Expression of the Japanese encephalitis virus NS3 and NS2b proteins as glutathione S-transferase fusions

Flaviviruses generate their structural and nonstructural proteins by proteolytic processing of a single large polyprotein precursor. These proteolytic events are brought about both by host cell signalase and a virally encoded protease. The virally encoded proteolytic activity has been shown to reside within the nonstructural protein 3 (NS3) and requires the product of the nonstructural 2b (NS2b) gene. In order to obtain sufficient quantities of pure NS2b and NS3 proteins for kinetic analysis, we have expressed both these proteins in recombinant systems as fusions to glutathione S-transferase (GST). The fusion constructs were driven by the strong bacteriophage T7 promoter. Transfection of these constructs into the African green monkey kidney cell line CV-1 previously infected with a recombinant vaccinia virus expressing the T7 RNA polymerase resulted in synthesis of the fusion proteins. Both the fusion proteins could be purified to homogeneity in a single step using a glutathione agarose affinity matrix.

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.

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.

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.)

The precautionary approach and the management of the European eel (Anguilla anguilla) – critical remarks

Recruitment and commercial catches of European eel have been in decline since the late 1970s. So far, the reasons are not well understood. A range of potential natural and anthropogenic reasons have been discussed, but the relative importance of the factors is unknown. As a consequence of the decline in recruitment an urgent need for protective management measures was concluded. The main approach is to restrict the fishery on eel, in particular with reference to the precautionary approach. However, in view of the lack of knowledge on the factors responsible for the recruitment decline and by considering that many yellow and silver eel stocks in freshwaters depend on restocking by the fishery, such simplified conclusions are critically discussed. A concept for the sustainable management of eel has to include 1) research on the factors determining the population dynamics, in particular during the oceanic stages, 2) a stronger consideration of socio-economic aspects, and 3) intensified research on artificial reproduction and rearing of eel.

Recovery of the stocks of the European eel (Anguilla anguilla) by spawner enhancement

The production of healthy high quality female European eel in recycle systems is proposed as a means to secure sufficient numbers of silver eel for spawning migration in order to meet the requirements of the European Commission’s proposal for a Regulation for the recovery of the stock of the European eel. Main advantages besides checks for parasites and viral diseases and avoidance of elevated levels of specific pollutants are the easily controllable numbers of spawners to be released and a reduction of labour and costs that will occur when acting along the lines of the Commission’s proposal.

Rapid identification of European (Anguilla anguilla) and North American eel (Anguilla rostrata) by polymerase chain reaction.

A rapid and cost effective DNA test is described to identify European eel (Anguilla anguilla) and North American eel (Anguilla rostrata). By means of polymerase chain reaction (PCR) technique parts of the mitochondrial cytochrome b gene are amplified with species specific primers which are designed to produce PCR fragments of different characteristic sizes for European and American eel. The size differences can easily be made visible by agarose gel electrophoresis