A Short Sequence Motif in the 5′ Leader of the HIV-1 Genome Modulates Extended RNA Dimer Formation and Virus Replication

The 5′ leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication.     

Functional Cross-talk between Distant Domains of Chikungunya Virus Non-structural Protein 2 Is Decisive for Its RNA-modulating Activity

Chikungunya virus (CHIKV) non-structural protein 2 (nsP2) is a multifunctional protein that is considered a master regulator of the viral life cycle and a main viral factor responsible for cytopathic effects and subversion of antiviral defense. The C-terminal part of nsP2 possesses protease activity, whereas the N-terminal part exhibits NTPase and RNA triphosphatase activity and is proposed to have helicase activity. Bioinformatics analysis classified CHIKV nsP2 into helicase superfamily 1. However, the biochemical significance of a coexistence of two functionally unrelated modules in this single protein remains unknown. In this study, recombinant nsP2 demonstrated unwinding of double-stranded RNA in a 5′–3′ directionally biased manner and RNA strand annealing activity. Comparative analysis of NTPase and helicase activities of wild type nsP2 with enzymatic capabilities of different truncated or N-terminally extended variants of nsP2 revealed that the C-terminal part of the protein is indispensable for helicase functionality and presumably provides a platform for RNA binding, whereas the N-terminal-most region is apparently involved in obtaining a conformation of nsP2 that allows for its maximal enzymatic activities. The establishment of the protocols for the production of biochemically active CHIKV nsP2 and optimization of the parameters for helicase and NTPase assays are expected to provide the starting point for a further search of possibilities for therapeutic interventions to suppress alphaviral infections.     

Perception of Human Papillomavirus Infection,Cervical Cancer and HPV Vaccination in North Indian Population

Background

Human Papillomavirus (HPV) -associated cervical cancer is the second-most common cancer in women worldwide but it is the most frequent gynaecological cancer and cancer associated death in India women. The objective of this study was to assess knowledge about cervical cancer, HPV, HPV vaccine, HPV vaccine acceptance among school and undergraduates students and their parent’s perception about acceptance of HPV vaccine in Northern part of India (Delhi and NCR regions).

Materials and Methods

A qualitative questionnaire based survey among 2500 urban/rural students aged 12–22 years was conducted.

Results

Overall, a low frequency (15%) of HPV and cervical cancer awareness was observed in students and their parents. However, the awareness was much higher in females belonging to urban setup compared to boys with a perception that HPV causes cervical cancer in women only. Additionally, only (13%) participants who were aware of cervical cancer and HPV) were willing to accept HPV vaccination. Apparently, parents of female students were two times more willing to accept HPV vaccination for their ward than male students (p<0.001; OR 95%CI = 2.09 (1.58–2.76).

Conclusion

Cervical cancer and HPV awareness among school, undergraduate students and also to their parents was found to be very low in this part of India. The level of awareness and education appears to be insignificant determinants in rural compared to urban setup. Better health education will be needed to maximize public awareness for cervical cancer prevention.     

Combining Cationic Liposomal Delivery with MPL-TDM for Cysteine Protease Cocktail Vaccination against Leishmania donovani : Evidence for Antigen Synergy and Protection

BackgroundWith the paucity of new drugs and HIV co-infection, vaccination remains an unmet research priority to combat visceral leishmaniasis (VL) requiring strong cellular immunity. Protein vaccination often suffers from low immunogenicity and poor generation of memory T cells for long-lasting protection. Cysteine proteases (CPs) are immunogenic proteins and key mediators of cellular functions in Leishmania. Here, we evaluated the vaccine efficacies of CPs against VL, using cationic liposomes with Toll like receptor agonists for stimulating host immunity against L. donovani in a hamster model.Conclusion/SignificanceThe present study is the first report of a comparative efficacy of leishmanial CPs and their cocktail using liposomal formulation with MPL-TDM against L. donovani. The level of protection attained has not been reported for any other subcutaneous single or polyprotein vaccination against VL.     

Five-Year Field Results and Long-Term Effectiveness of 20 mg/kg Liposomal Amphotericin B (Ambisome) for Visceral Leishmaniasis in Bihar,India

Background

Visceral Leishmaniasis (VL; also known as Kala-azar) is an ultimately fatal disease endemic in Bihar. A 2007 observational cohort study in Bihar of 251 patients with VL treated with 20 mg/Kg intravenous liposomal amphotericin B (Ambisome) demonstrated a 98% cure rate at 6-months. Between July 2007 and August 2012, Médecins Sans Frontières (MSF) and the Rajendra Memorial Research Institute (RMRI) implemented a VL treatment project in Bihar, India—an area highly endemic for Leishmania donovani—using this regimen as first-line treatment.

Methods and Principal Findings

Intravenous Ambisome 20 mg/kg was administered in four doses of 5 mg/kg over 4–10 days, depending on the severity of disease. Initial clinical cure at discharge was defined as improved symptoms, cessation of fever, and recession of spleen enlargement. This observational retrospective cohort study describes 8749 patients with laboratory-confirmed primary VL treated over a 5-year period: 1396 at primary healthcare centers, 7189 at hospital, and 164 at treatment camps. Initial clinical cure was achieved in 99.3% of patients (8692/8749); 0.3% of patients (26/8749) defaulted from treatment and 0.4% (31/8749) died. Overall, 1.8% of patients (161/8749) were co-infected with HIV and 0.6% (51/8749) with tuberculosis. Treatment was discontinued because of severe allergic reactions in 0.1% of patients (7/8749). Overall, 27 patients (0.3%) were readmitted with post Kala-azar dermal leishmaniasis (PKDL). Risk factors for late presentation included female sex, age >15 years and being from a scheduled caste.In 2012, a long-term efficacy survey in the same area of Bihar determined relapse rates of VL after 5 years'' intervention with Ambisome. Of 984 immunocompetent patients discharged between September 2010 and December 2011, 827 (84.0%) were traced in order to determine their long-term outcomes. Of these, 20 patients (2.4%) had relapsed or received further treatment for VL. Of those completing 6, 12, and 15 month follow-up, 0.3% (2/767), 3.7% (14/383), and 2.4% (4/164), respectively, had relapsed. The mean ±SD time-to-relapse was 9.6±3.0 months.

Significance

This is the largest cohort of VL patients treated with 20 mg/kg Ambisome worldwide. The drug has high initial and long-term efficacy, and a low rate of adverse reactions when administered under field conditions in Bihar, India. Although challenging, its use as first line treatment in rural settings in Bihar is safe and feasible.     

In vitro rooting of Psoralea corylifolia Linn: Peroxidase activity as a marker

Induction of rooting in microshoots of Psoraleacorylifolia was achieved within 6–8 days of cultureon half-strength basal Murashige and Skoog's(1962) medium supplemented with 0.005–0.01 mg/lindole-3-acetic acid (IAA) and 2% (w/v) sucrose. Rooting was drastically reduced and friable callusformed at the cut end of the microshoots when themedium was supplemented with a higher concentration ofauxin. Rooting was totally inhibited when themicroshoots were cultured in vitro undercontinuous light. However, the maximum percentage ofmicroshoots rooted when incubated in continuous lightfor 4 weeks before transfer to the rooting media.Peroxidase activity increased considerably duringroot induction indicating a key role of peroxidase inrooting of microshoots of Psoralea corylifolia invitro.