Development of a Taqman-based real-time PCR assay for detection of porcine sapelovirus infection in pigs.

The objective of the present study was to develop a rapid, simple, specific and sensitive Taqman-based real-time PCR assay for porcine sapelovirus (PSV) detection. Specific primers and probe were designed from the five untranslated regions (UTRs) of the viral genome. The detection limit of the real-time PCR was 102 copies. The specificity of the Taqman real-time PCR assay was evaluated using other animal viruses and nuclease free water as a negative control. Strong fluorescent signals were obtained only in the detection of PSV real-time PCR and conventional RT-PCR were preformed simultaneously on 90 faecal samples. Based on conventional RT-PCR study 17.7% (16/90) of the faecal samples were positive for PSV. Whereas 21 of 90 samples (23.3%) were positive by real-time RT-PCR. The results showed that real-time PCR was more sensitive than the conventional RT-PCR assay. In conclusion, the Taqman real-time PCR assay for detection of PSV developed, herein, is sensitive, specific, and reliable. This assay will be useful for clinical diagnosis, epidemiological, and pathogenesis studies.

Pathological and molecular investigation of porcine sapelovirus infection in naturally affected Indian pigs.

The aim of the present study was to pathological and molecular investigation of porcine sapelovirus (PSV) in naturally infected Indian pigs of various age groups. Eight samples (16%) out of 49 necropsied animals were positive for PSV on the basis of pathological and molecular investigation. Major lesions of PSV positive cases were thickening and clouding of meninges, congestion in brain, severe to moderate congestion in lungs along with froathy exudates in trachea, thickening of intestinal mucosa, especially mucosal folds of ileum. Microscopic lesions of PSV positive cases in CNS were perivascular cuffing, neuronophagia and focal gliosis. In lungs, interstitial pneumonia was noticed in all cases, and intestinal lesions comprised of sloughing of villi epithelium, moderate to severe congestion of blood vessels and infiltration of mononuclear cells mainly plasma cells in both large and small intestine. RT-PCR results of total cases examined for PSV were targeted for PSV 3D Polymerase, 5'UTR region and VP1 gene respectively. Genetic characterization was done on the basis of viral capsid protein 1 (VP1) gene of PSV. The sequencing and phylogenetic analysis of amplified VP1 gene product showed maximum identity 85-90% with South Korean, KJ821021.1 and Indian, KY053835.1 strain of PSV. Further explorative surveillance and epidemiological studies are suggested to find out the real impact of this economically important disease affecting pigs population of India.

Spatial prediction of landslide susceptibility in parts of Garhwal Himalaya, India, using the weight of evidence modelling.

Garhwal Himalaya in northern India has emerged as one of the most prominent hot spots of landslide occurrences in the Himalaya mainly due to geological causes related to mountain building processes, steep topography and frequent occurrences of extreme precipitation events. As this region has many pilgrimage and tourist centres, it is visited by hundreds of thousands of people every year, and in the recent past, there has been rapid development to provide adequate roads and building infrastructure. Additionally, attempts are also made to harness hydropower by constructing tunnels, dams and reservoirs and thus altering vulnerable slopes at many places. As a result, the overall risk due to landslide hazards has increased many folds and, therefore, an attempt was made to assess landslide susceptibility using 'Weights of Evidence (WofE)', a well-known bivariate statistical modelling technique implemented in a much improved way using remote sensing and Geographic Information System. This methodology has dual advantage as it demonstrates how to derive critical parameters related to geology, geomorphology, slope, land use and most importantly temporal landslide distribution in one of the data scarce region of the world. Secondly, it allows to experiment with various combination of parameters to assess their cumulative effect on landslides. In total, 15 parameters related to geology, geomorphology, terrain, hydrology and anthropogenic factors and 2 different landslide inventories (prior to 2007 and 2008-2011) were prepared from high-resolution Indian remote sensing satellite data (Cartosat-1 and Resourcesat-1) and were validated by field investigation. Several combinations of parameters were carried out using WofE modelling, and finally using best combination of eight parameters, 76.5 % of overall landslides were predicted in 24 % of the total area susceptible to landslide occurrences. The study has highlighted that using such methodology landslide susceptibility assessment can be carried out in vast stretches of Himalaya in short time in order to assess the impact of development as well as climate change/variability. The resultant map can play a critical role in selecting areas for remedial measures for slope stabilisation as well planning for future development of the region.

Geographic information system-based analysis of COVID-19 cases in India during pre-lockdown, lockdown, and unlock phases.

OBJECTIVE: The World Health Organization formally announced the global COVID-19 pandemic on March 11, 2020 due to widespread infections. In this study, COVID-19 cases in India were critically analyzed during the pre-lockdown (PLD), lockdown (LD), and unlock (UL) phases. METHOD: Analyses were conducted using geospatial technology at district, state, and country levels, and comparisons were also made with other countries throughout the world that had the highest infection rates. India had the third highest infection rate in the world after the USA and Brazil during UL2.0-UL3.0 phases, the second highest after the USA during UL4.0-UL5.0 phases, and the highest among South Asian Association for Regional Cooperation (SAARC) countries in PLD-UL5.0 period. RESULTS: The trend in the number of COVID-19 cases was associated with the population density where higher numbers tended to be record in the eastern, southern, and west-central parts of India. The death rate in India throughout the pandemic period under study was lower than the global average. Kerala reported the maximum number of infections during PLD whereas Maharashtra had the highest numbers during all LD and UL phases. Eighty percent of the cases in India were concentrated mainly in highly populous districts. CONCLUSION: The top 25 districts accounted for 70.99%, 69.38%, 54.87%, 44.23%, 40.48%, and 38.96% of the infections from the start of UL1.0 until the end of UL phases, respectively, and the top 26-50 districts accounted for 6.38%, 6.76%, 11.23%, 12.98%, 13.40%, and 13.61% of cases in these phase, thereby indicating that COVID-19 cases spread during the UL period. By October 31, 2020, Delhi had the highest number of infections, followed by Bengaluru Urban, Pune, Mumbai, Thane, and Chennai. No decline in the infection rate occurred, even in UL5.0, thereby indicating a highly alarming situation in India.

Immunocytochemistry assay in BHK-21 cell line infected with Porcine Sapelovirus.

The present study describes an immunocytochemistry (ICC) assay with self-raised hyperimmune sera and a Baby Hamster Kidney-21 (BHK-21) cell line infected with Porcine Sapelovirus (PSV). Sapelovivus/IVRI/SPF-c-6/2015 strain Indian PSV was isolated from the porcine IBRS-2 cell line and investigated for growth on non-porcine cell lines. After two passages, PSV was successfully grown in BHK-21 and produced the same cytopathic effects as in IBRS-2 such as shrinking of cytoplasm, rounding of cells and detachment of cells from the surface of flask within 24 h. For raising of hyperimmune sera, PSV was grown in IBRS-2 cell line up to the required volume and purified by ultracentrifugation. With self-raised hyperimmune sera in laboratory rats, ICC was performed in BHK-21 cells infected with PSV. Positive signals consisted of large granular aggregates of virus in the cytoplasm near the nucleus, suggesting that PSV can infect cell lines other than those of porcine origin.

Porcine sapelovirus among diarrhoeic piglets in India.

Porcine sapelovirus (PSV) A belongs to the genus Sapelovirus, family Picornaviridae. PSV infections in pigs have been reported from European countries, United States, Japan, China, Korea and Brazil. The virus has been isolated/detected from faeces of healthy pigs as well as those affected with diarrhoea, respiratory signs, encephalitis, skin lesions and fertility disorders. This study was planned to investigate whether PSV is prevalent among pigs in India and to characterize PSV encountered in the study population. The study revealed that five of 70 (7.14%) faecal samples were found positive for PSV using RT-PCR. Three viruses were successfully isolated from faecal samples using IB-RS-2 cell line. Complete genome sequencing and analysis of one Indian PSV isolate revealed highest homology (88%) with V13 strain from England. Phylogenetic analysis of the complete polyprotein nucleotide sequences of 14 strains of PSV classified the viruses into four distinct clades. This first report from India adds to our knowledge on genetic diversity of PSV detected so far among pigs in different countries. A large-scale surveillance of the virus is required to understand its genomic diversity and economic impact.

Low-dose cyclophosphamide inhibition of transplantable fibrosarcoma growth by augmentation of the host immune response.

Preinjection of a low dose of cyclophosphamide (Cy) (500 microgram/mouse) either delayed or inhibited tumor appearance following the inoculation of transplantable 3-methylcholanthrene-induced fibrosarcomas in inbred male C3H/HeJ mice. This dose of Cy decreased the spleen weight by 13% and the total spleen cell count by 23%. However, the same dose could potentiate the footpad swelling reaction (FPSR) measured against Staphylococcus aureus antigen. Splenic lymphocytes from Cy-treated animals showed increased blastogenic response against phytohemagglutinin-M and bacterial lipopolysaccharide. Thus 500 micrograms Cy/animal may have depleted suppressor cell populations leading to: a) an increase in FPSR, b) increased blastogenic transformation of lymphocytes, and c) tumor growth inhibition.

Antitumor immunity, V.BCG-induced growth inhibition of murine tumors. Effect of hydrocortisone, antiserum against theta antigen, and gamma-irradiated BCG.

Dimethylbenzdithionsaphthene (DBDN)-induced fibrosarcoma transplantable in syngeneic Swiss mice was a weakly immunogenic tumor and did not regress spontaneously. BCG from India, when incubated with the lethal dose of DBDN-induced fibrosarcoma and subsequently inoculated sc into the susceptible animal markedly inhibited growth of the tumor. Animals that did not show any tumors developed tumor-specific immunity. BCG, if administered separately and not inoculated with the tumor cells, did not show similar response. The close contact between the BCG organism and the tumor cell seemed to be an essential requirement for BCG-induced tumor-growth inhibition. Hydrocortisone-treated animals showed tumor growth even when a BCG-tumor admixture was incorporated, indicating the immunologic nature of the phenomenon. Animals preinoculated with anti-O-serum also showed tumor growth when the BCG-tumor admixture was given. This, however, pointed out a major role of O-bearing lymmphocytes in BCG-induced-tumor-growth inhibition.

Antitumor immunity. II. Viability, tumorigenicity, and immunogenicity of neuraminidase-treated tumor cells: effective immunization of animals with a tumor vaccine.

A dimethylbenzdithionaphthene (DBDN)-induced fibrosarcoma showed reduced transplantability if previously treated with Vibrio cholerae neuraminidase (VCN). However, the reduced transplantability of VCN-treated tumor cells was not associated with any loss of their viability or tumorigenic capability, but appeared to be due to their increase in immunogenicity. High doses of VCN-treated tumor cells could grow even in normal individuals. Lower doses, which did not induce tumor development in normal individuals, did so if injected into immunosuppressed animals. Although X-irradiation of VCN-treated tumor cells reduced their tumorigenic potential, it did not impair their increased immunogenic properties. Thus a suitable method for the preparation of a "tumor vaccine" was provided. The immunization of animals with the vaccine and a working hypothesis regarding its mechanism of action were described.

Mechanism of regression of mammary adenocarcinomas in rats following plasma adsorption over protein A-containing Staphylococcus aureus.

The plasma from 7,12-dimethylbenz(a)anthracene-induced mammary tumor-bearing rats was adsorbed ex vivo with nonviable protein A-containing Staphylococcus aureus Cowan I and then injected into the rats, along with its original blood cells. Tumors in the treated rats showed significant (p less than 0.005) growth inhibition. There were fewer metastatic nodules; cellular cytotoxicity in the presence of plasma was augmented, and there was increased antitumor cytotoxic antibody activity in treated rats. Plasma from sham-treated rats, however, showed blocking activity. It appears that plasma perfusion over S. aureus decreases blocking activity and augments antitumor immunoreactivity of plasma. The exact mechanism by which growth of 7,12-dimethylbenz(a)anthracene-induced mammary tumors was inhibited in these treated Sprague-Dawley rats is not known. However, it is hypothesized that the observed tumor regression is at least partly attributable to the augmentation of antitumor immunoreactivity in the treated animals.

Ability of nonviable Staphylococcus aureus Cowan I to bind ionized calcium in vitro and ex vivo in an extracorporeal perfusion system.

During immunoadsorption of plasma immunoglobulin G (IgG) and/or its complexes, using nonviable Staphylococcus aureus Cowan I (SA) as an immunoadsorbent, we observed a consistent drop in plasma calcium during every immunoadsorption procedure. The percentage of decrease in plasma calcium was directly dependent on the amount of SA adsorbent present. Although SA can bind both IgG and calcium, the degree of binding of IgG was greater than that of calcium. Calcium could not be detected in the IgG fraction of the plasma SA, however, could bind an appreciable amount of ionized calcium. Extracorporeal perfusion of plasma over SA greatly decreased plasma calcium in a hypercalcemic patient, indicating the feasibility of this method in such patients. Questions concerning the significance of lowering calcium levels as a concomitant of other effects observed in cancer patients undergoing immunoadsorption therapy using SA evolve from the above and become a new focus for attention.

Modulation of benzene toxicity by polyinosinic-polycytidilic acid, an interferon inducer.

Repeated intraperitoneal administration of benzene (1.0 ml/kg body wt.) for 3 days produced leucopenia, lymphocytopenia and significantly decreased body wt. (P less than 0.001) and organ weights of thymus (P less than 0.001) and spleen (P less than 0.001) in female albino rats. Total iron content, lipid peroxidation and superoxide dismutase activity of the liver and bone marrow were significantly increased as a result of benzene exposure. Low molecular weight (LMW) bleomycin detectable iron content was accumulated in bone marrow, whereas hepatic LMW iron was not detectable after benzene intoxication to rats. Prior administration of single dose (250 micrograms/100 g body wt.) of Poly IC, an interferon inducer with immunomodulating potential was found to be ameliorate some of the adverse effects of benzene as well as restoration of hepatic architecture histologically. Superoxide dismutase activity, lipid peroxidation, total iron content and LMW iron content (bone marrow) were normalised. Pretreatment of animals with Poly IC was able to enhance the SRBC antibody titre in benzene-treated animals. This study suggests that the beneficial effects of Poly IC in the amelioration of the acute toxicity of benzene has clinical significance.

Analgesic activity of Amorphophallus campanulatus tuber.

The methanol extract of Amorphophallus campanulatus tuber, given orally at the doses of 250 and 500 mg/kg, showed significant analgesic activity in mice.

Demonstration of two distinct antibodies in autoimmune hemolytic anemia with reticulocytopenia and red cell aplasia.

To determine the mechanism of the aplastic crisis in a patient with autoimmune hemolytic anemia (AIHA) and reticulocytopenia who developed red cell aplasia simultaneously, serum- and IgG-separated fractions were examined for the presence of erythroid progenitor cell inhibitors. The patient's red cell autoantibody was a complement-independent IgG that reacted with the little-e antigen of the Rh complex. A complement-dependent serum IgG inhibitor directed against erythroid colony- and burst-forming units but not granulocyte-macrophage units was detected in samples before treatment with extracorporeal staphylococcal protein-A immunoadsorption and corticosteroids. The erythroid progenitor cell inhibitor persisted in samples multiply adsorbed against type-ee red cells and was not detected in heat eluates prepared from these red cells. A reticulocytosis occurred when serum IgG levels were reduced to 27% of pretreatment values. At this point, the erythroid progenitor cell inhibitor was not detectable in vitro. These findings suggest that the development of the aplastic crisis in some patients with AIHA may be associated with the presence of two distinct IgG antibodies, one directed at the mature red cell and the other at the erythroid progenitor cells.

Specific immunoadsorption of IgG antibody in a patient with chronic lymphocytic leukemia and autoimmune hemolytic anemia. A new form of therapy for the acute critical stage.

Progressive and severe autoimmune hemolytic anemia developed in a patient with chronic lymphocytic leukemia (CLL) despite treatment with chlorambucil, high doses of corticosteroids and attempts to transfuse packed red blood cells. Splenectomy was not performed because of severe coronary artery disease. Direct antiglobulin tests revealed a warm red blood cell autoantibody of IgG-type with anti-e specificity. The patient was treated by extracorporeal immunoadsorption of plasma IgG using a cell separator and protein A as the immunoadsorbent. The patient responded by an increase in the hemoglobin levels and platelet counts after two treatments. Specificity of the procedure was shown by a decrease in the serum IgG and by the demonstration of the same reactivity to ficin-treated reagent red blood cell panel of the eluate from the protein A. Antibody titers of the patient's red blood cell eluate decreased from 1:128 to 1:64 and eventually the anti-e specificity was lost. This is a report of a novel approach to treatment of the acute phase of an autoimmune hemolytic anemia.

Mechanisms of protein A superantigen-induced signal transduction for proliferation of mouse B cell.

Apart from many of the biological properties of protein A (PA) of Staphylococcus aureus, it has been recognized recently as a B-cell superantigen. Therefore, we investigated the molecular mechanisms of PA superantigen-induced mice splenic B-cell proliferation. Treatment of resting B cells with PA-evoked cell proliferation. Binding of PA to B cells led to a cascade of signal transduction mechanisms involving tyrosine kinase that activated phospholipase C, which in turn activated protein kinase C (PKC), and translocated it from cytosol to membrane. Mitogen-activated protein (MAP) kinase has been found to be activated down-stream of PKC in this signal pathway, which ultimately caused an activation of serum-responsive factor (SRF). Inhibition at any step of this signaling cascade could block B-cell proliferation. PA could also stimulate the Bcl-2 gene expression at protein level thereby supporting the pro-proliferative effect of PA. Thus, the molecular mechanisms related to PA-induced B cell proliferation has been delineated in this report as tyrosine kinase > PLC > PKC > MAP kinase > SRF > Bcl-2. Knowledge gathered from these observations might be of immense help to study the immune cell proliferation as a part of immunoactivation process. Also, the development of suitable inhibitors of the signaling pathway outlined here might provide clues as to how to abrogate pathologic antibody production in many disease processes.

Mechanism of enhanced phagocytic response in protein a treated rat macrophages.

Protein A of S. aureus Cowan I has been shown to stimulate macrophage mediated phagocytosis. The present study was undertaken to understand the mechanism involved in the enhancement of phagocytosis of peritoneal macrophages by protein A. The lucigenin and luminol-dependent chemiluminescence (CL) of rat peritoneal macrophages, after incubation with various concentrations of protein A, flow-cytometric studies using DCFH-DA as a fluorescent compound and phagocytosis of sheep red blood cells (SRBCs) by rat peritoneal macrophages were studied. A significant increase in lucigenin dependent CL due to formation of superoxide anions (O2-.) and in luminol dependent CL due to formation of hydrogen peroxide (H2O2) was observed in protein A treated macrophages. A significant increase in intracellular hydrogen peroxide (H2O2) was also observed along with an increase in phagocytosis of SRBCs by protein A treated macrophages. The present findings indicate that protein A helps to increase phagocytosis and triggers respiratory burst of macrophages. Thus, both increased phagocytic response and respiratory burst of macrophages in protein A treated animals may be contributing to the antitumor property of protein A reported earlier.

Protein A of Staphylococcus aureus evokes Th1 type response in mice.

Protein A (PA) of Staphylococcus aureus is known to elicit several cytokines such as IFN gamma, TNF alpha and IL1. However, it has not been delineated yet as to which differentiation pathway lymphocytes follow after stimulation by PA. In this report, we attempted to collect such evidences. Cytokines, such as IFN gamma, IL2, IL4, IL6, IL10, TNF alpha, IL1alpha and IL1beta were measured in serum by ELISA. Our results show that 1 microg dose of PA stimulates the production of IFN gamma (115 +/- 5 pg/ml), TNF alpha (250 +/- 8 pg/ml) and IL1alpha (100 +/- 5 pg/ml) as compared to control levels of, 22 +/- 2, 20 +/- 2 and 35 +/- 3 pg/ml respectively whereas IL2 and IL1beta secretion were less (beyond the lower detection limit of the kit and 25 +/- 1 pg/ml, respectively) as compared to control (28 +/- 2 and 52 +/- 4 pg/ml, respectively). Larger dose of PA (10 microg) increases the expression of IL2 (75 +/- 3 pg/ml), TNF alpha (1380 +/- 120 pg/ml), IL1alpha (495 +/- 10 pg/ml) and IL1beta (110 +/- 7 pg/ml) as compared to controls described above. We also observed that 1 microg dose of PA decreases IL4, IL6 and IL10 secretion to 9 +/- 1, 10 +/- 1 and 10 +/- 2 pg/ml, respectively, whereas 10 microg dose also decreased them to 11 +/- 1, 12 +/- 2 and 30 +/- 4 pg/ml, respectively as compared to the background controls, i.e. 50 +/- 5, 50 +/- 2 and 215 +/- 9 pg/ml respectively. The ratio of IFN gamma to IL4 increased and the peak value at 4 h, came to 13 +/- 1 and 9.6 +/- 0.5 with 1 microg and 10 microg PA, respectively, which is an established parameter indicating a Th1 type response. Flow cytometry analysis of CD4+/CD8+ cells, and c-myc protein expression by splenocytes indicate that 1 microg dose of PA causes 2-fold increase of CD4+ cells with no change in CD8+ cells, and 10-fold increase in c-myc protein, whereas 10 microg dose increases CD4+ cells 4-fold, CD8+ cells 3-fold and c-myc protein 100-fold. The cell cycle data shows an induction of apoptosis in thymocytes and splenocytes with the large dose (10 microg), whereas the 1 microg dose does not show any apoptosis. This report indicates that a Th1 response is induced in mice, after PA inoculation at a dose of 1 microg animal. Thus, cytokine mediated therapeutic strategies should consider the fact that an induction of large concentration of some cytokines might become detrimental to the host.

Therapeutic and prophylactic uses of protein A in the control of Leishmania donovani infection in experimental animals.

The role of the immunomodulator Protein A (PA) (from Staphylococcus aureus, Cowan I strain) in the control of leishmanial infection was studied in experimental animals. Treatment of Leishmania donovani infected hamsters with PA led to a moderate level of reduction of parasite load in their spleen (68%) and liver (46%). However, combination therapy of PA with the antileishmanial drug stibanate induced a more marked reduction of the spleen (88%) and liver (85%) parasitemia compared to that induced by PA or drug treatment alone. Similar results were also obtained with L. donovani infected BALB/c mice as the combination therapy of PA and stibanate led to a significant reduction (84%) of liver parasite load in comparison to that induced by PA (38%) or drug (61%) treatment alone. Apart from its therapeutic use, PA could also be used as a prophylactic agent in the control of leishmanial infection. Thus, treatment of hamsters with PA before leishmanial challenge significantly reduced their organ parasite load (by 59-78%) compared to that observed in infected controls without prior PA treatment. The antileishmanial effect of PA was likely to be mediated through the activation of macrophages leading to an enhancement of their phagocytic as well as leishmaniacidal activities. Subsequent studies demonstrated that PA treatment led to an increased production of nitric oxide by macrophages which could primarily be responsible for their enhanced parasite killing ability.

Antitumour activity of protein A in a mouse skin model of two-stage carcinogenesis.

Protein A (PA) is an immunostimulating glycoprotein (mol. wt. 43,000 kDa) obtained from Staphylococcus aureus cowan I. The antitumour property of PA is well documented in the literature in various transplantable tumours of rats and mice. In the present set of investigations, the antitumour property of PA was tested in Swiss albino mice in a two-stage initiation-promotion mouse skin carcinogenesis model. The animals were initiated topically with a single subcarcinogenic dose (52 microgram) of 7,12-dimethylbenzanthracene (DMBA). PA was administered intraperitoneally (1 microgram/animal), twice weekly for 2 weeks. Promotion was performed by twice weekly applications of 12-O- tetradecanoyl phorbol-13-acetate (TPA) at a dose of 5 microgram/animal for 32 weeks. The result showed that the treatment schedule can effectively check the onset of tumorigenesis, the cumulative number of tumours and the average number of tumours per mouse. In the PA administered group, 30% of the animals remained tumour free until the termination of the experiments (i.e. 32 weeks of promotion). Thus the present study proves that protein A can effectively inhibit DMBA initiated and TPA promoted mouse skin carcinogenesis.