The global emergence of Chikungunya infection: An integrated view.

Chikungunya virus (CHIKV) is one of the emerging viruses around the globe. It belongs to the family Togaviridae and genus Alphavirus and is an arthropod borne virus that transmits by the bite of an infected mosquito, mainly through Aedes aegypti and Aedes albopcitus. It is a spherical, enveloped virus with positive single stranded RNA genome. It was first discovered during 1952-53 in Tanganyika, after which outbreaks were documented in many regions of the world. CHIKV has two transmission cycles; an enzootic sylvatic cycle and an urban cycle. CHIKV genome contains 11,900 nucleotides and two open reading frames and shows great sequence variability. Molecular mechanisms of virus host-cell interactions and the pathogenesis of disease are not fully understood. The disease involves three phases; acute, post-acute and chronic with symptoms including high-grade fever, arthralgia, macupapular rashes and headache. There is no licensed vaccine or specific treatment for CHIKV infection. This lack of specific interventions combined with difficulties in making a precise diagnosis together make the disease difficult to manage. In this review we aim to present the current knowledge of global epidemiology, transmission, structure, various aspects of diagnosis as well as highlight potential antiviral drugs and vaccines against CHIKV.

Induction of Profibrotic Microenvironment via TLR4 MyD88-Dependent and -Independent Inflammatory Signaling in Chronic Hepatitis C Virus Infection.

Hepatitis C virus (HCV) is a well-known pathogen to establish chronic infection leading to end-stage liver disease. The destruction of liver tissues takes its roots under chronic inflammation and proinflammatory signaling in liver microenvironment. The viral proteins interact with certain pattern recognition receptors, including Toll-like receptors, activating the innate immune system to clear the virus. HCV achieves immune evasion through other mechanisms and induce a continuous inflammatory microenvironment via Kupffer cells and Hepatic Stellate cells. This promotes disease progression. The current study aims to elucidate that the role of Toll-like receptor 4 (TLR4) induced innate immune response in chronic inflammation in patients chronically infected with HCV. For this purpose, changes in downstream signaling cascade of TLR4 during chronic HCV infection using peripheral blood mononuclear cells of chronic HCV patients were studied. We found significant increase in expression levels of proinflammatory and profibrotic genes induced by TLR4 Myeloid differentiation factor 88 (MyD88)-dependent pathway between treatment naive and healthy controls, while no significant difference between the expressions of genes involved in TLR4 signaling was found between treatment responders and healthy controls. Interestingly, both TLR4 MyD88-dependent and -independent pathways were found to be operational in nonresponders to interferon treatment. This further strengthens the involvement of innate immune signaling as a leading factor in HCV-mediated liver disease progression and the role of TLR4 MyD88-dependent and -independent pathway in ensuring the conditions for chronic inflammation.

Nanoniosome-encapsulated levoflaxicin as an antibacterial agent against Brucella.

A study was conducted to examine the prevalence of brucellosis (in animal farms) in the vicinity of Islamabad and Rawalpindi. A total of 170 milk samples were collected randomly from several farmhouses. The collected milk samples were initially screened by a Brucella selective medium. The bacterial isolates grown on the selective medium were subjected to biochemical identification for further confirmation of Brucella species. Among the tested samples, 28 (16.4%) were found positive for selective medium and 14 (8.2%) were found positive after biochemical confirmation. The antimicrobial susceptibility of several antibiotics performed by the disc-diffusion method did not yield any significant findings. Encapsulating antimicrobial drugs in unilamellar niosomes is an effective approach to treat the endemic infection. In this study, the antimicrobial activity of niosome-encapsulated levofloxacin is compared with free drug. The drug-encapsulating and empty niosomes were synthesized by using two surfactants Tween 80 and Span 40. Niosomal characterization included electron microscopy, dynamic light scattering, and zeta potential. The encapsulation efficiency was found to be 78% and 74% for Span 40 and Tween 80 niosomes, respectively. The antibacterial activity of niosomal levofloxacin was evaluated against the identified Brucella species and the antimicrobial activity of the free drug was increased many folds after encapsulation. In this study, levofloxacin niosomes were successfully synthesized against Brucellosis.

HCV non-structural NS4A protein of genotype 3a induces mitochondria mediated death by activating Bax and the caspase cascade.

Currently almost 170 million of the world population is suffering with Hepatitis C virus (HCV) that is the major cause of liver diseases, which leads to liver fibrosis, cirrhosis and hepatocellular carcinoma. Approximately 6% of the Pakistani population is chronically infected with HCV, with genotype 3a being the most prominent strain in Pakistan. Complex of HCV non-structural proteins NS3-4A plays an important role in the viral replication machinery that together has serine protease and helicase activity. Genetic heterogeneity within HCV genotypes makes it pertinent to assess the apoptotic pathway within different HCV genotypes. Findings of present study reveal that HCV genotype 3a NS4A and NS3-NS4A induce cell death in Huh-7 cells. Moreover, our results demonstrated that NS3-4A and NS4A proteins were not only localized on ER but also on the mitochondria. Bax a pro-apoptotic protein was found translocated to the mitochondria in the transfected cells, while up-regulated expression of Bax and down-regulated expression of anti-apoptotic Bcl-xL protein was also observed in the presence of NS4A and NS3-4A proteins. High level of mitochondrial superoxide generation was observed in the transfected cells and NS3-4A and NS4A triggered a cascade of activation starting from caspase-9, then caspase-7 and caspase-3 that ultimately led to the cleavage of poly (ADP-ribose) polymerase PARP. Collectively findings of the present study suggest that NS4A and co-expression of NS3-4A and NS4A of genotype 3a has similar capacity to induce apoptosis through a Bax-triggered, mitochondrial-mediated, caspase cascade.

Zika virus: what we need to know?

Zika virus is one of the emerging viruses and is of significant threat to human health globally. It is a mosquito borne flavivirus similar to dengue, yellow fever, and West Nile viruses. It was reported about 5 decades ago and then it spreads to different parts of the world. Large outbreaks were reported on Yap Islands in 2007. Now it has gained wide attention globally by health communities. Major vector for virus transmission is Aedes aegypti mosquito. ZIKV infection is mostly asymptomatic but it is also responsible to cause mild influenza like illness to serious manifestations. There is no specific anti-viral treatment is available for ZIKV infection. The virus disseminates very fast due to which it possesses a serious threat especially in those areas where there is lack of specific immunity against virus. Little knowledge is available on its transmission and pathogenicity. Although virus was discovered years ago but its genomic structure is not clearly understood yet. In this review we focus on the current knowledge of epidemiology of ZIKV, its transmission, its structural biology, different aspects of diagnosis and diagnostic challenges as well as highlighted appropriates antiviral drugs and vaccines regarding treatment.

In-Vitro Transcription analysis of NS5A from HCV-3a circulating in Pakistani patients with chronic hepatitis C and their differential response to antiviral therapy.

OBJECTIVE: Mutations in HCV nonstructural protein 5A (NS5A) play a vital role in virus resistance. The aim of this study was to develop a correlation between NS5A mutations (genotype 3a) and virological response towards interferon alpha (IFN-α) plus ribavirin therapy. METHODS: In this study, which was conducted from 09-02-2013 to 25-11-2015 in the rural area of Province Sindh - Pakistan, total patients' responses to peg-IFN therapy were investigated. Patients were given peg-IFN therapy for 24 to 48 weeks and categorized as sustained virologic responders (SVR) or non-responders (NR) to HCV infection. HCV NS5A region (2215-2335) of genotype 3a was identified in both responders and non-responders. RESULTS: Twenty-four NR with 24 SVR isolates showed significant mutations within the nonstructural protein 5A region in HCV genotype 3a. The New Zealand (NZL1) (GenBank D17763) differences were observed by using gene. The ISDR mutations for nonstructural protein 5A in non-responders have been reported as a possible explanation of HCV interferon resistance. CONCLUSION: Based on these results, it is suggested that decreased SVR is caused by the increased mutations in nonstructural protein 5A sequences. When the sequence outside the Protein kinases R binding domain (PKRBD) (2281-2335) was examined, significant differentiations were observed among the SVR and NR classes at few amino acid strains.

Correlation of OAS1 gene polymorphism at exon 7 splice accepter site with interferon-based therapy of HCV infection in Pakistan.

The most useful treatment for HCV infection worldwide is peg-interferon plus ribavirin, although the response varies from person to person. Hence, host genetics are significantly involved in the treatment response to HCV infection. The 2'-5' oligoadenylate synthetase (OAS) is one of the most important components of the immune system having significant antiviral functions. The aim of this study was to investigate the role of single nucleotide polymorphism (SNP) at the exon 7 splice acceptor site (SAS) of OAS1 to interferon-based therapy of HCV infection. OAS1 genotyping was performed in 140 HCV patients by restriction fragment length polymorphism polymerase chain reaction method (RFLP-PCR). These patients were enrolled for the study in 2010-2013. OAS1 SNP was also established in 120 healthy controls. Correlation of HCV genotypes, OAS1 SNP, and other factors with response to interferon therapy were statistically analyzed by SPSS 13 software. There were no significant differences in the distribution of OAS1 genotypes between healthy and patients subjects. The distribution of AG and AA genotypes of OAS1 genotypes between sustained virological responders (SVRs) and the non-responders (NRs) group were also comparable. However, Pearson chi square analysis indicated that the patients possessing a GG genotype of the OAS1 gene at exon 7 SAS demonstrated significantly positive association with treatment response to HCV infection (p=0.039). This study determined that SNP at exon 7 SAS of OAS1 was significantly associated with response to interferon-based therapy of HCV infection in our population.

An overview: in vitro models of HCV replication in different cell cultures.

Although much of productive research has been conducted in the field of molecular virology of Hepatitis C virus (HCV) regarding its genes, gene functions and proteins, development of an efficient cell culture model for its replication remained a focused area. Focus has been directed to establish HCV in vitro replication system. This replication system should mimic its intrahepatic pathogenesis so that antivirals should be screened and in vitro gene profiling of HCV induced pathogenesis should be worked out. Since 1990 various experimental approaches and strategies have been utilized in phase of development of a robust replication model for HCV, and success has been reported for a few genotypes. Still the work is going on to have more success in availing such robust replication models for all the genotypes. This will help to have a common antiviral strategy against HCV induced pathogenesis involving any genotype or subtype.

Phylogenetic and structural analysis of HCV nonstructural protein 4A from Pakistani patients.

Hepatitis C virus nonstructural protein, NS4A, is a small protein comprising of about 54 amino acids. Despite its small size, it plays key role in many viral and cellular functions. The most important of which is its role as the co-factor of viral serine protease and helicase (NS3). Our study examines the phylogenetic and structural analysis of this coding region after isolation from Pakistani HCV patient samples. Phylogenetic analysis of the gene revealed that Pakistani 3a HCV strains do not show significant divergence from those reported from the rest of the world. The findings of this study also depict that NS4A sequence is conserved within genotypes, whereas it shows variations among different genotypes. While predicting the tertiary structure of the protein two important mutations (H28Y & E32G) were observed when comparing the Pakistani sequences with that of a reference HCV (genotype 3a) strain NZL (D17763). These mutations were observed in the central domain of NS4A which is responsible for interaction with NS3. Taken together, these mutations within the NS4A coding region can play an important role in the binding capacity of NS4A with HCV serine protease NS3.

Hepatitis C virus infection: molecular pathways to insulin resistance.

Chronic Hepatitis C virus has the potential of inducing insulin resistance and type 2 Diabetes Mellitus in vitro as well as in vivo . Structural and non-structural proteins of HCV modulate cellular gene expression in such a way that insulin signaling is hampered, concomitantly leads toward diabetes mellitus. A number of mechanisms have been proposed in regard to the HCV induced insulin resistance involving the upregulation of Inflammatory cytokine TNF-α, hypophosphorylation of IRS-1 and IRS-2, phosphorylation of Akt, up-regulation of gluconeogenic genes, accumulation of lipids and targeting lipid storage organelles. This review provides an insight of molecular mechanisms by which HCV structural and non-structural proteins can induce insulin resistance.

Dengue fever in the Indian Subcontinent: an overview.

The Indian Subcontinent has emerged as a scene of many mosquito-borne infectious diseases, including malaria and dengue fever. After the 1990s, the rate of malaria declined owing largely to preventive measures, but at the same time dengue fever (DF) and dengue hemorrhagic fever (DHF) were increasing in the region. Outbreaks were recorded in all countries of the Indian Subcontinent with India, Pakistan, Bangladesh and Sri Lanka on the forefront and suffering from the largest number of cases and deaths. We discuss annual cases of DF/DHF in these four countries and possible factors involved in DF outbreaks. We also discuss prevalent serotypes in this region where data suggest the emergence of DEN2 and DEN3 as the most dominant and lethal serotypes. Climate is an important factor influencing DF outbreaks, and rainfall, temperature and humidity play a pivotal role in DF outbreaks. Finally the economic impact of DF/DHF cases is discussed showing that direct and indirect economic loss due to DF/DHF reaches millions of USD each year.

A molecular evaluation of dengue virus pathogenesis and its latest vaccine strategies.

More than one third of the world's population living in tropical and subtropical areas of the world is at risk of dengue infections and as many as 100 million people are yearly infected. This disease has reemerged during the past 20 years in the form of an epidemic. Dengue is caused by one of four related serotypes of dengue virus and often leads to severe forms of the disease, resulting commonly from secondary infections. Dengue virus is a mosquito borne virus, belongs to the family Flaviviridae and consists of a single stranded positive sense RNA genome. Like other RNA viruses it escapes defense mechanisms and neutralization attempts by mutations, which make it more resistant and adaptable to its environment. Antiviral strategies and vaccine development is thus impaired and hence to date there is no licensed vaccine available for dengue virus. Here we discuss various efforts made towards the identification of potential vaccine targets for dengue as well as various strategies employed by research groups/pharmaceutical companies towards the development of a successful dengue vaccine.