Molecular characterization of recombinant premembrane protein of dengue virus serotype-2 for development of diagnostic assay.

Dengue is an acute arboviral infection common in tropical and subtropical countries. Dengue has been highlighted as a public health concern in the last five decades, affecting almost 50% of the population in developing nations. Dengue infection results in a complex symptomatic disease that ranges from headache, fever, and skin rash to extreme hemorrhage fever and liver dysfunction. The diagnosis of the disease is essential for effective treatment. The early onset of the infection can be detected through viral structural peptides that act as markers for detection, including Pre-Membrane (Pre-M) protein. In the currently proposed research, the structural gene obtained from local isolates was targeted for studies. For this purpose, recombinant structural protein Pre-M was amplified, cloned, and expressed in the bacterial expression system. The expression of the structural protein (Pre-M) was scrutinized by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and validated by western blot and dot blot, and afterwards, the antigen was purified. The purified Pre-M protein carries the potential for the development of in-house diagnostic assay as well as for vaccine production. This study aimed to develop a highly specific, sensitive, and cost-effective in-house enzyme-linked immunoassay (ELISA) for the detection of antibodies of Pakistani most prevalent dengue virus serotype 2 (DENV-2). The success of this research would also pave the way toward developing novel vaccines for the future prevention of dengue infection.

Viruses as tools in gene therapy, vaccine development, and cancer treatment.

Using viruses to our advantage has been a huge leap for humanity. Their ability to mediate horizontal gene transfer has made them useful tools for gene therapy, vaccine development, and cancer treatment. Adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, alphaviruses, and herpesviruses are a few of the most common candidates for use as therapeutic agents or efficient gene delivery systems. Efforts are being made to improve and perfect viral-vector-based therapies to overcome potential or reported drawbacks. Some preclinical trials of viral vector vaccines have yielded positive results, indicating their potential as prophylactic or therapeutic vaccine candidates. Utilization of the oncolytic activity of viruses is the future of cancer therapy, as patients will then be free from the harmful effects of chemo- or radiotherapy. This review discusses in vitro and in vivo studies showing the brilliant therapeutic potential of viruses.

Novel Coronavirus: A Review from Origin to Current Status of Therapeutic Strategies.

Coronaviruses (CoVs) are continuously emerging, highly transmissible, and pathogenic agents that primarily target the human respiratory system. Previous outbreaks of severe acute respiratory syndrome-CoV and Middle East respiratory syndrome-CoV remain life-threatening and global public health concerns. A novel CoV outbreak that occurred in December 2019 in Wuhan, China was declared a pandemic outbreak that has since killed millions of individuals worldwide. Rapid transmission, genetic variations, and unavailability of specific therapeutic drugs are major factors that led to this alarming and deadly situation. Currently, > 200 clinical vaccine trials are underway to combat infection. This review summarizes reports related to CoV origin, genetic variations, drug options, status of nine vaccines that were in phase III trials, and novel therapies including convalescent plasma and stem cell treatment.

Genome-wide methylation profiling of HCV pathogenesis to develop diabetes and diabetic complications.

HCV is key pathological factor for inducting insulin resistance. Such HCV-induced insulin resistance is linked with metabolic syndrome, type 2 diabetes mellitus, extrahepatic manifestations, hepatic fibrosis progression and development of hepatocellular carcinoma. DNA methylation alterations can cause developmental abnormalities, tumours and other diseases. In our study, PBMCs were isolated and genomic DNA was extracted. DNA fragmentation was achieved by sonication to 200-400 bp; subsequently, end repair and adenylation was performed. Manufacturer's guidelines were followed to ligate Cytosine-methylated barcodes to sonicated DNA. EZ DNA Methylation-GoldTM Kit was then employed to treat these DNA segments twice with bisulphite. A Library was maintained, sequenced on an Illumina platform and 150/125 bp paired-end reads generated. GO seq R package was used to perform Gene Ontology (GO) enrichment analysis for genes linked to DMRs and DMPs; gene length bias was corrected. We identified 12 945 significant hypermethylated DMRs among all samples that were screened as those with at least 0.1 methylation level differences and P-value less than 0.05. Fisher's exact test with FDR multiple test correction is used for identification of DMPs and DMRs. High throughput bisulphite sequencing (Illumina) was carried out, and bioinformatics analysis was performed to analyse methylation status. Gene ontology (GO) and KEGG pathway enrichment analysis showed differentially methylated regions enriched in various pathways that include PI3K-AKT/IRS1 signalling pathway, metabolic pathway, oxidative phosphorylation, Renin-angiotensin system that are all involved in developing type-2 diabetes (T2D). Our study provides supporting evidence for significant involvement of HCV infection in development of epigenetic modifications in regulation of metabolic disorders like T2D and its complications.

Next-generation sequencing studies on the E1-HVR1 region of hepatitis C virus (HCV) from non-high-risk HCV patients living in Punjab and Khyber Pakhtunkhwa, Pakistan.

The incidence rate of hepatitis C virus (HCV) infection in Pakistan is very high. In this study, we evaluated the genetic heterogeneity of HCV hypervariable region 1 (HVR1) from the HCV-infected Pakistani population and compare the isolated genotypes with representative sequences from internationally diverse geographic regions. We also investigated potential transmission events in non-high-risk HCV patients. Next-generation sequencing (NGS) data from the E1-HVR1 region from 30 HCV patients were used for phylogenetic analysis. Reference sequences were retrieved from the Los Alamos HCV and GenBank databases. NGS data were analyzed to examine HCV HVR1 sequence diversity and identify transmission links among HCV-infected individuals using Global Hepatitis Outbreak and Surveillance Technology (GHOST). Phylogenetic analysis showed the predominance of HCV genotype 3a (86.6%), followed by 1a (6.6%), 1b (3.3%), and 3b (3.3%). NGS of HVR1 displayed significant genetic heterogeneity of HCV populations within each patient. The average nucleotide sequence diversity for HVR1 was 0.055. JR781281 was found to be the most diverse (0.14) of the specimens. Phylogenetic analysis demonstrated that all HCV specimens sequenced in this study were more similar to each other and showed variations from the representative sequences. The GHOST results suggested genetic relatedness between two (6.6%) HCV cases, possibly defining an incipient outbreak in a non-high-risk population. We urge rigorous countrywide investigation of outbreaks to identify transmission clusters and their sources to incorporate preventive measures for disease control.

Cosmopolitan A1 lineage of dengue virus serotype 2 is circulating in Pakistan: A study from 2017 dengue viral outbreak.

Dengue viral infection has become a challenge in tropical and subtropical countries where dengue virus is endemic. Its epidemics are occurring at higher rates amid its circulation throughout the year. Since the first documented outbreak in Pakistan in 1994, this region has reported many sporadic cases and epidemics. There is availability of small scale demographic and epidemiological studies on dengue viral infection in Pakistan. The year 2017 witnessed a huge dengue outbreak in Peshawar city of Pakistan with 69 deaths and 24 807 laboratory-confirmed cases. We suspect that the circulation of a different lineage or genotype could be responsible for the enhanced number of infected patients in Pakistan's 2017 outbreak since previous studies have already described this phenomenon in other countries. For this, we collected 1447 suspected blood samples and their epidemiological data. After serotyping through polymerase chain reaction nine samples of Dengue virus2 (DENV2) were randomly selected and were subjected to Sanger's sequencing for genotyping analysis. The mean distance, genetic diversity, and phylogenetic analysis were carried out using K2 model. The phylogenetic analysis split Pakistani isolates into two lineages, the sequences from 2017 outbreak in Peshawar grouped within A1 lineage of cosmopolitan genotype (IV) of DENV2. The difference in distance, genetic diversity, and amino acids composition strongly back the results that the new lineage is circulating in the region. This is significant as Pakistan is struggling to control dengue epidemics which have caused much loss in both monetary and health sectors.

Phytochemical profiling and antiviral activity of Ajuga bracteosa, Ajuga parviflora, Berberis lycium and Citrus lemon against Hepatitis C Virus.

Hepatitis C is a serious health issue and cause liver disorders in millions of people. Available therapeutic agents require long term administration with numerous side effects. Therefore, there is a dire need to find alternative treatment options for this disease. Since ancient times, medicinal plants are widely used to cure various diseases with no or less harmful effects. Therefore, this study was designed to find out phytochemicals and investigate antiviral activity of methanol extract of Ajuga bracteosa, Ajuga parviflora, Berberis lycium and Citrus lemon against Hepatitis C Virus (HCV infection). Phytochemical analysis of the plant extract was performed using various chemical tests. Toxicity of the plant extract was determined against using trypan blue exclusion method. Antiviral activity of the selected plant extract was find out against HCV infected HepG2 cells. For this purpose, HepG2 cells were seeded with HCV positive and negative serum and nontoxic doses of plant extract for 24 and 48 h. After this RNA was extracted and viral load was determined using Real-time PCR. Phytochemical analysis showed the presence of flavonoids and phenols in all plant extracts while amino acids, alkaloids and tannins were present in B. lycium and saponins were detected in C. lemon. Toxicity assay showed that all plant extracts were nontoxic at maximum concentration of 200 µg/ml except B. lycium, which showed mild toxicity at 40 µg/ml and were extremely toxic at 60 µg/ml and above doses. Real-time PCR quantitation result revealed that after 24 h treatments A. parviflora showed highest antiviral activity, followed by A. bracteosa, while B. lycium extract had low (35%) and C. lemon has no antiviral effects. The 48 h treatments showed an increase antiviral activity by A. bracteosa followed by A. parviflora and B. lycium while C. lemon showed negative effect. Our results depicted that mentioned plants might be used as an alternative therapeutic regime or in combination with existing treatments against HCV.

Role of altered immune cells in liver diseases: a review.

Immune cells play an important role in controlling liver tumorigenesis, viral hepatitis, liver fibrosis and contribute to pathogenesis of liver inflammation and injury. Accumulating evidence suggests the effectiveness of natural killer (NK) cells and Kupffer cells (KCs) against viral hepatitis, hepatocellular damage, liver fibrosis, and carcinogenesis. Activation of natural killer cells provides a novel therapeutic strategy to cure liver related diseases. This review discusses the emerging roles of immune cells in liver disorders and it will provide baseline data to scientists to design better therapies for treatment.

HCV-induced regulatory alterations of IL-1ß, IL-6, TNF-α, and IFN-ϒ operative, leading liver en-route to non-alcoholic steatohepatitis.

Over the course of time, Hepatitis C has become a universal health menace. Its deleterious effects on human liver encompass a lot of physiological, genetic as well as epigenetic alterations. Fatty liver (Hepatic steatosis) is an inflammation having multifactorial ancestries; one of them is HCV (steatohepatitis). HCV boosts several cellular pathways involving up-regulation of a number of cytokines. Current study reviews the regulation of some selective key cytokines during HCV infection, to help generate an improved understanding of their role. These cytokines, IL-1ß, IL-6, TNF-α, and IFN-ϒ, are inflammatory markers of the body. These particular markers along with others help hepatocytes against viral infestation. However, recently, their association has been found in degradation of liver on the trail heading to non-alcoholic steatohepatitis (NASH). Consequently, the disturbance in their equilibrium has been repeatedly reported during HCV infection. Quite a number of findings are affirming their up-regulation. Although these cell markers are stimulated by hepatocytes as their standard protection mechanism, but modern studies have testified the paradoxical nature of this defense line. Nevertheless, direct molecular or epigenetic research is needed to question the actual molecular progressions and directions commanding liver to steatosis, cirrhosis, or eventually HCC (Hepatocellular Carcinoma).

Immunotherapeutic strategies for sexually transmitted viral infections: HIV, HSV and HPV.

More than 1 million sexually transmitted infections (STIs) are acquired each day globally. Etiotropic drugs cannot effectively control infectious diseases therefore, there is a dire need to explore alternative strategies especially those based on the regulation of immune system. The review discusses all rational approaches to develop better understanding towards immunotherapeutic strategies based on modulation of immune system in an attempt to curb the elevating risk of infectious diseases such as HIV, HPV and HSV because of their high prevalence. Development of monoclonal antibodies, vaccines and several other immune based treatments are promising alternative strategies that are offering new opportunities to eradicate pathogens.

Studies on the role of neutralizing antibodies against envelope genes in resolving HCV pseudo-particles infection.

Characterization of antibodies targeting the attachment and entry of the viral particles into host cells is important for studding antibody mediated neutralization. Antibodies against the envelope glycoproteins (EGP) have neutralizing capacity and can prevent HCV infections. System based on HCV pseudo typed-particles (HCVpp) stably expressing EGP can be used for screening of HCV anti envelope neutralizing antibodies in the serum of patients with acute and chronic HCV infections. The aim of the current study was to check HCVpp as a useful tool for the detection of anti-HCV envelope antibodies in the serum of HCV infected patients and to test the binding potential of these antiviral molecules to EGP of HCV 3a. Previously developed HCVpp harboring unmodified glycoproteins from local isolates in 293T cell line were used in this study. HCVpp were pre incubated with different concentrations of anti E1 antibody and different E2 antibodies to check antiviral activity. Further we used serum samples with low/medium (≤800,000 IU/mL), and high (>800,000 IU/mL) viral titer from chronic HCV male and female patients. Infection was done in Huh-7 cells for 1 h at 37 oC. Infectivity was checked through Luciferase assay. Considerable decrease in HCVpp infectivity with anti-envelope antibodies was observed in dose dependent manner. Maximum inhibition was seen when 5 µg/ml of monoclonal anti E1 antibody used. Further increase in concentration exhibited no decrease in infectivity which suggests that other factors are also involved in causing infection. Various well characterized E2-specific monoclonal antibodies (mAbs) have been screened for their capability to reduce infection in Huh-7 cells. Three of the four mAbs specific for the E2 had no effect on the infectivity of HCVpp. Confirmation sensitive antibody H53 showed maximum inhibition of infectivity. HCV ELISA positive samples from both male and female patients were used to neutralize the HCVpp. The neutralizing antibody response was observed in both males and females patients and do not assemble the rapidly evolving HCV envelope glycoproteins. That is why in spite the presence of neutralizing antibodies in the blood they fail to resolve infections. Moreover E1 antibodies insignificantly (>0.05) inhibit HCVpp infectivity while E2 antibodies significantly (<0.05) inhibit HCVpp infection. Based on the results of this study it is concluded that anti-envelope antibodies particularly the anti-E2 could be extremely valuable for characterizing the humoral immune response to HCV and for evaluating the potential for developing passive and active immunization for hepatitis C along with interferon therapy.

Generation of infectious HCV pseudo typed particles and its utilization for studying the role of CD81 & SRBI receptors in HCV infection.

Hepatitis C virus (HCV) entry into isolated primary liver cells and cell lines requires interaction with the cell surface receptors. The study of HCV attachment with host cell surface receptors has been hindered by the unavailability of competent cell culture based system for HCV propagation. This problem has been overcome by the development of genetically tagged infectious HCV pseudo particles (HCVpp) harboring unmodified E1 and E2 glycoproteins. Studies using cell binding assays together with infection assays using HCVpp have shown that CD81 and scavenger receptor (SRBI) are actively involved in binding with envelope proteins facilitating the viral entrance process. This paper aimed to develop HCVpp of local HCV 3a Pakistani isolate and to study the viral tropism role of CD81 and SRBI receptors in HCV infectivity. HCV E1 and E2 genes were amplified and cloned in mammalian expression vector pcDNA 3.1/myc. The expressing plasmid of HCV E1-E2 glycoprotein in native form was co-transfected into 293FT cells with lentiviral packaging plasmid encoding the MLV Gag-Pol core proteins, and a packaging competent MLV-derived genome (pMLVYCMV-Luc) encoding the luciferase marker protein to produce infectious HCVpp. Anti-CD81 antibody (CBL579), anti-SRBI type II antibody (sc-20441) HCV anti-E2 mouse IgG1 (sc-65457) and HCV anti-E1 antibody mouse IgG1 (sc-65459) were used in this setup. We showed that primary site of viral replication is liver which involve CD81 and SRBI receptors for HCV gp-dependent infection with HCVpp. This is the preliminary reported cell cultured based mechanism from Pakistan which facilitated functional studies of different antiviral agents. Understanding of this technique will help in development of new antiviral therapeutics focusing on earlier steps of HCV life cycle. We have developed infectious pseudo particles of local 3a-isolate and concluded that a number of liver-specific surface proteins function along with CD81 and SRBI receptor regarding HCV infectivity. To endeavors and to identify this liver specific co-receptor molecule(s) will provide insights into the role of these molecules in the initial steps of HCV life cycle.

Regulatory pathways and therapeutic potential of PDE4 in liver pathophysiology.

Phosphodiesterase 4 (PDE4), crucial in regulating the cyclic adenosine monophosphate (cAMP) signaling pathway, significantly impacts liver pathophysiology. This article highlights the comprehensive effects of PDE4 on liver health and disease, and its potential as a therapeutic agent. PDE4's role in degrading cAMP disrupts intracellular signaling, increasing pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). This contributes to liver inflammation in conditions such as hepatitis and non-alcoholic steatohepatitis (NASH). Additionally, PDE4 is a key factor in liver fibrosis, characterized by excessive extracellular matrix deposition. Inhibiting PDE4 shows promise in reducing liver fibrosis by decreasing the activation of hepatic stellate cells, which is pivotal in fibrogenesis. PDE4 also influences hepatocyte apoptosis a common feature of liver diseases. PDE4 inhibitors protect against hepatocyte apoptosis by raising intracellular cAMP levels, thus activating anti-apoptotic pathways. This suggests potential in targeting PDE4 to prevent hepatocyte loss. Moreover, PDE4 regulates hepatic glucose production and lipid metabolism, essential for liver function. Altering cAMP levels through PDE4 affects enzymes in these metabolic pathways, making PDE4 a target for metabolic disorders like type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). Since PDE4 plays a multifaceted role in liver pathophysiology, influencing PDE4's mechanisms in liver diseases could lead to novel therapeutic strategies. Still, extensive research is required to explore the molecular mechanisms and clinical potential of targeting PDE4 in liver pathologies.

Assessment of ubiquitin specific Peptidase-18 gene in peripheral blood of chronic hepatitis C patients treated with direct-acting antiviral drugs.

Hepatitis C virus (HCV) infection remains one of the leading causes of liver complications globally. Ubiquitin Specific Peptidase-18 (USP18) is a ubiquitin-specific protease that cleaves interferon-stimulated gene 15 (ISG15) from ISGylated protein complexes and is involved in regulating interferon responsiveness. To study the effect of direct-acting antivirals (DAAs) on the USP18 gene using qPCR, 132 participants were recruited and classified into different groups based on treatment duration. USP18 expression was raised compared to rapid virologic response (RVR) and early virologic response (EVR) groups with P = 0.0026 and P = 0.0016, respectively. USP18 was found to be 7.36 folds higher in naïve patients than those with RVR and sustained viral response (SVR). In RVR and SVR groups where patients had cleared HCV RNA after treatment with direct-acting antiviral agents (DAA) therapy, the expression of USP18 was found to be low, with a fold change of 1.3 and 1.4 folds, respectively. Expression of USP18 was significantly higher in the non-RVR group than in the RVR group. In the No EVR group, gene expression was significantly higher than in the EVR group. It is concluded that targeting HCV proteins using DAAs can cause USP18 expression to be normalized more effectively. Moreover, USP18 is a vital marker indicating treatment resistance and distinguishing responders from non-responders during DAA therapy.

Improving Access to Anti-HDV Testing: Development and Validation of an Affordable In-House ELISA Assay.

In certain low-income nations, the hepatitis Delta virus and hepatitis B virus (HBV) pose a serious medical burden, where the prevalence of hepatitis B surface antigen (HBsAg) is greater than 8%. Especially in rural places, irregular diagnostic exams are the main restriction and reason for underestimation. Utilizing serum samples from a Pakistani isolate, an internal ELISA for the quick identification of anti-HDV was created, and the effectiveness of the test was compared to a commercial diagnostic kit. HDV-positive serum samples were collected, and a highly antigenic domain of HDAg antigen was derived from them. This antigenic HDAg was expressed in a bacterial expression system, purified by Ni-chromatography, and confirmed by SDS-PAGE and Western blot analysis. The purified antigen was utilized to develop an in-house ELISA assay for anti-HDV antibody detection of the patient's serum samples at very low cost. Purified antigens and positive and negative controls can detect anti-HDV (antibodies) in ELISA plates. The in-house developed kit's efficiency was compared with that of a commercial kit (Witech Inc., USA) by the mean optical density values of both kits. No significant difference was observed (a P value of 0.576) by applying statistical analysis. The newly developed in-house ELISA is equally efficient compared to commercial kits, and these may be useful in regular diagnostic laboratories, especially for analyzing local isolates.

Genotypic distribution of human papillomavirus and phylogenetic analysis of E6 and E7 gene of HR-HPV variants isolated from Pakistani population.

High-risk-human papillomavirus (HR-HPV)-induced cervical cancer is the second most common cause of death among females worldwide. HPV16 is the most prevalent HR-HPV infection worldwide. This study found the genotypic distribution of HR-HPV in the local population and investigated the sequence variations among the E6 and E7 oncogenes of the local HPV16 genotype to the E6 and E7 oncogenes of the foreign HPV16 genotypes and constructed a phylogenetic relationship based on nucleotide sequence comparison among the variants identified in our study along with previously reported isolates that were obtained from different regions of the world. The samples were collected from patients with cervical cancer. Genomic DNA was extracted, and HR-HPV genotypes were determined using real-time PCR. The HPV16 E6 and E7 genes were amplified and sequenced. A HPV16 phylogenetic tree was constructed using the maximum likelihood method with MEGA 7. HPV16 was the most prevalent human papillomavirus (HPV) type identified in the present study. HPV16 isolates belonged to the A1 sublineage of the European branch. Twenty-one nucleotide sequences were included in this analysis. The first, second, and third codon positions are also included. The final dataset included 776 positions.

Expression Profiling of the Tripartite Motif Family Genes in Chronic Hepatitis C Patients.

Hepatitis C virus (HCV) is one of the most prevalent pathogens which causes significant morbidity and mortality in 2% of the world's population. Several interferon-stimulated genes (ISGs) are involved in HCV clearance by interacting with the viral proteins. Among these ISGs, the tripartite motif (TRIM) family genes are elevated during HCV infection. This study aims to evaluate the expression of three TRIM family genes in chronic hepatitis C patients, distributed among different groups, including TRIM11, TRIM14, and TRIM25. A total of 242 participants were recruited in this study, including 182 infected patients, 37 naïve individuals, and 23 control individuals. Out of 182 infected patients, 100 achieved sustained virologic response (SVR), 61 achieved rapid virologic response (RVR), and 21 patients developed hepatocellular carcinoma (HCC), showing no response to the given treatments. Our results indicate highest expression levels of TRIM mRNA transcripts in the RVR group with the highest increase of 7.5 folds in TRIM25, 6.68 folds in TRIM14, followed by the data from patients of the SVR group. The elevation was also evident in other groups, i.e., SVR and HCC, in different patterns among all the three TRIM genes. In addition to elevation in expression levels, a linear correlation is observed between the TRIM mRNAs and viral loads of HCV. These results showed the potential role of TRIM family genes in HCV restriction.

Decoding the mystery of MMTV-like virus and its relationship with breast cancer metastasis.

BACKGROUND: MMTV causes mammary tumors in mice, and it is associated with invasive and aggressive forms of breast cancer in humans. However, the underlying mechanisms are yet unknown. We aimed to determine the MMTV-like virus (MMTV-LV) association with histological types of breast cancer, nodal involvement, and metastasis. METHODS: First, 105 breast cancer biopsies and 15 disease-free biopsies were collected. Details of clinicopathological characteristics were retrieved from patients' records. The status of MMTV-LV was already known for these biopsy samples. Associations of MMTV-LV prevalence with LNM status and metastatic history were determined. Next, quantitative PCR (qPCR) was used to quantify env gene mRNA in biopsies positive for MMTV-LV. Expression of the env gene was compared against different histopathological types of mammary tumor, LNM status, and metastasis by performing Ordinary One Way ANOVA followed by Tukey's multiple comparisons test. RESULTS: MMTV-LV prevalence was found to have no significant association with LNM or metastatic history. As compared to normal control, expression of the env gene was significantly higher (>2.8 folds) in invasive samples (P-value: < 0.01). Expression was also higher (3.28 and 2.89 folds) in patient samples with LNM (P-value: 0.0006) or metastatic history (P-value: < 0.0001), respectively. CONCLUSION: We conclude that MMTV-LV prevalence is not associated with LNM status or breast cancer metastasis; samples with invasive phenotypes, nodal involvement, and metastasis exhibit significantly higher expression of the MMTV-like env gene.

Seroprevalence of SARS-CoV-2: Insights into the epidemiology of the pandemic.

AIM: This study aimed to assess the seroprevalence of SARS-CoV-2 and disease symptoms in Malakand, Pakistan. MATERIALS & METHOD: 623 samples with suspected SARS-CoV-2 were collected from different regions of Malakand and analyzed to detect SARS-CoV-2 IgG antibodies using ELISA. RESULTS: 306 (49.1%) 0 f 623 patients were anti-SARS-CoV-2 IgG reactive, with a higher prevalence in males (75%) than females (25%). In this study, we enrolled two groups, subjects working in a non-medical setting and subjects working in a medical setting. Clinical symptoms were statistically linked with SARS-CoV-2. Four weeks of follow-up analysis of IgG titers in health care workers showed an increase in IgG antibodies titer. CONCLUSION: This study gives insights into the community-based spread of SARS-CoV-2 infection, associated immunity, and herd immunity in the studied population. This study can provide insights to the government about early vaccination of this population as most of the population is not yet vaccinated.

Transcriptional Analysis of TP53 Gene in Chronic Hepatitis C Patients Treated with Sofosbuvir, Daclatasvir, Pegylated Interferon, and Ribavirin.

Hepatitis C virus (HCV) is a major public health problem that affects more than 170 million people globally. HCV is a principal cause of hepatocellular carcinoma (HCC) around the globe due to the high frequency of hepatitis C infection, and the high rate of HCC is seen in patients with HCV cirrhosis. TP53 is considered as a frequently altered gene in all cancer types, and it carries an interferon response element in its promoter region. In addition to that, the TP53 gene also interacts with different HCV proteins. HCV proteins especially NS3 protein and core protein induce the mutations in the TP53 gene that lower the expression of this gene in HCV patients and leads to HCC development. In this study, we examined the transcriptional analysis of the TP53 gene in HCV-infected patients administered with different combinations of antiviral therapies including sofosbuvir + daclatasvir, sofosbuvir + ribavirin, and pegylated interferon + ribavirin. This study included 107 subjects; 15 treated with sofosbuvir + daclatasvir, 58 treated with sofosbuvir + ribavirin, 11 treated with interferon + ribavirin, 8 untreated, 10 HCC patients, and 5 were healthy controls. Total RNA was extracted from the PMBCs of HCV infected patients and reverse transcribed into cDNA using a gene specific reverse primer. The expression level of TP53 mRNA was analyzed using quantitative PCR. The expression of TP53 mRNA was notably upregulated in rapid virological response (RVR), early virological response (EVR), and sustained virological response (SVR) groups as compared to non-responders and naïve groups. The expression of TP53 mRNA was seen high in HCC as compared to control groups. Additionally, it has been demonstrated that sofosbuvir + daclatasvir treatment stimulates significant elevation in TP53 gene expression as compared to (sofosbuvir + ribavirin) and (IFN + ribavirin) treatment. This study indicates that the TP53 gene expression is highly upregulated in RVR, EVR, and SVR groups as compared to control groups. Moreover, sofosbuvir + daclatasvir therapy induces significant rise in TP53 mRNA expression levels as compared to (sofosbuvir + ribavirin) and (IFN + ribavirin) treatment. According to these results, it can be concluded that sofosbuvir + daclatasvir plays a significant role in preventing HCV patients from developing severe liver complications as compared to other administered therapies. This study is novel as no such type of study has been conducted previously on the expression of TP53 in local HCV-infected population treated with different combinations of therapies. This study is helpful for the development of new therapeutic strategies and for improving existing therapies.