs were present in 18.2% (n = 8/44) of the sequences of HBV-positive individuals with available sequences. IEM Q129H was detected in eight out of the 44 (18.2%) HBV isolates sequenced in this study; however, no DRMs were observed. This study confirms the circulation of HBV IEMs and reports the presence of Q129H IEM for the first time in Nigeria. Intensified research on the dynamics of IEM is necessary in order to enhance the elimination of HBV.

A novel class III endogenous retrovirus with a class I envelope gene in African frogs with an intact genome and developmentally regulated transcripts in Xenopus tropicalis.

BACKGROUND: Retroviruses exist as exogenous infectious agents and as endogenous retroviruses (ERVs) integrated into host chromosomes. Such endogenous retroviruses (ERVs) are grouped into three classes roughly corresponding to the seven genera of infectious retroviruses: class I (gamma-, epsilonretroviruses), class II (alpha-, beta-, delta-, lentiretroviruses) and class III (spumaretroviruses). Some ERVs have counterparts among the known infectious retroviruses, while others represent paleovirological relics of extinct or undiscovered retroviruses. RESULTS: Here we identify an intact ERV in the Anuran amphibian, Xenopus tropicalis. XtERV-S has open reading frames (ORFs) for gag, pol (polymerase) and env (envelope) genes, with a small additional ORF in pol and a serine tRNA primer binding site. It has unusual features and domain relationships to known retroviruses. Analyses based on phylogeny and functional motifs establish that XtERV-S gag and pol genes are related to the ancient env-less class III ERV-L family but the surface subunit of env is unrelated to known retroviruses while its transmembrane subunit is class I-like. LTR constructs show transcriptional activity, and XtERV-S transcripts are detected in embryos after the maternal to zygotic mid-blastula transition and before the late tailbud stage. Tagged Gag protein shows typical subcellular localization. The presence of ORFs in all three protein-coding regions along with identical 5' and 3' LTRs (long terminal repeats) indicate this is a very recent germline acquisition. There are older, full-length, nonorthologous, defective copies in Xenopus laevis and the distantly related African bullfrog, Pyxicephalus adspersus. Additional older, internally deleted copies in X. tropicalis carry a 300 bp LTR substitution. CONCLUSIONS: XtERV-S represents a genera-spanning member of the largely env-less class III ERV that has ancient and modern copies in Anurans. This provirus has an env ORF with a surface subunit unrelated to known retroviruses and a transmembrane subunit related to class I gammaretroviruses in sequence and organization, and is expressed in early embryogenesis. Additional XtERV-S-related but defective copies are present in X. tropicalis and other African frog taxa. XtERV-S is an unusual class III ERV variant, and it may represent an important transitional retroviral form that has been spreading in African frogs for tens of millions of years.

HERV-K Gag RNA and Protein Levels Are Elevated in Malignant Regions of the Prostate in Males with Prostate Cancer.

Heightened expression of human endogenous retrovirus (HERV) sequences has been associated with a range of malignancies, including prostate cancer, suggesting that they may serve as useful diagnostic or prognostic cancer biomarkers. We analysed the expression of HERV-K (Gag and Env/Np9 regions), HERV-E 4.1 (Pol and Env regions), HERV-H (Pol) and HERV-W (Gag) sequences in prostate cancer cells lines and normal prostate epithelial cells using qRT-PCR. HERV expression was also analysed in matched malignant and benign prostate tissue samples from men with prostate cancer (n = 27, median age 65.2 years (range 47-70)) and compared to prostate cancer-free male controls (n = 11). Prostate cancer epithelial cell lines exhibited a signature of HERV RNA overexpression, with all HERVs analysed, except HERV-E Pol, showing heightened expression in at least two, but more commonly all, cell lines analysed. Analysis of primary prostate material indicated increased expression of HERV-E Pol but decreased expression of HERV-E Env in both malignant and benign regions of the prostate in men with prostate cancer as compared to those without. Expression of HERV-K Gag was significantly higher in malignant regions of the prostate in men with prostate cancer as compared to matched benign regions and prostate cancer-free men (p < 0.001 for both), with 85.2% of prostate cancers donors showing malignancy-associated upregulation of HERV-K Gag RNA. HERV-K Gag protein was detected in 12/18 (66.7%) malignant tissues using immunohistochemistry, but only 1/18 (5.6%) benign tissue sections. Heightened expression of HERV-K Gag RNA and protein appears to be a sensitive and specific biomarker of prostate malignancy in this cohort of men with prostate carcinoma, supporting its potential utility as a non-invasive, adjunct clinical biomarker.

Screening and Identification of the First Non-CRISPR/Cas9-Treated Chinese Miniature Pig With Defective Porcine Endogenous Retrovirus pol Genes.

Pig to human xenotransplantation is considered to be a possible approach to alleviate the shortage of human allografts. Porcine endogenous retrovirus (PERV) is the most significant pathogen in xenotransplantation. We screened for pigs that consistently did not transmit human-tropic replication competent PERVs (HTRC PERVs), namely, non-transmitting pigs. Then, we conducted whole-genome resequencing and full-length transcriptome sequencing to further investigate the sequence characteristics of one non-transmitting pig. Using in vitro transmission assays, we found 5 (out of 105) pigs of the Chinese Wuzhishan minipig inbred line that did not transmit PERV to human cells, i.e., non-transmitting pigs. Whole-genome resequencing and full-length transcriptome sequencing of one non-transmitting pig showed that all of the pol genes were defective at both the genome and transcript levels. We speculate that the defective PERV pol genes in this pig might be attributable to the long-term inbreeding process. This discovery is promising for the development of a strain of highly homozygous and genetically stable pigs with defective PERV pol genes as a source animal species for xenotransplantation.

Mutational characterization of HBV reverse transcriptase gene and the genotype-phenotype correlation of antiviral resistance among Chinese chronic hepatitis B patients.

Background and Aims: The drug resistance of hepatitis B virus (HBV) originates from mutations within HBV reverse transcriptase (RT) region during the prolonged antiviral therapy. So far, the characteristics of how these mutations distribute and evolve in the process of therapy have not been clarified yet. Thus we aimed to investigate these characteristics and discuss their contributing factors. Methods: HBV RT region was direct-sequenced in 285 treatment-naive and 214 post-treatment patients. Mutational frequency and Shannon entropy were calculated to identify the specific mutations differing between genotypes or treatment status. A typical putative resistance mutation rtL229V was further studied using in-vitro susceptibility assays and molecular modeling. Results: The classical resistance mutations were rarely detected among treatment-naive individuals, while the putative resistance mutations were observed at 8 AA sites. rtV191I and rtA181T/V were the only resistance mutations identified as genotype-specific mutation. Selective pressure of drug usage not only contributed to the classical resistance mutations, but also induced the changes at a putative resistance mutation site rt229. rtL229V was the major substitution at the site of rt229. It contributed to the most potent suppression of viral replication and reduced the in-vitro drug susceptibility to entecavir (ETV) when coexisting with rtM204V, consistent with the hypothesis based on the molecular modeling and clinical data analysis. Conclusions: The analysis of mutations in RT region under the different circumstances of genotypes and therapy status might pave the way for a better understanding of resistance evolution, thus providing the basis for a rational administration of antiviral therapy.

Establishment of a system for finding inhibitors of ε RNA binding with the HBV polymerase.

Although several nucleo(s)tide analogs are available for treatment of HBV infection, long-term treatment with these drugs can lead to the emergence of drug-resistant viruses. Recent HIV-1 studies suggest that combination therapies using nucleo(s)tide reverse transcriptase inhibitors (NRTIs) and non-nucleo(s)tide reverse transcriptase inhibitors (NNRTIs) could drastically inhibit the viral genome replication of NRTI-resistant viruses. In order to carry out such combinational therapy against HBV, several new NRTIs and NNRTIs should be developed. Here, we aimed to identify novel NNRTIs targeting the HBV polymerase terminal protein (TP)-reverse transcriptase (RT) (TP-RT) domain, which is a critical domain for HBV replication. We expressed and purified the HBV TP-RT with high purity using an Escherichia coli expression system and established an in vitro ε RNA-binding assay system. Then, we used TP-RT in cell-free assays to screen candidate inhibitors from a chemical compound library, and identified two compounds, 6-hydroxy-DL-DOPA and N-oleoyldopamine, which inhibited the binding of ε RNA with the HBV polymerase. Furthermore, these drugs reduced HBV DNA levels in cell-based assays as well by inhibiting packaging of pregenome RNA into capsids. The novel screening system developed herein should open a new pathway the discovery of drugs targeting the HBV TP-RT domain to treat HBV infection.

The dual-target approach in viral HIV-1 viremia testing: An added value to virological monitoring?

New methods of HIV-1 RNA quantification based on dual-target detection are increasingly used in HIV viral load monitoring, but clinical implications and impact of dual-target detection on HIV-1 infection management are not established. Aptima HIV-1 Quant Dx assay is a last generation HIV viral load method, that uses pol and LTR as simultaneous target, providing quantitative results based mainly on pol target, while LTR target is used to report the results when pol signal is absent. In our laboratory, about 6% of results of all HIV-1 viral load tests performed with this platform in one year period resulted from LTR signal. Interestingly, LTR-based viremia (sometimes exceeding 1,000 copies/mL) was observed in a small proportion (up to 1%) of patients under ART, considered for long time virologically suppressed on the basis of a single target (pol-based) assay. Male gender, >700 vs <200 CD4 cell/mL and dual therapy including NRTI plus either NNRTI, or PI/b or INSTI were independently associated with increased risk of LTR-based HIV-1 viral load detection by multivariable logistic regression. A significant linear correlation was observed between LTR-based HIV-1 RNA levels and PBMC-associated proviral DNA. Moreover, in a small group of patients with HIV-1 RNA levels >200 copies/mL, longitudinal assessments showed parallel kinetics between plasma viremia and proviral DNA. Sequencing of pol region for drug resistance assessment in patients with LTR-based viremia failed on plasma HIV-1 RNA, while it was successful on proviral DNA. The detection/quantification of HIV-1 viremia based only on LTR signal with a dual target assay in samples resulting undetectable with the more conventional target pol needs accurate evaluation; unravelling the biological basis of this phenomenon, here described for the first time, is mandatory to establish relevance and implication by both pathogenetic (i.e. infectivity of LTR-detected viruses, reservoir turnover, immune activation, etc.) and clinical standpoint.

Modulation of human endogenous retroviruses -H, -W and -K transcription by microbes.

The human endogenous retroviruses (HERVs) are endogenous retroviruses that are inserted into the germ cell DNA of humans over 30 million years ago. Using real-time RT-PCR we describe HERV modulation by commensal microbes in the human gut. Infants, exclusively or predominant breast milk feeding, less than 12 weeks of age, during bacteria gut colonization, were assessed for eligibility. Our data demonstrate that the colonization with commensal microbes, in particular, Bifidobacterium spp., of the gut causes modulation of HERVs.

Koala retrovirus viral load and disease burden in distinct northern and southern koala populations.

Koala retrovirus (KoRV) displays features of both an endogenous and exogenous virus and is linked to neoplasia and immunosuppression in koalas. This study explores the apparent differences in the nature and impact of KoRV infection between geographically and genetically separated "northern" and "southern" koala populations, by investigating the disease status, completeness of the KoRV genome and the proviral (DNA) and viral (RNA) loads of 71 northern and 97 southern koalas. All northern animals were positive for all KoRV genes (gag, pro-pol and env) in both DNA and RNA forms, whereas many southern animals were missing one or more KoRV genes. There was a significant relationship between the completeness of the KoRV genome and clinical status in this population. The proviral and viral loads of the northern population were significantly higher than those of the southern population (P < 0.0001), and many provirus-positive southern animals failed to express any detectable KoRV RNA. Across both populations there was a positive association between proviral load and neoplasia (P = 0.009). Potential reasons for the differences in the nature of KoRV infection between the two populations are discussed.

Hepatitis B virus-triggered PTEN/ß-catenin/c-Myc signaling enhances PD-L1 expression to promote immune evasion.

Hepatitis B virus (HBV) exploits multiple strategies to evade host immune surveillance. Programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) signaling plays a critical role in regulating T cell homeostasis. However, it remains largely unknown as to how HBV infection elevates PD-L1 expression in hepatocytes. A mouse model of HBV infection was established by hydrodynamic injection with a vector containing 1.3-fold overlength HBV genome (pHBV1.3) via the tail vein. Coculture experiments with HBV-expressing hepatoma cells and Jurkat T cells were established in vitro. We observed significant decrease in the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and increase in ß-catenin/PD-L1 expression in liver tissues from patients with chronic hepatitis B and mice subjected to pHBV1.3 hydrodynamic injection. Mechanistically, decrease in PTEN enhanced ß-catenin/c-Myc signaling and PD-L1 expression in HBV-expressing hepatoma cells, which in turn augmented PD-1 expression, lowered IL-2 secretion, and induced T cell apoptosis. However, ß-catenin disruption inhibited PTEN-mediated PD-L1 expression, which was accompanied by decreased PD-1 expression, and increased IL-2 production in T cells. Luciferase reporter assays revealed that c-Myc stimulated transcriptional activity of PD-L1. In addition, HBV X protein (HBx) and HBV polymerase (HBp) contributed to PTEN downregulation and ß-catenin/PD-L1 upregulation. Strikingly, PTEN overexpression in hepatocytes inhibited ß-catenin/PD-L1 signaling and promoted HBV clearance in vivo. Our findings suggest that HBV-triggered PTEN/ß-catenin/c-Myc signaling via HBx and HBp enhances PD-L1 expression, leading to inhibition of T cell response, and promotes HBV immune evasion.NEW & NOTEWORTHY This study demonstrates that during HBV infection, HBV can increase PD-L1 expression via PTEN/ß-catenin/c-Myc signaling pathway, which in turn inhibits T cell response and ultimately promotes HBV immune evasion. Targeting this signaling pathway is a potential strategy for immunotherapy of chronic hepatitis B.

Evaluation of the pol/S Gene Overlapping Mutations in Chronic Hepatitis B Patients in Northern Cyprus.

Mutations associated with the pol and the S gene can emerge as a consequence of the high replication capacity and proofreading deficiencies of hepatitis B virus during replication. The current study was constructed to evaluate primary, partial, compensatory and the escape mutations in chronic hepatitis B patients in Northern Cyprus. The samples of HBsAg positive treatment naïve 100 patients were involved in this study. HBV pol gene region was sequenced, amplified and HBV pol/S gene mutations were determined. The samples of thirty-two patients were excluded because of their low viral load (HBV < 1000 iu/ml). Among the sequenced 68 samples, there was a partial mutation (1.5%) and 36.7% displayed a resistance profile to lamivudine, adevofir, and telbivudine. Immune response escape, vaccine escape, HBIg and diagnosis escape mutations were determined in 24%, 10%, 6%, and 4% samples of the patients, respectively. Additionally, there were six different combined mutations. These data underscored that there is no concern for primary mutations in Northern Cyprus, however, we have identified a compensatory mutation (rtV173M) that may have primary mutation characteristics by combining with other mutation patterns. Additionally, HBsAg escape mutants demonstrated that detection of the S gene together with the pol gene mutations might be beneficial and important to monitor the surveillance of S variants.Mutations associated with the pol and the S gene can emerge as a consequence of the high replication capacity and proofreading deficiencies of hepatitis B virus during replication. The current study was constructed to evaluate primary, partial, compensatory and the escape mutations in chronic hepatitis B patients in Northern Cyprus. The samples of HBsAg positive treatment naïve 100 patients were involved in this study. HBV pol gene region was sequenced, amplified and HBV pol/S gene mutations were determined. The samples of thirty-two patients were excluded because of their low viral load (HBV < 1000 iu/ml). Among the sequenced 68 samples, there was a partial mutation (1.5%) and 36.7% displayed a resistance profile to lamivudine, adevofir, and telbivudine. Immune response escape, vaccine escape, HBIg and diagnosis escape mutations were determined in 24%, 10%, 6%, and 4% samples of the patients, respectively. Additionally, there were six different combined mutations. These data underscored that there is no concern for primary mutations in Northern Cyprus, however, we have identified a compensatory mutation (rtV173M) that may have primary mutation characteristics by combining with other mutation patterns. Additionally, HBsAg escape mutants demonstrated that detection of the S gene together with the pol gene mutations might be beneficial and important to monitor the surveillance of S variants.

The piRNA Response to Retroviral Invasion of the Koala Genome.

Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a combination of horizontal and vertical transfer, allowing direct analysis of retroviral invasion of the germline genome. Gammaretroviruses produce spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome, but KoRV-A piRNAs are almost exclusively derived from unspliced genomic transcripts and are strongly sense-strand biased. Significantly, selective piRNA processing of unspliced proviral transcripts is conserved from insects to placental mammals. We speculate that bypassed splicing generates a conserved molecular pattern that directs proviral genomic transcripts to the piRNA biogenesis machinery and that this "innate" piRNA response suppresses transposition until antisense piRNAs are produced, establishing sequence-specific adaptive immunity.

Tenofovir is a more suitable treatment than entecavir for chronic hepatitis B patients carrying naturally occurring rtM204I mutations.

BACKGROUND: Hepatitis B virus (HBV) DNA polymerase mutations usually occur to long term use of nucleos(t)ide analogues (NAs), but they can occur spontaneously in treatment-naïve chronic hepatitis B (CHB) patients. The naturally occurring HBV DNA polymerase mutations might complicate antiviral therapy with NAs, leading to the generation of drug-resistant viral mutants and disease progression. The most common substitutions are known to be YMDD-motif mutations, but their prevalence and the influence on antiviral therapy is unclear. AIM: To investigate prevalence of the naturally occurring rtM204I mutations in treatment-naïve CHB genotype C2 patients and their influence on antiviral therapy. METHODS: A total of 410 treatment-naïve CHB patients infected with HBV genotype C2 strains were enrolled in this retrospective study. Among the 410 patients, 232 were treated with NAs for at least 12 mo. Significant fibrosis was defined as fibrosis-4 index > 3.25 or aspartate aminotransferase to platelet ratio index > 1.5. Complete viral response (CVR) during NAs was defined as undetectable serum HBV DNA (< 24 IU/mL). The rtM204I variants were analyzed by a newly developed locked nucleotide probe (LNA probe) based real-time PCR (LNA-RT-PCR) method. RESULTS: The LNA-RT-PCR could discriminate rtM204I mutant-type (17 patients, 4.2%) from rtM204 wild-type (386 patients, 95.8%) in 403 of 410 patients (98.3% sensitivity). Multivariate analysis showed that naturally occurring rtM204I variants were more frequently detected in patients with significant fibrosis [odd-ratio (OR) 3.397, 95% confidence-interval (CI) 1.119-10.319, P = 0.031]. Of 232 patients receiving NAs, multivariate analysis revealed that achievement of CVR was reversely associated with naturally occurring rtM204I variants prior to NAs treatment (OR 0.014, 95%CI 0.002-0.096, P < 0.001). Almost patients receiving tenofovir achieved CVR at 12 mo of tenofovir, irrespective of pre-existence of naturally occurring rtM204I mutations (CVR rates: patients with rtM204I, 100%; patients without rtM204I, 96.6%), whereas, pre-existence of naturally-occurring rtM204I-mutations prior to NAs significantly affects CVR rates in patients receiving entecavir (at 12 mo: Patients with rtM204I, 16.7%; patients without rtM204I, 95.6%, P < 0.001). CONCLUSION: The newly developed LNA-RT-PCR method could detect naturally occurring rtM204I mutations with high-sensitivity. Theses mutations were more frequent in patients with liver fibrosis. Tenofovir is a more suitable treatment than entecavir for CHB patients carrying the naturally occurring rtM204I mutations.

The Exaptation of HERV-H: Evolutionary Analyses Reveal the Genomic Features of Highly Transcribed Elements.

HERV-H endogenous retroviruses are thought to be essential to stem cell identity in humans. We embrace several decades of HERV-H research in order to relate the transcription of HERV-H loci to their genomic structure. We find that highly transcribed HERV-H loci are younger, more fragmented, and less likely to be present in other primate genomes. We also show that repeats in HERV-H LTRs are correlated to where loci are transcribed: type-I LTRs associate with stem cells while type-II repeats associate with embryonic cells. Our findings are generally in line with what is known about endogenous retrovirus biology but we find that the presence of the zinc finger motif containing region of gag is positively correlated with transcription. This leads us to suggest a possible explanation for why an unusually large proportion of HERV-H loci have been preserved in non-solo-LTR form.

[HBV pol/S gene mutations in chronic hepatitis B patients receiving nucleoside/nucleotide analogues treatment]. / Nükleozid/nükleotid analogu tedavisi alan kronik hepatit B hastalarinda hbv pol/S geni mutasyonlari.

Chronic hepatitis B (CHB) is an important public health problem affecting over 240 million people all around the world. The aim of the treatment in chronic hepatitis B is to prevent progression to cirrhosis and liver cancer. Interferons (standard and peginterferon) (Peg-IFN) and nucleoside/nucleotide analogues (NAs) are widely used in the treatment of CHB. The use of long-term therapy can however result in drug resistant mutations, which can lead to treatment failure. In patients with chronic hepatitis B, in addition to primary drug resistance mutations in the pol gene, compensatory mutations were reported. The genom of HBV polymerase (pol) gene overlaps with the envelope (S) gene. Nucleoside/nucleotide analogue (NA) resistance mutations in the pol gene of HBV, either from selection of primary or secondary resistance mutations, typically result in changes in HBsAg. Recent studies have conferred a new acronym for these HBV pol/S gene overlap mutants; ADAPVEMs, for antiviral drug-associated potential vaccine-escape mutants. The aim of this study was to investigate clinically and epidemiologically significant HBV pol/S gene mutations in NA treated CHB patients. In the study, a total of 100 patients who received nucleoside/nucleotide analogue therapy for one year or more were included. The levels of HBV DNA from serum samples were detected by the commercial real-time PCR assay and the mutations of pol/S genes by direct sequencing. Sixteen samples with low HBV DNA levels (> 200 IU/ml) could not be interpreted by sequencing due to insufficient amplification. Of the remaining 84 patients that could be sequenced HBV pol gene of HBV, 53 (63.09%) were males and 31 (36.91%) were women and the mean age was 47 ± 14.99 years (range: 20-67). Primary/secondary drug mutations (rtM204I/V, rtI169S, rtL180M, rtT184L, rtA194V, rtM204I/rtL91I, rtQ149K, rtQ215H/S, rtN238D) were detected in 38 (45.2%) of the patients. Because of the HBV pol/S gene overlapping, in 27 patients immun-selected amino acid substitutions (sI110L, sT127P, sS114A, sT123A), in nine patients HBIg selected escape mutants (sP120R, sT123N, sE164D, sY134F, sQ129H, sT118A, sP127K), in seven patients vaccine escape mutants (sT126I, sP120S, sG145A, s S193L) and in one patient misdiagnosis of HBsAg (sT131I) were detected. In addition, antiviral drug-associated potential vaccine-escape mutants were detected in 13 (15.4%) patients. In patients with chronic HBV, NAs including commonly used lamivudine were observed to have the potential for ADAPVEM to emerge during treatment. It was concluded that after determination of antiviral drug resistance and ADAPVEMs replanning of treatment should be done in the NA treatment of patients with CHB.

Association of HBsAg mutation patterns with hepatitis B infection outcome: Asymptomatic carriers versus HCC/cirrhotic patients.

INTRODUCTION AND OBJECTIVES: The hepatitis B virus (HBV) surface antigen (HBsAg) variations suggested having some effects on infection outcome. Due to some controversial issues, the aim of this study was to compare the pattern of HBsAg variation between asymptomatic carriers and HCC/cirrhosis patients. MATERIALS AND METHODS: In this cross-sectional study, 19 HCC/cirrhotic and 26 asymptomatic patients were enrolled. After viral DNA extraction, HBs gene was amplified using an in-house nested-PCR. Then, PCR products were introduced into bi-directional Sanger sequencing. The retrieved sequences were compared with references, to investigate the variation of immunologic sites, major hydrophilic region (MHR) of HBsAg as well as reverse transcriptase (RT), and also to determine genotype/subtype. RESULTS: The analysis of MHR and epitopes on HBsAg showed dozens of substitution, which occurred more prevalently in I110, P120, Y134, G159, S193, Y206, S207, I208, L213 and P214 positions. However, Y134N/F/L (P=0.04) and P120T/S (P=0.009) were significantly detected in MHR and B-cell epitope of HCC/Cirrhotic group. A number of truncation-related mutations were higher in HCC/Cirrhotic group (P>0.001), albeit only C69* stop codon was statistically significant (P=0.003). In RT, some potentially resistant substitutions such as Q215S, V191I and V214A, were revealed. Phylogenetic analysis showed that all of isolates belonged to genotype D, and the major serotype was ayw1. CONCLUSION: The higher frequency of substitutions in MHR and immune epitopes at positions such as Y134 and P120 as well as stop codons such as C69* in HCC/cirrhotic group might candidate them as predictive factors for infection outcome.

Risk factors for resistance development against lamivudine during long-term treatment of chronic hepatitis B virus infections.

BACKGROUND/AIM: The use of lamivudine for the treatment of chronic hepatitis B (CHB) is limited by high rates of lamivudine resistance. However, it is still in use in many regions. Factors associated with lamivudine resistance development have been studied in only a few European cohorts. The aim of our study was to assess the rate and risk factors for lamivudine resistance in a large real-life European cohort. PATIENTS AND METHODS: We retrospectively analyzed patients with CHB treated in three German University centers over up to 12 years. Lamivudine resistance was defined as virologic breakthrough and presence of genotypic lamivudine resistance. The probability of resistance was estimated by Kaplan-Meier analysis and resistance predictors by Cox regression. RESULTS: A total of 227 patients were included into the analysis (hepatitis B envelope antigen positive or negative). Rates of lamivudine resistance by years 1-7 were 7, 26, 35, 41, 46, 53, and 55%, respectively. Interestingly, two hepatitis B envelope antigen-negative patients developed resistance during the year 12 of treatment. Independent risk factors for resistance development were hepatitis B virus DNA levels of at least 10 copies/ml before and detectable hepatitis B virus DNA by month 6 of treatment. CONCLUSION: Even after long-term response to lamivudine more than 10 years, resistance may still develop. Our findings further discourage the use of lamivudine for the treatment of CHB.

Novel Modified Vaccinia Virus Ankara Vector Expressing Anti-apoptotic Gene B13R Delays Apoptosis and Enhances Humoral Responses.

Modified vaccinia virus Ankara (MVA), an attenuated poxvirus, has been developed as a potential vaccine vector for use against cancer and multiple infectious diseases, including human immunodeficiency virus (HIV). MVA is highly immunogenic and elicits strong cellular and humoral responses in preclinical models and humans. However, there is potential to further enhance the immunogenicity of MVA, as MVA-infected cells undergo rapid apoptosis, leading to faster clearance of recombinant antigens and potentially blunting a greater response. Here, we generated MVA-B13R by replacing the fragmented 181R/182R genes of MVA with a functional anti-apoptotic gene, B13R, and confirmed its anti-apoptotic function against chemically induced apoptosis in vitro In addition, MVA-B13R showed a significant delay in induction of apoptosis in muscle cells derived from mice and humans, as well as in plasmacytoid dendritic cells (pDCs) and CD141+ DCs from rhesus macaques, compared to the induction of apoptosis in MVA-infected cells. MVA-B13R expressing simian immunodeficiency virus (SIV) Gag and Pol and HIV envelope (SHIV) (MVA-B13R/SHIV) produced higher levels of envelope in the supernatants than MVA/SHIV-infected DF-1 cells in vitro Immunization of BALB/c mice showed induction of higher levels of envelope-specific antibody-secreting cells and memory B cells, higher IgG antibody titers, and better persistence of antibody titers with MVA-B13R/SHIV than with MVA/SHIV. Gene set enrichment analysis of draining lymph node cells from day 1 after immunization showed negative enrichment for interferon responses in MVA-B13R/SHIV-immunized mice compared to the responses in MVA/SHIV-immunized mice. Taken together, these results demonstrate that restoring B13R functionality in MVA significantly delays MVA-induced apoptosis in muscle and antigen-presenting cells in vitro and augments vaccine-induced humoral immunity in mice.IMPORTANCE MVA is an attractive viral vector for vaccine development due to its safety and immunogenicity in multiple species and humans even under conditions of immunodeficiency. Here, to further improve the immunogenicity of MVA, we developed a novel vector, MVA-B13R, by replacing the fragmented anti-apoptotic genes 181R/182R with a functional version derived from vaccinia virus, B13R Our results show that MVA-B13R significantly delays apoptosis in antigen-presenting cells and muscle cells in vitro and augments vaccine-induced humoral immunity in mice, leading to the development of a novel vector for vaccine development against infectious diseases and cancer.

CD8+ Cytotoxic T Lymphocyte Responses and Viral Epitope Escape in Acute HIV-1 Infection.

Epitope escape from HIV-1-targeted CD8+ cytotoxic T lymphocyte (CTL) responses occurs rapidly after acute infection and contributes to the eventual failure of effective immune control of HIV-1 infection. Because the early CTL response is key in determining HIV-1 disease outcome, studying the process of epitope escape is crucial for understanding what leads to failure of immune control in acute HIV-1 infection and will provide important implications for HIV-1 vaccine design. HIV-1-specific CD8+ T lymphocyte responses against viral epitopes were mapped in six acutely infected individuals, and the magnitudes of these responses were measured longitudinally during acute infection. The evolution of autologous circulating viral epitopes was determined in four of these subjects. In-depth testing of CD8+ T lymphocyte responses against index and all autologous-detected variant epitopes was performed in one subject. Among the four individuals examined, 10 of a total of 35 CD8+ T cell responses within Gag, Pol, and Nef showed evidence of epitope escape. CTL responses with viral epitope variant evolution were shown in one subject, and this evolution occurred with and without measurable CTL responses against epitope variants. These results demonstrate a dynamic period of viral epitope evolution in early HIV-1 infection due to CD8+ CTL response pressure.