OBJECTIVE: To study the correlation between miR-122 and early HBV infection and analyze its application value in early infection of voluntary blood donors. METHODS: A total of 150 samples from voluntary blood donors in Fujian Blood Center from May 2021 to July 2022 were collected and divided into group N (normal group), group E (ELISA single positive group), and group D (both ELISA and nucleic acid positive group), and the general information of the three groups of blood donors was collected. Total RNA was extracted from the three groups of samples, and the expression level of miR-122 was detected by qRT-PCR. The expression differences of miR-122 among the three groups of samples were statistically analyzed, and the correlation between the expression level of miR-122 in group D and its HBV DNA copy number was analyzed. The miRNA database was used to predict the potential target genes of miRNA and perform bioinformatics analysis. RESULTS: There was no statistical difference in gender, education level, and occupation distribution among the three groups, but the age distribution and number of blood donations among different groups were statistically significant. Compared with group N, the relative expression levels of miR-122 in the plasma of group E and group D were significantly downregulated (P < 0.05); the relative expression level of miR-122 in group D was more significantly downregulated than that in group E (P < 0.001). Pearson correlation analysis showed that the expression level of miR-122 in group D was negatively correlated with the HBV-DNA copy number (R ²=-0.804,P < 0.01). Two potential target genes were screened using the miRNA database: ALDOA(aldolase A) and PKM (pyruvate kinase). GO analysis results showed that the potential target genes of miRNA mainly involved in biological processes including cell homeostasis and regulation of transcriptional processes. CONCLUSION Downregulation of miR-122 expression is closely related to early HBV infection and replication activity.
Humans ; MicroRNAs ; Blood Donors ; Hepatitis B/diagnosis* ; Hepatitis B virus ; DNA, Viral/blood* ; Male ; Female ; Early Diagnosis ; Adult

OBJECTIVE: To investigate the pattern of hepatitis B virus (HBV) infection and the prevalence of hepatitis D virus (HDV) infection among voluntary blood donors who tested reactive for HBV in Wuhan, and to provide data support for the prevention and treatment of HBV and HDV infections. METHODS: Electrochemiluminescence (ECL) method was used to detect hepatitis B serological markers in the samples with HBsAg and/or HBV DNA reactivity, and the HBV infection in different groups was statistically analyzed. The HDV IgM and IgG antibodies were screened by ELISA, and the prevalence of HDV infection in the retained samples was analyzed. RESULTS: In 351 ELISA and/or nucleic acid test (NAT) reactive samples, the serological tests for hepatitis B revealed that 4 cases (1.1%) were positive for HBsAg, HBeAg, and anti-HBc, 182 cases (51.9%) were positive for HBsAg, anti-HBe, and anti-HBc, and 55 cases (15.7%) were negative for HBsAg but positive for anti-HBc. Among them, the HBsAg ELISA dual reagent reactive group (HBsAg R&R group) and the HBsAg ELISA single reagent reactive/HBV DNA reactive group (HBsAg R&NR/HBV DNA R group) had the highest rates of HBsAg(+), anti-HBe(+), and anti-HBc(+), accounting for more than 90% and 65%, respectively, followed by low activity of HBV acute infection or chronic carriers, accounting for about 5% and 20%, respectively. In the HBsAg R&NR/HBV DNA NR group, the combined proportion of individuals with anti-HBs single positive and all hepatitis B serological markers negative accounted for 78%, and those who were HBsAg negative but anti-HBc positive accounted for approximately 20%. In the HBsAg NR&NR/HBV DNA R group, there was nearly 9% of HBsAg(+), anti-HBe(+), and anti-HBc(+), the remaining were all HBsAg negative but anti-HBc positive, with a 100% anti-HBc positivity rate in this group. No HDV IgM or IgG antibodies were detected in the retained samples. CONCLUSION Blood donors with HBV-reactive results in blood screening exhibit multiple patterns of infection indicators. The prevalence rate of HDV infection among blood donors in Wuhan is extremely low. However, the risk of asymptomatic occult hepatitis B infection (OBI) blood donors being co-infected with HDV should not be overlooked in areas with high prevalence of HBV.
Humans ; Blood Donors ; Hepatitis B/blood* ; China/epidemiology* ; Adult ; Male ; Female ; Hepatitis D/epidemiology* ; Middle Aged ; Hepatitis B virus/immunology* ; Hepatitis B Antibodies/blood* ; Young Adult ; DNA, Viral/blood* ; Hepatitis B Surface Antigens/blood* ; Prevalence ; Adolescent

Gene synthesis is an enabling technology that supports the development of synthetic biology. The existing approaches for de novo gene synthesis generally have tedious operation, low efficiency, high error rates, and limited product lengths, being difficult to support the huge demand of synthetic biology. The assembly and error correction are the keys in gene synthesis. This study first designed the oligonucleotide sequences by reasonably splitting the virus genome of approximately 10 kb by balancing the parameters of sequence design software ability, PCR amplification ability, and assembly enzyme assembly ability. Then, two-step PCR was performed with high-fidelity polymerase to complete the de novo synthesis of 3.0 kb DNA fragments, and error correction reactions were performed with T7 endonuclease Ⅰ for the products from different stages of PCR. Finally, the virus genome was assembled by 3.0 kb DNA fragments from de novo synthesis and error correction and then sequenced. The experimental results showed that the proposed method successfully produced the DNA fragment of about 10 kb and reduced the probability of large fragment mutations during the assembly process, with the lowest error rate reaching 0.36 errors/kb. In summary, this study developed an efficient de novo method for synthesizing a viral genome of about 10 kb with T7 endonuclease Ⅰ-mediated error correction. This method enabled the synthesis of a 10 kb viral genome in one day and the correct plasmid of the viral genome in five days. This study optimized the de novo gene synthesis process, reduced the error rate, simplified the synthesis and assembly steps, and reduced the cost of viral genome assembly.
Genome, Viral/genetics* ; Polymerase Chain Reaction/methods* ; DNA, Viral/genetics* ; Bacteriophage T7/enzymology* ; Synthetic Biology/methods*

Foot-and-mouth disease (FMD) is one of the major animal infectious diseases in the world. All cloven-hoofed animals are susceptible to FMD. Vaccination is still the first choice for the prevention and control of FMD. mRNA vaccines can be rapidly designed, synthesized, and produced on a large scale in vitro, and they can induce effective protective immune responses, demonstrating the advantages of rapid development, easy preparation, and low biosafety risks. The design of untranslated regions is a key to enhancing the expression and efficacy of mRNA vaccines. In order to generate an efficient FMD mRNA vaccine, we designed three FMD P12A3C expression vectors with different untranslated regions and synthesized corresponding mRNAs. By comparing expression efficiency of these vectors and their mRNAs at different time points and in different cell lines, we found that the mRNA P12A3C-UTR3 had the best expression and universality. This study laid a foundation for the development of mRNA vaccines against FMD and provided a theoretical basis for the optimal sequence design of efficient mRNA.
Foot-and-Mouth Disease Virus/genetics* ; Animals ; RNA, Messenger/biosynthesis* ; Foot-and-Mouth Disease/immunology* ; Antigens, Viral/biosynthesis* ; Viral Vaccines/biosynthesis* ; Genetic Vectors/genetics* ; Cell Line ; Vaccines, DNA/immunology*

Geminiviruses cause substantial crop yield losses worldwide. The replication initiator protein (Rep) encoded by geminiviruses is indispensable for geminiviral replication. The Rep protein encoded by beet severe curly top virus (BSCTV, genus Curtovirus, family Geminiviridae) induces VARIANT IN METHYLATION 5 (VIM5) expression in Arabidopsis leaves upon BSCTV infection. VIM5 functions as a ubiquitination-related E3 ligase to promote the proteasomal degradation of methyltransferases, resulting in reduction of methylation levels in the BSCTV C2-3 promoter. However, the specific domains of Rep responsible for VIM5 induction remain poorly characterized. Although Rep proteins from several geminiviruses act as viral suppressors of RNA silencing (VSRs), whether BSCTV Rep also possesses VSR activity remains to be illustrated. In this study, we employed a transient expression system in the 16c-GFP transgenic and the wild-type Nicotiana benthamiana plants to analyze the VSR and the VIM5-inducing activities of different truncated Rep proteins haboring distinct domains. We found that the N-terminal domain (amino acids 1-180) of Rep suppressed GFP silencing in 16c-GFP transgenic N. benthamiana leaves. The minimal N-terminal fragment (amino acids 1-104) induced VIM5 expression upon co-infiltration, while C-terminal truncations lacked VIM5-inducing activity. Our results indicate that the N-terminal domain of Rep encoded by BSCTV mediates the suppression of RNA silencing and induces VIM5 expression. Thus, our findings contribute to a better understanding of interactions between geminiviral Rep and plant hosts.
Geminiviridae/genetics* ; Nicotiana/metabolism* ; Arabidopsis/metabolism* ; RNA Interference ; Viral Proteins/metabolism* ; Arabidopsis Proteins/metabolism* ; Plants, Genetically Modified/metabolism* ; Protein Domains ; Plant Diseases/virology* ; Methyltransferases/metabolism* ; Ubiquitin-Protein Ligases/metabolism* ; DNA Helicases/genetics*

Female ; Humans ; Adenocarcinoma in Situ ; Uterine Cervical Neoplasms/pathology* ; Papillomavirus Infections ; Cervix Uteri/pathology* ; Adenocarcinoma/pathology* ; Papillomaviridae ; DNA, Viral

Porcine deltacoronavirus (PDCoV) is a major pathogen causing fatal diarrhea in suckling piglets, and there is currently a lack of effective vaccines and drugs to prevent and control the virus. The nonstructural protein 13 (NSP13) serves as a virus-coded helicase and is considered to be a crucial target for antiviral drugs, making it imperative to investigate the helicase activity of NSP13. In this study, the NSP13 gene of PDCoV was synthesized and integrated into the prokaryotic expression vector pET-28a to construct the recombinant plasmid pET-28a-NSP13. NSP13 was successfully expressed in BL21 (DE3) and subsequently purified. The study also verified the helicase activity of the purified NSP13 and explored the factors that influence this activity. The results indicated that NSP13 from PDCoV was effectively expressed in the prokaryotic system and exhibited helicase activity, capable of unwinding double-stranded DNA with a tail at the 5' end. Additionally, NSP13 demonstrated an annealing function by promoting the complementary pairing of single-stranded nucleotide chains to form double strands. The helicase activity of NSP13 was affected by metal ions, but Mg²⁺concentrations in the range of 0.5-6.0 mmol/L had no significant effect on helicase activity of NSP13. When the solution pH was in the range of 4-9, there was no difference in helicase activity. ATP concentrations in the range of 0.25-6.00 mmol/L had a weak effect on helicase activity, and NSP13 concentration ≥80 nmol/L inhibited the helicase activity. We obtained the NSP13 of PDCoV and investigated its helicase activity. These findings provided a theoretical foundation for the further research on the regulatory mechanism of NSP13 in PDCoV replication and the development of anti-coronaviral drugs.
Viral Nonstructural Proteins/metabolism* ; Escherichia coli/metabolism* ; Recombinant Proteins/metabolism* ; Swine ; Animals ; DNA Helicases/metabolism* ; Genetic Vectors/metabolism*

Objective: To investigate the demographic characteristics and clinical influencing factors which associates with the occurrence probability of persistent or intermittent hypoviremia (LLV) in patients with chronic hepatitis B (CHB) treated with nucleos(t)ide analogues (NAs). Methods: A single-center retrospective analysis was performed on patients with CHB who received outpatient NAs therapy for≥48 ± 2 weeks. According to the serum hepatitis B virus (HBV) DNA load at 48±2 weeks treatment, the study groups were divided into LLV (HBV DNA < 20 IU/ml and < 2 000 IU/ml) and MVR group (sustained virological response, HBV DNA < 20 IU/ml). Demographic characteristics and clinical data at the start of NAs treatment (considered as baseline) were retrospectively collected for both patient groups. The differences in the reduction of HBV DNA load during treatment was compared between the two groups. Correlation and multivariate analysis were further conducted to analyze the associated factors influencing the LLV occurrence. Statistical analysis was performed using the independent samples t-test, c2 test, Spearman analysis, multivariate logistic regression analysis, or area under the receiver operating characteristic curve. Results: A total of 509 cases were enrolled, with 189 and 320 in the LLV and MVR groups, respectively. Compared to patients with MVR group at baseline: (1) the demographics characteristics of patients showed that LLV group was younger in age (39.1 years, P = 0.027), had a stronger family history (60.3%, P = 0.001), 61.9% received ETV treatment, and higher proportion of compensated cirrhosis (20.6%, P = 0.025) at baseline; (2) the serum virological characteristics of patients showed that LLV group had higher HBV DNA load, qHBsAg level, qHBeAg level, HBeAg positive rate, and the proportion of genotype C HBV infection but decreased HBV DNA during treatment (P < 0.001) at baseline; (3) the biochemical characteristics of patients showed that LLV group had lower serum ALT levels (P = 0.007) at baseline; (4) the noninvasive fibrosis markers of patients showed that LLV group were characterized by high aspartate aminotransferase platelet ratio index (APRI) (P = 0.02) and FIB-4 (P = 0.027) at baseline. HBV DNA, qHBsAg and qHBeAg were positively correlated with LLV occurrence (r = 0.559, 0.344, 0.435, respectively), while age and HBV DNA reduction were negatively correlated (r = -0.098, -0.876, respectively). Logistic regression analysis showed that ETV treatment history, high HBV DNA load at baseline, high qHBsAg level, high qHBeAg level, HBeAg positive, low ALT and HBV DNA level were independent risk factors for patients with CHB who developed LLV with NAs treatment. Multivariate prediction model had a good predictive value for LLV occurrence [AUC 0.922 (95%CI: 0.897 ~ 0.946)]. Conclusion: In this study, 37.1% of CHB patients treated with first-line NAs has LLV. The formation of LLV is influenced by various factors. HBeAg positivity, genotype C HBV infection, high baseline HBV DNA load, high qHBsAg level, high qHBeAg level, high APRI or FIB-4 value, low baseline ALT level, reduced HBV DNA during treatment, concomitant family history, metabolic liver disease history, and age < 40 years old are potential risk factors for developing LLV in patients with CHB during the therapeutic process.
Humans ; Adult ; Hepatitis B, Chronic/complications* ; Retrospective Studies ; Cross-Sectional Studies ; Hepatitis B e Antigens ; DNA, Viral ; Antiviral Agents/therapeutic use* ; Hepatitis B virus/genetics* ; Demography

Humans ; Squamous Cell Carcinoma of Head and Neck ; RNA, Messenger ; Immunohistochemistry ; Papillomavirus Infections/diagnosis* ; Oncogene Proteins, Viral/genetics* ; Head and Neck Neoplasms ; Cyclin-Dependent Kinase Inhibitor p16 ; Papillomaviridae ; Papillomavirus E7 Proteins/genetics* ; DNA, Viral

The resolution of the hepatitis C issue has raised expectations for a chronic hepatitis B cure, driving the industry to expand investment in research and development efforts to strengthen functional cure strategies. These strategies have a wide variety of types, and the published research findings are heterogeneous. The theoretical analysis of these strategies is of great significance for determining prioritized research orientations as well as sensibly allocating research and development resources. However, due to a paucity of necessary conceptual models, current theoretical analysis has not been able to unify various therapeutic strategies into a proper theoretical framework. In view of the fact that the decrease in the quantity of cccDNA is an inevitable core event accompanied by the process of functional cure, this paper intends to analyze several chronic hepatitis B cure strategies using cccDNA dynamics as a framework. Furthermore, there are currently few studies on the dynamics of the cccDNA field, hoping that this article can promote recognition and research in this field.
Humans ; Hepatitis B virus/genetics* ; Hepatitis B, Chronic/drug therapy* ; Antiviral Agents/therapeutic use* ; Virus Replication ; DNA, Circular/therapeutic use* ; DNA, Viral/genetics* ; Hepatitis B/drug therapy*