Biochemical and Biophysical Characterisation of the Hepatitis E Virus Guanine-7-Methyltransferase.

Hepatitis E virus (HEV) is an understudied pathogen that causes infection through fecal contaminated drinking water and is prominently found in South Asian countries. The virus affects ~20 million people annually, leading to ~60,000 infections per year. The positive-stranded RNA genome of the HEV genotype 1 has four conserved open reading frames (ORFs), of which ORF1 encodes a polyprotein of 180 kDa in size, which is processed into four non-structural enzymes: methyltransferase (MTase), papain-like cysteine protease, RNA-dependent RNA polymerase, and RNA helicase. MTase is known to methylate guanosine triphosphate at the 5'-end of viral RNA, thereby preventing its degradation by host nucleases. In the present study, we cloned, expressed, and purified MTase spanning 33-353 amino acids of HEV genotype 1. The activity of the purified enzyme and the conformational changes were established through biochemical and biophysical studies. The binding affinity of MTase with magnesium ions (Mg2+) was studied by isothermal calorimetry (ITC), microscale thermophoresis (MST), far-UV CD analysis and, fluorescence quenching. In summary, a short stretch of nucleotides has been cloned, coding for the HEV MTase of 37 kDa, which binds Mg2+ and modulate its activity. The chelation of magnesium reversed the changes, confirming its role in enzyme activity.

An insight on microbial degradation of benzo[a]pyrene: current status and advances in research.

Benzo[a]pyrene (BaP) is a high molecular weight polycyclic aromatic hydrocarbon produced as a result of incomplete combustion of organic substances. Over the years, the release of BaP in the atmosphere has increased rapidly, risking human lives. BaP can form bonds with DNA leading to the formation of DNA adducts thereby causing cancer. Therefore addressing the problem of its removal from the environment is quite pertinent though it calls for a very cumbersome and tedious process owing to its recalcitrant nature. To resolve such issues many efforts have been made to develop physical and chemical technologies of BaP degradation which have neither been cost-effective nor eco-friendly. Microbial degradation of BaP, on the other hand, has gained much attention due to added advantage of the high level of microbial diversity enabling great potential to degrade the substance without impairing environmental sustainability. Microorganisms produce enzymes like oxygenases, hydrolases and cytochrome P450 that enable BaP degradation. However, microbial degradation of BaP is restricted due to several factors related to its bio-availability and soil properties. Technologies like bio-augmentation and bio-stimulation have served to enhance the degradation rate of BaP. Besides, advanced technologies such as omics and nano-technology have opened new doors for a better future of microbial degradation of BaP and related compounds.

Estimating salivary carriage of severe acute respiratory syndrome coronavirus 2 in nonsymptomatic people and efficacy of mouthrinse in reducing viral load: A randomized controlled trial.

BACKGROUND: Many people infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) never develop substantial symptoms. With more than 34 million people in the United States already infected and highly transmissible variants rapidly emerging, it is highly probable that post- and presymptomatic people will form an important fraction of those seeking dental care. Salivary carriage rates in these populations are not known. Moreover, although preventing transmission is critical for controlling spread, the efficacy of mouthrinses in reducing oral viral load is poorly studied. METHODS: The authors recruited 201 asymptomatic, presymptomatic, postsymptomatic, and symptomatic people and measured copy numbers of SARS-CoV-2 in unstimulated saliva using real-time reverse transcriptase quantitative polymerase chain reaction. Subsequently, the authors inducted 41 symptomatic people into a randomized, triple-blinded study and instructed them to rinse with saline, 1% hydrogen peroxide, 0.12% chlorhexidine, or 0.5% povidone-iodine for 60 seconds. The authors measured viral load 15 and 45 minutes after rinsing. RESULTS: Salivary SARS-CoV-2 was detected in 23% of asymptomatic, 60% of postsymptomatic, and 28% of presymptomatic participants. Neither carriage rate nor viral load correlated with COVID-19 symptomatology, age, sex, or race or ethnicity. All 4 mouthrinses decreased viral load by 61% through 89% at 15 minutes and by 70% through 97% at 45 minutes. The extent of reduction correlated significantly with initial viral load. CONCLUSIONS: Nonsymptomatic people can pose a risk of transmitting the virus, and mouthrinses are simple and efficacious means of reducing this risk, especially when the load is less than 104 copies per milliliter. PRACTICAL IMPLICATIONS: At a time when resources are stretched, the findings of this study contribute to evidence-based selection of personal protection equipment and simple infection-control practices to reduce contagion at source. This clinical trial was registered at ClinicalTrials.gov. The registration number is NCT04603794.

Acquisition, Divergence, and Personalization of the Female Perineal Microbiomes Are Driven by Developmental Milestones and Disrupted by Urinary Tract Infection: A Pilot Study.

Introduction: The pediatric perineal microbiomes inhabit a dynamic environment with changes related to diet, toileting habits, and hormonal development. We hypothesized that next-generation sequencing would reveal different perineal bacterial signatures associated with developmental milestones in premenstrual females. Furthermore, we predicted that these microbial changes would be disrupted in premenstrual females with a history of urinary tract infection (UTI). Study Design: Healthy females were recruited at well-child visits. Subjects were divided into 4 developmental groups: (1) 0-3 month old newborns; (2) 4-10 month old infants transitioning to solid foods; (3) 2-6 year old toddlers peri-toilet training; and (4) 7-12 year old premenstrual girls. A separate group of females with a history of culture proven UTI and off antibiotics >1 month was also recruited. DNA was isolated from swabs of the perineum and subjected to 16S rRNA sequencing. The diversity and species changes between developmental cohorts and age matched children with history of UTI was determined. Results: A total of 75 subjects were recruited: 15 in each group. There was a clear evolution of the perineal microbiomes with development. There was a significant microbial disruption in girls with a history of UTI, irrespective of developmental milestone age group. The periurethral/perivaginal site displayed greater changes in microbiome structure than other sites in girls with a history of UTI. Discussion: This pilot study evaluates the normal microbiome of the premenstrual girl at specific developmental milestones. Although the number of children per cohort was limited to 15, we observed statistical significance corresponding with developmental milestones. This study provides the first, culture independent delineation of the development of the perineal microbiome in girls. Furthermore, the sites closest to the site of infection appear to be more sensitive to antibiotic remodeling than those more distant. The factors that remodel the perineal microbiomes and predispose females, particularly girls, to UTIs (e.g., increase in uropathogen presence, absence of protective organisms) are unclear. Identification of specific signatures that increase susceptibility to UTI and their sequelae will improve patient care and promote personalized medicine.

Preparedness for malaria elimination in the wake of climate change in the State of Uttarakhand (India).

BACKGROUND & OBJECTIVES: Climate change is an emerging issue particularly in the context of vector-borne diseases. A study was undertaken in Nainital and Almora districts of Uttarakhand to provide evidences of changing climatic conditions, abundance of vectors, and knocking of malaria in hilly areas. MATERIAL AND METHODS: Longitudinal data on temperature and relative humidity were procured from Tussar Silk Centre, Bhimtal, India as well as generated using HOBO device. Monthly density of malaria vectors, their positivity for sporozoite proteins of malaria parasite and fever surveys were conducted as per the standard procedures from 2010 to 2013. Epidemiological data were procured from the State Programme Officer of Uttarakhand state. RESULTS: It was found that the temperature has increased since 1990 resulting in extension in windows of malaria transmission, temporal distribution as well as man hour density of Anopheles culicifacies and An. fluviatilis in hilly districts of Uttarakhand state. Both the vectors were found in high density up to a maximum man hour density of 110 (An. culicifacies) and 69 (An. fluviatilis) as compared to 32 and 33, respectively during 1998. The field collected vector species were also found positive for sporozoite proteins of malaria parasites in the month of October and November. Evidence of occurrence of malaria cases was also found in areas hitherto free from malaria. INTERPRETATION & CONCLUSION: The findings reveal that Himalayan region needs attention to strengthen surveillance for malaria to identify emerging new foci of malaria transmission in view of climate change. Health education to communities about preventive measures to contain breeding of vectors and seeking timely treatment should be imparted so as to achieve the goal of malaria elimination in category-1 in the first instance.

Hepatitis E Virus Cysteine Protease Has Papain Like Properties Validated by in silico Modeling and Cell-Free Inhibition Assays.

Hepatitis E virus (HEV) has emerged as a global health concern during the last decade. In spite of a high mortality rate in pregnant women with fulminant hepatitis, no antiviral drugs or licensed vaccine is available in India. HEV-protease is a pivotal enzyme responsible for ORF1 polyprotein processing leading to cleavage of the non-structural enzymes involved in virus replication. HEV-protease region encoding 432-592 amino acids of Genotype-1 was amplified, expressed in Sf21 cells and purified in its native form. The recombinant enzyme was biochemically characterized using SDS-PAGE, Western blotting and Immunofluorescence. The enzyme activity and the inhibition studies were conducted using Zymography, FTC-casein based protease assay and ORF1 polyprotein digestion. To conduct ORF1 digestion assay, the polyprotein, natural substrate of HEV-protease, was expressed in E. coli and purified. Cleavage of 186 kDa ORF1 polyprotein by the recombinant HEV-protease lead to appearance of non-structural proteins viz. Methyltransferase, Protease, Helicase and RNA dependent RNA polymerase which were confirmed through immunoblotting using antibodies generated against specific epitopes of the enzymes. FTC-casein substrate was used for kinetic studies to determine Km and Vmax of the enzyme and also the effect of different metal ions and other protease inhibitors. A 95% inhibition was observed with E-64 which was validated through in silico analysis. The correlation coefficient between inhibition and docking score of Inhibitors was found to have a significant value of r2 = 0.75. The predicted 3D model showed two domain architecture structures similar to Papain like cysteine protease though they differed in arrangements of alpha helices and beta sheets. Hence, we propose that HEV-protease has characteristics of "Papain-like cysteine protease," as determined through structural homology, active site residues and class-specific inhibition. However, conclusive nature of the enzyme remains to be established.

Aquatic macrophytes mediated remediation of toxic metals from moderately contaminated industrial effluent.

The present study assessed Zn, Cr, Cd, and Pb removal efficiency of Colocasia esculenta, Hydrilla verticillata, Phragmitis australis, Typha latifolia, and Spirodella polyrhiza from sewage-mixed industrial effluent. The fresh/dry weight and relative growth rate of each macrophyte decreased with increasing effluent concentration. H. verticillata and C. esculenta exhibited better growth at 50% effluent over control. The maximum Zn, Cd, and Pb accumulation (1008.23, 28.03, and 483.55 mg/kg dry wt., respectively) was recorded in C. esculenta, whereas Cr (114.48 mg/kg dry wt.) in H. verticillata at 100% effluent. Metal accumulation in roots of all plants species was higher (≥50%) initially with increasing effluent concentration and later transferred to shoots. All plants exhibited BCF >1.0 for all heavy metals, highest being for Zn (91.2) and Cd (75.2) in H. verticillata, for Cr (97.9) and Pb (103) in C. esculenta. Except S. polyrhhiza, all other plants exhibited TF <1.0. Maximum removal efficiency of Zn was 82.8% by H. verticillata, whilst that of Cr, Cd, and Pb by C. esculenta at 50% effluent, demonstrating wide applicability of H. verticillata and C. esculenta for treatment of mixed industrial effluent having heavy metals.

Dysbiotic Subgingival Microbial Communities in Periodontally Healthy Patients With Rheumatoid Arthritis.

OBJECTIVE: Studies that demonstrate an association between rheumatoid arthritis (RA) and dysbiotic oral microbiomes are often confounded by the presence of extensive periodontitis in these individuals. This study was undertaken to investigate the role of RA in modulating the periodontal microbiome by comparing periodontally healthy individuals with RA to those without RA. METHODS: Subgingival plaque was collected from periodontally healthy individuals (22 with RA and 19 without RA), and the 16S gene was sequenced on an Illumina MiSeq platform. Bacterial biodiversity and co-occurrence patterns were examined using the QIIME and PhyloToAST pipelines. RESULTS: The subgingival microbiomes differed significantly between patients with RA and controls based on both community membership and the abundance of lineages, with 41.9% of the community differing in abundance and 19% in membership. In contrast to the sparse and predominantly congeneric co-occurrence networks seen in controls, RA patients revealed a highly connected grid containing a large intergeneric hub anchored by known periodontal pathogens. Predictive metagenomic analysis (PICRUSt) demonstrated that arachidonic acid and ester lipid metabolism pathways might partly explain the robustness of this clustering. As expected from a periodontally healthy cohort, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were not significantly different between groups; however, Cryptobacterium curtum, another organism capable of producing large amounts of citrulline, emerged as a robust discriminant of the microbiome in individuals with RA. CONCLUSION: Our data demonstrate that the oral microbiome in RA is enriched for inflammophilic and citrulline-producing organisms, which may play a role in the production of autoantigenic citrullinated peptides in RA.

Intricacies of using temperature of different niches for assessing impact on malaria transmission.

BACKGROUND & OBJECTIVES: The influence of temperature on the life cycle of mosquitoes as well as on development of malaria parasite in mosquitoes is well studied. Most of the studies use outdoor temperature for understanding the transmission dynamics and providing projections of malaria. As the mosquitoes breed in water and rest usually indoors, it is logical to relate the transmission dynamics with temperature of micro-niche. The present study was, therefore, undertaken to understand the influence of different formats of temperature of different micro-niches on transmission of malaria for providing more realistic projections. METHODS: The study was conducted in one village each of Assam and Uttarakhand s0 tates of India. Temperatures recorded from outdoor (air) as well as indoor habitats (resting place of mosquito) were averaged into daily, fortnightly and monthly and were used for determination of transmission windows (TWs) for Plasmodium vivax (Pv) and P. falciparum (Pf) based on minimum temperature threshold required for transmission. RESULTS: The daily temperature was found more useful for calculation of sporogony than fortnightly and monthly temperatures. Monthly TWs were further refined using fortnightly temperature, keeping in view the completion of more than one life cycle of malaria vectors and sporogony of malaria parasite in a month. A linear regression equation was generated to find out the relationship between outdoor and indoor temperatures and R [2] to predict the percentage of variation in indoor temperature as a function of outdoor temperature at both localities. INTERPRETATION & CONCLUSIONS: The study revealed that the indoor temperature was more than outdoors in stable malarious area (Assam) but fluctuating in low endemic area like Uttarakhand. Transmission windows of malaria should be determined by transforming outdoor data to indoor and preferably at fortnightly interval. With daily recorded temperature, sporogonic and gonotrophic cycles can also be calculated which is otherwise not possible with monthly data. The study highlights that the projections made for malaria in view of climate change need to be seen with limitation of difference in outdoor and indoor temperatures at different locations, highlighting the need for local data generation at least at sub-district level.

Expression and Characterization of Yeast Derived Chikungunya Virus Like Particles (CHIK-VLPs) and Its Evaluation as a Potential Vaccine Candidate.

Chikungunya virus (CHIKV) has emerged as a global health concern due to its recent spread in both old and new world. So far, no CHIKV specific drug or vaccine is licensed for human use. In this study, we report production of Chikungunya virus like particles (CHIK-VLPs) using novel yeast expression system (Pichia pastoris) and its evaluation as vaccine candidate. The gene encoding structural polyprotein of CHIKV from a recent epidemic strain was cloned into yeast expression system. The multicopy integrants were processed for expression of CHIK-VLPs. The VLPs were purified and confirmed through electron microscopic analysis for their morphological identity with CHIKV. The in vitro and in vivo evaluation of CHIK-VLPs as vaccine candidate was determined in Balb/c mice. Induction of both humoral and cellular immune response was observed with different doses of CHIK-VLPs. The humoral immune response was studied through different techniques like enzyme linked immunosorbent assay, IgG Isotyping and plaque reduction neutralization test. CHIK-VLPs were found to elicit high titer of antibodies that are able to recognize native CHIKV. Higher level of IgG2a and IgG1 subtypes was identified suggestive of balanced Th1/Th2 response. Both in vitro and in vivo neutralization activity of CHIK-VLPs antibodies was observed even with low concentration, which shows its high specificity and neutralizing activity against two different CHIKV strains. Neonatal mice receiving anti-CHIK-VLPs antibodies were protected from CHIKV challenge. Induction of cellular immune response was confirmed through higher level of TNF-α, IL-10 and substantial level of IL-2, IL-4 and IFN-γ indicating a balanced response. This is the first report, where CHIK-VLPs has been expressed by Pichia pastoris and evaluated for neutralizing activity against CHIKV. These promising results indicate the utility of CHIK-VLPs as a promising vaccine candidate against emerging CHIKV.

Emergence of influenza A (H1N1)pdm09 genogroup 6B and drug resistant virus, India, January to May 2015.

To investigate the aetiology of the 2015 A(H1N1)pdm09 influenza outbreak in India, 1,083 nasopharyngeal swabs from suspect patients were screened for influenza A(H1N1)pdm09 in the state of Madhya Pradesh. Of 412 positive specimens, six were further characterised by phylogenetic analysis of haemagglutinin (HA) sequences revealing that they belonged to genogroup 6B. A new mutation (E164G) was observed in HA2 of two sequences. Neuraminidase genes in two of 12 isolates from fatal cases on prior oseltamivir treatment harboured the H275Y mutation.

Characterization of pandemic influenza A (H1N1) virus hemagglutinin specific polyclonal antibodies for biosensor applications.

In this study, recombinant hemagglutinin protein (rH1N1HA) of Pandemic influenza virus and polyclonal antibodies against it for biosensor applications have been characterized. For rapid and high sensitive detection of H1N1 virus or its antibodies, PCR-free and label free detection method based on a surface plasmon resonance technique has been proposed. The glycosylated H1N1HA protein was expressed in yeast and the authenticity of the expressed protein was confirmed by Western blotting. Rabbit polyclonal antibodies developed against rH1N1HA protein were evaluated for their ability to neutralize H1N1 virus through plaque reduction neutralization test and indirect ELISA. Affinity purified anti-H1N1HA IgG were characterized further for their specificity, affinity of interaction, the association and dissociation rates at which they interact through surface plasmon resonance technique. The equilibrium constant and maximum binding capacity of analyte was found to be 49.7 nM and 47.28m°, respectively. The assay could detect a lowest IgG of 0.5 ng on a rH1N1HA coated chip. Combined with the high sensitivity of surface plasmon resonance technique and specificity of the reagents, it is possible to develop a rapid detection assay for monitoring influenza infections.

Altitude, temperature, and malaria vectors in Nainital and Udham Singh Nagar districts of Uttarakhand, India: an evidence-based study.

BACKGROUND & OBJECTIVES: The relationship between altitude, temperature and malaria are poorly understood. Hence, a study was undertaken at three sites of Udham Singh Nagar (erstwhile Nainital district) and Nainital district (Uttarakhand) during 2010- 11 for the generation of evidences in the context of potential threat of climate change. METHODS: Data on temperature and relative humidity (RH) were recorded through data-logger device in study villages at the altitudes of 166, 226 and 609 m were selected for detailed work. Mosquito collections were made fortnightly during 0600- 0800 hrs. Malaria incidence data were procured from concerned Primary Health Centres. RESULTS: The study provides evidences of decrease in temperature with increase in altitude, even within a district resulting in variation in temporal distribution of malaria vector. With the increase of 67 m altitude between plains and foothill village, there was a reduction in temperature to the tune of 1.1°C and with further increase in altitude of 416 m between foothill and hilly villages, the temperature decreased by 0.27°C. The difference in temperature at three altitudes affects the Transmission windows (TWs) of both Plasmodium vivax (Pv) and P. falciparum (Pf), and opening of TWs are inversely proportional to altitude. In the plains, the TW for Pv and Pf were open for 11 and 10 months respectively, while 10 and 9 months in the foothills and 9 and 8 months, respectively for both the parasites at hilly altitude. Comparison of malaria vectors in plains, foothills, and hilly villages showed that the availability of Anopheles culicifacies and An. fluviatilis decreased with an increase in altitude from foothills to hilly areas. INTERPRETATION & CONCLUSION: This study may be extrapolated to know the suitability of occurrence of malaria vectors and transmission of parasites at different altitudes from the viewpoint of temperature as limiting factor in unknown areas.

Optimization of Dengue-3 recombinant NS1 protein expression in E. coli and in vitro refolding for diagnostic applications.

Dengue non-structural protein (NS1) is known to be protective antigen and also has immense application for serodiagnosis. Several serodiagnostic assays available for dengue viral infection are dependent on tissue culture-grown viral proteins. This task is unsafe, laborious, more expensive that makes it unsuitable for routine large-scale production. Although bacterial expression is relatively simple and easy for recombinant protein expression, it is more challenging to make NS1 protein with native structural and immunological features using bacterial expression system. We have successfully developed a method leading to the purification and refolding of recombinant dengue virus type 3 (DENV3) NS1. The gene encoding NS1 was amplified and cloned in pET28a (+) vector. In order to increase the purity of the recombinant NS1, the transgene was engineered to carry 6× Histidine tags at both N and C-terminal ends. The recombinant construct (pETNS1) was transformed into E. coli Rosetta-gami cells and the expression conditions viz IPTG concentration, media type, temperature, and harvest time were optimized. The size of the expressed protein was found to be ~45 kDa and the authenticity of the expressed protein was confirmed using anti-His and anti-NS1 monoclonal antibodies. The NS1 protein was purified under denaturing conditions, to attain the native conformation, NS1 protein was in vitro refolded and dialyzed. The refolded NS1 protein was detected by commercial Immuno chromatographic strip and NS1 specific monoclonal antibodies. IgM antibody capture ELISA was performed using refolded recombinant NS1 protein which recognized the IgM antibodies in dengue-positive samples of acute phase of infection. Our result suggests that rNS1 protein has immense diagnostic potential and can be used in developing point of care diagnostic assays.

A simple Pichia pastoris fermentation and downstream processing strategy for making recombinant pandemic Swine Origin Influenza a virus Hemagglutinin protein.

The present Influenza vaccine manufacturing process has posed a clear impediment to initiation of rapid mass vaccination against spreading pandemic influenza. New vaccine strategies are therefore needed that can accelerate the vaccine production. Pichia offers several advantages for rapid and economical bulk production of recombinant proteins and, hence, can be attractive alternative for producing an effective influenza HA based subunit vaccine. The recombinant Pichia harboring the transgene was subjected to fed-batch fermentation at 10 L scale. A simple fermentation and downstream processing strategy is developed for high-yield secretory expression of the recombinant Hemagglutinin protein of pandemic Swine Origin Influenza A virus using Pichia pastoris via fed-batch fermentation. Expression and purification were optimized and the expressed recombinant Hemagglutinin protein was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blot and MALDI-TOF analysis. In this paper, we describe a fed-batch fermentation protocol for the secreted production of Swine Influenza A Hemagglutinin protein in the P. pastoris GS115 strain. We have shown that there is a clear relationship between product yield and specific growth rate. The fed-batch fermentation and downstream processing methods optimized in the present study have immense practical application for high-level production of the recombinant H1N1 HA protein in a cost effective way using P. pastoris.

Influence of copy number on the expression levels of pandemic influenza hemagglutinin recombinant protein in methylotrophic yeast Pichia pastoris.

The hemagglutinin (HA) gene of novel Swine Origin Influenza A/California/04/2009 (H1N1) was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA-synthetic gene having α secretory tag under the control of AOX1 promoter was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having single and multiple copy integrants of the expression cassettes were screened for the expression of full length HA protein in the culture supernatant. In order to completely exploit the expression potential of the P. pastoris expression system, a systematic investigation on the influence of gene copy number on the expression of the recombinant protein was made. A panel of Pichia clones carrying increasing copies of the heterologous gene was selected based on Geneticin resistance and SYBR green-based quantitative real-time PCR approach. Using these strategies, recombinant Pichia transformants carrying up to a maximum of four to six copies of the transgene were identified. After optimising the expression conditions for shaker flask culture, the resultant clones demonstrated that the increase in copy number results in a proportional elevation in the expression level of H1N1HA recombinant protein. Our findings clearly suggest that the gene dosage effect play a vital role in high level expression of the pandemic Influenza HA protein in yeast system.

Yeast expressed recombinant Hemagglutinin protein of novel H1N1 elicits neutralising antibodies in rabbits and mice.

Currently available vaccines for the pandemic Influenza A (H1N1) 2009 produced in chicken eggs have serious impediments viz limited availability, risk of allergic reactions and the possible selection of sub-populations differing from the naturally occurring virus, whereas the cell culture derived vaccines are time consuming and may not meet the demands of rapid global vaccination required to combat the present/future pandemic. Hemagglutinin (HA) based subunit vaccine for H1N1 requires the HA protein in glycosylated form, which is impossible with the commonly used bacterial expression platform. Additionally, bacterial derived protein requires extensive purification and refolding steps for vaccine applications. For these reasons an alternative heterologous system for rapid, easy and economical production of Hemagglutinin protein in its glycosylated form is required. The HA gene of novel H1N1 A/California/04/2009 was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA- synthetic gene having α-secretory tag was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having multiple copy integrants of the transgene expressed full length HA protein in the culture supernatant. The Recombinant yeast derived H1N1 HA protein elicited neutralising antibodies both in mice and rabbits. The sera from immunised animals also exhibited Hemagglutination Inhibition (HI) activity. Considering the safety, reliability and also economic potential of Pichia expression platform, our preliminary data indicates the feasibility of using this system as an alternative for large-scale production of recombinant influenza HA protein in the face of influenza pandemic threat.

Prospective application of Leucaena leucocephala for phytoextraction of Cd and Zn and nitrogen fixation in metal polluted soils.

The study deals with phytoextraction of Zn and Cd by Leucaena leucocephala grown on effluent fed and low nitrogen soils collected from S1, S2, and S3 sites, representing decreasing metal content with increasing distance from the effluent drain. Plant nitrogen fixation potential and soil micro-biochemical attributes against metal stress were also assessed. Increasing soil metal content and plant growth enhanced metal accumulation. Relatively greater amount of Zn than Cd was accumulated by L. leucocephala, which exceeded in roots with that of other parts. Remediation factor for Cd was maximum (3.6%) in S2 grown plant. Nodule numbers, their biomass, nitrogenase activity, and leghaemoglobin content were maximum in plants grown in S3 and minimum in S1 soil having maximum metals. Maximum soil organic C, total N, C(mic), and N(mic), respiration rate, ATP content, and enzymatic activities in response to phytoremediation was recorded in S3 followed by S2 and S1. Phytoremediation for a year enhanced extractable Zn and Cd by 36% and 45%, and their total removal by 20% and 30%, respectively from S2, which suggests the possible application of L. leucocephala for the remediation of metal contaminated sites and their fertility restoration by improving microbial functionalities and N-pool.