Evolutionary patterns and heterogeneity of Dengue Virus serotypes in Pakistan.

Comprehensive and systematic examination of Dengue virus (DENV) evolution is essential in the context of Pakistan as the virus presents a significant public health challenge with the ability to adapt and evolve. To shed light on intricate evolutionary patterns of all four DENV serotypes, we analyzed complete genome sequences (n=43) and envelope (E) gene sequences (n=44) of all four DENV serotypes collected in Pakistan from 1994 to 2023 providing a holistic view of their genetic evolution. Our findings revealed that all four serotypes of DENV co-circulate in Pakistan with a close evolutionary relationship between DENV-1 and DENV-3. Genetically distinct serotypes DENV-2 and DENV-4 indicate that DENV-4 stands out as the most genetically different, while DENV-2 exhibits greater complexity due to the presence of multiple genotypes and the possibility of temporal fluctuations in genotype prevalence. Selective pressure analysis in Envelope (E) gene revealed heterogeneity among sequences (n=44) highlighting 46 codons in the genome experiencing selective pressure, characterized by a bias towards balancing selection indicating genetic stability of the virus. Furthermore, our study suggested an intriguing evolutionary shift of DENV-4 towards the DENV-2 clade, potentially influenced by antibodies with cross-reactivity to multiple serotypes providing a critical insight into the complex factors shaping DENV evolution and contributing to the emergence of new serotype.

Comparative analysis of chicken cecal microbial diversity and taxonomic composition in response to dietary variation using 16S rRNA amplicon sequencing.

BACKGROUND: Antibiotic resistance poses a grave threat to One-Health. By replacing antibiotics with non-antibiotic additives (are alternatives to antibiotics, ATAs) like phytogenic feed additives and organic acids in poultry feed. ATAs are a potential alternative as these decline the proliferation of pathogenic bacteria and strengthen gut function in broiler chickens. In this study, we use 16S rRNA amplicon sequencing of the V3-V4 region to evaluate phytogenic feed additives and organic acids on the cecal microbial diversity of broiler chickens. METHODS AND RESULTS: Two hundred & forty broiler chicks were divided into five treatments comprising: a controlled basal diet (CON), antibiotic group (AB), phytogenic feed additives (PHY), organic acids (ORG), and a combination of PHY + ORG (COM). A distinctive microbial community structure was observed amongst different treatments with increased microbial diversity in AB, ORG, and COM (p < 0.05). The synergistic effects of PHY and ORG increased bacterial population of phyla: Firmicutes, Bacteroides, and Proteobacteria in the cecum. The presence of species, Akkermansia muciniphila (involved in mucin degradation) and Bacillus safensis (a probiotic bacterium) were noticed in COM and PHY, respectively. Clustering analysis revealed a higher relative abundance of similar microbial community composition between AB and ORG groups. CONCLUSIONS: Treatments with PHY and ORG modified the relative abundance and presence/absence of specific microbiota in the chicken cecum. Hence, cecal microbiota modulation through diet is a promising strategy to reduce cross-contamination of zoonotic poultry pathogens, led to healthy and economical broiler meat.

Paradoxical Role of Dengue Virus Envelope Protein Domain III Antibodies in Dengue Virus Infection.

Every year, approximately 100 million individuals are infected with dengue viral infections. Severe dengue infection, characterized as dengue hemorrhagic fever, leads to loss of intravascular fluids and severe bleeding. During dengue virus (DENV) secondary infection, the body produces neutralizing antibodies that cause a strong immune response, resulting in severe hemolysis and plasma leakage. DENV infections in humans stimulate production of virus serotype-specific and cross-reactive antibodies. The envelope (E) protein of DENV contains potent antigenic sites, with one known as E protein domain III (EDIII). Studies of DENV EDIII in mouse models have shown that strongly neutralizing mouse monoclonal antibodies (mAbs) are DENV-serotype specific and bind to an epitope on EDIII that is unique to each serotype. Unlike DENV-serotype-specific mouse mAbs, cross-reactive mAbs that bind to EDIII have moderate-to-weak neutralizing activity. Studies with mouse mAbs resulted in identification and mapping of different epitopes on the lateral ridge of DENV EDIII.

CRISPR-CAS Replacing Antiviral Drugs against HIV: An Update.

Various antiretroviral drugs do not kill or cure the human immunodeficiency virus (HIV) but do prevent the replication of the virus. The combination of antiretroviral drugs is known as highly active antiretroviral therapy (HAART). Current drug therapies effectively suppress HIV-1 replication but do not inactivate the provirus that persists in latent reservoirs. Guide RNA (gRNA)-directed CRISPR/Cas9 system can be used for sequence-specific attacks on this proviral DNA. The biggest achievement might be the complete elimination of HIV from infected cells. A study revealed that the tail injection, in transgenic mice and rats having HIV-1 genome, of an adenoassociated virus (AAV) vector expressing a short version of the Cas9 endonuclease (saCas9) and the gRNAs resulted in the cleavage of integrated HIV-1 DNA and excision of a DNA fragment spanning between the LTR and Gag gene in the spleen, liver, heart, kidney, and circulating lymphocytes. HIV-1 has capacity to escape the attack on its genome from most of inhibitors. Thus, to achieve successful antiretroviral treatment, combinations of several antiviral therapies have been applied that are based on two important facts. The first is that multiple drugs lead to synergistic or additive inhibition, and the second is that the combinational therapy increases chances of drug resistance. The success that has been achieved with the help of the genetically engineered tool CRISPR is that dCas9 protein alone can efficiently silence viral gene expression in bacteria with sgRNA. All the reported investigations have indicated that CRISPR/Cas9 can be used as immune machinery into human cells in the form of novel antivirus tools.

Conserved B and T cell epitopes prediction of ebola virus glycoprotein for vaccine development: An immuno-informatics approach.

Ebola virus (EBOV), a non-segmented single-stranded RNA virus, is often-most transmitted through body fluids like sweat, tears, saliva, and nasal secretions. Till date, there is no licensed vaccine of EBOV is available in the market; however, the world is increasingly vulnerable to this emerging threat. Hence, it is the need of time to develop a vaccine for EBOV to hinder its dissemination. The current study has been designed for identification and characterization of the potential B and T-cell epitopes using the Immuno-informatics tools, and it helped in finding the potent vaccine candidates against EBOV. Prediction, antigenicity and allergenicity testing of predicted B and T cells' epitopes was done as well to identify their potential as a vaccine candidate and to measure their safety level respectively. Among B-cell epitopes "WIPAGIGVTGVIIA" showed a high antigenicity score and it would play an important role in evoking the immune response. In T-cell epitopes, peptides "AIGLAWIPY" and "IRGFPRCRY" presented high antigenicity score, which binds to MHC class-I and MHC class-II alleles respectively. All predicted epitopes were analyzed and compared with already reported peptides carefully. Comparatively, Peptides predicted in the present study showed more immunogenicity score than already reported peptides, used as positive control, and are more immunogenic as compared to them. Peptides reported in the present study do not target only Zaire EBOV (ZEBOV), as in previous studies, but also other species, i.e. Tai Forest EBOV (TAFV), Sudan EBOV (SUDV), Bundibugyo EBOV (BDBV), and Reston EBOV (RESTV) and would bring the promising results as potent vaccine candidates.

Crimean-Congo Hemorrhagic Fever (CCHF) in Pakistan: The "Bell" is Ringing Silently.

Pakistan is being hit by communicable and noncommunicable diseases over time. Among these, tickborne viral disease, Crimean-Congo hemorrhagic fever (CCHF) is one of the most fatal infections. Rapid climate change aroused by industrial, occupational, and agricultural activities to support ever-growing human population has been considered the single most causative basis for emergence or re-emergence of CCHF in Pakistan, where it has biannual peaks between the months of March-May and August-October. Many factors, including poor sanitation at farms, villages, and cities, unhygienic transportation and slaughter of animals at numerous sites within a city, inefficient tick-control programs, post-slaughter piles of animal remains other than meat, nomadic lifestyle, and lack of trained animal and human healthcare staff, are contributing to the spread of CCHF. Pakistan has confirmed cases of CCHF in almost every province: Sindh (Karachi), Punjab (Faisalabad, Multan, and Rawalpindi), Balochistan (Quetta) and Khyber Pakhtunkhwa (Peshawar). The root cause behind the spread of CCHF in Pakistan seems to be the absence of an effective disease surveillance system in the human as well as the animal populations. Most of the time, CCHF cases are not diagnosed, and if they are diagnosed they are not reported. If these cases are reported, there are not enough effective measures by the relevant provincial and district authorities. There is a need to educate the general public, farmers, and healthcare workers about the causes, transmission, and dangers of CCHF. An immediate plan for the implementation of a surveillance system, standard preventive measures, early detection, proper treatment, and timely response is urgently needed. Without such a plan, the accumulation of factors responsible for the sudden outbreak of CCHF may pose a serious threat to humans and animals in different geographical regions of the country.

Deadly outbreak of chickenpox at district Faisalabad, Pakistan: possible causes, and preventive way forward.

This article details our recommendations for the deadly outbreak of chickenpox to consider the additional referral of the absence of a monitoring system of prevention and control along with poor vaccination system for children in low-resource settings. The recent spread of chickenpox outbreak in Pakistan has claimed dozens of lives. The deaths in this current outbreak in quick successions are beyond understanding. Re-emergence of chickenpox in the area has raised many questions. Keeping in view the spread of chickenpox mainly in Faisalabad and its international reputation in trading, chickenpox breakout needs international attention to control its spread. It should be taken as an eye opener for the Government of Pakistan and government should develop and implement Centralized Infectious Disease Reporting Information Management System that will help to narrow down the pathogens as far as the epidemics are concerned and also for early preventive and countermeasure response.

Genetically Modified Aedes aegypti to Control Dengue: A Review.

Dengue is an acute infectious disease of viral etiology characterized by lymphadenopathy, leucopenia, headache, biphasic fever, pain in various parts of the body, rashes, and extreme physical weakness. It is a vector-borne disease caused by a positive-stranded RNA virus of the family Flaviviridae, genus Flavivirus. Dengue inflicts a significant health, economic, and social burden on populations of endemic areas. Dengue virus is transmitted to humans by the mosquito vector Aedes aegypti. Vaccines against dengue viruses have been claimed to be developed, but as yet no effective treatment is available. Alternative therapeutic strategies to overcome this disease and its spread are direly needed. A traditional sterile insect technique (SIT) harms the health of male insects, leading to their reduced ability to compete for wild-type female insects for breeding. Oxitec (Abingdon, UK) has developed genetically modified (GM) strains of A. aegypti via the release of insects carrying a dominant lethal (RIDL) strategy. RIDL male mosquitoes offer a resolution to many of the limitations of traditional SIT, which has resulted in reduced application of SIT in mosquitoes. The technique using RIDL mosquitoes is considered to be ecologically friendly and specific. Homing endonuclease genes, also called selfish genes, can also be used in genetic modification methods in such a way that the vector population and its competency can be reduced. GM mosquitoes carrying a gene that transcribes RNA interference can also be crucial to control expression of RNA viruses. The RNA virus interference pathway is one of the most critical components of the innate immune system of insects that can frustrate a variety of RNA viruses such as Flaviviruses. Here, we summarize and focus on alternative techniques used to control dengue spread.

Deep learning guided prediction modeling of dengue virus evolving serotype.

Evolution remains an incessant process in viruses, allowing them to elude the host immune response and induce severe diseases, impacting the diagnostic and vaccine effectiveness. Emerging and re-emerging diseases are among the significant public health concerns globally. The revival of dengue is mainly due to the potential for naturally arising mutations to induce genotypic alterations in serotypes. These transformations could lead to future outbreaks, underscoring the significance of studying DENV evolution in endemic regions. Predicting the emerging Dengue Virus (DENV) genome is crucial as the virus disrupts host cells, leading to fatal outcomes. Deep learning has been applied to predict dengue fever cases; there has been relatively less emphasis on its significance in forecasting emerging DENV serotypes. While Recurrent Neural Networks (RNN) were initially designed for modeling temporal sequences, our proposed DL-DVE generative and classification model, trained on complete genome data of DENV, transcends traditional approaches by learning semantic relationships between nucleotides in a continuous vector space instead of representing the contextual meaning of nucleotide characters. Leveraging 2000 publicly available DENV complete genome sequences, our Long Short-Term Memory (LSTM) based generative and Feedforward Neural Network (FNN) based classification DL-DVE model showcases proficiency in learning intricate patterns and generating sequences for emerging serotype of DENV. The generated sequences were analyzed along with available DENV serotype sequences to find conserved motifs in the genome through MEME Suite (version 5.5.5). The generative model showed an accuracy of 93 %, and the classification model provided insight into the specific serotype label, corroborated by BLAST search verification. Evaluation metrics such as ROC-AUC value 0.818, accuracy, precision, recall and F1 score, all to be around 99.00 %, demonstrating the classification model's reliability. Our model classified the generated sequences as DENV-4, exhibiting 65.99 % similarity to DENV-4 and around 63-65 % similarity with other serotypes, indicating notable distinction from other serotypes. Moreover, the intra-serotype divergence of sequences with a minimum of 90 % similarity underscored their uniqueness.

Prospects of Microfluidic Technology in Nucleic Acid Detection Approaches.

Conventional diagnostic techniques are based on the utilization of analyte sampling, sensing and signaling on separate platforms for detection purposes, which must be integrated to a single step procedure in point of care (POC) testing devices. Due to the expeditious nature of microfluidic platforms, the trend has been shifted toward the implementation of these systems for the detection of analytes in biochemical, clinical and food technology. Microfluidic systems molded with substances such as polymers or glass offer the specific and sensitive detection of infectious and noninfectious diseases by providing innumerable benefits, including less cost, good biological affinity, strong capillary action and simple process of fabrication. In the case of nanosensors for nucleic acid detection, some challenges need to be addressed, such as cellular lysis, isolation and amplification of nucleic acid before its detection. To avoid the utilization of laborious steps for executing these processes, advances have been deployed in this perspective for on-chip sample preparation, amplification and detection by the introduction of an emerging field of modular microfluidics that has multiple advantages over integrated microfluidics. This review emphasizes the significance of microfluidic technology for the nucleic acid detection of infectious and non-infectious diseases. The implementation of isothermal amplification in conjunction with the lateral flow assay greatly increases the binding efficiency of nanoparticles and biomolecules and improves the limit of detection and sensitivity. Most importantly, the deployment of paper-based material made of cellulose reduces the overall cost. Microfluidic technology in nucleic acid testing has been discussed by explicating its applications in different fields. Next-generation diagnostic methods can be improved by using CRISPR/Cas technology in microfluidic systems. This review concludes with the comparison and future prospects of various microfluidic systems, detection methods and plasma separation techniques used in microfluidic devices.

Rabies molecular virology, diagnosis, prevention and treatment.

Rabies is an avertable viral disease caused by the rabid animal to the warm blooded animals (zoonotic) especially human. Rabies occurs in more than 150 countries and territories. According to an estimation by WHO, almost 55,000 people die because of rabies every year. The Dogs are the major reason behind this, approximately 99% human deaths caused by dog's bites. Developing and under developing countries, both are the victims of rabies. With the post-exposure preventive regimes, 327,000 people can prevent this disease annually.The current article mainly covers the genome, virology, symptoms, epidemiology, diagnostic methods, and the high risk countries around the globe.

Isolation and characterization of two new hepatitis E virus genotype 1 strains from two mini-outbreaks in Lahore, Pakistan.

BACKGROUND: Pakistan is a highly endemic area for hepatitis E virus (HEV) infection. The aim of the current study was to isolate and characterize strains of HEV in two mini outbreaks. RESULTS: RNA was extracted and reverse transcribed to cDNA. Nested PCR was done for the detection of HEV RNA. The positive bands were eluted, cloned in TA vector and sequenced in both directions using genetic Analyzer (Applied Biosystem Inc, USA). Phylogenetic analysis was done using MEGA4 software. We isolated two new HEV genotype-1 strains from Lahore, Pakistan, based on cloning and sequencing of ORF2 region. CONCLUSIONS: Our study suggest that both the human HEV strains were closely related to the Sar-55 but different from the Abb-2B and 87-Pakistan-B HEV isolates sharing 88-91% sequence identity to Pakistani isolate Sar-55. These results indicated that Sar-55 is the main endemic HEV strain in various areas of the country.

Molecular investigation of mental retardation locus gene PRSS12 by linkage analysis.

The present study was carried out to determine the prevalence of families having mental retardation in Pakistani population. We enrolled seven mentally retarded (MR) families with two or more affected individuals. Family history was taken to minimize the chances of other abnormalities. Pedigrees were drawn using the Cyrillic software (version 2.1). The structure of pedigrees shows that all the marriages are consanguineous and the families have recessive mode of inheritance. All the families were studied by linkage analysis to mental retardation locus (MRT1)/gene PRSS12. Three STR markers (D4S191, D4S2392, and D4S3024) in vicinity of mental retardation (MR) locus (MRT1)/gene PRSS12 were amplified on all the sample of each family by PCR. The PCR products were then genotyped on non denaturing polyacrylamide gel electrophoresis (PAGE). The Haplotype were constructed to determine the pattern of inheritance and also to determine that a family was linked or unlinked to gene PRSS12. One out of the seven families was potentially linked to gene PRSS12, while the other six families remain unlinked.

Thymoquinone attenuates rheumatoid arthritis by downregulating TLR2, TLR4, TNF-α, IL-1, and NFκB expression levels.

BACKGROUND: Thymoquinone (TQ), the most important active principle of Nigella sativa is known to have anti-inflammatory, analgesic, antimicrobial and antioxidant properties. AIM: The present study was designed to see the anti-arthritic effect of TQ in rat model of arthritis. METHODS: In the current research, anti-arthritic effect of TQ was determined in Freund's Complete Adjuvant (FCA)-induced arthritic rats by measuring TLRs expression levels. The mRNA expression levels of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), interleukin-1 (IL-1), nuclear factor-kappa B (NFκB) and tissue necrosis factor-α (TNF-α) were measured by reverse-transcription polymerase chain reaction. Arthritic signs were observed by macroscopic criteria. Hematoxylin and Eosin staining was used to perform ankle joint histopathology and agglutination method was used for measuring C-reactive protein (CRP) levels. Rheumatoid factor, alanine transaminase, aspartate aminotransferase, urea and creatinine were also determined in serum. RESULTS: TQ treatment reduced the macroscopic arthritic score, levels of CRP, synovial inflammation, pannus formation and bone erosion. It also reduced the mRNA levels of TLR2, TLR4, IL-1, NFκB and TNF-α. Methotrexate, used as a reference drug also significantly decreased their expression levels. TQ also normalized the hematological markers and did not depict any signs of hepatotoxicity and nephrotoxicity as determined by serum levels of alanine transaminase, aspartate aminotransferase, urea and creatinine. Our results showed that TQ possesses significant anti-arthritic activity which can be due to its anti-inflammatory and immunomodulatory effects. CONCLUSION: Results indicate that TQ has got the potential to ameliorate rheumatoid arthritis by downregulating TLR2, TLR4, TNF-α, IL-1, and NFκB expression levels.

Recent Advances in Nanoparticle-Based Targeted Drug-Delivery Systems Against Cancer and Role of Tumor Microenvironment.

Cancer is one of the major causes of death worldwide. The silent activation of cellular factors responsible for deviation from normal regulatory pathways leads to the development of cancer. Nano-biotechnology is a novel drug-delivery system with high potential of efficacy and accuracy to target lethal cancers. Various biocompatible nanoparticle (NP)-based drug-delivery systems such as liposomes, dendrimers, micelles, silica, quantum dots, and magnetic, gold, and carbon nanotubes have already been reported for successful targeted cancer treatment. NPs are functionalized with different biological molecules, peptides, antibody, and protein ligands for targeted drug delivery. These systems include a hydrophilic central core, a target-oriented biocompatible outer layer, and a middle hydrophobic core where the drug destined to reach target site resides. Most of the NPs have the ability to maintain their structural shape and are constructed according to the cancer microenvironment. The self-assembling and colloidal properties of NPs have caused them to become the best vehicles for targeted drug delivery. The tumor microenvironment (TME) plays a major role in cancer progression, detection, and treatment. Due to its continuous complex behavior, the TME can hinder delivery systems, thus halting cancer treatment. Nonetheless, a successful biophysiological interaction between the NPs and the TME results in targeted release of drugs. Currently, a number of drugs and NP-based delivery systems against cancer are in clinical and preclinical trials and a few have been approved by Food and Drug Administration (FDA); for example: taxol, doxil, cerubidine, and adrucil. This review summarizes topical advances about the drugs being used for cancer treatment, their targeted delivery systems based on NPs, and the role of TME in this connection.

Ex vivo expansion of functional human UCB-HSCs/HPCs by coculture with AFT024-hkirre cells.

Kiaa1867 (human Kirre, hKirre) has a critical role in brain development and/or maintenance of the glomerular slit diaphragm in kidneys. Murine homolog of this gene, mKirre expressed in OP9 and AFT024 cells could support hematopoietic stem cells/hematopoietic progenitor cells (HSC/HPC) expansion in vitro. HKirre is also expressed in human FBMOB-hTERT cell line and fetal liver fibroblast-like cells but its function has remained unclear. In this paper, we cloned a hKirre gene from human fetal liver fibroblast-like cells and established a stably overexpressing hKirre-AFT024 cell line. Resultant cells could promote self-renewal and ex vivo expansion of HSCs/HPCs significantly higher than AFT024-control cells transformed with mock plasmid. The Expanded human umbilical cord blood (hUCB) CD34(+) cells retained the capacity of multipotent differentiation as long as 8 weeks and successfully repopulated the bone marrow of sublethally irradiated NOD/SCID mice, which demonstrated the expansion of long-term primitive transplantable HSCs/HPCs. Importantly, hkirre could upregulate the expressions of Wnt-5A, BMP4, and SDF-1 and downregulate TGF- ß with other hematopoietic growth factors. By SDS-PAGE and Western Blot analysis, a ~89 kDa protein in total lysate of AFT024-hKirre was identified. Supernatants from AFT024-hkirre could also support CD34(+)CD38(-) cells expansion. These results demonstrated that the AFT024-hKirre cells have the ability to efficiently expand HSCs/HPCs.

Multifunctional Fe5 C2 nanoparticles: a targeted theranostic platform for magnetic resonance imaging and photoacoustic tomography-guided photothermal therapy.

Fe5 C2 NPs exhibit a high contrast in magnetic resonance imaging (MRI), superior photoacoustic tomography improvements, and efficient photothermal therapy (PTT) due to their unique core/shell structure, with a magnetic core and carbon shell. By conjugating a new class of affinity proteins (ZHER2:342), they can target to tumor cells with low cytotoxicity, and kill them through laser irritation. It is also possible to ablate tumors under guidance by MRI and PTT without noticeable side effects.

Evaluation of three different hepatitis C virus typing methods for detection of mixed-genotype infections.

OBJECTIVE: To evaluate the clinical applicability of an eligible assay for the true prevalence of hepatitis C virus (HCV) mixed-genotype infections. METHODS: A newly developed HCV genotyping method targeting all six major HCV genotypes and 12 subtypes, restriction fragment length polymorphism (RFLP) and a serotyping assay were utilized for the detection of HCV mixed-genotype infections using known HCV genotypes and unknown samples. RESULTS: In a defined mix of HCV genotypes, a genotype present at levels as low as 8.3% was detected by our newly developed assay, showing a threefold increase in sensitivity over that of direct deoxyribonucleic (DNA) sequencing. A comparative study of the accuracy among the three genotyping methods was carried out on samples obtained from 50 thalassemic patients who received multiple blood transfusions. The results showed that viruses in approximately 42% of the samples from this group were determined to be infected with mixed genotypes by our newly developed method. A serotyping assay and RFLP analysis, performed with poor results, could identify only 18% and 10% of mixed-genotype infections, respectively. CONCLUSION: The newly developed assay may be the method of choice when detection of genotypes present at low levels in mixed-genotype infections due to its higher level of sensitivity.

Hepatitis C virus genotype 3a infection and hepatocellular carcinoma: Pakistan experience.

AIM: To assess the association between chronic hepatitis C virus (HCV) infection and hepatocellular carcinoma (HCC) in Pakistan, and the genotype distribution among these HCC patients. METHODS: One hundred and sixty-one subjects with HCC were included in this study. Liver biopsy was performed on 145 of the patients; sixteen were excluded because they failed to fulfill the inclusion criteria. Qualitative polymerase chain reaction (PCR) was performed for hepatitis B virus and HCV. Samples positive for HCV RNA were genotyped using genotype-specific PCR and confirmed by HCV 5' noncoding region sequencing analysis. RESULTS: Chronic HCV infection was identified a major risk factor (63.44% of tested HCC patients) for the development of HCC. The time from HCV infection to appearance of cancer was 10-50 years. In the HCC patient population, broader distributions of genotypes were present with genotype 3a as the predominant genotype. Using the type-specific genotyping method, we found HCV genotype 3a in 40.96%, 3b in 15.66%, 1a in 9.63%, and 1b in 2.40% of HCC tissue samples. About 28% of cases were found with mixed genotypes. Two cases were unable to be genotyped because of low viral load. Sixty-six percent of treated patients with cirrhosis had an end of treatment response, but unfortunately they relapsed quickly when the treatment was discontinued, and HCC developed during a median 3.8 years. CONCLUSION: There was a strong association between chronic HCV infection and HCC in Pakistan, and between HCV genotype 3a and HCC.