Identification of a putative Gag binding site critical for feline immunodeficiency virus genomic RNA packaging.

The retroviral Gag precursor plays a central role in the selection and packaging of viral genomic RNA (gRNA) by binding to virus-specific packaging signal(s) (psi or ψ). Previously, we mapped the feline immunodeficiency virus (FIV) ψ to two discontinuous regions within the 5' end of the gRNA that assumes a higher order structure harboring several structural motifs. To better define the region and structural elements important for gRNA packaging, we methodically investigated these FIV ψ sequences using genetic, biochemical, and structure-function relationship approaches. Our mutational analysis revealed that the unpaired U85CUG88 stretch within FIV ψ is crucial for gRNA encapsidation into nascent virions. High-throughput selective 2' hydroxyl acylation analyzed by primer extension (hSHAPE) performed on wild type (WT) and mutant FIV ψ sequences, with substitutions in the U85CUG88 stretch, revealed that these mutations had limited structural impact and maintained nucleotides 80-92 unpaired, as in the WT structure. Since these mutations dramatically affected packaging, our data suggest that the single-stranded U85CUG88 sequence is important during FIV RNA packaging. Filter-binding assays performed using purified FIV Pr50Gag on WT and mutant U85CUG88 ψ RNAs led to reduced levels of Pr50Gag binding to mutant U85CUG88 ψ RNAs, indicating that the U85CUG88 stretch is crucial for ψ RNA-Pr50Gag interactions. Delineating sequences important for FIV gRNA encapsidation should enhance our understanding of both gRNA packaging and virion assembly, making them potential targets for novel retroviral therapeutic interventions, as well as the development of FIV-based vectors for human gene therapy.

Expression, purification, and functional characterization of soluble recombinant full-length simian immunodeficiency virus (SIV) Pr55Gag.

The simian immunodeficiency virus (SIV) precursor polypeptide Pr55Gag drives viral assembly and facilitates specific recognition and packaging of the SIV genomic RNA (gRNA) into viral particles. While several studies have tried to elucidate the role of SIV Pr55Gag by expressing its different components independently, studies using full-length SIV Pr55Gag have not been conducted, primarily due to the unavailability of purified and biologically active full-length SIV Pr55Gag. We successfully expressed soluble, full-length SIV Pr55Gag with His6-tag in bacteria and purified it using affinity and gel filtration chromatography. In the process, we identified within Gag, a second in-frame start codon downstream of a putative Shine-Dalgarno-like sequence resulting in an additional truncated form of Gag. Synonymously mutating this sequence allowed expression of full-length Gag in its native form. The purified Gag assembled into virus-like particles (VLPs) in vitro in the presence of nucleic acids, revealing its biological functionality. In vivo experiments also confirmed formation of functional VLPs, and quantitative reverse transcriptase PCR demonstrated efficient packaging of SIV gRNA by these VLPs. The methodology we employed ensured the availability of >95% pure, biologically active, full-length SIV Pr55Gag which should facilitate future studies to understand protein structure and RNA-protein interactions involved during SIV gRNA packaging.

A Stretch of Unpaired Purines in the Leader Region of Simian Immunodeficiency Virus (SIV) Genomic RNA is Critical for its Packaging into Virions.

Simian immunodeficiency virus (SIV) is an important lentivirus used as a non-human primate model to study HIV replication, and pathogenesis of human AIDS, as well as a potential vector for human gene therapy. This study investigated the role of single-stranded purines (ssPurines) as potential genomic RNA (gRNA) packaging determinants in SIV replication. Similar ssPurines have been implicated as important motifs for gRNA packaging in many retroviruses like, HIV-1, MPMV, and MMTV by serving as Gag binding sites during virion assembly. In examining the secondary structure of the SIV 5' leader region, as recently deduced using SHAPE methodology, we identified four specific stretches of ssPurines (I-IV) in the region that harbors major packaging determinants of SIV. The significance of these ssPurine motifs were investigated by mutational analysis coupled with a biologically relevant single round of replication assay. These analyses revealed that while ssPurine II was essential, the others (ssPurines I, III, & IV) did not significantly contribute to SIV gRNA packaging. Any mutation in the ssPurine II, such as its deletion or substitution, or other mutations that caused base pairing of ssPurine II loop resulted in near abrogation of RNA packaging, further substantiating the crucial role of ssPurine II and its looped conformation in SIV gRNA packaging. Structure prediction analysis of these mutants further corroborated the biological results and further revealed that the unpaired nature of ssPurine II is critical for its function during SIV RNA packaging perhaps by enabling it to function as a specific binding site for SIV Gag.

A purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77Gag.

Retroviral RNA genome (gRNA) harbors cis-acting sequences that facilitate its specific packaging from a pool of other viral and cellular RNAs by binding with high-affinity to the viral Gag protein during virus assembly. However, the molecular intricacies involved during selective gRNA packaging are poorly understood. Binding and footprinting assays on mouse mammary tumor virus (MMTV) gRNA with purified Pr77Gag along with in cell gRNA packaging study identified two Pr77Gag binding sites constituting critical, non-redundant packaging signals. These included: a purine loop in a bifurcated stem-loop containing the gRNA dimerization initiation site, and the primer binding site (PBS). Despite these sites being present on both unspliced and spliced RNAs, Pr77Gag specifically bound to unspliced RNA, since only that could adopt the native bifurcated stem-loop structure containing looped purines. These results map minimum structural elements required to initiate MMTV gRNA packaging, distinguishing features that are conserved amongst divergent retroviruses from those perhaps unique to MMTV. Unlike purine-rich motifs frequently associated with packaging signals, direct involvement of PBS in gRNA packaging has not been documented in retroviruses. These results enhance our understanding of retroviral gRNA packaging/assembly, making it not only a target for novel therapeutic interventions, but also development of safer gene therapy vectors.

Identification of Pr78Gag Binding Sites on the Mason-Pfizer Monkey Virus Genomic RNA Packaging Determinants.

How retroviral Gag proteins recognize the packaging signals (Psi) on their genomic RNA (gRNA) is a key question that we addressed here using Mason-Pfizer monkey virus (MPMV) as a model system by combining band-shift assays and footprinting experiments. Our data show that Pr78Gag selects gRNA against spliced viral RNA by simultaneously binding to two single stranded loops on the MPMV Psi RNA: (1) a large purine loop (ssPurines), and (2) a loop which partially overlaps with a mostly base-paired purine repeat (bpPurines) and extends into a GU-rich binding motif. Importantly, this second Gag binding site is located immediately downstream of the major splice donor (mSD) and is thus absent from the spliced viral RNAs. Identifying elements crucial for MPMV gRNA packaging should help in understanding not only the mechanism of virion assembly by retroviruses, but also facilitate construction of safer retroviral vectors for human gene therapy.

Purification and Functional Characterization of a Biologically Active Full-Length Feline Immunodeficiency Virus (FIV) Pr50Gag.

The feline immunodeficiency virus (FIV) full-length Pr50Gag precursor is a key player in the assembly of new viral particles. It is also a critical component of the efficient selection and packaging of two copies of genomic RNA (gRNA) into the newly formed virus particles from a wide pool of cellular and spliced viral RNA. To understand the molecular mechanisms involved during FIV gRNA packaging, we expressed the His6-tagged and untagged recombinant FIV Pr50Gag protein both in eukaryotic and prokaryotic cells. The recombinant Pr50Gag-His6-tag fusion protein was purified from soluble fractions of prokaryotic cultures using immobilized metal affinity chromatography (IMAC). This purified protein was able to assemble in vitro into virus-like particles (VLPs), indicating that it preserved its ability to oligomerize/multimerize. Furthermore, VLPs formed in eukaryotic cells by the FIV full-length Pr50Gag both in the presence and absence of His6-tag could package FIV sub-genomic RNA to similar levels, suggesting that the biological activity of the recombinant full-length Pr50Gag fusion protein was retained in the presence of His6-tag at the carboxy terminus. Successful expression and purification of a biologically active, recombinant full-length Pr50Gag-His6-tag fusion protein will allow study of the intricate RNA-protein interactions involved during FIV gRNA encapsidation.

Genetic characterization of hemagglutinin (HA) gene of influenza A viruses circulating in Southwest India during 2017 season.

Molecular surveillance of influenza viruses is essential for early detection of novel variants. The aim of the present study was to analyze the hemagglutinin gene of influenza A(H1N1)pdm09 and A(H3N2) viruses circulating during the 2017 season. To investigate the genetic diversity of hemagglutinin gene of influenza A(H1N1)pdm09 and A(H3N2) viruses from 2017 season, ten samples from each subtype were sequenced and analyzed. The season was predominated by influenza A(H1N1)pdm09 viruses. Ten samples were sequenced from each subtype and all sequenced influenza A(H1N1)pdm09 and A(H3N2) viruses belonged to clades 6B.1 and 3C.2a, respectively. Sequence analysis of H1 gene in comparison to 2010-2016 vaccine strain showed mutations K166Q and S188T (K180Q and S202T here) that most likely resulted in antigenic drift and emergence of variant viruses. H3 gene substitutions N137K, N187K, I422V, and G500E that define clade 3C.2a1 were detected during analysis of sequences in comparison to 2017-2018 vaccine strain of northern hemisphere. These substitutions contributed to the change of WHO's recommendation of the 2018-2019 vaccine strain for northern hemisphere. The results of this study provide insights about the continuous genetic variability of the HA gene.

Detection of genital chlamydial and gonococcal infection using urine samples: A community-based study from India.

Sexually transmitted infections (STI) have a major impact on the reproductive health of women. Among the different etiological agents of STIs, Chlamydia trachomatis and Neisseria gonorrhoeae are the main bacterial pathogens that cause sexually transmitted infections in women. The aim of the study was to estimate the prevalence of genital chlamydial and gonococcal infection among women in the age group of 18-65 years from a community-based setting. A community-based cross-sectional study was performed using the archived urine samples (n=811) of women in the age group of 18-65 years for C. trachomatis and N. gonorrhoeae using a multiplex conventional Polymerase Chain Reaction (PCR). Out of 811 samples tested in the present study, 2 (0.24%) were tested positive for C. trachomatis and none were positive for N. gonorrhoeae. The study demonstrates the very low prevalence of C. trachomatis and N. gonorrhoeae infection in a rural community. For large population-based screening, urine samples were observed to be more socially acceptable and cost-effective.

Molecular characterization of neuraminidase genes of influenza A(H3N2) viruses circulating in Southwest India from 2009 to 2013.

Molecular characterization of neuraminidase (NA) gene of 25 influenza A(H3N2) virus isolates (2009-2013) archived at the Manipal Centre for Virus Research was carried out. The annual rate of amino acid substitutions in the N2 gene of influenza A(H3N2) virus isolates was 0.2-0.6%. Out of the 25 NA sequences analyzed, catalytic site mutations were observed in three isolates. Two of the mutations (D151G and E276G) were detected in functional catalytic residues, and an E227V mutation was detected in the framework residues. To the best of our knowledge, NA inhibitor resistance associated with the mutations E276G and E227V has not been reported. However, the mutation D151G, which is commonly associated with culturing of influenza A(H3N2) virus in Madin-Darby canine kidney (MDCK) cells, has been reported to result in a reduction in virus susceptibility to NA inhibitor drugs. Our study also detected mutations in antigenic residues. Some of the mutations (except D197G, K249E, A250T, S334C, and H347R/N) remained conserved in isolates of succeeding seasons. Antigenic residue mutations (D197G and S334C) have not been reported globally to date. The effect of these catalytic and antigenic mutant residues on drug and antibody binding was analyzed using three-dimensional structural analysis and biochemical assays. Antigenic variability of influenza A(H3N2) viruses is a major concern, and vaccine failures are mainly due to genetic variations in the HA gene. Our study documents that genetic changes in N2 occur at a slower rate, and this information is useful for the consideration and standardization of NA in influenza vaccines.

Life Cycle Assessment of Solar Photovoltaic Microgrid Systems in Off-Grid Communities.

Access to a reliable source of electricity creates significant benefits for developing communities. Smaller versions of electricity grids, known as microgrids, have been developed as a solution to energy access problems. Using attributional life cycle assessment, this project evaluates the environmental and energy impacts of three photovoltiac (PV) microgrids compared to other energy options for a model village in Kenya. When normalized per kilowatt hour of electricity consumed, PV microgrids, particularly PV-battery systems, have lower impacts than other energy access solutions in climate change, particulate matter, photochemical oxidants, and terrestrial acidification. When compared to small-scale diesel generators, PV-battery systems save 94-99% in the above categories. When compared to the marginal electricity grid in Kenya, PV-battery systems save 80-88%. Contribution analysis suggests that electricity and primary metal use during component, particularly battery, manufacturing are the largest contributors to overall PV-battery microgrid impacts. Accordingly, additional savings could be seen from changing battery manufacturing location and ensuring end of life recycling. Overall, this project highlights the potential for PV microgrids to be feasible, adaptable, long-term energy access solutions, with health and environmental advantages compared to traditional electrification options.

Detection of D151G/N mutations in the neuraminidase gene of influenza A (H3N2) viruses by real-time RT-PCR allelic discrimination assay.

Single nucleotide polymorphisms (SNPs) at D151 position of neuraminidase (NA) gene of influenza A (H3N2) virus has been associated with drug resistance and increased binding affinity. NA-D151G/N-substitutions of influenza A (H3N2) viruses are frequently induced and selected by culturing in Madin-Darby canine kidney (MDCK) cell lines. It is important to consider and exclude D151G/N mutants after isolation of influenza virus in MDCK cell line; since, the substitutions can highly influence the results of experimental research. The study aims to develop an allelic discrimination real-time reverse transcriptase polymerase chain reaction (RT-PCR) for the screening of D151G/N mutants. Thirty-six influenza A (H3N2) virus isolates were included and screened for D151G/N mutants using allelic discrimination assay. Out of the 36 isolates, 11 isolates (30.5%) were detected as heterozygous for D and G/N substitutions. Twenty-one (58.3%) isolates were identified as homozygous wild type and four isolates (11.1%) were undetermined. Isolates with substitutions at D151 position were sequenced by Sanger sequencing method. The present study demonstrates a rapid and convenient method for primary screening of the mutation after culturing of the influenza virus in MDCK cell lines in order to avoid potential misinterpretations of results and improve the quality of experimental research.

Detection of Genital HPV Infection Using Urine Samples: a Population Based Study in India.

BACKGROUND: Cervical cancer is the second commonest cancer among Indian women and its association with human papilloma virus (HPV) is well established. This preventable cancer accounts for the maximum number of cancer related deaths among rural Indian women. Unlike in developed countries there are no organized cervical cancer screening programmes in India due to lack of resources and manpower. OBJECTIVE: To detect genital HPV infection using urine samples among asymptomatic rural women in the age group of 18-65 years. MATERIALS AND METHODS: The study area chosen was Perdoor village in Udupi Taluk, Karnataka State and all the women in the age group of 18-65 years formed the study cohort. A cross sectional study was conducted by house visits and 1,305 women were enrolled in the study. After taking written informed consent a data sheet was filled and early stream random urine samples were collected, transported to a laboratory at 4OC and aliquoted. Samples were tested using nested HPV PCR with PGMY09/11 and GP5+/6+ primers. Positive cases were genotyped by sequence analysis. RESULTS: Study participants included 1,134 sexually active and 171 unmarried women with a mean age at marriage of 22.1 (SD=3.9) years. Study area showed high female literacy rate of 86.6%. Five urine samples tested positive for HPV DNA (0.4%). CONCLUSIONS: We found very low genital HPV infection rate among women from monogamous community. This is the first major population based study carried out among asymptomatic rural women to detect genital HPV infectio from Karnataka using urine samples.

An unusual presentation of Pseudomonas aeruginosa blebitis following combined surgery.

We report a case of blebitis that occurred 3 years later following a combined glaucoma and cataract surgery. It was an atypical presentation, as patient had no classical fiery looking signs of blebitis despite the isolated organism being Pseudomonas aeruginosa. Improvized surgical techniques like use of Mitomycin C, releasable flap sutures though considered as part of the recommended procedure for better surgical outcomes, their role as potential risk factors for visually blinding complications like endophthalmitis are often overlooked. This case report throws light on such risk factors for bleb associated infections and recommends removal or trimming of all releasable sutures and the need for a regular postoperative follow-up.

Design and testing of a percutaneously implantable fetal pacemaker.

We are developing a cardiac pacemaker with a small, cylindrical shape that permits percutaneous implantation into a fetus to treat complete heart block and consequent hydrops fetalis, which can otherwise be fatal. The device uses off-the-shelf components including a rechargeable lithium cell and a highly efficient relaxation oscillator encapsulated in epoxy and glass. A corkscrew electrode made from activated iridium can be screwed into the myocardium, followed by release of the pacemaker and a short, flexible lead entirely within the chest of the fetus to avoid dislodgement from fetal movement. Acute tests in adult rabbits demonstrated the range of electrical parameters required for successful pacing and the feasibility of successfully implanting the device percutaneously under ultrasonic imaging guidance. The lithium cell can be recharged inductively as needed, as indicated by a small decline in the pulsing rate.