Japanese encephalitis virus infection of mouse cell lines: ability to prime mice for generation of virus specific cytotoxic T lymphocytes and differences in CTL recognisable viral determinants

Ten different mouse cell lines were examined for Japanese encephalitis virus (JEV) infection in vitro and then tested for their ability to generate virus specific cytotoxic T lymphocytes (CTL). Among all cell lines examined, Neuro La (a neuroblastoma) was readily infected with JEV as examined by immunofluorescence and viral replication. Among other cells, P388D1, RAW 264.7 (Macrophage origin), Sp2/0 (B-cell Hybridoma), YAC-1 (T-cell lymphoma), and L929 (Fibroblast) were semipermissive to JEV infection. The cytopathic effects caused by progressive JEV infection varied from cell line to cell line. In the case of YAC-1 cells long-term viral antigen expression was observed without significant alterations in cell viability. Intermediate degrees of cytopathicity are seen in RAW 264.7 and L929 cells while infection of PS, Neuro 2a, P388D1 and Sp2/0 caused major viability losses. All infected cell lines were able to prime adult BALB/c (H-2(d)) mice for the generation of secondary JEV specific CTL. In contrast to YAC-1, the permissive neuroblastoma cell line Neuro 2a (H-2K(k)D(d)) was found to be least efficient in its ability to stimulate anti-viral CTL generation. Cold target competition studies demonstrated that both Neuro 2a and YAC-1 (H-2K(k)D(d)) cells expressed similar viral determinants that are recognised by CTL, suggesting that the reason for the lower ability of Neuro 2a to stimulate anti-viral CTL was not due to lack of viral CTL determinants. These findings demonstrate that a variety of mouse cell lines can be infected with Japanese encephalitis virus, and that these infected cells could be utilised to generate virus specific CTL in BALB/c mice.

In vivo clearance of Japanese encephalitis virus by adoptively transferred virus specific cytotoxic T lymphocytes

Japanese encephalitis virus (JEV) is a positive stranded RNA virus that belongs to the flavivirus group, JEV infection damages the central nervous system (CNS) and is one of the main causative agents of acute encephalitis, H-2 restricted virus-specific cytotoxic T lymphocytes (CTL) have been generated specifically against JEV in our laboratory and these CTL have been shown to protect mice against lethal challenge with JEV, Virus replication was found to be inhibited in the brains of animals that mere adoptively transferred with JEV specific CTL as revealed by immunohistological staining as,veil as viral plaque assays. We further show that virus specific CTL could be recovered from such protected mice as long as 45 days after adoptive transfer.

Discrimination of alpha-Amino Acids Using Green Tea Flavonoid (-)-Epigallocatechin Gallate as a Chiral Solvating Agent

We report a special, hitherto-unexplored property of (-)-epigallocatechin gallate (EGCG) as a chiral solvating agent for enantiodiscrimination of alpha-amino acids in the polar solvent DMSO. This phenomenon has been investigated by H-1 NMR spectroscopy. The mechanism of the interaction property of EGCG with alpha-amino acids has been understood as arising out of hydrogen-bonded noncovalent interactions, where the -OH groups of two phenyl rings of EGCG play dominant roles. The conversion of the enantiomeric mixture into diastereomers yielded well-resolved peaks for D and L amino acids permitting the precise measurement of enantiomeric composition. Often one encounters complex situations when the spectra are severely overlapped or partially resolved hampering the testing of enantiopurity and the precise measurement of enantiomeric excess (ee). Though higher concentration of EGCG yielded better discrimination, the use of lower concentration being economical, we have exploited an appropriate 2D NMR experiment in overcoming such problems. Thus, in the present study we have successfully demonstrated the utility of the bioflavonoid (-)-EGCG, a natural product as a chiral solvating agent for the discrimination of large number of alpha-amino acids in a polar solvent DMSO. Another significant advantage of this new chiral sensing agent is that it is a natural product and does not require tedious multistep synthesis unlike many other chiral auxiliaries.

Infection of Human Endothelial Cells by Japanese Encephalitis Virus: Increased Expression and Release of Soluble HLA-E

Japanese encephalitis virus (JEV) is a single stranded RNA virus that infects the central nervous system leading to acute encephalitis in children. Alterations in brain endothelial cells have been shown to precede the entry of this flavivirus into the brain, but infection of endothelial cells by JEV and their consequences are still unclear. Productive JEV infection was established in human endothelial cells leading to IFN-beta and TNF-alpha production. The MHC genes for HLA-A, -B, -C and HLA-E antigens were upregulated in human brain microvascular endothelial cells, the endothelial-like cell line, ECV 304 and human foreskin fibroblasts upon JEV infection. We also report the release/shedding of soluble HLA-E (sHLA-E) from JEV infected human endothelial cells for the first time. This shedding of sHLA-E was blocked by an inhibitor of matrix metalloproteinases (MMP). In addition, MMP-9, a known mediator of HLA solubilisation was upregulated by JEV. In contrast, human fibroblasts showed only upregulation of cell-surface HLA-E. Addition of UV inactivated JEV-infected cell culture supernatants stimulated shedding of sHLA-E from uninfected ECV cells indicating a role for soluble factors/cytokines in the shedding process. Antibody mediated neutralization of TNF-alpha as well as IFNAR receptor together not only resulted in inhibition of sHLA-E shedding from uninfected cells, it also inhibited HLA-E and MMP-9 gene expression in JEV-infected cells. Shedding of sHLA-E was also observed with purified TNF-alpha and IFN-beta as well as the dsRNA analog, poly (I:C). Both IFN-beta and TNF-alpha further potentiated the shedding when added together. The role of soluble MHC antigens in JEV infection is hitherto unknown and therefore needs further investigation.

RNA-dependent RNA polymerase of Japanese encephalitis virus binds the initiator nucleotide GTP to form a mechanistically important pre-initiation state

Flaviviral RNA-dependent RNA polymerases (RdRps) initiate replication of the single-stranded RNA genome in the absence of a primer. The template sequence 5'-CU-3' at the 3'-end of the flaviviral genome is highly conserved. Surprisingly, flaviviral RdRps require high concentrations of the second incoming nucleotide GTP to catalyze de novo template-dependent RNA synthesis. We show that GTP stimulates de novo RNA synthesis by RdRp from Japanese encephalitis virus (jRdRp) also. Crystal structures of jRdRp complexed with GTP and ATP provide a basis for specific recognition of GTP. Comparison of the jRdRp(GTP) structure with other viral RdRp-GTP structures shows that GTP binds jRdRp in a novel conformation. Apo-jRdRp structure suggests that the conserved motif F of jRdRp occupies multiple conformations in absence of GTP. Motif F becomes ordered on GTP binding and occludes the nucleotide triphosphate entry tunnel. Mutational analysis of key residues that interact with GTP evinces that the jRdRp(GTP) structure represents a novel pre-initiation state. Also, binding studies show that GTP binding reduces affinity of RdRp for RNA, but the presence of the catalytic Mn2+ ion abolishes this inhibition. Collectively, these observations suggest that the observed pre-initiation state may serve as a check-point to prevent erroneous template-independent RNA synthesis by jRdRp during initiation.

Infection of human amniotic and endothelial cells by Japanese encephalitis virus: Increased expression of HLA-F

Productive infection of human amniotic and endothelial cell lines with Japanese encephalitis virus (JEV) was established leading to the induction of NF kappa B and HLA-F, a non-classical MHC molecule. Induction of the HLA-F gene and protein in JEV-infected cells was shown to be NF kappa B dependent since it was blocked by inhibitors of NF kappa B activation. ShRNA targeting lentivirus-mediated stable knockdown of the p65 subunit of NF kappa B inhibited JEV-mediated induction of HLA-F both in the amniotic cell line, AV-3 as well as the human brain microendothelial cell line, HBMEC. The induction of HLA-F by treatment of AV-3 with TNF-alpha was also inhibited by ShRNA mediated knockdown of NF kappa B. TNF-alpha treatment of HEK293T cells that were transfected with reporter plasmids under the control of HLA-F enhancer A elements resulted in significant transactivation of the luciferase reporter gene. NF kappa B-mediated induction of HLA-F following JEV infection and TNF-alpha exposure is being suggested for the first time. (C) 2014 Elsevier Inc. All rights reserved.

Inhibition of ERK and proliferation in NK cell lines by soluble HLA-E released from Japanese encephalitis virus infected cells

Productive infection of human endothelial cells with Japanese encephalitis virus (JEV), a single stranded RNA virus induces shedding of sHLA-E. We show here that sHLA-E that is released upon infection with this flavivirus can inhibit IL-2 and PMA mediated ERK 1/2 phosphorylation in two NK cell lines, Nishi and NKL. Virus infected or IFN-gamma treated cell culture supernatants containing sHLA-E were found to partially inhibit IL-2 mediated induction of CD25 molecules on NKL cells. It was also found that sHLA-E could inhibit IL-2 induced H-3]-thymidine incorporation suggesting that, similar to cell surface expressed HLA-E, sHLA-E could also inhibit NK cell responses. Hence JEV-induced shedding of sHLA-E needs further investigation to better understand immune responses in JEV infections since it may have a role in viral evasion of NK cell responses. (C) 2014 Elsevier B.V. All rights reserved.

A TDMA-based Energy Aware MAC (TEA-MAC) Protocol for Reliable Multicast in WSNs

Multicast in wireless sensor networks (WSNs) is an efficient way to spread the same data to multiple sensor nodes. It becomes more effective due to the broadcast nature of wireless link, where a message transmitted from one source is inherently received by all one-hop receivers, and therefore, there is no need to transmit the message one by one. Reliable multicast in WSNs is desirable for critical tasks like code updation and query based data collection. The erroneous nature of wireless medium coupled with limited resource of sensor nodes, makes the design of reliable multicast protocol a challenging task. In this work, we propose a time division multiple access (TDMA) based energy aware media access and control (TEA-MAC) protocol for reliable multicast in WSNs. The TDMA eliminates collisions, overhearing and idle listening, which are the main sources of reliability degradation and energy consumption. Furthermore, the proposed protocol is parametric in the sense that it can be used to trade-off reliability with energy and delay as per the requirement of the underlying applications. The performance of TEA-MAC has been evaluated by simulating it using Castalia network simulator. Simulation results show that TEA-MAC is able to considerably improve the performance of multicast communication in WSNs.

电流测量线圈及其在TEA脉冲激光器中的应用

本文提供了电流测量线圈的误差公式。用电流测量线圈测量了TEA脉冲CO_2激光器的放电电流。

Size composition of catches of Yellowfin tuna in the Japanese long-line fishery in the eastern tropical Pacific east of 130°W

ENGLISH (pgs. 267-283): In the spring of 1963, the senior author, who is a member of the staff of the Nankai Regional Fisheries Research. Laboratory, Fisheries Agency, Japanese Government, came to the Institute of Marine Resources of the University of California as a visiting investigator, bringing with him catch statistical data from the fishery in the eastern Pacific, which had been collected at the Nankai Regional Fisheries Research Laboratory (NRFRL) through September 1962, in order to conduct studies of these data in collaboration with the junior author, and with investigators of the InterAmerican Tropical Tuna Commission. A general review of the long-line fishery, based on the catch statistics of the commercial fishing fleet has been published by Suda and Schaefer (1965). In this paper we present an analysis of data respecting the size-composition of yellowfin tuna taken on long-line gear throughout the eastern Pacific between 1958 and 1962, and make some comparisons with data on size-composition of yellowfin tuna taken in the near-surface fishery, by bait boats and purse-seiners, in waters adjacent to the American coast. As has been shown by Suda and Schaefer (1965), the long-line fishery in the eastern Pacific is primarily directed toward the capture of bigeye tuna. However, considerable quantities of yellowfin tuna are also taken on this gear, and, in addition, there are substantial catches of albacore and of several species of spearfishes in some parts of the range of this fishery. Information respecting the catch rates of yellowfin tuna, and information respecting the size-composition of the stock of yellowfin tuna being exploited by the long-line fishery, is of particular interes~" because the yellowfin tuna population of the eastern Pacific is also subject to an intensive fishery by baitboats and purse-seiners which capture this species, together with skipjack, near the surface along the coast of the Americas, and around the outlying islands, in the region of California to northern Chile. SPANISH (pgs. 311-329): En la primavera de 1963, el autor principal, quien es miembro del personal del Nankai Regional Fisheries Research Laboratory, Fisheries Agency del gobierno japonés, vino al Institute of Marine Resources de la Universidad de California en calidad de investigador visitante y trajo consigo datos estadísticos de las capturas de la pesquería en el Pacífico oriental, que habían sido recolectados en el Nankai Regional Fisheries Research Laboratory (NRFRL) hasta septiembre de 1962, con el fin de hacer estudios de esos datos en colaboración con el coautor y con investigadores de la Comisión Interamericana del Atún Tropical. Una revisión general de la pesquería con palangre, basada sobre las estadísticas de captura de la flota pesquera comercial, ha sido publicada por Suda y Schaefer (1965). En este trabajo presentamos un análisis de los datos correspondientes a la composición de tamaños del atún aleta amarilla capturado con equipo palangrero en todo el Pacífico oriental, entre 1958 y 1962, y hacemos algunas comparaciones con los datos sobre la composición de tamaños del atún aleta amarilla cogido en la pesquería superficial cercana, por barcos de carnada y rederos en aguas adyacentes a la costa americana. Como ha sido demostrado por Suda y Schaefer (1965) la pesquería con palangre en el Pacífico oriental tiene como principal objeto la captura del atún ojo grande. Sin embargo, considerables cantidades de atún aleta amarilla son capturadas también por este equipo y, además, hay también considerables capturas de albacora y de diversas especies de peces-espada en algunas partes de la región que abarca esta pesquería. La información respecto a las tasas de captura del atún aleta amarilla, y la relativa a la composición de tamaños del stock de esta especie que explota la pesquería con palangre, es de particular interés, a causa de que la población de atún aleta amarilla del Pacífico oriental es también objeto de una pesca intensiva por barcos de carnada y rederos que capturan esta especie, junto con el barrilete, cerca de la superficie a 10 largo de la costa de las Américas y alrededor de las islas mar afuera, en la región desde California hasta el norte de Chile.

Size composition, growth and sexual maturity of bigeye tuna, Thunnus obesus (Lowe), from the Japanese long-line fishery in the Eastern Pacific Ocean

Size composition data of bigeye tuna taken from the eastern tropical Pacific Ocean by Japanese Prefectural experimental training vessels from 1958 to 1964 are examined. A gradient of increasing fish size from east to west is noted. Males increase in ratio over females for the entire range of lengths examined, and beyond 170 cm comprise more than 75 per cent of the total. The first semester of the year is important as a bigeye spawning season. A general relationship between sexual maturity and thermal structure of the water is discussed. At the end of their 12th quarter of life bigeye are about 114 cm long, by the 16th quarter, 137 cm and at the end of 20 quarters, about 153 cm. The long-line fishery in the eastern Pacific has had a marked effect on the size composition of the stocks of bigeye, but whether the fishing has driven the stocks below a point which could afford a maximum sustainable yield could not be determined. (PDF contains 55 pages.)

Studies on the Japanese long-line fishery for tuna and marlin in the Eastern Tropical Pacific Ocean during 1963

ENGLISH: During 1963, the area of operation of the Japanese long-line fishery in the eastern Pacific further expanded, and total fishing effort increased. Charts are presented showing the distribution of effort and of catch-rates, by species, for major fishing areas; and showing catch-rates by species, by quarters of the year, by areas of one-degree of latitude and longitude. In the newly-exploited region north of 10°N, yellowfin and striped marlin are principal elements of the catch, while in the newly-fished region south of the former fishing areas, south of about 20°S, albacore dominate in the catch. Continued decline in catch rates of bigeye tuna, associated with increased fishing effort, indicates that there has been a real decrease in abundance of this species, and that it may have resulted from effects of the fishery on the stock. Changes in catch rates of yellowfin tuna seem to be associated with effects on the stock by both the long-line fishery and by the near-surface fishery by purse-seiners and bait-boats. Over the short series of years for which data are available, there are no discernible trends in apparent abundance of striped marlin or albacore. Information is presented concerning seasonal and geographical distributions of spawning yellowfin tuna, based on examination of gonads. Analysis of data on size composition of yellowfin tuna for 1958-1964 indicates that the long-line fishery is becoming increasingly dependent on the most recently recruited year class, further confirming the effect of the fishery on the stock. The long-line fishery now takes nearly all of its catch of yellowfin tuna from two year-classes each year, during their third and fourth years of life. SPANISH: Durante 1963 el área de operación de la pesca palangrera japonesa en el Pacífico oriental se extendió más y se incrementó el esfuerzo total de pesca. Se presentan cartas indicando la distribución del esfuerzo y las tasas de captura por especies, para las áreas principales de pesca; y se muestran las tasas de captura por especies y por trimestres del año en áreas de un grado de latitud y longitud. En la región recientemente explotada al norte de los 10°N, los principales elementos de la captura son el atún aleta amarilla y el marlín rayado, mientras aproximadamente al sur de los 20°S en la región recientemente pescada al sur de las primeras áreas de pesca la albacora predomina en la captura. La continua merma en las tasas de captura del patudo en asociación con el incremento en el esfuerzo de pesca, indica que ha habido una real reducción en la abundancia de esta especie y que puede ser el resultado de los efectos de la pesquería sobre el stock. Los cambios en las tasas de captura del atún aleta amarilla parecen estar asociados con los efectos sobre el stock tanto de la pesquería palangrera como de la pesca de superficie por barcos rederos y de carnada. No hay tendencias perceptibles en la abundancia aparente del marlín rayado o la albacora en el corto período de años de los que se disponen datos. Se presenta información referente a la distribución estacional y geográfica del desove del atún aleta amarilla basado en el examen de las gónadas. El análisis de los datos sobre la composición de tallas del atún aleta amarilla correspondiente a 1958-1964 indica que la pesca palangrera depende cada vez más de las clases anuales recientemente reclutadas, confirmando aún más el efecto que tiene la pesquería sobre el stock. La pesquería palangrera coge casi toda su pesca de atún aleta amarilla de dos clases anuales cada año, durante su tercer y cuarto año de vida. (PDF contains 70 pages.)