Search

Most of the thalassemic children of Bangladesh are receiving repeated blood transfusion. But they do not receive chelation therapy due to financial constraints. As a result, iron overload occurs in various organs of these children. Extra iron that is loaded in thyroid gland causes thyroid dysfunction. This study was undertaken to evaluate thyroid status in children with transfusion dependent Thalassemia patient. This cross-sectional analytical study was conducted in the Department of Pediatrics, Mymensingh Medical College Hospital, Bangladesh from September 2016 to April 2018. Children having thalassemia diagnosed by Hb electrophoresis, aged 3-12 years of both sexes were included as study group. Children of same age and sex admitted in indoor of Mymensingh Medical College Hospital with minor illness and without thalassemia were taken as comparison group. Purposive Sampling technique was applied. Serum FT₄, TSH and ferritin level were estimated in all children. Data analysis was done with Statistical Package for Social Science (SPSS) version 21.0. A total of 60 patients were enrolled as study group and another 60 patients were compared as comparison group. Mean ages of study group was 7.88±2.55 years and comparison group were 7.22±2.48 years. The mean pre-transfusion hemoglobin, serum ferritin, serum FT₄ and serum TSH level were found 6.23±0.60 gm/dl, 2658.33±879.39 ng/ml, 15.14±4.40 fmol/mL, 4.29±4.60 μIU/mL respectively in study group. The mean serum FT₄ was found significantly lower and mean serum TSH was significantly higher in thalassemic children in comparison to non-thalassemic children (p= <0.05). Frequency of subclinical hypothyroidism was found significantly higher in study group (25.0%) compared to comparison group (3.3%) (p=0.001). Mean serum ferritin level was found significantly higher in hypothyroid cases. Mean FT₄ level was significantly lower and mean TSH level was significantly higher in hypothyroid thalassemic patients (p= <0.001). Significant positive correlation between serum ferritin level and serum TSH level was found. Higher serum ferritin level was found significantly associated with the development of hypothyroidism in thalassemic patients.

The influenza virus neuraminidase (NA) protein is responsible for actively cleaving the sialic acid (SA) bound to the viral hemagglutinin. In the present study, we identified a combination of five novel amino acid substitutions in the NA, conferring increased substrate binding and altered surface characteristics to a low pathogenic avian influenza (LPAI) H9N2 virus strain. The H9N2 strain reported from India, A/Environmental/India/1726265/2017 (H9N2-1726265) showed the combination of amino acid substitutions T149I, R249W, G346A, W403R and G435R, which were in the vicinity of the enzyme active site cavity. The strain A/chicken/India/99321/2009 (H9N2-99321) did not show these substitutions and was used for comparison. Virus elution was studied using turkey red blood cells (tRBCs). NA enzyme kinetics assays were carried out using the MUNANA substrate, which is an SA analogue. Homology modelling and molecular docking were performed to determine alterations in the surface characteristics and substrate binding. H9N2-1726265 showed enhanced elution from tRBCs. Enzyme kinetics revealed a lower K M of H9N2-1726265 (111.5 μM) as compared to H9N2-99321 (135.2 μM), indicating higher substrate binding affinity of H9N2-1726265, due to which the NA enzyme cleaved the SA more efficiently, leading to faster elution. Molecular docking revealed a greater number of binding interactions of H9N2-1726265 to SA as compared to H9N2-99321 corroborating the greater substrate binding affinity. Changes in the surface charge, hydrophobicity, and contour, were observed in H9N2-1726265 NA due to the five substitutions. Thus, the novel combination of five amino acids near the sialic acid binding site of NA, resulted in altered surface characteristics, higher substrate binding affinity, and virus elution., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The MS-H vaccine strain (Vaxsafe MS®; Bioproperties Pty. Ltd., Australia) is a live attenuated temperature sensitive derivative of a virulent strain of M. synoviae, 86079/7NS, and is used to prevent diseases from M. synoviae challenges in poultry farms. The genome sequence of MS-H includes 32 single nucleotide polymorphisms (SNPs) compared to that of 86079/7NS. To investigate the nature of mutations responsible for temperature sensitivity, MS-H strain was subjected to thermal adaptation in vitro and in vivo. The only observed variation detected in the MS-H culture following sequential passages with incremental incubation temperature from 33 °C to 39.5 °C was an Ala210Val variation in Obg protein, associated with loss of temperature sensitivity phenotype. An identical variation was detected in the MS-H culture reisolated from one out of five bird 28 days after inoculation with MS-H. These findings suggest that M. synoviae is capable of thermoadaptive evolution and Obg plays a significant role in this trait., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: This study was funded by the Australian Research Council and Bioproperties Pty. Ltd., Australia through the ARC Linkage Project grant LP180100762. The MS-H vaccine is produced by Bioproperties Pty. Ltd., Australia., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Despite recent advances in neonatal care, early detection of neonatal sepsis still remains challenging. Positive blood culture is the gold standard for definitive diagnosis of neonatal sepsis but is time consuming and demands a well equipped laboratory setting. Therefore, it becomes imperative to evaluate usefulness of white blood cell count, Immature to total (IT) ratio and C-reactive protein as potential markers in the early diagnosis of neonatal sepsis. The objective of the study was to evaluate role of white blood cell count, IT ratio and C-reactive protein in early detection of clinically suspected neonatal sepsis. This cross-sectional descriptive study was conducted from January 2017 to December 2018 at Special Care Newborn Unit (SCANU) of Rangpur Medical College Hospital, Rangpur, Bangladesh. After parental permission and ethical clearance, a total of 70 eligible neonates were included into the study. Estimation of total white blood cell count, IT ratio and C-reactive protein as well as blood culture were done for each case. Significance for Chi-Square test and Pearson's correlation coefficient test was predetermined as p<0.05. Of the total 70 neonates studied, 19(27.14%) were blood culture positive and most common organism was Escherichia coli (7/14, 37.0%). Among individual and combination tests, CRP was highly sensitive (100%) followed by WBC count (74.94%). Highly specific tests in diagnosing sepsis were combination test of IT ratio and CRP (88.23%) followed by combination test of WBC count and CRP (82.35%). Positive predictive value (PPV) was high for combination test of WBC count and CRP (90.90%) followed by combination test of IT ratio and CRP (90.47%). Negative predictive value (NPV) was high in CRP (100.0%) followed by WBC count (89.19%). IT ratio positively correlated with CRP (p=0.002) and there was significant association between raised CRP and WBC count (p=0.005) in neonatal sepsis. Diagnostic role of both individual and combination tests were significant in early detection of clinically suspected neonatal sepsis while awaiting results of blood culture. However, none of the combination tests were able to achieve 100.0% sensitivity.

Background: A massive outbreak of dengue-like illness was reported from Pune district of Maharashtra, India during May-June 2022. Isolation and characterization of the etiological agent at genomic level for possible mutations that led to higher transmissibility is the topic of the study. Methods: Entomological investigations were carried out by ICMR-National Institute of Virology (Pune, India); Aedes aegypti mosquitoes were collected and processed for virus detection by molecular techniques. Positive mosquito pools were processed for virus isolation in cell culture. Sanger sequencing and whole-genome sequencing (WGS) using Oxford Nanopore Technology platform were used for genomic characterization. Results: Reverse transcriptase RT-PCR and qRT-PCR analysis detected chikungunya virus (CHIKV) in mosquito samples. Six CHIKV isolates were obtained. WGS revealed four nonsynonymous mutations in the structural polyprotein region, and five in the nonstructural polyprotein encoding region when compared with Yawat-2000 and Shivane-2016 strains. Sixty-four nucleotide changes in the nonstructural polyprotein region and 35 in the structural polyprotein region were detected. One isolate had an exclusive amino acid change, T1123I, in the nsP2 (protease) region. Conclusion: Abundant Ae. aegypti breeding and detection of CHIKV RNA in mosquitoes confirmed it as a chikungunya outbreak. Novel mutations detected in the epidemic strain warrants investigations to address their role in disease severity, transmission, and fitness.

Background & Objectives: Sandflies are implicated as vectors of Chandipura virus (CHPV) (Vesiculovirus: Rhabdoviridae). The virus is prevalent in central India including Vidarbha region of Maharashtra. CHPV causes encephalitis in children below 15 yr of age with case fatality rates ranging from 56 to 78 per cent. The present study was undertaken to determine the sandfly fauna in the CHPV endemic Vidharba region., Methods: A year round survey of sandflies was conducted at 25 sites in three districts of Vidarbha region. Sandflies were collected from their resting sites using handheld aspirators and identified using taxonomical keys., Results: A total of 6568 sandflies were collected during the study. Approximately 99 per cent of the collection belonged to genus Sergentomyia, which was represented by Ser. babu, Ser. bailyi and Ser. punjabensis. Genus Phlebotomus was represented by Ph. argentipes and Ph. papatasi. Ser. babu was the predominant species (70.7%) collected during the study. Ph. argentipes was detected in four villages with 0.89 per cent, whereas Ph. papatasi was detected in only one village with 0.32 per cent of the total collection. CHPV could not be isolated despite processing all the sandflies for virus isolation in cell culture., Interpretation & Conclusions: The present study showed influence of higher temperature and relative humidity on sandfly population dynamics. An important observation during the study was the absence or decline in the population of Ph. papatasi and Ph. argentipes in the study area. Surge in Sergentomyia population and their breeding/resting in close vicinity to humans pose a concern as they are known to harbour CHPV and other viruses of public health importance.

The Mycoplasma synoviae live attenuated vaccine strain MS-H (Vaxsafe MS; Bioproperties Pty., Ltd., Australia) is commonly used around the world to prevent chronic infections caused by M. synoviae in birds and to minimize economic losses in the poultry industry. MS-H is a temperature-sensitive strain that is generated via the chemical mutagenesis of a virulent M. synoviae isolate, 86079/7NS. 32 single nucleotide polymorphisms have been found in the genome of MS-H compared to that of 86079/7NS, including 25 in predicted coding sequences (CDSs). There is limited information on the stability of these mutations in MS-H in vitro during the propagation of the vaccine manufacturing process or in vivo after the vaccination of chickens. Here, we performed a comparative analysis of MS-H genomes after in vitro and in vivo passages under different circumstances. Studying the dynamics of the MS-H population can provide insights into the factors that potentially affect the health of vaccinated birds. The genomes of 11 in vitro laboratory passages and 138 MS-H bird reisolates contained a total of 254 sequence variations. Of these, 39 variations associated with CDSs were detected in more than one genome (range = 2 to 62, median = 2.5), suggesting that these sequences are particularly prone to mutations. From the 25 CDSs containing previously characterized variations between MS-H and 86079/7NS, 7 were identified in the MS-H reisolates and progenies examined here. In conclusion, the MS-H genome contains individual regions that are prone to mutations that enable the restoration of the genotype or the phenotype of wild-type 86079/7NS in those regions. However, accumulated mutations in these regions are rare. IMPORTANCE Preventative measures, such as vaccination, are commonly used for the control of mycoplasmal infections in poultry. A live attenuated vaccine strain (Vaxsafe MS; MS-H; Bioproperties Pty. Ltd., Australia) is used for the prevention of disease caused by M. synoviae in many countries. However, information on the stability of previously characterized mutations in the MS-H genome is limited. In this study, we performed a comparative analysis of the whole-genome sequences of MS-H seeds used for vaccine manufacturing, commercial batches of the vaccine, cultures minimally passaged under small-scale laboratory and large-scale manufacturing conditions, MS-H reisolated from specific-pathogen-free (SPF) chickens that were vaccinated under controlled conditions, and MS-H reisolated from vaccinated commercial poultry flocks around the world. This study provides a comprehensive assessment of genome stability in MS-H after in vitro and in vivo passages under different circumstances and suggests that most of the mutations in the attenuated MS-H vaccine strain are stable.

Infection and immunity studies involving genetically modified organisms (GMOs), such as gene knockout bacterial mutants, require stringent physical containment to prevent the accidental spread of these organisms into the environment. Experimental respiratory tract infection models often require the animals, for example birds, to be transported several times between a negative pressure housing isolator and a bespoke aerosol exposure chamber under positive pressure. While the exposure chamber is sealed and fitted with HEPA filters, the repeated movements of infected animals and opening of the chamber can still pose a serious risk of breaching containment of the organism in the experimental facility. In the current study, the ability of two aerosol infection protocols that expose birds to avian pathogenic E. coli (APEC) aerosols directly within the housing isolator was evaluated. Young chicks were exposed to APEC E956 within the negative pressure housing isolators using either a nebulizer or an atomizer. Birds exposed twice (days 1 and 4) to aerosols of APEC E956 produced by the nebulizer developed a rapidly progressing disease mimicking field cases of avian colibacillosis. However, birds exposed to aerosols of APEC E956 produced by an atomizer did not develop colibacillosis even after three exposures to APEC E956 on days 1, 4 and 7. Consequently, the current study reports the nebulizer was more efficacious in producing avian colibacillosis under stricter bacterial containment settings. RESEARCH HIGHLIGHTS Two aerosol exposure methods were evaluated to develop avian colibacillosis.Nebulizer method found to be more efficient in reproducing avian colibacillosis.Refined infection method can be used to study genetically modified organisms (GMOs).

Infections caused by Mycoplasma synoviae are major welfare and economic concerns in poultry industries worldwide. These infections cause chronic respiratory disease and/or synovitis in chickens and turkeys leading to reduced production and increased mortality rates. The live attenuated vaccine strain MS-H (Vaxsafe ® MS), commonly used for protection against M. synoviae infection in many countries, contains 32 single nucleotide variations compared to its wildtype parent strain, 86079/7NS. Genomic analysis of vaccine strains reisolated from flocks following the administration of MS-H has identified reversions to the original 86079/7NS sequence in the obgE , oppF and gapdh genes. Here, three MS-H field reisolates containing the 86079/7NS genotype in obgE (AS2), obgE and oppF (AB1), and obgE , oppF and gapdh (TS4), as well as the vaccine MS-H and the parental strain 86079/7NS were experimentally inoculated to chickens. The strains were assessed for their ability to infect and elicit immune responses in the recipient chickens, as well as in naïve in-contact chickens. Despite the loss of temperature sensitivity phenotype and colonization of the reisolates in the lower respiratory tract, there was no significant differences detected in the microscopic mucosal thickness of the middle or lower trachea of the inoculated chickens. Concurrent reversions in ObgE, OppF and GAPDH proteins were associated with higher gross air sac lesion scores and increased microscopic upper-tracheal mucosal thickness in chickens directly inoculated with the reisolates following intratracheal administration of a virulent strain of infectious bronchitis virus. The gross air sac lesions of the chickens in-contact with those inoculated with reisolates were not significantly different to those of chickens in-contact with MS-H inoculated chickens, suggesting that horizontal transmission of the reisolates in the poultry flock will not lead to higher pathogenicity or clinical signs. These results suggest a significant role of GAPDH and/or cumulative effect of ObgE, OppF and GAPDH on M. synoviae pathogenicity. Future experiments will be required to investigate the effect of single mutations in gapdh or oppF gene on pathogenicity of M. synoviae ., Competing Interests: This study was funded by the Australian Research Council and Bioproperties Pty. Ltd. through the ARC Linkage Project grant LP180100762. The MS-H vaccine is produced by the Bioproperties Pty. Ltd., Australia. The authors declare that this study received funding from Bioproperties Pty. Ltd. The funder had the following involvement in the study: study design, decision to publish, and reviewing the manuscript., (Copyright © 2022 Klose, Omotainse, Zare, Vaz, Armat, Shil, Wawegama, Kanci Condello, O'Rourke, Disint, Andrews, Underwood, Morrow, Marenda and Noormohammadi.)

Background: Studies on antigenic proteins for arboviruses are important for providing diagnostics and vaccine development. India and its neighboring countries have a huge burden of arboviral diseases. Data mining for country-specific sequences from existing bioinformatics databases is cumbersome and time-consuming. This necessitated the development of a database of antigenic proteins from arboviruses isolated from the countries of the Indian subcontinent., Methods: Arboviral antigenic protein sequences were obtained from the NCBI and other databases. In silico antigenic characterization was performed (Epitope predictions) and data was incorporated into the database. The front end was designed and developed using HTML, CSS, and PHP. For the backend of the database, we have used MySQL., Results: A database, named ArVirInd, is created as a repository of information on curated antigenic proteins. This enlists sequences by country and year of outbreak or origin of the viral strain. For each entry, antigenic information is provided along with functional sites, etc . Researchers can search this database by virus/protein name, country, and year of collection (or in combination) as well as peptide search for epitopes. It is available publicly via the Internet at http://www.arvirind.co.in. ArVirInd will be useful in the study of immune informatics, diagnostics, and vaccinology for arboviruses., Competing Interests: The authors declare there are no competing interests., (©2022 Atre et al.)

India is one of the worst affected countries during the COVID-19 pandemic. We carried out comparative analyses of the COVID-19 situation in the Maharashtra state, India for the first and second waves. Epidemiological and demographics data were obtained from open sources and the Government of Maharashtra. Mathematical modeling and analyses were conducted to estimate the epidemiological parameters like basic reproduction number (R0) for the first wave at different times. The districts with a higher percentage of the urban population recorded a higher attack rate during the first wave. However, during the second wave, the rural population was more affected. The effective reproduction number (Re) was estimated for the second wave at different times. The second wave affected more individuals than the first wave due to various factors such as strictness of restrictions or the lack of it and the emergence of new strains., (Copyright © 2022. Published by Elsevier Ltd.)

Infectious laryngotracheitis virus (ILTV) is the causative agent of an economically important disease of chickens causing upper respiratory tract infection. Strains of ILTV are commonly identified by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and/or PCR high resolution melt (PCR-HRM) curve analysis targeting several genes. However, these techniques examine only a limited number of mutations present inside the target regions and may generate unreliable results when the sample contains more than one strain. Here, we attempted to sequence the whole genome of ILTV with known identity (class 9) directly from tracheal scrapings to circumvent in vitro culturing, which can potentially introduce variations into the genome. Despite the large number of quality reads, mapping was compromised by poor overlapping and gaps, and assembly of the complete genome sequence was not possible. In a map-to-reference alignment, the regions with low coverage were deleted, those with high coverage were concatenated and a genome sequence of 139,465 bp was obtained, which covered 91% of the ILTV genome. Sixteen single-nucleotide polymorphisms (SNPs) were found between the ILTV isolate examined and ILTV class 9 (JN804827). Despite only 91% genome coverage, using sequence analysis and comparison with previously sequenced ILTVs, we were able to classify the isolate as class 9. Therefore, this technique has the potential to replace the current PCR-HRM technique, as it provides detailed information about the ILTV isolates., (© 2022. The Author(s).)

Chandipura virus (CHPV) is an emerging pathogen responsible for acute encephalitic syndrome (AES) in pediatric population in India. Several outbreaks of CHPV have been reported from different states of India since the year 2003. At present there is no vaccine or therapeutic measures available to curtail the disease. In this study, we have identified both T-cell and B-cell epitopes of different antigenic proteins of CHPV like Nucleoprotein (N), Phosphoprotein (P) and Matrix protein (M) along with the immuno-dominant glycoprotein (G) and conducted in silico characterization for the same. The idea is to design a multi-epitope peptide construct using the epitopes, which were found to be non-toxic, non-allergenic and possessing high immunogenicity. The final multi-epitope construct named as: MEC-CHPV, comprised of β-defensin adjuvant at N-terminal for enhancement of immunogenicity followed by fourteen B-cell epitopes, four Helper T-cell epitopes and six Cytotoxic T-cell epitopes. The characterization of designed construct was carried out in terms of physicochemical parameters, antigenicity and allergenicity. The 3D structure prediction was performed. Molecular docking and molecular-dynamics simulation of MEC-CHPV with Toll like receptors (TLR-3 and TLR-8) showed stable interactions. In silico cloning of MEC-CHPV in pET30a(+) expression vector was also conducted using codon optimization. The in silico immune-simulation indicated a typical immune response against MEC-CHPV when used as a potential vaccine. This study provides a cost-effective and time-saving way to design a peptide vaccine candidate against CHPV using immuno-informatics approach. Development of the MEC-CHPV construct may pave the way for future laboratory experiments.Communicated by Ramaswamy H. Sarma.

Fowlpox (FP) is an economically important viral disease of commercial poultry. The fowlpox virus (FPV) is primarily characterised by immunoblotting, restriction enzyme analysis in combination with PCR, and/or nucleotide sequencing of amplicons. Whole-genome sequencing (WGS) of FPV directly from clinical specimens prevents the risk of potential genome modifications associated with in vitro culturing of the virus. Only one study has sequenced FPV genomes directly from clinical samples using Nanopore sequencing, however, the study didn't compare the sequences against Illumina sequencing or laboratory propagated sequences. Here, the suitability of WGS for strain identification of FPV directly from cutaneous tissue was evaluated, using a combination of Illumina and Nanopore sequencing technologies. Sequencing results were compared with the sequence obtained from FPV grown in chorioallantoic membranes (CAMs) of chicken embryos. Complete genome sequence of FPV was obtained directly from affected comb tissue using a map to reference approach. FPV sequence from cutaneous tissue was highly similar to that of the virus grown in CAMs with a nucleotide identity of 99.8%. Detailed polymorphism analysis revealed the presence of a highly comparable number of single nucleotide polymorphisms (SNPs) in the two sequences when compared to the reference genome, providing essentially the same strain identification information. Comparative genome analysis of the map to reference consensus sequences from the two genomes revealed that this field isolate had the highest nucleotide identity of 99.5% with an FPV strain from the USA (Fowlpox virus isolate, FWPV-MN00.2, MH709124) and 98.8% identity with the Australian FPV vaccine strain (FWPV-S, MW142017). Sequencing results showed that WGS directly from cutaneous tissues is not only rapid and cost-effective but also provides essentially the same strain identification information as in-vitro grown virus, thus circumventing in vitro culturing., Competing Interests: The authors have declared that no competing interests exist.

Avian hepatitis E virus (aHEV) is the causative agent of an important disease of broiler breeders and layers. aHEV cannot be readily propagated in cell culture and is characterised primarily by sequencing of amplicons generated through several RT-PCRs that target individual genes. This study aims to uncover the origin of current Australian aHEV isolates based on whole genome sequencing using clinical liver tissues. Complete genome sequences of the two aHEV isolates were assembled using Nanopore and Illumina reads. The two isolates possessed only four single nucleotide polymorphisms to each other. Comparison of the sequences with aHEV genome sequences available in the GenBank showed the highest nucleotide sequence identity of 88% with the prototype USA strain (AY535004), 82% with the European (AM943647) and genotype 1 Australian strains (AM943647). Recombination analysis suggested that aHEV isolates characterised in this study are progeny of a cross between a US and a Hungarian strain. Phylogenetic tree and phylogenetic networks constructed using complete genome and individual coding sequences revealed that Australian aHEV isolates formed a distinct clade closer to the USA strains and classified as genotype 2 whereas genotype 1 Australian strain clustered together with South Korean strains., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Chandipura virus (CHPV), belonging to the genus Vesiculovirus of the family Rhabdoviridae, has been identified as one of the causes of pediatric encephalitis in India. Currently, neither vaccines nor therapeutic drugs are available against this agent. Considering that the disease progresses very fast with a high mortality rate, working towards the development of potential therapeutics against it will have a public health impact. Although the use of viral inhibitors as antiviral agents is the most common way to curb virus replication, the mutation-prone nature of viruses results in the development of resistance to antiviral agents. The recent development of proteomic platforms for analysis of purified viral agents has allowed certain upregulated host proteins that are involved in the morphogenesis and replication of viruses to be identified. Thus, the alternative approach of inhibition of host proteins involved in the regulation of virus replication could be explored for their therapeutic effectiveness. In the current study, we have evaluated the effect of inhibition of cyclophilin A (CypA), an immunophilin with peptidyl-prolyl cis/trans-isomerase activity, on the replication of CHPV. Treatment with cyclosporin A, used in vitro for the inhibition of CypA, resulted in a 3-log reduction in CHPV titer and an undetectable level of CypA in comparison to an untreated control. An in silico analysis of the interaction of the CHPV nucleoprotein with the human CypA protein showed stable interaction in molecular docking and molecular dynamics simulations. Overall, the results of this study suggest a possible role of CypA in facilitating CHPV replication, thus making it one of the potential host factors to be explored in future antiviral studies., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Characterisation of the entire genome of Fowl aviadenoviruses (FAdV) requires isolation and propagation of the virus in chicken embryo liver or kidney cells, a process which is not only time consuming but may occasionally fail to result in viral growth. Furthermore, in a mixed infection, isolation in cell culture may result in the loss of viral strains. In this study, we optimised a FAdV DNA extraction technique directly from affected liver tissues using kaolin hydrated aluminium silicate treatment. The whole genome of FAdV was sequenced directly from extracted DNA without any targetted PCR based enrichment. The extraction method was also tested on avian liver tissues affected with the RNA virus Avian hepatitis E virus and demonstrated to yield sequencing grade RNA. Therefore, the method described here is a simple technique which is potentially useful for the extraction of sequencing grade DNA/RNA from tissues with high fat content., Competing Interests: Declaration of Competing Interest We have no conflicts of interest to disclose., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Genomic comparison of Mycoplasma synoviae vaccine strain MS-H and the MS-H parental strain 86,079/7NS established a preliminary profile of genes related to attenuation of MS-H. In this study we aimed to identify the stability of mutations found in MS-H after passage in experimental or field chickens, and to evaluate if any reverse mutation may be associated with changes in characteristics of MS-H in vitro or in vivo., Results: Whole genome sequence analysis of 5 selected MS-H field reisolates revealed that out of 32 mutations reported previously in MS-H, 28 remained stable, while four found to be reversible to the wild-type. Each isolate possessed mutations in one to three of the genes obg, oppF 1 and gap and/or a non-coding region. Examination of the 4 reversible mutations by protein modeling predicted that only two of them (in obg and oppF 1 genes) could potentially restore the function of the respective protein to that of the wild-type., Conclusions: These results suggest that the majority of the MS-H mutations are stable after passage in vaccinated chickens. Characterisation of stable mutations found in MS-H could be utilised to develop rapid diagnostic techniques for differentiation of vaccine from field strains or ts- MS-H reisolates.

The Mycoplasma synoviae (MS) vaccine strain MS-H harbours a frameshift mutation in oppF 1 (oligopeptide permease transporter) which results in expression of a truncated OppF 1 . The effect of this mutation on growth and attenuation of the MS-H is unknown. In this study, the impact of the mutation on the vaccine phenotype was investigated in vitro by introducing a wild-type copy of oppF 1 gene in the MS-H genome. Wild-type oppF 1 was cloned under the vlhA promoter into an oriC vector carrying a tetracycline resistance gene. MS-H was successfully transformed with the final construct pMS-oppF 1 -tetM or with a similar vector lacking oppF 1 coding sequence (pMS-tetM). The MS-H transformed with pMS-oppF 1 -tetM exhibited smaller colony size than MS-H transformed with pMS-tetM. Monospecific rabbit sera against C-terminus of OppF 1 detected bands of expected size for full-length OppF 1 in the 86079/7NS parental strain of MS-H and the MS-H transformed with pMS-oppF 1 -tetM, but not in MS-H and MS-H transformed with pMS-tetM. Comparison of the growth curve of MS-H transformants harvested from media with/without tetracycline was conducted using vlhA Q-PCR which revealed that MS-H transformed with pMS-tetM had a higher growth rate than MS-H transformed with pMS-oppF 1 -tetM in the media with/without tetracycline. Lastly, the whole genome sequencing of MS-H transformed with pMS-oppF 1 -tetM (passage 27) showed that the chromosomal copy of the mutated oppF 1 had been replaced with a wild-type version of the gene. This study reveals that the truncation of oppF 1 impacts on growth characteristics of the MS-H and provides insight into the molecular pathogenesis of MS and perhaps broader mycoplasma species. RESEARCH HIGHLIGHTS The full-length OppF 1 was expressed in Mycoplasma synoviae MS-H vaccine.Truncation of oppF 1 impacts on growth characteristics of the MS-H.Chromosomal copy of the mutated oppF 1 in MS-H was replaced with wild-type oppF 1 .

Rice (Oryza sativa L) is a most important staple food crop of the world because more than half of the World's population is dependent on it for their livelihood. Global rice production must be doubled by 2050 to cope up with the situation of population growth. Narrow genetic base in the released varieties has made the improvement in plateaus. Widening the genetic base is necessary to overcome the yield barrier. Hybridization and pre-breeding has been carried out to broaden the genetic base. Heritability and genetic advances were measured in the F5 lines (Tulaipanji × IR64), F3 lines (Tulaipanji × IR64 × PB1460), and F3 lines (Badshabhog × Swarna sub1). Some of the breeding lines were showing promising field performance with high yield potentiality. Wide crosses were performed to widen the genetic base between (Ranjit × O. rufipogon) and (Badshabhog × O. rufipogon) and the heritability pattern of the morphological characteristics in the progeny lines was evaluated. Nutritional quality of the rice grain is totally dependent on the morphology and histological characteristics of the caryopsis which are genetically determined. Caryopses ultrastructural analyses were carried out in seventeen different rice breeding lines through SEM. SEM analysis showed distinguishing ultrastructure in respect to pericarp, testa, aleurone layer, protein bodies and starchy endosperm in the breeding lines with distinctive inheritance pattern. This study provides information about the cross compatibility of the wide hybridization and heritability measures of the morphological traits which may supplement the breeding program to break the yield plateaus.

Background & Objectives: An outbreak of dengue-like illness was reported from Wadi area within the Nagpur Municipal Corporation during September-October 2017 with five deaths. Major symptoms reported were high fever (103-106 o F), acute joint pains, myalgia, drowsiness, breathlessness, etc. An investigation was conducted to confirm the etiological agent, its characterization and the vectors involved in the outbreak., Methods: Serological analysis was conducted to detect dengue (DEN)/chikungunya IgM antibodies in 158 sera samples. Nested-PCR was carried out to serotype eight ELISA positive samples. Adult and larval mosquito collections were conducted in the affected areas to determine species composition and mosquito density., Results: Dengue IgM antibodies were detected in 44 sera samples. Molecular typing revealed involvement of DEN-2 and DEN-3 serotypes. Dengue hemorrhagic fever symptoms were observed in two patients. Aedes aegypti breeding was found rampant with Breteu index and house index ranging from 23 to 70 and 17 to 56, respectively. Major breeding habitats encountered were, used tyres, cement tanks and refrigerator trays., Interpretation & Conclusion: Clinical symptoms, detection of anti-DEN IgM antibodies in high number of samples and heavy breeding of Ae. aegypti confirmed it was a dengue outbreak.

Background and Aim: The most widely adopted technique to preserve the gross specimen of the parasite is immersions and storage in liquid preservatives. The present study aimed to describe the dry method of the preservation of Toxocara vitulorum using plastination technique., Materials and Methods: Acetone dehydrated parasites were incubated at -20°C for 1 month in five different plastination solutions, prepared by mixing melamine and turpentine oil with clove oil (MTCl)/chloroform (MTC)/isopropanol (MTI)/benzene (MTB)/xylene (MTX) in 1:1:1 ratio to infiltrate the polymer. Technical personnel was asked to assign weekly score for dryness, stickiness, shrinkage, glossiness, flexibility, and odor of the prepared model on a 5-point scale., Results: Overall, the plastinated parasites were dry, non-sticky, glossy, odorless, chemical-free, harmless, to some extent flexible, with detectable morphological structure including natural form but lost their natural color, and cuticle became translucent. A varying level of shrinkage was noted in all types of plastinated model, but it was least in MTCl model. One month post-plastination, the mean evaluation score for glossiness was maximum in the parasite plastinated in MTCl solution (4.50±0.17), followed by MTC (3.72±0.32), MTX (3.56±0.38), MTB (2.83±0.37), and MTI (2.31±0.33). Likewise, for flexibility, the score was maximum in the parasite plastinated in MTCl solution (4.36±0.16), followed by MTB (3.11±0.14), MTC (2.94±0.41), MTX (2.75±0.41), and MTI (1.97±0.28). The degree of dryness, stickiness, and odor of the prepared model varies non-significantly (p>0.05) with the polymer mixtures. Maximum shrinkage percentage in terms of length and width was 4.24% and 50%, respectively, in the parasites plastinated in MTB solution. Shrinkage percentage was minimal (1.81% in length and 25% in width) in the MTCl plastinated parasites. Shrinkage percentage in terms of dimension was statistically non-significant among the different polymer solutions. Plastinated models withstand the process of microbial decomposition. There were 5 and 11 odd points in favor of plastination and formalin preservation technique, respectively., Conclusion: The prepared T . vitulorum model in MTCl can be used as an adjunct to the parasite preserve in 10% formalin solution. The plastination technique can be used as an alternative method of liquid preservation., (Copyright: © Kumar, et al.)

Background & Objectives: Due to the emergence of Kyasanur forest disease (KFD) virus to new regions in India, there is an urgent need to develop an early diagnostic system, which is cost-effective and can be efficiently used with minimum paraphernalia. The non-structural-1 (NS1) protein is known to be an early diagnostic marker for flaviviruses. Furthermore, NS1 antigen capture ELISA kits developed using bacterially expressed dengue NS1 protein are commercially available., Methods: Based on the data available on dengue virus, West Nile virus and other flaviviruses, bacterially expressed Kyasanur forest disease virus (KFDV) NS1 protein and polyclonal serum raised against the NS1 protein in mice and rabbit were used to develop an antigen capture ELISA for early diagnosis of the virus. The feasibility of this ELISA was further tested using in silico predictions., Results: KFDV NS1 gene was cloned, expressed and confirmed by SDS-PAGE and western blotting. An antigen detection ELISA was standardized and sensitivity and specificity was tested with other flaviviruses. KFDV acute phase 43 samples were tested and only two were found to be positive for KFDV NS1 antigen. Superimposition of KFDV NS1 and TBEV NS1 revealed a root mean square distance (RMSD) of ~0.79 Å covering 1220 backbone atoms. This implies that the structures are very similar in terms of 3D fold. The identity of amino acid composition between these proteins was 73.4% and similarity was 92.9%, as revealed from the pairwise comparison., Interpretation & Conclusion: The study points out that the half-life, expression and secretion levels of KFDV NS1 protein are not sufficient enough for its use as early diagnostic marker. The protein may have to be expressed in eukaryotic host to counter the lack of glycosylation in bacterial plasmid based expression of proteins. Hence, bacterially expressed KFDV NS1 protein may not be an ideal early diagnostic marker for the virus., Competing Interests: None

Background & Objectives: Chikungunya virus (CHIKV), a mosquito-borne arthritogenic virus causes infections ranging from febrile illness to debilitating polyarthralgia in humans. Re-emergence of the virus has affected millions of people in Africa and Asia since 2004. During the outbreak, a new lineage of the virus has evolved as an adaptation for enhanced replication and transmission by Aedes albopictus mosquito. A study was designed to compare the susceptibility of four vertebrate cell lines, namely Vero E6 (African green monkey kidney), BHK-21 (Baby hamster kidney), RD (human rhabdomyosarcoma), A-549 (human alveolar basal epithelial cell) and C6/36 (Ae. albopictus) to Asian genotype and two lineages of East, Central and South African (E1:A226 and E1:A226V) of CHIKV., Methods: One-step growth kinetics of different CHIKV strains was carried out in the above five cell lines to determine the growth kinetics and virus yield. Virus titre was determined by 50 per cent tissue culture infectious dose assay and titres were calculated by the Reed and Muench formula. Growth and virus yield of the three strains in Ae. aegypti mosquitoes was studied by intrathoracic inoculation and virus titration in Vero E6 cell line., Results: Virus titration showed Vero E6, C6/36 and BHK-21 cell lines are high virus yielding with all the three lineages while RD and A-549 yielded low virus titres. C6/36 cell line was the most sensitive and yielded the maximum titre. Ae. aegypti mosquitoes, when inoculated with high titre virus, yielded an almost equal growth with the three strains while rapid growth of E1:A226V and Asian strain was observed with 1 log virus., Interpretation & Conclusions: C6/36 cell line was found to be the most sensitive and high yielding for CHIKV irrespective of lineages while Vero E6 and BHK-21 cell lines yielded high titres and may find application for vaccine/diagnostic development. Infection of Ae. aegypti mosquitoes with the three CHIKV strains gave almost identical pattern of growth., Competing Interests: None

Mycoplasma synoviae (MS) is a major pathogen of poultry globally, causing chronic respiratory disease and arthritis. Vaccination is an effective means for the control of the disease. The MS-H vaccine is an attenuated strain developed through chemical mutagenesis of an Australian field strain, 86079/7NS. Analysis of whole genome of MS-H and its comparison with that of 86079/7NS has revealed a frameshift mutation early in a gene (oppF) that codes for an oligopeptide transporter permease, OppF. Monospecific antibodies raised against peptides upstream and downstream of the mutation in OppF revealed that only N-terminus of the OppF was expressed in MS-H while the full version was expressed in 86079/7NS. Also, examination of the recombinant N- (OppF-N) and C termini (OppF-C) of OppF, upstream and downstream of the mutation site respectively, as well as the full length OppF in Western immunoblotting experiments showed that serum from MS-H vaccinated chicken strongly bound OppF-N while serum from 86079/7NS challenged chicken detected OppF, OppF-N and OppF-C. The potential of the recombinant OppF, OppF-N and OppF-C to discriminate antibody responses to MS-H reisolates with wild or vaccine type OppF was assessed against 88 chicken sera in indirect ELISA and ratios were calculated between optical densities (OD) over those obtained in MS major membrane protein MSPB ELISA. Comparison of the OD ratios revealed that the MSPB/OppF and MSPB/OppF-C OD ratios of the sera against isolates with vaccine type OppF were significantly higher than those against isolates with wild type OppF. These results are in accordance with oppF gene mutation in MS-H and confirms that MS-H does not express OppF beyond the frame shift mutation found in its oppF gene. Also, the indirect ELISA based on OppF-C in combination with the MSPB has the potential to differentiate between MS-H and field strain antibody responses., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

An unknown virus was repeatedly isolated from hard tick (Haemaphysalis spinigera) during a proactive arbovirus survey in ticks conducted in 1957, in India. The virus remained uncharacterized for a long time. The passages of this virus in different vertebrate and invertebrate cells along with human and monkey-derived cell culture showed no cytopathic effect. It was identified later to be a member of Kaisodi group among Phlebovirus genus in the family Phenuiviridae (Order: Bunyavirales) by serological methods. Due to its genomic diversity, sequencing of this virus was a challenge for a while. In this study, we were able to sequence the complete genome of this virus isolate using next-generation sequencing (NGS) platform. The unknown virus was identified to be Kaisodi virus (KASDV) using NGS analysis. De novo genome assembly derived three genomic segments for the KASDV which encode for RNA-dependent RNA polymerase, glycoprotein precursor, and nucleoprotein. Functional as well as conserved domains for Kaisodi serogroup viruses were predicted and compared to a known representative of the genus Phlebovirus. The phylogenetic tree revealed its closeness to Silverwater virus, of Kaisodi serogroup with nucleotide (69%, 62%, and 61%) and amino acid (52%, 51%, and 62%) identity for L, M, and S segment, respectively. The study demonstrates the presence of a conserved motif ( 72 TRGNK 76 ) around the RNA binding motif region in tick-borne phleboviruses. The intergenic region encompassing the S segment of Kaisodi serogroup was GC-rich whereas the other Phlebovirus had AT-rich genome. KASDV has the largest intergenic region and larger loops, suggesting stem-loops formed due to larger loops as a possible factor for instability and cause of transcription termination. This paper also describes the real-time RT-PCR and RT-PCR assays developed and used for the detection of KASDV RNA in ticks from Karnataka, Kerala and Maharashtra State, India. The KASDV positivity observed in the recently collected tick pools indicates that the KASDV, isolated from Karnataka state in 1957, is also circulating in the adjoining Kerala state. On the basis of the current study, it should be possible to develop diagnostic assays which would facilitate an in-depth field survey exploring the veterinary and medical significance of KASDV., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Chandipura virus (CHPV), associated with an encephalitic illness in humans, has caused multiple outbreaks with high mortality in central and western India in recent years. The present study compares surface glycoprotein (G-protein) from prototype and recent outbreak strains using in silico tools and in vitro experiments. In silico epitope predictions (B-cell and T-helper cell) for the sequences, 3D structure prediction and comparison of the G-proteins of the strains: I653514 (Year 1965), CIN0327 (Year 2003) and 148974 (Year 2014) revealed that the CHPV G-protein is stable and antigenic determinants are conserved. A monoclonal antibody developed against strain CIN0327 (named NAbC) was found to neutralize prototype I653514 as well as the currently circulating strain 148974. In silico antigen-antibody interaction studies using molecular docking of predicted structures of NAbC and G-proteins of various CHPV strains led to the identification of a conserved neutralizing epitope in the fusion domain of G-protein, which also contained a putative T-helper peptide. The identification of a conserved neutralizing epitope in domain IV (fusion domain amino acids 53 to 172) of CHPV G-protein is an important finding that may have the scope towards the development of protective targets against CHPV infection.

Influenza A virus infection induces type I interferons (IFNs α/β) which activate host antiviral responses through a cascade of IFN signaling events. Herein, we compared highly pathogenic H5N1 and low pathogenic H11N1 avian influenza viruses isolated from India, for their replication kinetics and ability to induce IFN-β and interferon-stimulating genes (ISGs). The H5N1 virus showed a higher replication rate and induced less IFN-β and ISGs compared to the H11N1 virus when grown in the human lung epithelial A549 cells, reflecting the generation of differential innate immune responses during infection by these viruses. The non-structural 1 (NS1) protein, a major IFN-antagonist, known to help the virus in evading host innate immune response was compared from both the strains using bioinformatics tools. Analyses revealed differences in the composition of the NS1 proteins from the two strains that may have an impact on the modulation of the innate immune response. Intriguingly, H5N1 virus attenuated IFN-β response in a non-NS1 manner, suggesting the possible involvement of other viral proteins (PB2, PA, PB1/PB1-F2) of H5N1 in synergy with NS1. Preliminary analyses of the above proteins of the two strains by sequence comparison show differences in charged residues. The insight gained will be useful in designing experimental studies to elucidate a probable role of the polymerase protein(s) in association with NS1 in inhibiting the IFN signaling and understanding the molecular mechanism governing the difference.

Changing climate scenario has resulted in recent emergence and re-emergence of various arboviral diseases including Chikungunya. This disease is caused by Chikungunya virus (CHIKV), which belongs to Togaviridae family of viruses and spread by Aedes mosquitoes. A resurgence of CHIKV and its rapid global spread has been observed since 2004. The disease reemerged in India in 2005, after a gap of 32 years, causing massive outbreaks in some states and circulating thereafter. In the present paper we analyze CHIKV incidence data from India (2010-2014) with a view to understand association with environmental parameters, if any. Data on country-wide occurrences of CHIKV cases were considered from the National Vector Borne Disease Control Board, India. Meteorological data for different climatic subdivisions of India were obtained and processed mathematically. State-wise association of number of cases with rainfall, if any, were studied by statistical analyses. We observe that prevailing temperature range was favorable for CHIKV propagation and the occurrences were modulated by average rainfall. Most affected states were West Bengal, Maharashtra and Karnataka. Overall for India, favorable climatic conditions have contributed to incidences of CHIKV during the study period. There is strong positive association between rainfall variations and occurrence of CHIKV cases.

Background & Objectives: Kyasanur Forest disease (KFD) is a febrile illness characterized by haemorrhages and caused by KFD virus (KFDV), which belongs to the Flaviviridae family. It is reported to be an endemic disease in Shimoga district of Karnataka State, India, especially in forested and adjoining areas. Several outbreaks have been reported in newer areas, which raised queries regarding the changing nature of structural proteins if any. The objective of the study was to investigate amino acid composition and antigenic variability if any, among the envelope glycoprotein (E-proteins) from old and new strains of KFDV., Methods: Bioinformatic tools and techniques were used to predict B-cell epitopes and three-dimensional structures and to compare envelope glycoprotein (E-proteins) between the old strains of KFDV and those from emerging outbreaks till 2015., Results: The strain from recent outbreak in Thirthahalli, Karnataka State (2014), was similar to the older strain of KFDV (99.2%). Although mutations existed in strains from 2015 in Kerala KFD sequences, these did not alter the epitopes., Interpretation & Conclusions: The study revealed that though mutations existed, there were no drastic changes in the structure or antigenicity of the E-proteins from recent outbreaks. Hence, no correlation could be established between the mutations and detection in new geographical areas. It seems that KFDV must be present earlier also in many States and due to availability of testing system and alertness coming into notice now., Competing Interests: None

Aedes vittatus (Bigot) mosquito is a voracious biter of humans and has a geographical distribution throughout tropical Asia, Africa and the Mediterranean region of Europe. It is predominantly a rock-hole breeder, though it can breed in diverse macro- and micro-habitats. The mosquito plays an important role in the maintenance and transmission of yellow fever (YFV), dengue (DENV), chikungunya (CHIKV) and Zika (ZIKV) viruses. It has been implicated as an important vector of YFV in several African countries as evidenced by repeated virus isolations from the mosquito and its potential to transmit the virus experimentally. Similarly, DENV-2 has been isolated from wild caught Ae. vittatus mosquitoes in Senegal, Africa which has been shown to circulate the virus in sylvatic populations without causing human infection. Experimental studies have shown replication of the virus at a low scale in naturally infected mosquitoes while high rate of infection and dissemination have been reported in parenterally infected mosquitoes. Natural isolation of ZIKV has been reported from Senegal and Cote d'Ivoire from these mosquitoes. They were found highly competent to transmit the virus experimentally and the transmission rate is at par with Ae. leuteocephalus, the primary vector of ZIKV. A few CHIKV isolations have also been reported from the mosquitoes in Senegal and other countries in Africa. Experimental studies have demonstrated high susceptibility, early dissemination and efficient transmission of CHIKV by Ae. vittatus mosquitoes. The mosquitoes with their high susceptibility and competence to transmit important viruses, viz. YFV, DENV, CHIKV and ZIKV pose a major threat to public health due to their abundance and anthropophilic behaviour., Competing Interests: None

An outbreak of influenza A(H1N1)pdm09 was detected during the ongoing community-based surveillance of influenza-like illness (ILI). Among reported 119 influenza A(H1N1)pdm09 cases (59 cases in the year 2012 and 60 cases in 2015) in summer months, common clinical features were fever (100%), cough (90·7%), sore throat (85·7%), nasal discharge (48·7%), headache (55·5%), fatigue (18·5%), breathlessness (3·4%), and ear discharge (1·7%). Rise in ILI cases were negatively correlated with the seasonal factors such as relative humidity (Karl Pearson's correlation coefficient, i.e. r = -0·71 in the year 2012 and r = -0·44 in the year 2015), while rise in ILI cases were positively correlated with the temperature difference (r = 0·44 in the year 2012 and r = 0·77 in the year 2015). The effective reproduction number R, was estimated to be 1·30 in 2012 and 1·64 in 2015. The study highlights the rise in unusual influenza activity in summer month with high attack rate of ILI among children aged ⩽9 years. Children in this age group may need special attention for influenza vaccination. Influenza A(H1N1)pdm09 outbreak was confirmed in inter-seasonal months during the surveillance of ILI in Pune, India, 2012-2015.

Human Cathelicidin antimicrobial peptide LL-37 is known to have antiviral activity against many viruses. In the present study, we investigated the in-vitro effect of LL-37 on dengue virus type 2 (DENV-2) infection and replication in Vero E6 cells. To study the effect of pretreatment of virus or cells with LL-37, the virus was pretreated with different concentrations of LL-37 (2.5μM-15μM) or scrambled (Scr) LL-37(5μM-15μM) and used for infection or the cells were first treated with LL-37 and infected. To study the effect of LL-37 post infection (PI), the cells were infected first followed by addition of LL-37 to the culture medium 24h after infection. In all conditions, after the incubation, the culture supernatant was assessed for viral RNA copy number by real time RT-PCR, infectious virus particles by focus forming unit assay (FFU) and non structural protein 1 (NS1) antigen levels by ELISA. Percentage of infection was assessed using immunoflourescence assay (IFA). The results revealed that pretreatment of virus with 10-15μM LL-37 significantly reduced its infectivity as compared to virus control (P<0.0001). Moreover, pretreatment of virus with 10-15μM LL-37 significantly reduced the levels of viral genomic RNA and NS1 antigen (P<0.0001). Treatment of virus with 10-15μM LL-37 resulted in two to three log reduction of mean log 10 FFU/ml as compared to virus control (P<0.0001). Treatment of the virus with scrambled LL-37 had no effect on percentage of infection and viral load as compared to virus control cultures (P>0.05). Pretreatment of cells before infection or addition of LL-37 to the culture 24h PI had no effect on viral load. Molecular docking studies revealed possible binding of LL-37 to both the units of DENV envelope (E) protein dimer. Together, the in-vitro experiments and in-silico analyses suggest that LL-37 inhibits DENV-2 at the stage of entry into the cells by binding to the E protein. The results might have implications for prophylaxis against DENV infections and need further in-vivo studies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The emergence of Zika virus (ZiV), a mosquito borne Flavivirus like dengue (DEN) and chikungunya (CHIK), in Brazil in 2014 and its spread to various countries have led to a global health emergency. Aedes aegypti is the major vector for ZiV. Fast dissemination of this virus in different geographical areas posses a major threat especially to regions where the population lacks herd immunity against the ZiV and there is abundance of Aedes mosquitoes. In this review, we focus on current global scenario, epidemiology, biology, diagnostic challenges and remedial measures for ZiVconsidering the Indian perspective.

Emerging evidences indicate that diminished activity of the vasoprotective axis of the renin-angiotensin system, constituting angiotensin-converting enzyme 2 (ACE2) and its enzymatic product, angiotensin-(1-7) [Ang-(1-7)] contribute to the pathogenesis of pulmonary hypertension (PH). However, long-term repetitive delivery of ACE2 or Ang-(1-7) would require enhanced protein stability and ease of administration to improve patient compliance. Chloroplast expression of therapeutic proteins enables their bioencapsulation within plant cells to protect against gastric enzymatic degradation and facilitates long-term storage at room temperature. Besides, fusion to a transmucosal carrier helps effective systemic absorption from the intestine on oral delivery. We hypothesized that bioencapsulating ACE2 or Ang-(1-7) fused to the cholera nontoxin B subunit would enable development of an oral delivery system that is effective in treating PH. PH was induced in male Sprague Dawley rats by monocrotaline administration. Subset of animals was simultaneously treated with bioencapsulaed ACE2 or Ang-(1-7) (prevention protocol). In a separate set of experiments, drug treatment was initiated after 2 weeks of PH induction (reversal protocol). Oral feeding of rats with bioencapsulated ACE2 or Ang-(1-7) prevented the development of monocrotaline-induced PH and improved associated cardiopulmonary pathophysiology. Furthermore, in the reversal protocol, oral ACE2 or Ang-(1-7) treatment significantly arrested disease progression, along with improvement in right heart function, and decrease in pulmonary vessel wall thickness. In addition, a combination therapy with ACE2 and Ang-(1-7) augmented the beneficial effects against monocrotaline-induced lung injury. Our study provides proof-of-concept for a novel low-cost oral ACE2 or Ang-(1-7) delivery system using transplastomic technology for pulmonary disease therapeutics., (© 2014 American Heart Association, Inc.)

Delivering neurotherapeutics to target brain-associated diseases is a major challenge. Therefore, we investigated oral delivery of green fluorescence protein (GFP) or myelin basic protein (MBP) fused with the transmucosal carrier cholera toxin B subunit (CTB), expressed in chloroplasts (bioencapsulated within plant cells) to the brain and retinae of triple transgenic Alzheimer's disease (3×TgAD) mice, across the blood-brain barriers (BBB) and blood-retinal barriers (BRB). Human neuroblastoma cells internalized GFP when incubated with CTB-GFP but not with GFP alone. Oral delivery of CTB-MBP in healthy and 3×TgAD mice shows increased MBP levels in different regions of the brain, crossing intact BBB. Thioflavin S-stained amyloid plaque intensity was reduced up to 60% by CTB-MBP incubation with human AD and 3×TgAD mice brain sections ex vivo. Amyloid loads were reduced in vivo by 70% in hippocampus and cortex brain regions of 3×TgAD mice fed with bioencapsulated CTB-MBP, along with reduction in the ratio of insoluble amyloid β 42 (Aβ42) to soluble fractions. CTB-MBP oral delivery reduced Aβ42 accumulation in retinae and prevented loss of retinal ganglion cells in 3×TgAD mice. Lyophilization of leaves increased CTB-MBP concentration by 17-fold and stabilized it during long-term storage in capsules, facilitating low-cost oral delivery of therapeutic proteins across the BBB and BRB.

Japanese encephalitis (JE) remains a major public health threat with vaccination as the only measure for its prevention. Epitope-based vaccination is a promising approach for achieving protective immunity and avoid immunopathology in Japanese encephalitis virus (JEV) infection due to flavivirus cross-reactivity. We have mapped B-cell epitopes from JEV envelope protein, responsible for elicitation of neutralizing antibodies. Incorporation of T helper (T(H)) epitopes, along with these, imparted protective immunity to the host. In the present study, based on in silico epitope selection we optimized and proposed a polytope DNA construct (P-JEV) consisting B-cell and T(H) epitopes from the JEV envelope (E) protein as well as non-structural protein-1 (NS1). The immunogenicity and protective efficacy of P-JEV was assessed by in vitro and in vivo experiments. The expressed P-JEV showed reactivity in in vitro assays with JEV monoclonal antibodies. Protective efficacy of P-JEV was assessed in BALB/c mice. Our findings indicate that P-JEV may be a candidate vaccine for the prevention of JEV infection., (Copyright © 2012 Elsevier B.V. All rights reserved.)

West Nile virus (WNV) and Japanese encephalitis virus (JEV), the members of JEV serocomplex group are pathogens of global health concern. The co-circulation of these viruses poses challenges in effective diagnostics due to antigenic similarity between the E-protein of these viruses. The present study aimed to design chimeric peptides and study the immune response against the same. B-cell epitopes were predicted on structural proteins of WNV and JEV based on bioinformatics tools. The peptides representing to these B-cell epitopes were synthesized and subjected to ELISA. Two peptides, one each from WNV (named WE147) and JEV (named JE40) E-protein, showed virus-specific and strong reactivity to the immune mice sera and human clinical samples. The chimeric peptides for WNV and JEV were constructed by synthesizing the B-cell epitope of WNV (WE147) or JEV (JE40) with T-helper epitope (JM17) separated by diglycine spacer in between. The immune response generated against these chimeric peptides was found to be specific to the respective B-cell epitopes. The anti-peptide sera showed virus-specific reactivity in ELISA and in immunofluorescence assay with no cross-reactivity. Also, the anti-peptide sera could neutralize JE and WN viruses in an in vitro virus neutralization assay. The B-cell epitopes identified in the present study may be used as diagnostic markers for differentiating between WN and JE virus infections. The present study can form a basis for future design of vaccines., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The Envelope glycoprotein (E-protein) of Japanese encephalitis virus (JEV) is the major structural component on the virion surface and is a primary target for the host immune system. Two monoclonal antibodies (MAbs) NHA-I (IgG2b) and NHA-II (IgM) against JEV (Indian strain 733913) were earlier developed in the authors' laboratory and found to be cross-reactive to nuclear histones. However, the epitope specificity of these MAbs has remained unknown. The present study was carried out to delineate the epitopes recognised by these MAbs on the E-protein of JEV strain 733913. The variable regions of the NHA-I and NHA-II were sequenced and the tertiary structures predicted. Molecular docking of the MAbs with the structural model of the JEV E-protein demonstrated that NHA-I binds to a predicted antigenic determinant (residue position 18-33) in domain-I. To understand the epitope specificity and check for possible cross-reactivity of these MAbs, comparative analysis of interactions with the known crystallographic structure of the West Nile virus (WNV) E-protein was also carried out. The studies predicted a differential binding of NHA-I but not of NHA-II between JEV and WNV. Mutagenesis studies could help analyse the specificity of NHA-I. The NHA-II appears to be cross-reactive as it docked in the groove region between domains I and III of both the JEV and WNV E-proteins. In laboratory assays, namely, ELISA and immunofluorescence assay both the MAbs reacted equally with JEV while the NHA-I did not show any reactivity with WNV. In silico results were thus validated by laboratory experiments. The present study would help in better understanding of virus-host interactions at the molecular level, and also be useful for the future design of vaccines as well as peptide based diagnostics., (Copyright © 2011 Elsevier B.V. All rights reserved.)

To understand the reported cross-reactivity of the 2009 H1N1 and the 1918 H1N1 pandemic viruses we docked the crystal structure of 2D1, an antibody derived from a survivor of the 1918 pandemic, to the structures of hemaglutinin (HA) of the 2009 strain and seasonal H1 vaccine strains. Our studies revealed that 2D1 binds to the 2009 HA at antigenic site 'Sa', with stabilizing contacts, similar to that in an available co-crystal structure of 2D1-1918 HA. However, 2D1 failed to bind to the known antigenic sites in the HAs of seasonal strains. Our study thus reveals the molecular basis for pre-existing immunity in elderly people to the 2009 pandemic virus.

The emergence of new strains of Influenza virus have caused several pandemics over the last hundred years with the latest being the H1N1 Swine flu pandemic of 2009. The Hemagglutinin (HA) protein of the Influenza virus is the primary target of human immune system and is responsible for generation of protective antibodies in humans. Mutations in this protein results in change in antigenic regions (antigenic drift) which consequently leads to loss of immunity in hosts even in vaccinated population (herd immunity). This necessitates periodic changes in the Influenza vaccine composition. In this paper, we investigate the molecular basis of the reported loss of herd immunity in vaccinated population (vaccine component: Influenza A/X-31/1968 (H3N2)) which resulted in the outbreak due to strain Influenza A/Port Chalmers/1/1973 (H3N2). Also, the effects of antigenic drift in HA protein (H3N2 vaccine strains 1968-2007) on the 3D structures as well as interactions with BH151, a 1968 antibody, has been studied. Rigid body molecular docking protocol has been used to study the antigen-antibody interactions. We believe that the present study will help in better understanding of host-pathogen interactions at the molecular level.

Antigenic drift and shift involving the surface proteins of Influenza virus gave rise to new strains that caused epidemics affecting millions of people worldwide over the last hundred years. Variations in the membrane proteins like Hemagglutinin (HA) and Neuraminidase (NA) necessitates new vaccine strains to be updated frequently and poses challenge to effective vaccine design. Though the HA protein, the primary target of the human immune system, has been well studied, reports on the antigenic variability in the other membrane protein NA are sparse. In this paper we investigate the molecular basis of antigenic drift in the NA protein of the Influenza A/H3N2 vaccine strains between 1968 and 2009 and proceed to establish correlation between antigenic drift and antigen-antibody interactions. Sequence alignments and phylogenetic analyses were carried out and the antigenic variability was evaluated in terms of antigenic distance. To study the effects of antigenic drift on the protein structures, 3D structure of NA from various strains were predicted. Also, rigid body docking protocol has been used to study the interactions between these NA proteins and antibody Mem5, a 1998 antibody.

Sequence based homology studies play an important role in evolutionary tracing and classification of proteins. Various methods are available to analyze biological sequence information. However, with the advent of proteomics era, there is a growing demand for analysis of huge amount of biological sequence information, and it has become necessary to have programs that would provide speedy analysis. ISHAN has been developed as a homology analysis package, built on various sequence analysis tools viz FASTA, ALIGN, CLUSTALW, PHYLIP and CODONW (for DNA sequences). This JAVA application offers the user choice of analysis tools. For testing, ISHAN was applied to perform phylogenetic analysis for sets of Caspase 3 DNA sequences and NF-kappaB p105 amino acid sequences. By integrating several tools it has made analysis much faster and reduced manual intervention.

Treatment of cancer patients is subject to limitations in radiotherapy and chemotherapy. This necessitates development of new protocols, and the present work reports on the effects of a combination of local electroporation with ionizing radiation and/or anticancer drug doxorubicin hydrochloride (DOX) on subcutaneous solid tumor murine fibrosarcoma. Localized treatment of fibrosarcoma tumor, grown in right hind leg of Swiss mice, has been carried out using DOX (0.6 mg/kg body weight), radiation (Co 60 gamma-rays, dose rate 0.37 Gy/min) and electroporation (1 kV/cm, 200 micros, 8 pulses per burst, 10 bursts) individually or in combinations. Measurements of the tumor growth kinetics after treatment with combinations have revealed significant growth delay. The treatment groups, (i) radiation and electroporation, (ii) DOX and electroporation, and (iii) radiation, DOX and electroporation, have yielded tumor growth delays (TGDs) of 1.22, 1.5, and 1.73 days, respectively, compared to control with the tumor volumes being 53%, 57%, and 49% that of control on the final day of observation. These results suggest that the antitumor effects of a moderate dose of gamma radiation and low concentration of DOX can be significantly enhanced by combination with electroporation.

Increasing evidence has accumulated in recent years to suggest that the cell membrane forms the vital common target for action by ionizing radiation and electroporation. The present work describes the use of electric pulses for enhancement of radiation-induced cytotoxicity of cancer cells both in vitro and in vivo. In vitro: low dose rate (0.37 Gy/min) Co60 gamma-rays (2 Gy) in combination with electric pulses of square wave (2 kV/cm, 200 micros duration, 8 pulses/burst, 10 times) significantly enhanced the cytotoxicity in Ehrlich ascites carcinoma cells (EAC), probably through enhanced production of intracellular reactive oxygen species (ROS). The intracellular generation of ROS and changes in oxidative damage-mediated membrane fluidity were determined by fluorescence using DCH-FDA and DPH, respectively, as probes. Both radiation and electroporation, separately, have been observed to produce ROS in a dose-dependent fashion. We show that the combined treatment of cells with radiation and electroporation significantly increased intracellular ROS and changed membrane fluidity of EAC cells as compared to the effects by each individual treatment. In vivo studies have been carried out with murine fibrosarcoma as a model system. The localized treatment of a fibrosarcoma tumor, grown in the right hind leg of Swiss mice, had been carried out using radiation (Co60 gamma-rays, 2 Gy, dose rate: 0.37 Gy/min) and electric pulses (1 kV/cm, 200 micros, 8 pulses/burst, 10 times). Studies on tumor growth kinetics have shown a significant growth delay (by 50% to that of control) 7 days after treatment of tumor with radiation and electroporation. The results suggest that radiocytotoxicity of tumor cells in vitro as well as in vivo were enhanced significantly by electric pulses, which may offer a potentially improved treatment of cancer.