Shazia Rafique

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.

Hepatitis C virus (HCV) causes various liver complications, including fibrosis, cirrhosis, and steatosis, and finally progresses toward hepatocellular carcinoma (HCC). The current study aimed to explore the antiviral activity of the traditional Pakistani medicinal plant Salix nigra (S. nigra) known as black willow against the hepatitis C virus (HCV). The anti-HCV activity of S. nigra was established against stable Hep G2 cell lines expressing the HCV NS3 gene. Various plant-derived compounds with anti-HCV activity were identified, making phytotherapy a promising alternative to conventional treatments due to their cost-effectiveness and milder side effects. The two extraction methods (Maceration and Soxhlet) and four solvents (n-hexane, methanol, ethyl acetate, and water) were used to obtain crude extracts from S. nigra. Cytotoxicity testing showed that methanol (CC₅₀ 25 μg/mL) and water (CC₅₀ 30 μg/mL) extracts were highly toxic, while ethyl acetate and n-hexane (CC₅₀ > 200 μg/mL) extracts were nontoxic at low concentrations (10-50 μg/mL), making them suitable for further anti-HCV investigations. Stable transfection of the NS3 gene was successfully performed in Hep G2 cells, creating a cellular expression system for studying virus-host interaction. The ethyl acetate extract of S. nigra exhibited significant inhibition of NS3 gene expression (mRNA and protein levels). The phytochemical analysis of S. nigra was also performed using the high-performance liquid chromatography (HPLC) technique. The phytochemical analysis identified several polyphenolic substances in the extracts of S. nigra. Our results concluded that the extracts of S. nigra have significantly reduced the expression of the NS3 gene at mRNA and protein levels. These findings contribute to the global efforts to combat hepatitis C by offering plant-based treatment options for HCV management.

Crimean–Congo haemorrhagic fever (CCHF), caused by Crimean–Congo haemorrhagic fever virus (CCHFV), is a disease of worldwide importance (endemic yet not limited to Asia, Middle East, and Africa) and has triggered several outbreaks amounting to a case fatality rate of 10–40% as per the World Health Organization. Genetic diversity and phylogenetic data revealed that the Asia-1 genotype of CCHFV remained dominant in Pakistan, where 688 confirmed cases were reported between the 2012–2022 period. Currently, no approved vaccine is available to tackle the viral infection. Epitope-based vaccine design has gained significant attention in recent years due to its safety, timeliness, and cost efficiency compared to conventional vaccines. In the present study, we employed a robust immunoinformatics-based approach targeting the structural glycoproteins G1 and G2 of CCHFV (Asia-1 genotype) to design a multi-epitope vaccine construct. Five B-cells and six cytotoxic T-lymphocytes (CTL) epitopes were mapped and finalized from G1 and G2 and were fused with suitable linkers (EAAAK, GGGS, AAY, and GPGPG), a PADRE sequence (13 aa), and an adjuvant (50S ribosomal protein L7/L12) to formulate a chimeric vaccine construct. The selected CTL epitopes showed high affinity and stable binding with the binding groove of common human HLA class I molecules (HLA-A*02:01 and HLA-B*44:02) and mouse major histocompatibility complex class I molecules. The chimeric vaccine was predicted to be an antigenic, non-allergenic, and soluble molecule with a suitable physicochemical profile. Molecular docking and molecular dynamics simulation indicated a stable and energetically favourable interaction between the constructed antigen and Toll-like receptors (TLR2, TLR3, and TLR4). Our results demonstrated that innate, adaptive, and humoral immune responses could be elicited upon administration of such a potent muti-epitope vaccine construct. These results could be helpful for an experimental vaccinologist to develop an effective vaccine against the Asia-1 genotype of CCHFV.

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.

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.

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.