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The O2- scavenging activity of some ligands of asymmetrical triazine seems to be enhanced when complexed with copper. S1 Cu complex in concentration of 10(-8) M inhibits the multiplication of type 1 (Sendai) parainfluenza virus cultivated on MCA fragments. The possible mechanism of inhibition is discussed.
Although several procedures for isolating viral proteins have been described, the simultaneous separation of all the viral macromolecules in a single step has not yet been reported. We now describe TUA (Triton X-100, urea, acetic acid, pH 4.2)-SP (Sulphopropyl)-Trisacryl cation exchange chromatography, which proved to be ideal for this purpose. Optimal conditions for chromatography were established by screening on TUA-PAGE (polyacrylamide gel electrophoresis) using a horizontal linear 0-8.5 M urea gradient followed by identification of the proteins by SDS-PAGE in the second dimension. Segregation of the constituents of Semliki Forest virus cultivated in two cell lines (chicken embryo fibroblasts and Aedes albopictus cells) was studied, considering that the proteins have identical primary sequences but diverse post-translational modifications, thereby allowing the efficacy of the procedure and its applicability to different viruses to be tested. The results show that the RNA and lipids did not bind to the cation exchange in TUA and were eluted in the flow-through fractions. The proteins were fractionated using 3 linear NaCl gradients in TUA. SDS-PAGE revealed that all the proteins could be purified by this procedure. Furthermore, a direct correlation was obtained between the distance migrated by the proteins in the TUA-PAGE and their order of elution from the TUA-cation exchange column.
Studies of potent antiretroviral combination regimens were undertaken in young infants to evaluate the potential for long-term suppression of viral replication and to evaluate the immune consequences of such therapies. Early combination antiretroviral therapy led to a loss of plasma viremia, cultivable virus, and labile extrachromosomal replication intermediates. Despite preservation of immune function, persistent human immunodeficiency type 1 (HIV-1)-specific immune responses were not detected in most infants. The absence of detectable, persisting immune responses in most HIV-1-infected infants treated early contrasts with what is typically seen in adults who are treated early. These results are consistent with the notion that early combination antiretroviral therapy of HIV-1-infected infants allows the long-term suppression of viral replication.
The continuous cell line 293 was evaluated as a replacement for primary human embryo kidney (HEK) cells in the cultivation of ocular viruses. The 293 cells were found to be as sensitive as HEK cells and human embryo fibroblast (HEF) cells for the cultivation of adenoviruses and herpes simplex virus (HSV) respectively. As a continuous cell line, 293 cells are preferable to HEK and HEF cells for the routine isolation of ophthalmic viruses.
The growth characteristics of Japanese encephalitis virus cultivated in Novikoff hepatoma cells grown in shaker culture can be differentially altered by the presence of 6-cis or cis-9-octadecenoic acid in Swim's 67-G medium. The addition of 125 mug of the 6-isomer per ml medium reduced the number of infectious particles produced, whereas the same amount of the 9-isomer enhanced virus production. The virus was found to be more stable in cell-free spent medium than in fresh medium. The presence of 125 mug 6-18:1 per ml in fresh medium resulted in a rapid loss of virus infectivity.
Human monoclonal antibodies specific for human cytomegalovirus (CMV) antigens have been established using peripheral blood lymphocytes from a seropositive donor. Immortalization of antigen-specific B cells was achieved by Epstein-Barr virus transformation followed by somatic cell fusion of antigen-specific lymphoblastoid cells. Four clones producing high-affinity antibodies (0.2-7 x 10(9) M-1) specific for the viral matrix protein pp65 have been further characterized with respect to epitope specificity of secreted antibodies. The studied antigen represents a major protein produced by in vitro-cultivated virus, and is important in the serodiagnosis of CMV infection. The human monoclonal antibodies recognized different epitopes, some of which proved to be overlapping. The fine specificity of these antibodies was evaluated using synthetic peptides covering the sequence of pp65. The antibody MO58 recognized a linear epitope (residues 283-288) whereas antibody MO53 recognized a discontinuous epitope involving residues 208-216 and 280-285. Despite the close proximity of these epitopes, the antibodies did not compete with each other for the same binding site on intact antigen.
The flavivirus genome codes for structural and nonstructural proteins which are produced from a common polyprotein precursor by proteolytic processing. The nonstructural protein NS1 is hypothesized to be involved in virus assembly and maturation as well as in protective immunity. This paper describes the synthesis of several forms of yellow fever (YF) virus NS1 protein during virus cultivation in vitro. These include cell-associated and secreted NS1 forms, the major difference among these being their N-linked glycosylation status. High-mannose and complex (or hybrid)-type moieties were identified on YF NS1 by analysis with endoglycosidases of known specificity. These studies are the basis for a systematic approach to determining the role of NS1 in the virus cycle and possibly in pathogenesis.
Deep groundwater, even if generally protected, could be contaminated by surface or rain water infiltration through soil fractures, septic tanks, cesspits, land irrigation, disposal of wastewater and disposal of muds from depuration systems. The sanitary importance of such possible contamination is related to the different uses of the water and it is at the maximum level when it is intended for human use. Routine microbiological analyses do not consider viruses, only bacterial parameters, as contamination indicators. However, it is known that enteric viruses can survive a long time in deep aquifers and that they may not always be associated with bacterial indicators. The virological analysis of waters intended for drinking use is provided only as an occasional control exercised at the discretion of the sanitary authority. Technological difficulties with obtaining data about groundwater viral contamination led to a study to devise rapid and efficient methods for their detection and the application of these methods to samples from different sources. Four acid nucleic extraction techniques have been tested (classic proteinase K- phenol/chloroform, QIAamp Viral RNA Kit (Qiagen), SV Total RNA Isolation System (Promega) and NucleoSpin Virus L (Macherey-Nagel). Sensitivity and specificity of RT-PCR protocols for entero- (EV), hepatitis A (HAV) and small round structured (SRSV) viruses have been verified. Deep groundwater samples (100 L) were concentrated (2-step tangential flow ultrafiltration) and the concentrate contaminated with serial 10-fold dilutions of a known titre of poliovirus type 3. Extracted RNA was concentrated (microcon-100) and analysed by RT-PCR using specific EV primers and visualising amplification products by agarose gel electrophoresis. In addition, two different methods of RT-PCR for non-cultivable viruses have been tested: (a) RT-PCR and nested RT-PCR for HAV and (b) RT-PCR with generic primers and RT-PCR with specific primers for SRSV. Different specificity tests have been carried out in the presence of some of the commoner microorganisms. The most efficient, sensitive and specific protocols were used to test 35 x 100L deep groundwater samples. Sample concentrates were split with one part treated with chloroform and analysed by cell culture (BGM and Frp/3, derived from FrHK/4, cells) and the other tested by RT-PCR for HAV, EV and SRSV. Results demonstrated the high efficiency of the classic and QIAamp methods. Microcon-100 did not increase the sensitivity of the technique used. The highest sensitivity was observed for RT-PCR with specific primers for SRSV and for nested RT-PCR for HAV. One sample showed a cytopathic effect, not confirmed at the third subculture, while the RT-PCR allowed the detection of echovirus 7. Cell culture did not allow detection of the majority of the enteric viruses while PCR gave sensitive, specific and rapid detection of a range of agents in the same samples. Even if it was impossible to fix a virological quality standard, it would be necessary to find a viral indicator in order to achieve a complete preventive check which would be particularly useful in some cases (e.g. water never used before, after pollution accidents, for seasonal checking).
The capsid protein of hepatitis E virus (HEV) is encoded by open reading frame 2 (ORF 2) and exhibits variable processing when expressed in insect and COS cells, but nothing is known of its processing in cells relevant to its replication. The full-length ORF 2 protein was expressed at high levels in mammalian cells by insertion of ORF 2 in the Semliki Forest virus (SFV) replicon to generate rSFV/HEV ORF 2K. Expression of the capsid protein was detected readily by metabolic labelling and indirect immunofluorescence in BHK-21 cells transfected with RNA transcripts derived from rSFV/HEV ORF 2K. ORF 2 protein was also expressed at high levels in cells of diverse origin, including liver-derived cell lines Huh7 and HepG2, following infection with recombinant virus derived from cotransfection of BHK-21 cells with the rSFV/HEV ORF 2K and helper SFV replicon RNAs. The addition of hypertonic KCl during metabolic labelling reduced the level of host cell protein synthesis and enhanced the detection of intermediates in ORF 2 protein processing. The wide host range and high level expression directed by SFV replicon particles has particular utility in the analysis of cell-specific factors in the protein processing and assembly of non-cultivable viruses such as HEV.
The immunogenicity and protective efficacy of formalin-inactivated vaccines prepared from influenza A (H1N1) viruses grown in MDCK cells and in eggs was compared in animal models. The A/Chr/157/83 virus grown in MDCK cells (157M) differed by two amino acid substitutions in the HA molecule from the corresponding virus grown in eggs (157E) and the two viruses could be distinguished antigenically by monoclonal and polyclonal antibodies. Following two intramuscular injections of vaccine in ferrets, guinea pigs, and hamsters, both vaccines were equally immunogenic when antibody was analyzed by hemagglutination inhibition using homologous virus. However, single radial hemolysis analysis following antibody cross-adsorption showed that antibody stimulated by 157E vaccine was exclusively strain specific whereas that produced by the 157M vaccine was more broadly reactive. When immunized hamsters were challenged with virus cultivated on mammalian (MDCK) cells, the homologous vaccine induced a higher degree of protection than the corresponding egg-grown vaccine.
The results obtained at the first passage of ceruloplasmin-incubated influenza virus A/PR8/34 (H0N1) in chorioallantoic membrane fragments demonstrate the "trap effect" of ceruloplasmin and are characterized by the low values of the ratios between the hemagglutinating and neuraminidase activities and the infectivity of the resulted progens. At the 2nd passage, both in the absence and in the presence of ceruloplasmin the previously observed changes are accompanied by severe modifications in the antigenicity of the progens, as shown by the HAI titres obtained against a rabbit antiserum to influenza virus A/PR8/34 (H0N1).
Non-infectious virus-like particles of SIVsmB7 that expresses env and gag gene products but are defective in pol and vpx/vpr were assessed for their ability to induce protective immunity against infection with pathogenic SIVsmE660 in rhesus macaques. Animals were immunized in three groups: group A was primed with cell-associated SIVsmB7 and boosted with cell-free SIVsmB7; group B was primed with cell-free SIVsmB7 and boosted with cell-free SIVsmB7 conjugated to iron oxide microbeads; group C was primed with cell-free SIVsmB7 mixed with Titer Max adjuvant and boosted with cell-free SIVsmB7 mixed with SAF-M adjuvant followed by secondary boosting with cell-free SIVsmB7 conjugated to microbeads. Animals were challenged intravenously with 20 animal infectious doses of SIVsmE660 grown in rhesus peripheral blood mononuclear cells 3 weeks after final boosting. All animals became infected as evidenced by quantitative virus cultivation. Sera from immunized animals contained low-titer antibodies by ELISA and low or undetectable neutralizing antibodies on the day of challenge but strong anamnestic antibody responses were observed following challenge. Interestingly, 2 of 3 animals in group A showed evidence of transient viremia and more stable CD4 counts following challenge as compared to the other immunized animals and to non-immunized controls. Thus, immunization with cell-associated SIVsmB7 did not provide sterilizing immunity against challenge with a highly pathogenic SIV strain but might have caused virus clearance later in infection.
From a structural point of view an essential distinction between complete and defective rabies viruses is difference in size. In addition, isoelectric properties differ. The complete virus has an isoelectric point approaching neutrality, whereas the defective virus focuses between pH 3-4.7. The isoelectric points of the glycoprotein from complete and defective viruses differ in a corresponding fashion. The Pasteur virus cultivated on BHK21C13 cells, contains glycoproteins, the glycopeptides of which have a structure containing the following five monosaccharides: sialic acid, D-galactose, (N-acetyl)D-glucosamine, D-mannose and L-fucose. The glycosylation of the glycoproteins is different, at least in so far as the relative sialic acid/glucosamine ratio is concerned.
In order to improve the sensitivity of the diagnosis of viral encephalopathy and retinopathy (VER) in sea bass, a nested reverse transcriptase-polymerase chain reaction (RT-PCR) detection method was developed. The reverse transcription step and the first stage PCR were performed using outer primers specific for the coat protein gene, whereas a new primer set was used as inner primers for the second stage PCR. Fish were collected just before, during and after a VER outbreak occurring in a mediterranean fish farm. For each time point, ten different fish were analysed individually by nested RT-PCR, single step PCR and virus cultivation. The results showed that the frequency of positive samples was always higher using the nested RT-PCR assay. In particular, it was possible to detect nodavirus specific signals 1 month before the appearance of the first mortalities, but only by nested RT-PCR. Altogether these results showed that the sensitivity of nodavirus detection is greatly improved using a nested RT-PCR method. In particular, it was possible to monitor the presence of viral genome in asymptomatic carrier fish using this method.
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