Fish viruses serologic comparison of the golden shiner and infectious pancreatic necrosis viruses

High titer neutralizing antiserum to golden shiner virus (GSV) was produced in a rabbit. The GSV antiserum had significant neutralization indices in tests with the original strain of GSV and with another more recent isolate. Neutralization indices were not significant when two strains of infectious pancreatic necrosis virus (IPNV) were reacted with GSV antiserum, and neither isolate of GSV showed significant reactivity in neutralization tests with polyvalent IPNV antiserum. Our results indicate that IPNV and GSV are serologically unrelated.     

Complement-mediated enhancement of antibody function for neutralization of pseudotype virus containing hepatitis C virus E2 chimeric glycoprotein

We previously reported a number of features of hepatitis C virus (HCV) chimeric glycoproteins related to pseudotype virus entry into mammalian cells. In this study, pseudotype virus was neutralized by HCV E2 glycoprotein-specific antibodies and infected human sera. Neutralization (50% reduction of pseudotype virus plaque formation) was observed with two human immunoglobulin G1 monoclonal antibodies (MAbs) at concentrations of between 2.5 and 10 microg/ml. A hyperimmune rabbit antiserum to an E2 hypervariable region 1 (HVR1) mimotope also exhibited an HCV E2 pseudotype virus neutralization titer of approximately 1/50. An E1 pseudotype virus used as a negative control was not neutralized to a significant level (<1/10) by these MAbs or rabbit antiserum to E2 HVR1. Since HCV probably has a lipid envelope, the role of complement in antibody-mediated virus neutralization was examined. Significant increases in the neutralization titers of the human MAbs (approximately 60- to 160-fold higher) and rabbit antiserum to HVR1 mimotopes (approximately 10-fold higher) were observed upon addition of guinea pig complement. Further, these studies suggested that complement activation occurred primarily by the classical pathway, since a deficiency in the C4 component led to a significant decrease in the level of virus neutralization. This same decrease was not observed with factor B-deficient complement. We also determined that 9 of 56 HCV-infected patient sera (16%) had detectable pseudotype virus neutralization activity at serum dilutions of between 1/20 and 1/50 and that complement addition enhanced the neutralization activity of some of the HCV-infected human sera. Taken together, these results suggest that during infection, HCV E2 glycoprotein induces a weak neutralizing antibody response, that those antibodies can be measured in vitro by the surrogate pseudotype virus plaque reduction assay, and that neutralization function can be augmented by complement.     

Antigenic determinants of the 70,000 molecular weight glycoprotein of woolly monkey type C RNA virus.

The 70,000 molecular weight glycoprotein (gp70) of a type-C RNA virus originally isolated from a woolly monkey has been partially purified and immunologically characterized. Evidence that this viral protein is viral coded was derived from studies showing its antigenic properties to be unaltered by virus passage in cells of different species. A broadly reactive competition immunoassay was developed utilizing antiserum prepared against feline leukemia virus to precipitate 125I-labeled woolly monkey virus gp70. Gibbon and woolly viruses, as well as feline and several mouse type-C viruses, all reacted with equal efficiency in this assay. In contrast, an endogenous virus of the baboon failed to cross-react, suggesting that viruses of this latter group are less immunologically related to the others. In a homologous competition immunoassay for the woolly viral glycoprotein, the woolly virus was readily distingusihed from otherwise colsely related viruses of gibbon apes. These findings demonstrate the pronounced type-specific antigenic dterminants possessed by this viral protein. The antigenic determinants of gp70 responsible for neutralization have also been investigated.     

Virus Aggregation as the Cause of the Non-neutralizable Persistent Fraction

The non-neutralizable or persistent fraction of virus populations has been found to be caused by aggregated virus. Detailed investigation was performed with the prototype strain of echovirus type 4 (Pesascek), as this virus is notorious for its large non-neutralizable fraction. When Pesascek virus was clarified by low-speed centrifugation, homologous antiserum hardly neutralized the virus. However, when the virus was filtered through membranes having a porosity only twice the diameter of the virus, monodispersed virus was obtained which was efficiently neutralized. Serum titers were up to 1,000 times higher if the neutralization test was carried out with monodispersed virus. Virus in non-neutralizable aggregates was found to constitute 30% of the infective units of unfiltered Pesascek virus but only 0.1% of the antigenically related DuToit strain. This explains why DuToit strain has been a more satisfactory indicator strain for detecting type 4 antibodies, regardless of the echo 4 strain used for inducing the antibodies. Clarified suspensions and ultrafiltrates of viruses belonging to the picorna-, reo-, myxo-, adeno-, herpes-, and poxvirus groups were studied. Clarified suspensions yielded persistent fractions of 0.005% for poliovirus, of 0.1% for reovirus, of 0.6% for influenza virus, of <0.001% for adenovirus, of 0.06% for herpesvirus, and of 10 to 30% for vaccinia virus. In all cases the persistent fractions were removed by membrane filters which had a pore diameter no larger than twice that of the virus under test, and the high concentration of virus in each ultrafiltrate was completely neutralized by antiserum.     

The effect of specific antiserum on the resistance of Neisseria gonorrhoeae to intracellular killing by phagocytes of human blood.

The high natural resistance of gonococci showing a characteristic 'double highlight' (DH) colonial morphology (Penn, Veale & Smith, 1977b) to intracellular killing by human phagocytes was markedly reduced by addition of rabbit antiserum to the phagocytosis medium or by preincubation of organisms with antiserum. Antisera raised to three different DH gonococcal strains showed a complex pattern of specificity in phagocytosis tests with the homologous organisms and three other DH strains. The effect of antiserum could be neutralized by adsorption with intact organisms or with extracts, prepared ultrasonically, of the homologous strain. Antiserum also promoted the intracellular killing of a strain which had a 'single highlight' colonial morphology (Penn et al., 1977b) and a low natural resistance to phagocytic killing, but adsorption with this strain neutralized the antiserum less consistently than the DH strain. The neutralization of antiserum-mediated promotion of intracellular killing by extracts of organisms naturally resistant to such killing may provide an assay for the aggressins responsible for this resistance.     

Serum enhancement of human immunodeficiency virus (HIV) infection correlates with disease in HIV-infected individuals.

The sera from 16 individuals infected with the human immunodeficiency virus (HIV) at different clinical stages were evaluated for antibody-dependent neutralization and/or enhancement of infectivity by HIV. The HIV isolate from each individual (homotypic) and established laboratory strains showing broad cellular host range and cytopathicity were used. All sera could neutralize one of the laboratory-passaged isolates, whereas only two could neutralize the corresponding homotypic strain. Seven homotypic isolates were enhanced by serum from the respective individual. This activity was primarily observed in patients with acquired immune deficiency syndrome. Moreover, the tropism for macrophages of four of these seven viral isolates was found to be enhanced by the homotypic sera. Finally, sequential pairs of HIV and sera obtained from five HIV-infected individuals with different clinical progression were studied over time. The enhancing activity of three of the five sera appeared to increase over time, indicating changes in both the host virus population and the type of antibodies produced. These results suggest that enhancing antibodies contribute to the spread and pathogenesis of HIV in vivo. They emphasize the necessity of studying further the association of enhancing antibodies and disease progression in infected individuals.     

Differentiation of type D virus from continuous human cells (Il'in-Bykovski? virus) and Mason-Pfizer monkey virus by the antigens of the viral envelopes]

For differentiation of Ilvin-Bykovsky virus (IBV) and monkey Meson-Pfeizer virus (M-PMV) the method of virus neutralization with antibodies against the envelope virus antigen was used. The viruses were cultivated in similar human embryo cells. The results of the virus neutralization were determined by presence or absence of the gs-antigen in the infected cells. The antiserum to M-PMV envelope antigens did not neutralize the IBV antigen. It has been concluded that IBV and M-PMV differ by their envelope antigens and should be regarded as different viruses.     

Serum neutralization of nucleopolyhedrosis viruses (Baculovirus subgroup A) pathogenic for Orgyia pseudotsugata

The preparation of antisera to intracellular nonoccluded virions and an in vivo neutralization test procedure (constant serum and virus in dilutions) are described. Results of homologous neutralization tests showed that rabbit antisera to two multicapsid viruses pathogenic for Orgyia pseudotsugata had higher neutralization indices than antiserum to a unicapsid Baculovrus from O. pseudotsugata. Based on reciprocal tests, the three viruses are antigenically distinguishable. Blood serum of rats which had been exposed by inhalation to 25 projected acre doses of a technical-grade Baculovirus preparation demonstrated no viral neutralizing activity. Since the neutralization test used in this study does not require availability of susceptible cell lines and is sensitive and accurate, it could find application in quality control programs and in field monitoring of Baculovirus strains.     

Mechanism of formation of pseudotypes between vesicular stomatitis virus and murine leukemia virus.

Pseudotypes of vesicular stomatitis virus (VSV) and Moloney murine leukemia virus (MuLV), defined by their resistance to neutralization by anti-VSV antiserum, are released preferentially at early times after infection of MuLV-producing cells with VSV. At later times, after synthesis of MuLV proteins has been inhibited by the VSV infection, neither MuLV virions nor the VSV (MuLV) pseudotypes are made. Infection of MuLV-producing cells with mutants of VSV having temperature-sensitive lesions in either G or M protein does not generate pseudotypes at nonpermissive temperature, indicating that both proteins are needed for pseudotypes to form. Although the pseudotypes resist neutralization by anti-VSV serum, they are inactivated by anti-VSV serum plus complement, and they can be precipitated by rabbit anti-VSV serum plus goat anti-rabbit IgG. These results, coupled with experiments using a temperature-sensitive mutant of VSV G protein grown at partly restrictive temperature, suggest that small numbers of VSV G protein are obligately incorporated into VSV(MuLV) pseudotypes. There appears to be a stringent requirement for recognition of the viral core by homologous envelope components as the nucleating step in the budding process. Only after such a nucleation can the envelope components of the second virus substitute into the membrane of the budding particle.     

Identification of gp120 regions targeted by a highly potent neutralizing antiserum elicited in a chimpanzee inoculated with a primary human immunodeficiency virus type 1 isolate

We have previously reported that a chimpanzee infected with a primary human immunodeficiency virus type 1 (HIV-1) isolate (HIV-1(DH12)) developed an extremely potent virus-neutralizing antibody. Immunoglobulin G purified from this animal conferred sterilizing immunity following passive transfer to macaques which were subsequently challenged with simian immunodeficiency virus/HIV-1 chimeric virus strain DH12. In addition to being highly strain specific, the chimpanzee antiserum did not bind to the V3 loop peptide of HIV-1(DH12), nor did it block the interaction of gp120 with the CD4 receptor. When neutralization was examined in the context of virus particles carrying chimeric envelope glycoproteins, the presence of all five hypervariable regions (V1 to V5) was required for optimal neutralization. Virions bearing chimeric gp120 containing the V1-V2 and V4 regions of HIV-1(DH12) could also be neutralized, but larger quantities of the chimpanzee antiserum were needed to block infection. These results indicate that the HIV-1 gp120 epitope(s) targeted by the chimpanzee antiserum is highly conformational, involving surface elements contributed by all of the hypervariable domains of the envelope glycoprotein.     

Association of the parainfluenza virus fusion and hemagglutinin-neuraminidase glycoproteins on cell surfaces.

We previously observed that cell fusion caused by human parainfluenza virus type 2 or type 3 requires the expression of both the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins from the same virus type, indicating that a type-specific interaction between F and HN is needed for the induction of cell fusion. In the present study we have further investigated the fusion properties of F and HN proteins of parainfluenza virus type 1 (PI1), type 2 (PI2), and type 3 (PI3), Sendai virus (SN), and simian virus 5 (SV5) by expression of their glycoprotein genes in HeLa T4 cells using the vaccinia virus-T7 transient expression system. Consistent with previous results, cell fusion was observed in cells transfected with homotypic F/HN proteins; with one exception, coexpression of any combination of F and HN proteins from different viruses did not result in cell fusion. The only exception was found with the closely related PI1 HN and SN HN glycoproteins, either of which could interact with SN F to induce cell fusion upon coexpression as previously reported. By specific labeling and coprecipitation of proteins expressed on the cell surface, we observed that anti-PI2 HN antiserum coprecipitated PI2 F when the homotypic PI2 F and PI2 HN were coexpressed, but not the F proteins of other paramyxoviruses when heterotypic F genes were coexpressed with PI2 HN, suggesting that the homotypic F and HN proteins are physically associated with each other on cell surfaces. Furthermore, we observed that PI3 F was found to cocap with PI3 HN but not with PI2 HN, also indicating a specific association between the homotypic proteins. These results indicate that the homotypic F and HN glycoproteins are physically associated with each other on the cell surface and suggest that such association is crucial to cell fusion induced by paramyxoviruses.     

Studies on the Neutralization of Herpes Simplex Virus

A portion of the seed virus remained active even in the presence of excess antibody. With an early serum or relatively low concentrations of a hyperimmune serum, the presence of such unneutralizable virus was uncovered by the addition of complement. This unneutralizable persistent fraction (PF) was distinguished from sensitized virus surviving in insufficient antibody, because the latter was inactivated by a high concentration of antibody as quickly as the control virus. The level of the PF was constant regardless of the serum species, serum lot, dilution of serum, antibody class and complement requirement of antibody, being approximately 0.1% of the seed virus. Millipore filtration of the seed virus lowered this PF level to varying degrees depending upon the porosity. However, when filtered virus-serum mixtures were incubated at 37 C for a sufficient time and then filtered again, a portion of the unneutralized virus did pass a 0.22 μ membrane. Furthermore, virus sensitized with insufficient antibody showed no increase in resistance to complement after 10 hr incubation at 4 C. These results proved that viral aggregation was not the essential cause of the PF. It was confirmed, on the other hand, that the neutralization of virus by hyperimmune IgG followed a one-hit curve. Analysis of these data appeared to support Rappaport's postulation that the PF represented antibody-coated virus whose critical antigenic sites were all left unbound.     

Serological relationship and detection of bean common and bean yellow mosaic viruses in agar gel

Microprecipitin tests demonstrated a distant serological relationship between intact virus particles of BCMV and BYMV. Antiserum titres of 2048–4096 and 16–128 (reciprocals of dilution end-points) were found for homologous and heterologous antigens, respectively. Cross-reactivity, however, was not obtained in agar immunodiffusion tests when the antigens, in purified preparations or crude infective sap, were treated with pyrrolidine or when the reactants were placed in agar gels containing sodium dodecyl sulphate. Only homologous antigens produced a precipitin line; homologous antiserum titre was 16. In comparative immunodiffusion trials, single-radial-diffusion tests (antiserum incorporated in agar) were much more sensitive than double-diffusion tests for detecting low virus concentrations. Analyses of virus proteins by polyacrylamide-gel electrophoresis demonstrated that coat protein of each virus was composed of a single polypeptide chain with a molecular weight of 35000.     

Serological Studies on Mung Bean Yellow Mosaic Virus

Particles of mung bean yellow mosaic virus (MYMV) were purified by a method that yields up to 3 mg per kg of systemically infected Phaseolus vulgaris“Top Crop” and used to prepare antiserum. MYMV antiserum prepared gave a single precipitin line and had a titre of 1/512 with homologous virus in gel double-diffusion tests. MYMV was shown to be serologically related to other whitefly-transmitted viruses, bean golden mosaic virus, tobacco leaf curl virus and cassava latent virus.     

Rotavirus serotype G9 strains belonging to VP7 gene phylogenetic sequence lineage 1 may be more suitable for serotype G9 vaccine candidates than those belonging to lineage 2 or 3

A safe and effective group A rotavirus vaccine that could prevent severe diarrhea or ameliorate its symptoms in infants and young children is urgently needed in both developing and developed countries. Rotavirus VP7 serotypes G1, G2, G3, and G4 have been well established to be of epidemiologic importance worldwide. Recently, serotype G9 has emerged as the fifth globally common type of rotavirus of clinical importance. Sequence analysis of the VP7 gene of various G9 isolates has demonstrated the existence of at least three phylogenetic lineages. The goal of our study was to determine the relationship of the phylogenetic lineages to the neutralization specificity of various G9 strains. We generated eight single VP7 gene substitution reassortants, each of which bore a single VP7 gene encoding G9 specificity of one of the eight G9 strains (two lineage 1, one lineage 2 and five lineage 3 strains) and the remaining 10 genes of bovine rotavirus strain UK, and two hyperimmune guinea pig antisera to each reassortant, and we then analyzed VP7 neutralization characteristics of the eight G9 strains as well as an additional G9 strain belonging to lineage 1; the nine strains were isolated in five countries. Antisera to lineage 1 viruses neutralized lineage 2 and 3 strains to at least within eightfold of the homotypic lineage viruses. Antisera to lineage 2 virus neutralized lineage 3 viruses to at least twofold of the homotypic lineage 2 virus; however, neutralization of lineage 1 viruses was fourfold (F45 and AU32) to 16- to 64-fold (WI61) less efficient. Antisera to lineage 3 viruses neutralized the lineage 2 strain 16- to 64-fold less efficiently, the lineage 1 strains F45 and AU32 8- to 128-fold less efficiently, and WI61 (prototype G9 strain) 128- to 1024-fold less efficiently than the homotypic lineage 3 viruses. These findings may have important implications for the development of G9 rotavirus vaccine candidates, as the strain with the broadest reactivity (i.e., a prime strain) would certainly be the ideal strain for inclusion in a vaccine.     

A new microplate neutralization test for typing of herpes simplex virus.

A microplate serum neutralization test for estimation of complement-requiring neutralizing (CRN) antibody was established as the first step for simplification of typing of herpes simplex virus (HSV). When guinea pigs were immunized with type 2 HSV, the late sera could mostly differentiate the types of HSV better than hyperimmune rabbit sera, the CRN titer against the heterologous type 1 HSV being much lower than the homologous titer. Sera of guinea pigs immunized with type 1 HSV showed about the same level of cross reaction against type 2 HSV as did rabbit antisera. Guinea pig sera having minimal levels of cross reaction were selected, and their high dilution (1:160) and complement were added to serial 10-fold dilutions of virus in the microplate titration of virus infectivity. Selective reduction of virus titer by either antiserum could determine the type of HSV. No equivocal intermediate case was found among a number of stock strains including many fresh isolates. The typing result coincided with that determined by a modification of Yang et al's method based on virus titers obtained with Vero and primary chick embryo cells. The typing based on plaquing in chick embryo cells sometimes failed to identify type 1 HSV.     

The use of protein A-sandwich ELISA as a means for quantifying serological relationships between members of the tobamovirus group

Indirect protein A sandwich ELISA (PAS-ELISA) was used to determine the serological relationship between eight tobamoviruses with antisera to 26 viruses and virus strains within the group. Very distant relationships were determined by trapping virus with heterologous antiserum and detecting it with homologous antiserum, while near and close relationships were differentiated by using heterologous antiserum each time. The results were esssentially consistent with previously recorded relationships determined by tube precipitin and other serological tests. Since PAS-ELISA requires much less antiserum than many conventional tests and does not require the purification of IgG or virus, it may offer many advantages in the detection of serological relationships.     

Bioassay of mosquito iridescent virus of Aedes taeniorhynchus in cell cultures of Aedes aegypti.

A bioassay of mosquito iridescent virus (MIV) of Aedes taeniorhynchus was developed using cell cultures of Aedes aegypti. The dilution end point technique was based on the occurrence of cytopathic effects which were optimum at 31°C. Peleg's A. aegypti cell line was more sensitive and reliable than Singh's A. aegypti cell line for infectivity titration of the “R” and “T” strains of MIV. The highest tissue culture infectivity dose 50s (TCID₅₀) were elicited by virion:cell ratios of approximately 10. TCID₅₀ titers were significantly reduced by virus neutralization with either homologous or heterologous antiserum to either RMIV or TMIV. The virus propagated in either cell line was not infectious to A. taeniorhynchus larvae, or to the respective cells from which the virus was produced. All plaque assay attempts were unsuccessful.     

Herpes Simplex Virus Type 1 ICP27 Protein： Its Expression, Purification and Specific Antiserum Production

Herpes simplex virus type 1 (HSV-1) is the causative agent of cold sores and other more serious diseases. HSV-1 infected-cell protein 27 (ICP27) is an immediate-early regulatory phosphoprotein homologous to gene products identified in all classes of herpesviruses so far. To raise the antiserum to ICP27 for further characterization of its biological function, the ICP27 gene was cloned into the pET-28a (+) vector, then ICP27 protein was expressed in E. Coli and purified by nickel-nitrilotriacetic acid (Ni2+-NTA) affinity resin column,finally the purified protein was used to raise antiserum. Western blot analysis demonstrated that the antiserum recognized the recombinant protein, and the antiserum was able to probe the ICP27 in HSV-1 infected cells with high specificity by immunofluorescence assay (IFA). Therefore, the specific antiserum will provide a valuable tool for further studies investigating ICP27's biological function during HSV-1 infection.     

Immunodiffusion test in avian encephalomyelitis. II. Detection of precipitating antibody in infected chickens in comparison with neutralizing antibody.

The sensitivities of double-immunodiffusion (DID) and neutralization tests to detect avian encephalomyelitis (AE) antibody in chickens were studied. Two antigens were employed in the tests. Concentrated antigen gave a higher titer of antiserum than crude antigen, which reacted only to serum having a neutralization log-index (NI) of 3.4approximately4.0 or more. Antibody responses were examined in four growing chick groups inoculated with AE virus by the intracerebral, subcutaneous and oral routes by the DID test with concentrated antigen and by the neutralization test for 1 or over 2 years after inoculation. When concentrated antigen was used, most sera having an NI of over 1.0 were positive for precipitating antibody. Therefore, the sensitivity of the DID test was nearly equal to that of the neutralization test. The DID test was considered to be applicable to the diagnosis of AE and an antibody survey in the field.