Comparison of antibody titers determined by hemagglutination inhibition and enzyme immunoassay for JC virus and BK virus

A comparison of antibody titers to JC virus (JCV) or BK virus (BKV) was made by hemagglutination inhibition (HI) and enzyme immunoassay (EIA) with 114 human plasma samples. Antibody titers to JCV or BKV determined by HI were lower than those determined by EIA. Nevertheless, as HI titers increased so did EIA titers. When antibody data were compared by the Spearman rank correlation test, highly significant correlations were found between HI and EIA titers. Results obtained by plotting EIA antibody titers for JCV against those for BKV generally showed a reciprocal relationship, i.e., samples with high antibody titers to JCV had lower antibody titers to BKV and vice versa. Some samples, however, had antibody titers to both viruses. Of the samples tested, 25.4% (25 of 114) had HI and EIA antibody titers to JCV and BKV which were identical or closely related. This is not the scenario one would expect for cross-reactive epitopes shared by the two viruses, but one suggesting that these samples were from individuals who had experienced infections by both viruses. Adsorption with concentrated JCV or BKV antigen of sera with high antibody titers to both JCV and BKV and testing by JCV and BKV EIA gave results which support this conclusion. Although 52.6% (51 of 97) of the samples from the Japanese population tested had very high antibody titers (>/=40,960) to either JCV or BKV, none of the samples were found by a dot blot immunoassay to have antibodies which cross-reacted with simian virus 40. The results from this study, in agreement with those of others, suggest that humans infected by JCV or BKV produce antibodies to species-specific epitopes on their VP1 capsid protein, which is associated with hemagglutination and cellular binding.     

Herpes simplex virus UL17 protein is associated with B capsids and colocalizes with ICP35 and VP5 in infected cells

Summary.  A previous study using a mutant lacking the UL17 gene has suggested that the UL17 protein of herpes simplex virus type 1 (HSV-1) is required for the cleavage/packaging of viral DNA. In this study, we have raised a rabbit polyclonal antiserum which specifically reacted with the UL17 protein which has an apparent molecular mass of 78-kDa in the lysates of HSV types 1- and 2-infected Vero cells. Western blot analysis of intracellular capsids demonstrates that the UL17 protein was associated with B and C capsids. Indirect immunofluorescence studies reveal that it colocalized with the major capsid protein VP5 and the scaffoling protein ICP35 within the nucleus. These results suggest that the association of the UL17 protein with immature B-type capsids is important for its role in cleavage/packaging. Accepted June 11, 1999/Received April 26, 1999     

Antigenicity of VP60 structural proteinof rabbit haemorrhagic disease virus

Summary.  Five overlapping segments of the VP60 capsid protein gene of rabbit haemorrhagic disease virus have been expressed in E. coli under the control of the T7 RNA polymerase. After purification, the antigenicity of these denatured protein segments has been studied by reactivity with sera from both naturally infected and vaccinated animals in Western blot analysis. The amino terminus segments of the protein (comprising the first 175 amino acids) are highly reactive with the tested sera, between 10 and 100 fold more than any of the segments reproducing the carboxy half of VP60, which is believed to be solvent-exposed in the virus particles. These results strongly suggest that the antigenic structure of the carboxy moiety of VP60 is mainly based on conformation-dependent B-cell epitopes whereas the amino terminal region of VP60 contains continuous antigenic determinants for the immune response elicited during both virus infection and exposure to the inactivated vaccine. Received April 21, 1997 Accepted May 16, 1997     

Expression and antigenic characterization of the major capsid proteins of human polyomaviruses BK and JC in Saccharomyces cerevisiae

BK and JC viruses are ubiquitous human polyomaviruses that are associated with post-transplant interstitial nephritis (BK virus) and progressive multifocal leucoencephalopathy (JC virus). The use of a yeast system to express the major capsid protein (VP1) of two antigenic variants of BKV (strains SB and AS) and JCV is described. VP1s of AS and JCV expressed in Saccharomyces cerevisiae produced proteins of expected molecular weight as determined by gel electrophoresis whereas that of SB appeared to be lower than anticipated. However, all VP1s self-assembled into virus-like particles (VLP) retaining sialic acid-binding and antigenic properties of native virions. This method is highly efficient for producing recombinant proteins and therefore provides an alternative to the baculovirus system.     

Induction of neutralizing antibodies and immunity in vaccinated guinea pigs by cyanogen bromide-peptides of VP3 of foot-and-mouth disease virus.

The specificity of guinea pig antisera against large cyanogen bromide-cleaved peptides of the virus capsid protein VP3 of foot-and-mouth disease virus type O1, strain Kaufbeuren has been characterized by double immunodiffusion, virus neutralization and protection tests. Antibodies to purified 146S particles and the cleavage peptides of VP3 showed an incomplete cross-section against VP3 peptide antigen when reacted in immunodiffusion tests, indicating that new antigenic determinants are exhibited by the peptides which are not recognized by the antiserum against the native virus proteins. The immune response against the reduced, unfolded chain constituents of VP3 was lower in comparison to that of native virus particles but still some immunological determinants remained actively capable of inducing virus-neutralizing antibodies in immunized guinea pigs.     

Parent-to-child transmission is relatively common in the spread of the human polyomavirus JC virus.

JC polyomavirus (JCV), the causative agent of progressive multifocal leukoencephalopathy, is ubiquitous in the human population, infecting children asymptomatically and then persisting in renal tissue. In most adults, renal JCV replicates and the progeny are excreted in urine. We used this urinary JCV to elucidate the routes of JCV transmission. A 610-bp JCV DNA region (IG region) encompassing the 3'-terminal sequences of both T-antigen and VP1 (major capsid protein) genes was amplified by means of PCR from urine specimens collected from all members of seven families. JCV strains were then unequivocally identified by the nucleotide sequences of the amplified IG regions. We could identify 18 distinctive JCV strains from 27 individuals. Different JCV strains were detected from all unrelated persons. However, the same viral strain was detected from one (four families), two (one family), or three offspring (one family) as well as from the fathers (three families) or from the mothers (three families). In total, the JCV strains detected in half of the JCV-positive children were identified in their parents. Since most humans are infected during childhood, these findings indicated that JCV is transmitted frequently from parents to children. We roughly estimated that 50% of JCV transmission occurs by this route and that the other 50% occurs outside the family.     

Undenatured parvovirus B19 antigens are essential for the accurate detection of parvovirus B19 IgG

Recombinant versions of parvovirus B19 capsid proteins VP1 and VP2 are used for immunodiagnostic assays for detection of antiviral antibodies. The immune response to B19 is characterized by a gradual loss of antibodies directed against linear epitopes of VP2. A similar occurrence for antibodies raised against VP1 protein would represent a limitation to serological assays incorporating denatured versions of either viral antigen. Four detection systems for B19 Ig detection have been developed, including an IgG enzyme immunoassay (EIA) based on undenatured VP2, an immunofluorescence assay (IFA) based on undenatured VP1, a Western blot assay incorporating denatured VP1 and VP2, and an alternative blot system using denatured VP1 but undenatured VP2. Specimens (n = 108) were tested by all four systems and identical results were obtained by EIA, IFA, and alternative blot systems, whereby 75/108 (69%) were B19 IgG-positive. Twelve B19 IgG-positive specimens, representing 16% (12/75) of the confirmed positives, did not react to either viral antigens when tested by Western blot. It is concluded that these sera do not react with linear epitopes of VP1 and VP2 antigens. Eighty-five different specimens, which had previously been shown to be both B19 IgM- and IgG-positive by EIA and IFA, were positive by B19 IgM and IgG Western blot. In the IgG Western blot assay, 69 reacted with both VP1 and VP2 and 16 with VP1 only. It is concluded that there is a requirement for at least one undenatured antigen for the immunological detection of B19 IgG. J. Med. Virol. 57:179–185, 1999. © 1999 Wiley-Liss, Inc.     

Propagation of JC virus in human neuroblastoma cell line IMR-32

JC virus (JCV), the causative agent of a human demyelinating disease, progressive multifocal leukoencephalopathy, has a very narrow host range. Cells permissive for infection by JCV have been essentially limited to primary human fetal glial cells, which are difficult to obtain and maintain. In pilot studies, it was found that JCV can multiply in an established cell line of human neuroblastoma. JCV strains Mad-1 and Tokyo-1 were inoculated, respectively, into two cell lines, IMR-32 (neuroblastoma) and A-172 (glioblastoma). Viral infection with cytopathic effect was observed only in IMR-32 cells, and the most efficient viral proliferation was obtained in cells cultured in medium containing 2% fetal calf serum (FCS). Both Mad-1 and Tokyo-1 strains propagated well, with the former being more efficient than the latter. Viral replication was confirmed by immunofluorescence, electron microscopy, and a hemagglutination assay. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophore-sis and Western blot analysis of the purified virus revealed the characteristic JCV protein profile. Thus, IMR-32 cells have been found to be permissive for JCV, which should provide a useful system for further studies of virus proliferation and viral tissue tropism. © 1994 Wiley-Liss, Inc.     

Development of a monoclonal antibody specific for serotype 3 rotavirus strains

Monoclonal antibodies were prepared against serotype 3 simian rotavirus SA11. Antigenic analysis of 18 hybridoma cell lines secreting monoclonal antibodies by radioimmunoprecipitation and Western blot revealed that seven monoclonals were directed against the major inner capsid protein VP6, four against VP3, an outer capsid protein with hemagglutinating activity, and one against VP7, the main outer capsid protein of the virus. The specificity of six monoclonals could not be determined. One monoclonal (1P14E2) directed against VP3 showed serotype 3-specific neutralizing activity. This monoclonal, which recognized only serotype 3 viruses in an enzyme-linked immunosorbent assay, could be useful in assays for serotyping rotavirus directly in stool samples.     

Recombinant Newcastle Disease virus capsids displaying enterovirus 71 VP1 fragment induce a strong immune response in rabbits

The complete VP1 protein of EV71 was truncated into six segments and fused to the C-terminal ends of full-length nucleocapsid protein (NPfl) and truncated NP (NPt; lacks 20% amino acid residues from its C-terminal end) of newcastle disease virus (NDV). Western blot analysis using anti-VP1 rabbit serum showed that the N-terminal region of the VP1 protein contains a major antigenic region. The recombinant proteins carrying the truncated VP1 protein, VP1(1-100), were expressed most efficiently in Escherichia coli as determined by Western blot analysis. Electron microscopic analysis of the purified recombinant protein, NPt-VP(1-100) revealed that it predominantly self-assembled into intact ring-like structures whereas NPfl-VP(1-100) recombinant proteins showed disrupted ring-like formations. Rabbits immunized with the purified NPt-VP(1-100) and NPfl-VP(1-100) exhibited a strong immune response against the complete VP1 protein. The antisera of these recombinant proteins also reacted positively with authentic enterovirus 71 and the closely related Coxsackievirus A16 when analyzed by an immunofluorescence assay suggesting their potential as immunological reagents for the detection of anti-enterovirus 71 antibodies in serum samples.     

Antibody response to gp E of tick-borne encephalitis virus: comparison between natural infection and vaccination breakdown

Human sera obtained after tick-borne encephalitis (TBE) without prior vaccination were compared with sera from patients after a vaccination breakdown. Most sera previously shown to have high titers of IgG and IgM against TBE virus as detected in the ELISA and hemagglutination inhibition (HI) tests also reacted in Western blot with TBE virus E protein which is involved in virus neutralization. The serum of a patient with a vaccination breakdown, however, reacted only very weakly with the E protein in the Western blot in spite of a high amount of antibodies detectable in ELISA. Using SDS-denaturated virus as an antigen in ELISA (imitating the blotting condition), this serum revealed a significant reduction in its reactivity with denatured virus compared to the control sera. This indicates that the patient had an insufficient immune response against certain denaturation resistant epitopes which might contribute to development of disease despite vaccination. The analysis of the immune response of human sera at the epitope level revealed a characteristic "fingerprint" for each serum reflecting the genetic control of the production of antibody populations against different antigenic determinants.     

The open reading frame L2 of cottontail rabbit papillomavirus contains antibody-inducing neutralizing epitopes.

Polyclonal antisera were generated against bacterially derived fusion proteins of the open reading frames (ORFs) of the capsid proteins of cottontail rabbit papillomavirus (CRPV). The carboxy-terminal two-thirds of CRPV L1 and the carboxy-terminal half of CRPV L2 were cloned into a bacterial expression vector and induced proteins were used as antigen and immunogen. The polyclonal antisera were tested in a series of immunological assays, including ELISA, Western blot, and neutralization of CRPV. ELISA demonstrated that the polyclonal antisera raised against expressed L1 proteins reacted strongly to disrupted CRPV virion antigen and weakly both to intact CRPV virion and disrupted BPV-1 virion. Anti-CRPV L2 antisera reacted strongly only to intact and disrupted CRPV virion antigen. Viral capsid proteins of CRPV were detected in Western blots of HPV-11, BPV-1, and CRPV virus particles by these polyclonal antisera. The anti-L1 sera recognized the major capsid protein (60 kDa) and the anti-L2 sera identified a 76-kDa viral protein of CRPV. Only the antisera generated against expressed L2 neutralized CRPV. The neutralizing titer of the anti-L2 sera, however, was several orders of magnitude lower than the titer of a neutralizing polyclonal antiserum that was generated by immunizations with intact CRPV virions.     

Identification of a primate cytomegalovirus group-common protein antigen

A monospecific rabbit antiserum has been prepared to the denatured 145K major capsid protein of strain Colburn cytomegalovirus (CMV). Using this antiserum to analyze proteins electrophoretically transferred to nitrocellulose, it was found that infected cell preparations from all primate CMVs examined contain a single cross-reactive protein band that ranged in molecular weight from 144K to 153K, depending upon the isolate. No reactivity was observed when similar preparations of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), and herpes zoster virus (HZV) were tested, suggesting that this antiserum may be CMV group-specific. A second antiserum prepared to the denatured Colburn 66K matrix protein was found to be more selective in its reactivity toward other simian CMVs, and without reactivity to human CMV preparations.     

Identification of a DNA encapsidation sequence for human polyomavirus pseudovirion formation.

Human polyomavirus is a naked capsid virus containing a closed circular double-stranded DNA genome. The mechanism of DNA encapsidation for the viral progeny formation is not fully understood. In this study, DNA encapsidation domain of the major capsid protein, VP1, of the human polyomavirus JCV was investigated. When the first 12 amino acids were deleted, the E. coli expressed VP1 (Delta N12VP1) failed to encapsidate the host DNA although the integrity of the capsid-like structure was maintained. In addition, capsid-like particles of Delta N12VP1 did not package exogenous DNA in vitro, which is in contrast to that of the full-length VP1 protein. These findings suggest that the N-terminal of the first 12 amino acids of VP1 were responsible for DNA encapsidation. The importance of amino acids in the DNA encapsidation domain was determined further using site-directed mutagenesis. All of the positively charged amino acids at the N-terminal region of VP1 were essential for DNA encapsidation. The results indicate that the N-terminal region of the human polyomavirus major capsid protein VP1 may be involved in viral genome encapsidation during progeny maturation.     

Prevalence and genotype identification of human JC virus in colon cancer in Taiwan

Although JC virus (JCV), a human polyomavirus, has been detected in colon cancers, the association between JCV and colon cancer remains controversial. In Taiwan, the prevalence of JCV infection in colon cancer patients has not been reported. Thus, the purpose of this study was to investigate JCV infection in colon cancers in Taiwan. Formalin-fixed, paraffin-embedded tissues from 22 colon cancer patients were examined in this study. Nested PCR was performed to detect viral genomic DNA. The product of the nested PCR flanking the JCV regulatory region was sequenced further. Viral large tumor protein, LT, and late capsid protein, VP1, were examined by immunohistochemistry (IHC). Nested PCR revealed JCV genomic DNA in 86.4% (19/22) of the colon cancer tissue samples. Both rearranged and archetypal genotypes of JCV were identified. Expression of JCV LT was positive in 63.6% (14/22) of the examined colon cancer tissue samples but not in any adjacent normal region. Expression of viral capsid protein VP1 was not detected in any of the tissues examined. The current study demonstrates that JCV genomic DNA was present in the examined colon cancer tissues. The genotypes of JCV in colon cancer tissues were also identified. Expression of viral early protein but not structural capsid protein was detected in the examined colon cancer tissues. Furthermore, a high prevalence of JCV infection in colon cancer tissues in Taiwan was also demonstrated.     

Characterization of polyclonal antibodies against the capsid proteins of Ljungan virus

Ljungan virus (LV) is a suspected human pathogen isolated from voles in Sweden and North America. To enable virus detection and studies of localization and activity of virion proteins, polyclonal antibodies were produced against bacterially expressed capsid proteins of the LV strain, 87-012G. Specific detection of proteins corresponding to viral antigens in lysates of LV infected cells was demonstrated by immunoblotting using each one of the generated polyclonal antibodies. In addition, native viral antigens present in cell culture infected with LV strains 87-012G or 145SLG were detected in ELISA and by immunofluorescence using the antibodies against the VP0 and VP1 proteins. The anti-VP3 antibody did not react with native proteins of the LV virion, suggesting that the VP3 is less potent in evoking humoral response and may have a less exposed orientation in the virus capsid. No activity of the antibodies was observed against the closely related human parechovirus type 1. The polyclonal antibody against the VP1 protein was further used for detection of LV infected myocytes in a mouse model of LV-induced myocarditis. Thus, polyclonal antibodies against recombinant viral capsid proteins enabled detection of natural LV virions by several different immunological methods.     

Immune responses in humans and animals to meningococcal transferrin-binding proteins: implications for vaccine design.

The results reported here show that the two meningococcal transferrin-binding proteins (TBP1 and TBP2) generate different immune responses in different host species and that there is variation in response dependent on the method of antigen preparation and possibly the route of administration. Mice immunized with either whole cells of Neisseria meningitidis SD (B:15:P1.16) or the isolated TBP1-TBP2 complex from the same strain produced antisera which, when tested against a representative panel of meningococcal isolates by Western blotting (immunoblotting), recognized some but not all heterologous TBP2 molecules. In contrast, rabbit antisera raised to the same preparations were cross-reactive with almost all the TBP2 molecules. The immune response to TBP1 was also host species dependent. Western blot analysis with denatured TBP1 failed to detect antibodies in antisera raised in mice to whole cells or in a rabbit to the TBP1-TBP2 complex but detected broadly cross-reactive antibodies in mouse anti-TBP1-TBP2 complex sera and strain-specific antibodies in rabbit anti-whole-cell serum. Human convalescent-phase sera obtained from five patients infected with meningococci of different serogroups and serotypes contained fully cross-reactive antibodies to TBP2 but no anti-TBP1 antibodies, when examined on Western blots. However, on dot immunoblots, the same patients' sera, as well as the mouse anti-whole cell and the rabbit anti-TBP1-TBP2 complex sera, reacted with purified biologically active TBP1 of strain SD. This indicates that native TBP1, a protein which loses its biological and some of its immunological activities when denatured, is immunogenic and that humans generate cross-reactive antibodies to native epitopes. These observations have important implications for assessing the vaccine potential of TBPs and other meningococcal antigens. Conclusions regarding the usefulness of TBPs as candidate components of meningococcal serogroup B vaccines based on results from certain animal species such as mice, or on methods such as Western blotting, may have little bearing on the situation in humans and may lead to some potentially useful antigens being disregarded.     

Self-assembly,antigenicity, and immunogenicity of the rabbit haemorrhagic disease virus (Czechoslovakian strain V-351) capsid protein expressed in baculovirus

Summary Rabbit haemorrhagic disease virus (RHDV) capsid protein was expressed in a baculovirus system. Analysis of the expressed product showed that the recombinant protein, which is 60 kDa in size, was antigenic as revealed by its reactions in ELISA and Western blot with the antibodies raised against RHDV. Direct electron microscopy of the cell culture supernatant and the purified protein demonstrated that the capsid protein expressed in insect cells self-assembled to form empty virus-like particles (VLP) which are similar in size and morphology to that of native virus. These particles were immunoreactive with polyclonal anti-RHDV antibodies and with four monoclonal antibodies which recognise conformational epitopes of the virus. The results indicated that the VLPs were morphologically and antigenically indistinguishable from native virus. The recombinant VLPs induced high levels of RHDV-specific antibodies in rabbits and mice following immunisation. The immune response to the VLPs protected the rabbits following challenge with the virulent RHDV. In haemagglutination assays, the VLPs bound to human red blood cells similar to the native virus particles. The recombinant protein and or VLPs is suitable for the development of a rapid, sensitive and reliable test for detection of antibodies to RHDV and for use as a vaccine for domestic rabbits.     

Antiserum to the recombinant truncated VP22 protein of herpes simplex virus type 1 that also recognizes full-length VP22

The herpes simplex virus type 1 (HSV-1) tegument protein VP22 encoded by the UL49 gene is essential for HSV-1 infection. However, its precise functions in the virus life cycle are unknown. A relatively important tool for disclosing these functions is an antiserum specifically detecting VP22 in the infected cell. To this end, a recombinant truncated VP22 protein consisting of C-terminal 45 aa fused to EYFP (enhanced yellow fluorescent protein) and His-tag was expressed in Escherichia coli, purified by the Ni2+-NTA affinity chromatography, and used for the preparation of antiserum in rabbits. Western blot and immunofluorescence assay showed that this antiserum specifically detected purified truncated VP22 as well as full-length VP22 in the HSV-1 infected cells. These results indicate that the prepared antiserum could serve as a valuable tool for further studies of VP22 functions.     

The high incidence of JC virus infection in urothelial carcinoma tissue in Taiwan

Human polyomaviruses, JC virus (JCV) and BK virus (BKV), usually remain latent in kidney and urothelial tissue after primary infection. Infection with human polyomavirus has still not been correlated conclusively with malignancy of kidney and urothelial tissue. The present study investigated further the possible relationship between JCV/BKV infection and urothelial carcinoma. Tissue samples were examined from 33 urothelial carcinomas and 5 renal cell carcinomas for JCV/BKV infection, using nested PCR with primers common to both JCV and BKV. The viral genotypes were further verified by endonuclease digestion and DNA sequencing following the PCR. In addition, immunohistochemistry and Western blotting were also performed to detect viral large tumor protein (LT) and the late capsid protein (VP1) in the tissue samples. The results from nested PCR showed that 90.1% (30/33) of the urothelial carcinomas samples and all of the renal cell carcinomas samples (5/5) were JCV DNA positive. Both archetypal and re-arranged JCV genotypes were detected. On the other hand, BKV DNA was detected in only one (3%) of the urothelial carcinoma tissue samples. The immunohistochemical results showed that 30% (10/33) of urothelial carcinoma tissues was stained positive for large tumor antigen (LT). However, the structural protein VP1 was not detectable in any of the tissue samples examined. The present study demonstrated that JCV is highly prevalent in urothelial carcinoma tissue as is the expression of large tumor antigen. Therefore, the findings support the hypothesis that JCV infection is associated with urothelial carcinoma.