A sensitive method to detect the hepatitis B virus mutations by using solid phase PCR on oligonucleotide array

A sensitive method based on solid phase PCR on oligonucleotide array was established to detect two point mutations 1896G-A and 1901G-A in hepatitis B virus (HBV) DNA, in which 6 probes including these two point mutations were immobilized on modified glass slides through 5' terminal linker, while the 3' terminal was made to be free. The mutated loci were designed to locate on the last nucleotides of 3' terminal respectively, and the positive control probes lacked the last nucleotide of 3' terminal in comparison with the probes used for detection. Probes fixed on oligonucleotide array were also the solid phase amplification primers. One pair of liquid primers was used to amplify the short template product from whole HBV DNA. Using target DNA as template, the solid primers were extended under the action of Taq DNA polymerase and incorporated with Cy-3dCTP as marker. During the thermal cycling reaction, probes served as solid phase amplification primers and amplification products bound to the oligonucleotide array, which could be visualized by incorporation with fluorescent dyes. After amplification, the oligonucleotide array was washed, performed with laser scanning, and then used for quantitative analysis to obtain the information for mutations. The hybridization results were compared with DNA sequencing. It was demonstrated that in case of sample A, the ratios of fluorescence intensities in wide type and in the mutated types of 1896G-A and 1901G-A mutations in HBV were 3.81:1 and 1:3.79 respectively, while, in case of sample B, those were 1:2.89 and 1:3.03 respectively, indicating the presence of point mutations in these two loci. These results correlated to those obtained from DNA sequencing analysis in which the fluorescence intensity ratios in wide type and in the mutated types of 1996G-A and 1901D-A mutations in HBV were 1.26:1 and 1.67:1 respectively. From the above observations, it is evident that the method using solid phase PCR based on oligonucleotide array appears to be a sensitive and promising way to detect mutations with low-density.

Emerging role of liquid biopsy in rat sarcoma virus mutated metastatic colorectal cancer:A case report

BACKGROUND In patients with metastatic colorectal cancer(mCRC),the treatment options are limited and have been proved to be affected by rat sarcoma virus(RAS)mutational status.In RAS wild-type(wt)patients,the combination of antiepidermal growth factor receptor(EGFR)monoclonal antibodies with chemotherapy(CT)is more effective than CT alone.On the other hand,RAS-mutated patients are not eligible for treatment with anti-EGFR antibodies.CASE SUMMARY Eleven patients with initially RAS-mutated mCRC were followed from diagnosis to May 2022.At the time of cell-free DNA determination,five patients had undergone one CT line,five patients had undergone two CT lines,and one patient had undergone three CT lines(all in combination with bevacizumab).At the second and third treatment lines[second line(2L),third line(3L)],patients with neo-RAS wt received a combination of CT and cetuximab.In neo-RAS wt patients treated with anti-EGFR,our findings indicated an increase in progression-free survival for both 2L and 3L(14.5 mo,P=0.119 and 3.9 mo,P=0.882,respectively).Regarding 2L overall survival,we registered a slight increase in neo-RAS wt patients treated with anti-EGFR(33.6 mo vs 32.4 mo,P=0.385).At data cut-off,two patients were still alive:A RAS-mutated patient undergoing 3L treatment and a neo-RAS wt patient who received 2L treatment with anti-EGFR(ongoing).CONCLUSION Our case series demonstrated that monitoring RAS mutations in mCRC by liquid biopsy may provide an additional treatment line for neo-RAS wt patients.

Association of miRNA-122-binding site polymorphism at the interleukin-1 a gene and its interaction with hepatitis B virus mutations with hepatocellular carcinoma risk

This study was designed to investigate the contribution of miRNA-122-binding site polymorphism at the IL-1A gene and its multiplicative interactions with hepatitis B virus （HBV） mutations in the risk of hepatocellular carcinoma （HCC）. A total of 1021 healthy controls, 302 HBV surface antigen （HBsAg） seroclearance subjects, and 2011 HBsAg-positive subjects （including 1021 HCC patients） were enrolled in this study. Quantitative PCR was used to genotype rs3783553. HBV mutations were determined by direct sequencing. Multivariate logistic regression analyses were performed to test the associations of rs3783553, mutations, and their interactions with the risk of HCC. No significant association was found between rs3783553 and the risk of HCC among healthy controls, HBsAg seroclearance subjects, HBsAg-positive subjects without HCC, and all controls. Additionally, rs3783553 was not significantly associated with chronic HBV infection, liver cirrhosis, HBV e antigen seroconversion, abnormal alanine aminotransferase, and high viral load （ 〉 10^4 copies/ml）. However, the TTCA insertion allele of rs3783553 was significantly associated with an increased frequency of HBV C7A mutation compared with homozygous TTCA deletion carriers [（del/ins ＋ ins/ins） vs. del/del, adjusted odds ratio （OR）= 1.48, 95% confidence interval （CI）= 1.09-2.02, P = 0.013]. Multiplicative interaction of rs3783553 with HBV preS deletion significantly reduced the risk of HCC in males, with an adjusted OR of 0.64 （95% CI = 0.42-0.98; P = 0.041） after age and HBV genotype were adjusted. Although rs3783553 did not significantly affect genetic susceptibility to HBV-related HCC, its variant allele may predispose the host to selecting HBV C7A mutation during evolution and significantly reduce the risk of HCC caused by HBV preS deletion. This study provides an insight into the complex host-virus interaction in HBV-induced hepatocarcinogenesis and is helpful in determining HBsAg-positive subjects who are likely to develop HCC.

EFFECTS OF MUTATIONS IN THE POLYMERASE GENE OF HEPATITIS B VIRUS GENOME ON LAMIVUDINETHERAPY

Objective To explore the offects of mutations in tyrosine-methionine-aspartic acid-aspartic acid (YMDD) motif of the polymerase in the hepatitis B virus (HBV) genome on lamivudine antiviral therapy. Methods Partial HBV DNA segment containing the YMDD motif in the P gene wes obtained through amplifi- cation by polymerase chain reaction (PCR )from 19 chronic hepatitis B patients with serum HBV DNA positive at the 48th week treatment with lamivudine and subjected to automatic sequencing. Influences of vartants with YMDD mutations on lamivudine therapy were seen by observing the dynamic changes of serum HBV DNA and ALT levels. Results Serum HBV DNA breakthrough was found in 3 out of 10 individuals with detection of the YMDD mutations at the 48th week and in 5 at the 52th week, 2 of the 5 patients accompanied by serum ALT re-elevation, whereas of 9 subjects without YMDD mutations, 2 experenced an HBV DNA breakthrough at the 48th week and 1 of them had a conversion from HBV DNA positive DNA positive to negative at the 52th week. Patients with detectable HBV DNA level had a fluctuating level of serum ALT all time during the treatment. Conclusion Detection of mutations in the YMDD motif of polymerase gene in HBV genome during the lamivudine therapy will be helpful to monitoring its therapeutic outcomes.

The Sunspot Cycle Leads to Origin and Epidemic Mechanism of Novel Coronavirus COVID-19

Listed examples of virus transmission epidemics that can be strongly transmitted through the air caused by sunspot change cycle, analyzed the mechanism that promotes the generation of new viruses. From the schematic diagram of the changes in the combined force of the hydrodynamic effect of the sun sweeping the earth and the sweeping force, we obtain the places that are prone to light vortices are 30 degrees north latitude and 30 degrees south latitude on the east coast of the mainland creatively. The curved continental lines are perfect, the range of the light vortex generated is more obviously, and the effect is stronger. And the curved continental lines are perfect, the range of the light vortex generated is more obviously, and the effect is stronger. It is inferred that the light vortex produces the special amplified energy so that can make the virus mutate to produce a new highly infectious novel coronavirus. The earliest known place and time of the novel coronavirus origin are consistent with the reasoning of the new theory. Because the radius and frequency of the light vortex are different, the resulting virus strains are also different. Moreover, the fatality rate in the light vortex area is much higher than that in the non-light vortex area, indicating that the virus’s toxicity and lethality are higher in the light vortex area, so it can explain why Russia, India, and countries in the African equatorial region mortality are much lower than the United States, Italy, Spain and Brazil. Finally, preventive and recommended measures are proposed.

Adaptive immune response during hepatitis C virus infection

Hepatitis C virus(HCV)infection affects about 170 million people worldwide and it is a major cause of liver cirrhosis and hepatocellular carcinoma.HCV is a hepatotropic non-cytopathic virus able to persist in a great percentage of infected hosts due to its ability to escape from the immune control.Liver damage and disease progression during HCV infection are driven by both viral and host factors.Specifically,adaptive immune response carries out an essential task in controllingnon-cytopathic viruses because of its ability to recognize infected cells and to destroy them by cytopathic mechanisms and to eliminate the virus by non-cytolytic machinery.HCV is able to impair this response by several means such as developing escape mutations in neutralizing antibodies and in T cell receptor viral epitope recognition sites and inducing HCV-specific cytotoxic T cell anergy and deletion.To impair HCV-specific T cell reactivity,HCV affects effector T cell regulation by modulating T helper and Treg response and by impairing the balance between positive and negative co-stimulatory molecules and between pro-and antiapoptotic proteins.In this review,the role of adaptive immune response in controlling HCV infection and the HCV mechanisms to evade this response are reviewed.

Two-year Observation of the Clinical Efficacy in Treating Chronic Hepatitis B Patients with Ganxian Recipe (肝纤方) and Lamivudine

Objective: To evaluate the clinical efficacy of Ganxian recipe (肝纤方, GXR) and lamivudine (LVD) in a two-year treatment of chronic hepatitis B (CHB). Methods: One hundred and twenty patients with CHB were randomly divided into the combinedly treated group (combined group) of 40 CHB patients who were treated with GXR combined with LVD. Another 40 CHB patients were treated with LVD alone (WM group), and still another 40 CHB patients were treated with GXR alone (TCM group). All these cases were randomly controlled and observed for two years. Results: Comprehensive efficacy: Total effective rate of the combined group (complete response and partial response) was 92.5%, while that of the WM group was 67.5% and TCM group 57.5%, respectively, with the difference between them was significant ( P <0.01); after treatment, the hepatic functions (AST, ALT, SB) of the three groups were all reduced, and the reduction in the combined group was particularly significant in comparison with the WM group or TCM group, P <0.05 or P < 0.01 respectively, suggesting that the effect in the combined group was better than that in the other two groups; the rate of tyrosine-methionine-aspartate-aspartate (YMDD) virus mutation: it was 7.5% in the combined group, 40.0% in the WM group, and 5.0% in the TCM group; liver fibrosis improvement parameter: after treatment, the results in the combined group got better than those in the other two groups. Conclusion: GXR could inhibit the appearance of YMDD after long-term application of LVD, and combined use has marked synergism.

Diagnostic and therapeutic progress of multi-drug resistance with anti-HBV nucleos(t)ide analogues

Nucleos(t)ide analogues(NA) are a breakthrough in the treatment and management of chronic hepatitis B.NA could suppress the replication of hepatitis B virus(HBV) and control the progression of the disease.However,drug resistance caused by their long-term use becomes a practical problem,which influences the long-term outcomes in patients.Liver transplantation is the only choice for patients with HBV-related end-stage liver disease.But,the recurrence of HBV after transplantation often caused by the development of drug resistance leads to unfavorable outcomes for the recipients.Recently,the multi-drug resistance(MDR) has become a common issue raised due to the development and clinical application of a variety of NA.This may complicate the antiviral therapy and bring poorly prognostic outcomes.Although clinical evidence has suggested that combination therapy with different NA could effectively reduce the viral load in patients with MDR,the advent of new antiviral agents with high potency and high genetic barrier to resistance brings hope to antiviral therapy.The future of HBV researches relies on how toprevent the MDR occurrence and develop reasonable and effective treatment strategies.This review focuses on the diagnostic and therapeutic progress in MDR caused by the anti-HBV NA and describes some new research progress in this field.

The virological impacts of SARS-CoV-2 D614G mutation

The coronavirus diseases 2019(COVID-19)caused by the infection of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)emerged in December 2019 has caused more than 140 million infections worldwide by the end of April 2021.As an enveloped single-stranded positive-sense RNA virus,SARS-CoV-2 underwent constant evolution that produced novel variants carrying mutation conferring fitness advantages.The current prevalent D614G variant,with glycine substituted for aspartic acid at position 614 in the spike glycoprotein,is one of such variants that became the main circulating strain worldwide in a short period of time.Over the past year,intensive studies from all over the world had defined the epidemiological characteristics of this highly contagious variant and revealed the underlying mechanisms.This review aims at presenting an overall picture of the impacts of D614G mutation on virus transmission,elucidating the underlying mechanisms of D614G in virus pathogenicity,and providing insights into the development of effective therapeutics.