Monoclonal antibodies against simian virus 40 nuclear large T tumour antigen: epitope mapping, papova virus cross-reaction and cell surface staining.

Thirty six cloned hybridomas have been isolated which produce monoclonal antibodies directed against simian virus 40 (SV40) large T tumour antigen. They have been shown to recognize at least six different epitopes along the T antigen polypeptide according to their reaction with the various truncated forms of T antigen expressed by adenovirus-SV 40 hybrid viruses. Sixteen antibodies cross-react with cells infected by the closely related human BK virus. Only two antibodies, PAb1604 and PAb1614, directed against different epitopes of the SV40 T antigen, cross-react with polyoma large T tumour antigen which has a more limited amino acid sequence homology. This cross-reaction is rarely seen with polyclonal antibodies. Monoclonal antibody PAb1620 gave nuclear immunofluorescence only with murine cells transformed by SV40 and was found to react with a complex of T-antigen and 53 000-dalton host-coded protein. All the monoclonal antibodies react with nuclear T antigen and all but four antibodies stained the surface of SV40-transformed cells. These were four of the five antibodies directed against the central third of the T antigen. Thus the monoclonal antibodies show that cell surface T antigen differs from nuclear T antigen, either in accessibility or structure.     

Characterization of a canine parvovirus strain isolated from an adult dog

A CPV-2b strain was detected from an adult vaccinated dog, affected with severe gastroenteritis. The faeces of the dog were positive to canine parvovirus by a hemagglutination assay and gave a CPV-2b-like pattern by a hemagglutination inhibition test using monoclonal antibodies. In vitro-cultivation of the virus was difficult and after a few passages on canine and feline cells, the presence of the virus was detectable only by an immunofluorescence assay on the feline cells, since hemagglutinating activity had disappeared. Characterization of the virus, by an indirect immunofluorescence assay with monoclonal antibodies, confirmed the antigenic CPV-2b-like pattern of the nonhemagglutinating virus.     

Enhanced neutralization of flavivirus by monoclonal antibodies against Negishi virus

Ten monoclonal antibodies against Negishi virus were classified into 3 groups and 7 types according to the reaction patterns to Negishi virus by the hemagglutination inhibition (HI) test and by several kinds of neutralization tests. When kinetic neutralization was applied to these monoclonal antibodies at the same HI titer of 1:64, the initial slope and the persistent fraction of the kinetic curve was varied in each antibody. In the double-kinetic neutralization test, neutralization did not proceed further when the second antibody was the same type as the first one. When the second antibody was a different type from that of the first one, neutralization proceeded further. The mixtures of 4 monoclonal antibodies classified as different groups and types remarkably enhanced neutralization in the kinetic assay. These results suggested that an assortment of antibodies is needed for effective neutralization of Negishi virus and that a multi-hit model is likely operating in the neutralization of Negishi virus.     

五种国产与进口小鼠病毒ELISA抗体检测试剂盒的比较研究

目的评价国产小鼠病毒抗体ELISA检测试剂盒。方法选择国产与进口小鼠淋巴细胞脉络丛脑膜炎病毒（LCMV）、肝炎病毒（MHV）、仙台病毒（SV）、腺病毒（MAV）、细小病毒（MPV）ELISA抗体检测试剂盒,进行敏感性、特异性、精密性、稳定性、可信度试验比较。结果国产与进口试剂盒：同种试剂盒之间灵敏度相差最低为2倍,差异显著（P〈0.05）,最高为16倍,差异极显著（P〈0.01）;特异性试验显示每种试剂盒,与其他4种病毒均无交叉反应;精密性试验显示5种试剂盒批内平均变异系数均小于10%;稳定性试验显示5种试剂盒相对偏差均小于25%;分别选择已知36份小鼠血清进行检测,国产和进口LCMV、MHV、SV、MPV符合率均为100%;国产MAV符合率为86.1%,进口MAV符合率均为100%,二者之间差异极显著（P〈0.01）。结论除国产MAV试剂盒敏感性、可信度低于进口外,国产LCMV、MHV、SV、MPV试剂盒与进口同种试剂盒相比,在敏感性、特异性、精密性、稳定性和可信度方面均良好。     

Immunobiochemical characterization with monoclonal antibodies of Epstein-Barr virus-associated early antigens in chemically induced cells.

Five monoclonal antibodies which are reactive to early antigens of Epstein-Barr virus have been produced by using somatic cell hybridization techniques. The specificity of the monoclonal antibodies to early antigens was demonstrated by indirect immunofluorescence, which showed that the antigens were localized to the nucleus of early antigen-induced Raji cells. Additional indirect immunofluorescence studies showed that like patient antisera to diffuse-staining early antigen, the monoclonal antibodies gave positive staining reactions after methanol fixation. One of the antibodies, 1150-4, was positive by the anti-complement immunofluorescence technique but differed with Epstein-Barr virus-associated nuclear antigen-positive patient sera in that it only stained induced cells. Different fixation methods were found to alter dramatically the appearance of the nuclear staining reactions produced by the monoclonal antibodies. Immunoprecipitation and immunoblot experiments revealed that monoclonal antibodies 1108-1 and 1129-1 recognized two polypeptides of 55,000 and 50,000 daltons (p55;50), 1173-6 and 1180-2 recognized just p50, and 1150-4 identified a 65,000-dalton nuclear protein. Immunobiochemical characterization of these viral antigens showed that p55 is a phosphoprotein, and p55;50 has strong DNA-binding activity preferentially to single-stranded DNA. Elucidation of the role of these nuclear proteins in Epstein-Barr virus infection and the events associated with Epstein-Barr virus-directed lymphocyte transformation may provide significant information on the pathogenicity of this important human virus.     

Induction of a neutralizing immune response to human respiratory syncytial virus with anti-idiotypic antibodies.

Anti-idiotypic (anti-Id) antibodies were raised in rabbits against monoclonal antibodies that recognized either F glycoprotein 47F or G glycoprotein 63G, 62G, or 74G of the human respiratory syncytial virus Long strain. Anti-Id sera inhibited the virus binding of the immunizing monoclonal antibodies and in some cases the binding of other antibodies reacting with overlapping epitopes. The anti-Id sera also inhibited virus neutralization mediated by the original monoclonal antibodies. Affinity purified anti-Id antibodies were subsequently used to raise a homologous anti-anti-Id response in rabbits. One of the rabbits, inoculated with anti-Id 63G, generated antibodies that reacted with the G protein of respiratory syncytial virus and neutralized the virus to high titers. The antiviral antibodies induced by anti-Id 63G were broadly cross-reactive with strains of the A and B subtypes. However, the specificities of monoclonal antibody 63G and anti-anti-Id 63G were not exactly the same, as indicated by their reaction with escape mutants to antibody 63G. These results demonstrate for the first time the induction of an anti-respiratory syncytial virus response by anti-Id antibodies.     

Monoclonal antibodies to pseudorabies virus produced by mouse hybridomas

Monoclonal antibodies to pseudorabies viral capsid protein were prepared by fusing Sp2/0-Ag14 myeloma cells with pseudorabies virus (PrV)-primed Balb/c splenocytes. These monoclonal antibodies were specific for PrV. No crossreaction with Herpes Simplex Virus (HSV, type I) or infectious bovine rhinotrachitis virus (IBR) was found. The reaction between these monoclonal antibodies and PrV was demonstrated by immunofluorescent staining technique and enzyme-linked immunoblotting assay. These antibodies could be a useful diagnostic reagent for PrV as well as a good tool for the study of the capsid protein of PrV.     

Optimization of conditions for preparation of the solid phase of infected cells in the immunoenzyme analysis of monoclonal antibodies

The conditions of immunoenzyme assay have been studied on the solid state phase of infected cells using the model of monoclonal antibodies MAK-14-7 to the virus of Venezuelan equine encephalomyelitis (VVEE) and monoclonal antibodies OKA-1 to vaccine virus in the systems of VNK-21 cells or 4647 cells infected by VVEE, or HeLa cells infected by vaccine virus. The titer of monoclonal antibodies detected grows with the dose of infected cells fixed in the holes of micropanel used for reaction and with the multiplicity of infection. The most intensive and contrasting dyeing of conjugate has been registered when the cells have been fixed with 0.25% glutaraldehyde 24 h after infection. The titers of ascytic preparations of monoclonal antibodies MAK-14-7 and OKA-1 under the optimal conditions of immunoenzyme assay reaction on the solid phase of infected cells present 1 : 10 000 and 1 : 100 000.     

Contribution of measles virus fusion protein in protective immunity: anti-F monoclonal antibodies neutralize virus infectivity and protect mice against challenge.

To study the contribution of the measles virus fusion (F) protein in the immune response, anti-F monoclonal antibodies were prepared by using a vaccinia-measles virus F recombinant. In contrast to previously described anti-F monoclonal antibodies, these antibodies not only neutralized virus infectivity and inhibited fusion but also passively protected mice. Since these monoclonal antibodies recognize a configurational epitope, presentation of the antigen during infection may play an important role in the immune response. These factors are discussed in relation to vaccination.     

Formalin inactivation of the lactate dehydrogenase-elevating virus reveals a major neutralizing epitope not recognized during natural infection.

Five hybridomas that secrete monoclonal antibodies which neutralize the infectivity of lactate dehydrogenase-elevating virus (LDV) were isolated from BALB/c mice primed with Formalin-inactivated LDV. Competition analyses indicated that all five neutralizing monoclonal antibodies recognize contiguous, if not identical, epitopes on the envelope glycoprotein of LDV (VP-3) which are not recognized by nonneutralizing VP-3-specific monoclonal antibodies isolated from the same fusion. Despite the presence of neutralizing activity, polyclonal anti-LDV antibodies obtained from persistently infected mice did not compete for binding to LDV with four of the five neutralizing monoclonal antibodies tested. The results indicate that the envelope glycoprotein of LDV possesses a major neutralizing epitope which is poorly recognized, if at all, by mice during a natural infection but is rendered immunogenic by Formalin inactivation of the virus. The epitope was also not immunogenic in a rabbit, since its polyclonal LDV-neutralizing antibodies did not inhibit binding of the mouse monoclonal antibodies to LDV. Passive immunization with the neutralizing monoclonal antibodies did not protect mice from LDV infection and did not alter the course of infection. Neutralizing monoclonal antibodies have been used to select a neutralization escape variant by a novel combination of in vitro and in vivo isolation.     

Uukuniemi virus maturation: immunofluorescence microscopy with monoclonal glycoprotein-specific antibodies.

Monoclonal antibodies directed against Uukuniemi virus glycoproteins G1 and G2 in combination with polyclonal antibodies against the nucleoprotein (N) were used to study the maturation of the virus in Golgi complexes of infected chicken embryo fibroblasts and BHK cells. Of 25 monoclonal antibodies obtained, 10 were shown to be G1 specific and 15 were shown to be G2 specific by immunoblotting and immunoprecipitation. In double-staining experiments, some of the monoclonal antibodies gave similar distributions of fluorescence as compared with the staining obtained from polyclonal rabbit anti-G1-G2 antibodies. Others, however, preferentially stained either the glycoproteins in the Golgi complex or those at the cell surface. This may indicate that the glycoproteins underwent conformational changes during their transport. Uukuniemi virus infection resulted in the vacuolization of the membranes of Golgi complexes where the maturation of the virus was taking place. Double-staining experiments with monoclonal antibodies which preferentially stained the Golgi-associated viral glycoproteins and with anti-N polyclonal rabbit antiserum showed a correlation between the progressive vacuolization of the Golgi complex and the accumulation of viral nucleoprotein in the Golgi region, suggesting that a morphological alteration of the Golgi complex may be a prerequisite for intracellular maturation of the virus. Treatment of Uukuniemi virus-infected cells with tunicamycin, a drug which inhibits N-linked glycosylation, resulted in the accumulation of both glycoproteins at an intracellular location, apparently representing the endoplasmic reticulum. Double-staining experiments showed a parallel accumulation of nucleoprotein at these sites, indicating that local accumulation of glycoproteins is required for nucleoprotein binding to intracellular membranes.     

Neutralizing monoclonal antibodies specific for herpes simplex virus glycoprotein D inhibit virus penetration.

Nine monoclonal antibodies specific for glycoprotein D (gD) of herpes simplex virus type 1 were selected for their ability to neutralize virus in the presence of complement. Four of these antibodies exhibited significant neutralization titers in the absence of complement, suggesting that their epitope specificities are localized to site(s) which contribute to the role of gD in virus infectivity. Each of these antibodies was shown to effectively neutralize virus after virion adsorption to cell surfaces, indicating that neutralization did not involve inhibition of virus attachment. Although some of the monoclonal antibodies partially inhibited adsorption of radiolabeled virions, this effect was only observed at concentrations much higher than that required to neutralize virus and did not correlate with complement-independent virus-neutralizing activity. All of the monoclonal antibodies slowed the rate at which virus entered cells, further suggesting that antibody binding of gD inhibits virus penetration. Experiments were carried out to determine the number of different epitopes recognized by the panel of monoclonal antibodies and to identify epitopes involved in complement-independent virus neutralization. Monoclonal antibody-resistant (mar) mutants were selected by escape from neutralization with individual gD-specific monoclonal antibodies. The reactivity patterns of the mutants and antibodies were then used to construct an operational antigenic map for gD. This analysis identified a minimum of six epitopes on gD that could be grouped into four antigenic sites. Antibodies recognizing four distinct epitopes contained in three antigenic sites were found to neutralize virus in a complement-independent fashion. Moreover, mar mutations in these sites did not affect the processing of gD, rate of virus penetration, or the ability of the virus to replicate at high temperature (39 degrees C). Taken together, these results (i) confirm that gD is a major target antigen for neutralizing antibody, (ii) indicate that the mechanism of neutralization can involve inhibition of virus penetration of the cell surface membrane, and (iii) strongly suggest that gD plays a direct role in the virus entry process.     

First record in America of Aedes albopictus naturally infected with dengue virus during the 1995 outbreak at Reynosa, Mexico

Abstract Mosquito collections were conducted during a dengue outbreak in Reynosa, Tamaulipas, Mexico, July-December 1995. A total of 6694 adult mosquitoes (four genera and nine species) were captured, of which 2986 (78.3% females and 21.7% males) were Aedes albopictus and 2339 (39.7% females and 60.3% males) were Ae.aegypti. These two species comprised 84.2% of the total collection. Specimens were grouped into pools, nearly 50% of them processed for detection of virus by cythopathic effect in C6-36 and VERO cell cultures and by haemagglutination test. Five pools gave positive haemagglutin-ation reactions and were examined by immunofluorescence using monoclonal antibodies to flavivirus and to dengue virus. One pool of ten Ae.albopictus males was positive for dengue virus: serotypes 2 and 3 were identified by serotype-specific monoclonal antibodies arid confirmed by RT-PCR. This is the first report of Ae.albopictus naturally infected with dengue virus in America. Also, it is the very first time Ae.albopictus males have been found infected with dengue virus in the wild.     

Small intestinal chromaffin cells and carcinoid tumours: a study with silver stains, formalin-induced fluorescence and monoclonal antibodies to serotonin

Summary The enterochromaffin cells of the human small intestinal mucosa were stained immunocytochemically with monoclonal antibodies against serotonin. The staining results were compared with those obtained with other methods for identifying serotonin-containing endocrine cells such as the argentaffin reaction, formalin-induced fluorescence and the argyrophil reaction of Grimelius. The different techniques gave similar, but not identical, results. The serotonin-immunoreactive cells outnumbered the argentaffin cells by 7%. Almost all (99%) serotonin-immunoreactive cells showed formalin-induced fluorescence but only a small population (5%) were fluorescent. In a subsequent study, these techniques were applied to 14 small intestinal carcinoids. It was shown that formalin-induced fluorescence and the argentaffin reaction were positive in 14 and 13 tumours, respectively, while the monoclonal serotonin antibodies failed to stain seven of the tumours. It is concluded that formalin-induced fluorescence and the argentaffin reaction are more useful techniques than serotonin immunocytochemistry for defining these tumours in routine formalin-fixed surgical specimens.     

Antigenic analysis of flaviviruses with monoclonal antibodies against Negishi virus

Twelve monoclonal antibodies against Negishi virus were obtained and characterized by hemagglutination inhibition (HI) and neutralization (NT) tests using five flaviviruses isolated in the pan-Pacific region. The reaction pattern of the antibodies showed that Negishi virus was most closely related to Langat virus, followed by 3-Arch, JE and Apoi viruses in that order. Hemagglutinating (HA) antigen of the virus had distinct HI relating sites which were Negishi virus specific, tick borne encephalitis (TBE) virus complex specific and flavivirus cross-reactive. Monoclonal anti-Negishi antibodies cross-reactive to Japanese encephalitis (JE) virus in the HI test had neutralizing activity to JE virus but no activity to homologous Negishi virus.     

Infection of a macrophage-like cell line, P388D1 with reovirus; effects of immune ascitic fluids and monoclonal antibodies on neutralization and on enhancement of viral growth

We studied the effect of antibody on the growth of reovirus, serotypes 1 and 3, in P388D1, a continuous mouse macrophage-like cell line. Enhanced growth of virus was observed when cells were infected in the presence of nonneutralizing monoclonal antibodies or subneutralizing concentrations of either immune ascitic fluids or neutralizing monoclonal antibodies. Both enhancement of viral growth and neutralization were accompanied by an antibody-mediated increase in binding of radiolabeled virus to P388D1 cells. Although neutralization was seen only with monoclonal antibodies directed toward the sigma-1 surface protein of the virus, enhancement was observed with two monoclonal antibodies directed toward other surface proteins. Trypsin treatment of P388D1 cells abrogated enhanced growth of virus mediated by a mouse IgG2a antibody; preincubation with P388D1 with human IgG1 but not IgG2 myeloma proteins also abrogated enhancement by immune ascitic fluid or monoclonal antibody. These observations are compatible with known properties of P388D1 Fc receptors and support the role of the Fc receptor in antibody-mediated infection.     

Development of ELISA using recombinant antigens for specific detection of mouse parvovirus infection.

Nucleotide sequences of mouse parvovirus (MPV) isolate, named MPV/UT, and mouse minute virus (MMV) were analyzed and used for expressing recombinant proteins in E. coli. ELISA tests using recombinant major capsid protein (rVP2) and recombinant major non-structural protein (rNS1) as antigens were developed and their performance in serologic detection of rodent parvovirus infection was assessed. MPV-rVP2 and MMV-rVP2 ELISAs reacted specifically with anti-MPV and anti-MMV mouse sera, respectively. MMV-rNS1 antigen had a wide reaction range with antisera to rodent parvoviruses including MPV, MMV, Kilham rat virus (KRV) and H-1 virus. All mice oronasally infected with MPV were seropositive at 4 weeks post-infection in screening by ELISAs using MPV-rVP2 and MMV-rNS1 antigens, but were negative by conventional ELISA using whole MMV antigen. A contact transmission experiment revealed that transmission of MPV occurred up to 4 weeks post-infection, and all cage mates were seropositive in screening with MPV-rVP2 and MMV-rNS1 ELISAs. These results indicate that MPV-rVP2 and MMV-rVP2 are specific ELISA antigens which distinguish between MPV and MVM infection, while MMV-rNS1 antigen can be used in generic ELISA for a variety of rodent parvoviruses. The higher sensitivity of MPV-rVP2 ELISA than conventional ELISA for detecting seroconversion to MPV in oronasally infected mice as well as in cage mates suggests the usefulness of MPV-rVP2 ELISA in quarantine and microbiological monitoring of MPV infection in laboratory mice.     

Monoclonal antibodies specific to yellow-head virus (YHV) of Penaeus monodon

Monoclonal antibodies specific to 22, 67 and 135 kDa proteins of yellow-head virus (YHV) were produced from a mouse immunized with partially purified YHV isolated from the haemolymph of experimentally YHV-infected Penaeus monodon. Four groups of monoclonal antibodies were identified. One group of antibodies bound only to native protein of YHV while the others were specific to 135, 67 and 22 kDa proteins in both native and denatured forms. All antibodies could be used to detect YHV infection by means of dot blot and immunohistochemistry. However, antibodies specific to the 22 kDa protein gave the best immunohistochemistry results in terms of intensity and sharpness of staining.     

Polyclonal Antibodies to the Bacterially Expressed Coat Protein of Faba Bean Necrotic Yellows Virus

Specific rabbit polyclonal antibodies against bacterially expressed coat protein of Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) were produced using a recombinant DNA approach. The FBNYV capsid protein (CP) gene located on component 5 was cloned in an expression vector pQE‐9 (Qiagen, QIAGEN Inc., Chatswortch, CA91311, USA). Expression of the CP with an N‐terminal hexahistidine tag in Escheri‐ chia coli M15 cells was induced by adding isopropyl‐3‐D ‐1‐thiogalactoside (IPTG) to a final concentration of 2 mM . About 8 mg of bacterially expressed CP (BE‐CP) was purified from 1 litre of bacterial liquid culture using a Ni‐NTA resin column (Qiagen). The expressed CP which migrated as a protein of approximately 23 kDa in sodium dodecyl sulphate (SDS)‐polyacrylamide gel electrophoresis (PAGE) was identified by its strong reaction with polyclonal antibodies produced against FBNYV particles and 2‐5H9 FBNYV‐monoclonal in Western blots. Expressed and purified CP (SDS‐PAGE 23 kDa band) was injected into a white rabbit, using seven intramuscular injections at weekly intervals. The antiserum produced was evaluated for FBNYV detection in double antibody sandwich (DAS)‐enzyme‐linked immunosorbent assay (ELISA), triple antibody sandwich (TAS)‐ELISA, tissue blot immunoassay (TBIA), dot blot, Western blot and goat antimouse coating (GAMC)‐ELISA using 13 different FBNYV monoclonal antibodies. The antiserum raised against the BE‐CP gave strong FBNYV‐specific TBIA reactions and very weak background reactions with non‐infected tissue, similar to those produced by monoclonal antibodies. Furthermore, BE‐CP polyclonal antibody reacted weakly with FBNYV‐infected tissue and strongly with BE‐CP in DAS‐ELISA, but not with FBNYV‐infected tissue in TAS‐ELISA when 13 detecting monoclonal antibodies were used. In addition, BE‐CP polyclonal antibody reacted strongly with BE‐CP in TAS‐ELISA only when 2‐5H9 detecting monoclonal was used. When monoclonals were used as primary antibody and BE‐CP polyclonal as detecting antibody (GAMC‐ELISA), FBNYV‐infected tissue gave moderate reactions with 2‐5H9 and strong reactions with 3‐2E9 monoclonal, whereas BE‐CP gave equally strong reactions with both monoclonals. These results showed that the BE‐CP polyclonal antibody is useful for the detection of FBNYV in infected tissue by TBIA and dot blot tests.     

Detection of mouse parvovirus in Mus musculus gametes, embryos, and ovarian tissues by polymerase chain reaction assay

We used primary and nested polymerase chain reaction (PCR) assays to determine the presence of mouse parvovirus (MPV) in mouse sperm, oocytes, preimplantation embryos, and ovarian tissues collected from MPV-infected mice. The primary PCR assay detected MPV in 56% of the sperm samples. MPV was not eliminated by passing sperm samples through a Percoll gradient. After Percoll treatment, MPV was still present in 50% of the samples according to primary PCR assay. Oocyte samples that did not undergo extensive washing procedures had detectable MPV in 7% of the samples based on the primary PCR assay, but nested PCR assay detected higher (28%) infection rate. However, MPV was not detected in oocytes that underwent extensive washing procedures, as assessed by either primary or nested PCR assay. Although primary PCR did not detect MPV in embryos, a nested PCR assay determined that 50% of the embryos were positive for the virus. In addition, ovarian tissues were collected from 3 different mouse colonies with enzootic MPV infection. Ovarian tissue collected from 129CT, 101/R1, and Sencar mice had high incidence (38%, 63%, and 65%, respectively) of MPV infection on the basis of nested PCR amplification. These results demonstrate that mouse gametes, embryos, and ovarian tissues may be contaminated with MPV and therefore caution is necessary when infected germplasm is used for assisted reproductive technologies such as embryo transfer, establishing embryonic stem cell lines, in vitro fertilization, ovary transplantation, and intracytoplasmic sperm injection.