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PubMed1992

Specific interactions between rotavirus outer capsid proteins VP4 and VP7 determine expression of a cross-reactive, neutralizing VP4-specific epitope.

We previously reported that the expression of rotavirus phenotypes by reassortants was affected by recipient genetic background and proposed specific interactions between the outer capsid proteins VP4 and VP7 as the basis for the phenotypic effects (D. Chen, J. W. Burns, M. K. Estes, and R. F. Ramig, Proc. Natl. Acad. Sci. USA 86:3743-3747, 1989). A neutralizing, cross-reactive VP4-specific monoclonal antibody (MAb), 2G4, was used to probe the protein-protein interactions. The VP4 specificity of 2G4 was confirmed by immunoblot analysis. MAb 2G4 reacted with both standard (SA11-C13) and variant rotavirus SA11 (SA11-4F) but did not react with bovine rotavirus B223 as determined by plaque reduction neutralization (PRN) and enzyme-linked immunosorbent assay (ELISA). When a panel of SA11-4F/B223 and SA11-Cl3/B223 reassortants in purified or crude lysate form that had been grown in the presence or absence of trypsin was analyzed with MAb 2G4 by PRN and ELISA, the results with some reassortants were unexpected. That is, MAb 2G4 reacted with VP4 of SA11 parental origin (4F or C13) when it was assembled into capsids with the homologous SA11 VP7 but failed to react with VP4 of SA11 assembled into capsids with heterologous B223 VP7. Conversely, MAb 2G4 failed to react with VP4 of B223 parental origin when it was assembled into capsids with homologous B223 VP7 but did react with B223 VP4 assembled into capsids with the heterologous SA11 VP7. Similar reactivity was observed when 2G4 was used to immunoprecipitate purified double-shelled virions. When soluble unassembled viral proteins were analyzed by ELISA, the 2G4 reactive pattern was as predicted from the parental origin of VP4. That is, 2G4 reacted with the soluble VP4 of reassortants having VP4 from SA11-Cl3 or SA11-4F and failed to react with VP4 of B223 origin, regardless of the origin of VP7. PRN and ELISA results obtained with nonglycosylated viruses revealed that the unexpected reactivity of 2G4 with virus particles was not the result of differential glycosylation of VP7 and epitope masking. These results indicate that the 2G4 epitope existed in the soluble form of VP4 encoded by SA11-Cl3 or SA11-4F but not in soluble B223 VP4. On the other hand, in assembled virions, the presentation of the 2G4 epitope on VP4 was unexpected in some reassortants and was affected by the specific interactions between VP4 and VP7 of heterologous parental origin.

Animals↗
PubMed1996

Assemblons: nuclear structures defined by aggregation of immature capsids and some tegument proteins of herpes simplex virus 1.

In cells infected with herpes simplex virus 1 (HSV-1), the viral proteins ICP5 (infected-cell protein 5) and VP19c (the product of UL38) are associated with mature capsids, whereas the same proteins, along with ICP35, are components of immature capsids. Here we report that ICP35, ICP5, and UL38 (VP19c) coalesce at late times postinfection and form antigenically dense structures located at the periphery of nuclei, close to but not abutting nuclear membranes. These structures were formed in cells infected with a virus carrying a temperature-sensitive mutation in the UL15 gene at nonpermissive temperatures. Since at these temperatures viral DNA is made but not packaged, these structures must contain the proteins for immature-capsid assembly and were therefore designated assemblons. These assemblons are located at the periphery of a diffuse structure composed of proteins involved in DNA synthesis. This structure overlaps only minimally with the assemblons. In contrast, tegument proteins were located in asymmetrically distributed structures also partially overlapping with assemblons but frequently located nearer to nuclear membranes. Of particular interest is the finding that the UL15 protein colocalized with the proteins associated with viral DNA synthesis rather than with assemblons, suggesting that the association with DNA may take place during its synthesis and precedes the involvement of this protein in packaging of the viral DNA into capsids. The formation of three different compartments consisting of proteins involved in viral DNA synthesis, the capsid proteins, and tegument proteins suggests that there exists a viral machinery which enables aggregation and coalescence of specific viral protein groups on the basis of their function.

Animals↗
PubMed1997

Hexon-only binding of VP26 reflects differences between the hexon and penton conformations of VP5, the major capsid protein of herpes simplex virus.

VP26 is a 12-kDa capsid protein of herpes simplex virus 1. Although VP26 is dispensable for assembly, the native capsid (a T=16 icosahedron) contains 900 copies: six on each of the 150 hexons of VP5 (149 kDa) but none on the 12 VP5 pentons at its vertices. We have investigated this interaction by expressing VP26 in Escherichia coli and studying the properties of the purified protein in solution and its binding to capsids. Circular dichroism spectroscopy reveals that the conformation of purified VP26 consists mainly of beta-sheets (approximately 80%), with a small alpha-helical component (approximately 15%). Its state of association was determined by analytical ultracentrifugation to be a reversible monomer-dimer equilibrium, with a dissociation constant of approximately 2 x 10(-5) M. Bacterially expressed VP26 binds to capsids in the normal amount, as determined by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cryoelectron microscopy shows that the protein occupies its usual sites on hexons but does not bind to pentons, even when available in 100-fold molar excess. Quasi-equivalence requires that penton VP5 must differ in conformation from hexon VP5: our data show that in mature capsids, this difference is sufficiently pronounced to abrogate its ability to bind VP26.

Capsid↗
PubMed2002

Cellular transcription factor Sp1 recruits simian virus 40 capsid proteins to the viral packaging signal, ses.

Simian virus 40 (SV40) capsid assembly occurs in the nucleus. All three capsid proteins bind DNA nonspecifically, raising the dilemma of how they attain specificity to the SV40 minichromosome in the presence of a large excess of genomic DNA. The SV40 packaging signal, ses, which is required for assembly, is composed of multiple DNA elements that bind transcription factor Sp1. Our previous studies showed that Sp1 participates in SV40 assembly and that it cooperates in DNA binding with VP2/3. We hypothesized that Sp1 recruits the capsid proteins to the viral minichromosome, conferring upon them specific DNA recognition. Here, we have tested the hypothesis. Computer analysis showed that the combination of six tandem GC boxes at ses is not found at cellular promoters and therefore is unique to SV40. Cooperativity in DNA binding between Sp1 and VP2/3 was not abolished at even a 1,000-fold excess of cellular DNA, providing strong support for the recruitment hypothesis. Sp1 also binds VP1 and cooperates with VP1 in DNA binding. VP1 pentamers (VP1(5)) avidly interact with VP2/3, utilizing the same VP2/3 domain as described for polyomavirus. We conclude that VP1(5)-VP2/3 building blocks are recruited by Sp1 to ses, where they form the nucleation center for capsid assembly. By this mechanism the virus ensures that capsid formation is initiated at a single site around its minichromosome. Sp1 enhances the formation of SV40 pseudovirions in vitro, providing additional support for the model. Analyses of Sp1 and VP3 deletion mutants showed that Sp1 and VP2/3 bind one another and cooperate in DNA binding through their DNA-binding domains, with additional contacts outside these domains. VP1 contacts Sp1 at residues outside the Sp1 DNA-binding domain. These and additional data allowed us to propose a molecular model for the VP1(5)-VP2/3-DNA-Sp1 complex.

Binding Sites↗
PubMed2002

Inhibition of herpes simplex virus replication by WAY-150138: assembly of capsids depleted of the portal and terminase proteins involved in DNA encapsidation.

Studies were carried out to examine the mechanism of action of WAY-150138, a member of a novel group of thiourea compounds recently shown to inhibit replication of herpes simplex virus type 1 (HSV-1). Previous studies have shown that the drug acts by preventing DNA encapsidation and that resistant mutants map to U(L)6, the gene encoding the protein subunit of the portal complex through which DNA enters the capsid. We tested the idea that WAY-150138 acts by preventing the incorporation of DNA-packaging proteins into capsids as they are assembled. Capsids were isolated from HSV-1-infected, drug-treated cells and examined by Western immunoblotting for the presence of two packaging proteins, the portal subunit (U(L)6) and a candidate terminase subunit (U(L)15). The results showed that both proteins were depleted in the capsids, suggesting that WAY-150138 antagonizes DNA encapsidation by depriving capsids of packaging proteins during the assembly process.

Animals↗
PubMed2003

Stability and morphology comparisons of self-assembled virus-like particles from wild-type and mutant human hepatitis B virus capsid proteins.

Instead of displaying the wild-type selective export of virions containing mature genomes, human hepatitis B virus (HBV) mutant I97L, changing from an isoleucine to a leucine at amino acid 97 of HBV core antigen (HBcAg), lost the high stringency of selectivity in genome maturity during virion export. To understand the structural basis of this so-called "immature secretion" phenomenon, we compared the stability and morphology of self-assembled capsid particles from the wild-type and mutant I97L HBV, in either full-length (HBcAg1-183) or truncated core protein contexts (HBcAg1-149 and HBcAg1-140). Using negative staining and electron microscopy, full-length particles appear as "thick-walled" spherical particles with little interior space, whereas truncated particles appear as "thin-walled" spherical particles with a much larger inner space. We found no significant differences in capsid stability between wild-type and mutant I97L particles under denaturing pH and temperature in either full-length or truncated core protein contexts. In general, HBV capsid particles (HBcAg1-183, HBcAg1-149, and HBcAg1-140) are very robust but will dissociate at pH 2 or 14, at temperatures higher than 75 degrees C, or in 0.1% sodium dodecyl sulfate (SDS). An unexpected upshift banding pattern of the SDS-treated full-length particles during agarose gel electrophoresis is most likely caused by disulfide bonding of the last cysteine of HBcAg. HBV capsids are known to exist in natural infection as dimorphic T=3 or T=4 icosahedral particles. No difference in the ratio between T=3 (78%) and T=4 particles (20.3%) are found between wild-type HBV and mutant I97L in the context of HBcAg1-140. In addition, we found no difference in capsid stability between T=3 and T=4 particles successfully separated by using a novel agarose gel electrophoresis procedure.

Capsid↗
PubMed2003

Disulfide bonding among micro 1 trimers in mammalian reovirus outer capsid: a late and reversible step in virion morphogenesis.

We examined how a particular type of intermolecular disulfide (ds) bond is formed in the capsid of a cytoplasmically replicating nonenveloped animal virus despite the normally reducing environment inside cells. The micro 1 protein, a major component of the mammalian reovirus outer capsid, has been implicated in penetration of the cellular membrane barrier during cell entry. A recent crystal structure determination supports past evidence that the basal oligomer of micro 1 is a trimer and that 200 of these trimers surround the core in the fenestrated T=13 outer capsid of virions. We found in this study that the predominant forms of micro 1 seen in gels after the nonreducing disruption of virions are ds-linked dimers. Cys679, near the carboxyl terminus of micro 1, was shown to form this ds bond with the Cys679 residue from another micro 1 subunit. The crystal structure in combination with a cryomicroscopy-derived electron density map of virions indicates that the two subunits that contribute a Cys679 residue to each ds bond must be from adjacent micro 1 trimers in the outer capsid, explaining the trimer-dimer paradox. Successful in vitro assembly of the outer capsid by a nonbonding mutant of micro 1 (Cys679 substituted by serine) confirmed the role of Cys679 and suggested that the ds bonds are not required for assembly. A correlation between micro 1-associated ds bond formation and cell death in experiments in which virions were purified from cells at different times postinfection indicated that the ds bonds form late in infection, after virions are exposed to more oxidizing conditions than those in healthy cells. The infectivity measurements of the virions with differing levels of ds-bonded micro 1 showed that these bonds are not required for infection in culture. The ds bonds in purified virions were susceptible to reduction and reformation in situ, consistent with their initial formation late in morphogenesis and suggesting that they may undergo reduction during the entry of reovirus particles into new cells.

Animals↗
PubMed2004

The last C-terminal residue of VP3, glutamic acid 257, controls capsid assembly of infectious bursal disease virus.

Infectious bursal disease virus (IBDV) is a nonenveloped virus with an icosahedral capsid composed of two proteins, VP2 and VP3, that derive from the processing of the polyprotein NH(2)-pVP2-VP4-VP3-COOH. The virion contains VP1, the viral polymerase, which is both free and covalently linked to the two double-stranded RNA (dsRNA) genomic segments. In this study, the virus assembly process was studied further with the baculovirus expression system. While expression of the wild-type polyprotein was not found to be self-sufficient to give rise to virus-like particles (VLPs), deletion or replacement of the five C-terminal residues of VP3 was observed to promote capsid assembly. Indeed, the single deletion of the C-terminal glutamic acid was sufficient to induce VLP formation. Moreover, fusion of various peptides or small proteins (a green fluorescent protein or a truncated form of ovalbumin) at the C terminus of VP3 also promoted capsid assembly, suggesting that assembly required screening of the negative charges at the C terminus of VP3. The fused polypeptides mimicked the effect of VP1, which interacts with VP3 to promote VLP assembly. The C-terminal segment of VP3 was found to contain two functional domains. While the very last five residues of VP3 mainly controlled both assembly and capsid architecture, the five preceding residues constituted the VP1 (and possibly the pVP2/VP2) binding domain. Finally, we showed that capsid formation is associated with VP2 maturation, demonstrating that the protease VP4 is involved in the virus assembly process.

Amino Acid Sequence↗
PubMed2005

Virulent variants emerging in mice infected with the apathogenic prototype strain of the parvovirus minute virus of mice exhibit a capsid with low avidity for a primary receptor.

The mechanisms involved in the emergence of virulent mammalian viruses were investigated in the adult immunodeficient SCID mouse infected by the attenuated prototype strain of the parvovirus Minute Virus of Mice (MVMp). Cloned MVMp intravenously inoculated in mice consistently evolved during weeks of subclinical infection to variants showing altered plaque phenotypes. All the isolated large-plaque variants spread systemically from the oronasal cavity and replicated in major organs (brain, kidney, liver), in sharp contrast to the absolute inability of the MVMp and small-plaque variants to productively invade SCID organs by this natural route of infection. The virulent variants retained the MVMp capacity to infect mouse fibroblasts, consistent with the lack of genetic changes across the 220-to-335 amino acid sequence of VP2, a capsid domain containing main determinants of MVM tropism. However, the capsid of the virulent variants shared a lower affinity than the wild type for a primary receptor used in the cytotoxic infection. The capsid gene of a virulent variant engineered in the MVMp background endowed the recombinant virus with a large-plaque phenotype, lower affinity for the receptor, and productive invasiveness by the oronasal route in SCID mice, eventually leading to 100% mortality. In the analysis of virulence in mice, both MVMp and the recombinant virus similarly gained the bloodstream 1 to 2 days postoronasal inoculation and remained infectious when adsorbed to blood cells in vitro. However, the wild-type MVMp was cleared from circulation a few days afterwards, in contrast to the viremia of the recombinant virus, which was sustained for life. Significantly, attachment to an abundant receptor of primary mouse kidney epithelial cells by both viruses could be quantitatively competed by wild-type MVMp capsids, indicating that virulence is not due to an extended receptor usage in target tissues. We conclude that the selection of capsid-receptor interactions of low affinity, which favors systemic infection, is a major evolutionary process in the adaptation of parvoviruses to new hosts and in the cause of disease.

Animals↗
PubMed2005

The coiled-coil domain of the adenovirus type 5 protein IX is dispensable for capsid incorporation and thermostability.

The 14.4-kDa hexon-associated protein IX (pIX) acts as a cement in the capsids of primate adenoviruses and confers a thermostable phenotype. Here we show that deletion of amino acids 100 to 114 of adenovirus type 5 pIX, which eliminates the conserved coiled-coil domain, impairs its capacity to self-associate. However, pIXDelta100-114 is efficiently incorporated into the viral capsid, and the resulting virions are thermostable. Deletion of the central alanine-rich domain, as in pIXDelta60-72, does not impair self-association, incorporation into the capsid, or the thermostable phenotype. These data demonstrate, first, that the self-association of pIX is dispensable for its incorporation into the capsid and generation of the thermostability phenotype and, second, that the increased thermostability results from pIX monomers binding to different hexon capsomers rather than capsid stabilization by pIX multimers.

Amino Acid Sequence↗
PubMed2025

Cell-free assays reveal that the HIV-1 capsid protects reverse transcripts from cGAS immune sensing.

Retroviruses can be detected by the innate immune sensor cyclic GMP-AMP synthase (cGAS), which recognizes reverse-transcribed DNA and activates an antiviral response. However, the extent to which HIV-1 shields its genome from cGAS recognition remains unclear. To study this process in mechanistic detail, we reconstituted reverse transcription, genome release, and innate immune sensing of HIV-1 in a cell-free system. We found that wild-type HIV-1 capsids protect viral genomes from cGAS even after completing reverse transcription. Viral DNA could be "deprotected" by thermal stress, capsid mutations, or reduced concentrations of inositol hexakisphosphate (IP6) that destabilize the capsid. Strikingly, the capsid inhibitor lenacapavir also disrupted viral cores and dramatically potentiated cGAS activity, both in vitro and in cellular infections. Our results provide biochemical evidence that the HIV-1 capsid lattice conceals the genome from cGAS and that chemical or physical disruption of the viral core can expose HIV-1 DNA and activate innate immune signaling.

HIV-1↗
PubMed1999

Induction of HPV16 capsid protein-specific human T cell responses by virus-like particles.

It has been postulated that upon binding to a cell surface receptor, papilloma virus-like particles (VLPs) gain entry into the cytosol of infected cells and the capsid proteins L1 and L2 can be processed in the MHC class I presentation pathway. Vaccination of mice with human papilloma virus-like particles consisting of capsid proteins L1 and L2 induced a CD8-mediated and perforin dependent protective immune response against a tumor challenge with human papilloma virus transformed tumor cells, which express only minute amounts of L1 protein. Here we show that HPV16 capsid proteins stimulate a MHC class I restricted CTL response with human peripheral blood lymphocytes (PBL) in vitro. The vigorous response was specific for VLP-infected target cells and was MHC class I restricted. Moreover we show the presence of at least one HLA-A*0201 restricted CTL epitope within the HPV-16 capsid proteins by using a VLP-'infected' HLA-A*0201 transfected human cell line as target cells. These results demonstrated that VLPs can induce a HPV16 capsid protein-specific immune response in humans, allowing the monitoring of immune responses induced by vaccines based on chimeric VLPs carrying additional immunogenic peptides or proteins in therapeutical applications in human patients.

Capsid↗
PubMed2005

Classical nucleation theory of virus capsids.

A fundamental step in the replication of a viral particle is the self-assembly of its rigid shell (capsid) from its constituent proteins. Capsids play a vital role in genome replication and intercellular movement of viruses, and as such, understanding viral assembly has great potential in the development of new antiviral therapies and a systematic treatment of viral infection. In this article, we assume that nucleation is the underlying mechanism for self-assembly and combine the theoretical methods of the physics of equilibrium polymerization with those of the classical nucleation to develop a theory for the kinetics of virus self-assembly. We find expressions for the size of the critical capsid, the lag time, and the steady-state nucleation rate of capsids, and how they depend on both protein concentration and binding energy. The latter is a function of the acidity of the solution, the ionic strength, and the temperature, explaining why capsid nucleation is a sensitive function of the ambient conditions.

Binding Sites↗
PubMed2003

[The structure and virus-like particle vaccine of the HIV-1 capsid protein].

The HIV-1 capsid protein (CA) plays an essential role in viral core assembly and maturation. Proteolytic cleavage at the MA-CA junction of the retroviral gag polyprotein refolds the amino-terminal end of capsid into a beta-helix structure that is stabilized by a salt bridge between the protein's processed amino-terminus and a conserved acidic residue. The refolded capsid aminoterminus then creates a new CA-CA interface, allowing assembly of the mature capsid core. Recently, researches focus on assembly of CA in vitro and development of CA vaccine. CA vaccine will provide widely immune protection because CA is comparatively conserved. Experiments demonstrate that fusing as few as four matrix residues onto the amino-terminus of capsid redirects protein assembly from cylinder to spheres in vitro. Evaluation of immunogenicity showed that immunization with virus-like particles induced both cellular and neutralizing antibody responses. Furthermore, mucosal administration of virus-like particles effectively induced both mucosal and systemic immune responses. These results indicate that virus-like particles consisting of HIV structural proteins are an attractive vaccine platform for eliciting anti-viral immune responses, especially neutralizing antibody responses. The production of antigens for vaccines in plants indicates that plant-based transgenic expression represents a viable means of producing CA vaccine for the development of HIV vaccine and for use in HIV diagnostic procedures and it has the potential as a safe and cost-effective alternative to traditional production systems.

AIDS Vaccines↗
PubMed2002

Kinetics of thermal denaturation of human rhinoviruses in the presence of anti-viral capsid binders analyzed by capillary electrophoresis.

In vivo, the icosahedral capsid of human rhinoviruses undergoes well-defined transitions during the infection pathway. Native virus, sedimenting at 150S, is converted to subviral particles with a sedimentation coefficient of 135S, which have lost the innermost capsid protein VP4. Upon release of the genomic RNA empty 80S capsids remain. Similar structural modifications are observed in vitro upon exposure to low pH and/or elevated temperature. Virions are stabilized against these transitions by various antiviral compounds, which bind to a hydrophobic pocket in the capsid protein VP1. Using capillary electrophoresis the kinetics of viral decay in the presence of such hydrophobic drugs was investigated. Assuming first-order kinetics, the increase of the time constant reflects the extent of stabilization. Exposure of the virions to 55 degrees C after presaturation with the antivirals increased the time constants (as compared to native virus) by a factor of 8-30, from a few minutes to several ten minutes. Denaturation of the stabilized capsid gave rise to heterogeneous material rather than to defined subviral particles. This was confirmed by electron microscopy and indicates that the structural modification of the virus follows a kinetically well-defined pathway which is disturbed by the drugs resulting in disorganized disruption of the virion.

Antiviral Agents↗
PubMed2006

Initial capsid-specific CD4(+) T cell responses protect against Theiler's murine encephalomyelitisvirus-induced demyelinating disease.

Central nervous system (CNS) infection by Theiler's murine encephalomyelitis virus (TMEV) causes an immune-mediated demyelinating disease similar to human multiple sclerosis in susceptible mice. To understand the pathogenic mechanisms, we analyzed the level, specificity, and function of CD4(+) Th cells in susceptible SJL/J and resistant C57BL/6 mice. Compared to resistant mice, susceptible mice have three- to fourfold higher levels of overall CNS-infiltrating CD4(+) T cells during acute infection. CD4(+) T cells in the CNS of both strains display various activation markers and produce high levels of IFN-gamma upon stimulation with anti-CD3 antibody. However, susceptible mice display significantly fewer (tenfold) IFN-gamma-producing Th1 cells specific for viral capsid epitopes as compared to resistant mice. Furthermore, preimmunization with capsid-epitope peptides significantly increased capsid-specific CD4(+) T cells in the CNS during the early stages of viral infection and delayed the development of demyelinating disease in SJL/J mice. This suggests a protective role of capsid-reactive Th cells during early viral infection. Therefore, a low level of the protective Th1 response to viral capsid proteins, in conjunction with Th1 responses to unknown epitopes may delay viral clearance in susceptible mice leading to pathogenesis of demyelination during acute infection, as compared to resistant mice.

Acute Disease↗
PubMed2003

IgG antibodies to human papillomavirus 16, 52, 58, and 6 L1 capsids: case-control study of cervical intraepithelial neoplasia in Japan.

In Japan, human papillomavirus (HPV) 16, 52, and 58 are most commonly associated with cervical intraepithelial neoplasia (CIN). By contrast, HPV6 is primarily associated with genital warts. This study was designed to evaluate the association between IgG antibody responses to common HPVs and the risk of CIN development within a Japanese population. CIN cases (n = 141) and controls (n = 109) were tested for cervical HPV DNA and serum IgG antibodies to L1 capsids of HPV16, 52, 58, and 6. Seropositivity to HPV16, 52, and 58 L1 capsids was significantly higher in CIN cases than in controls: 27%, 21%, and 31% versus 16%, 11%, and 11%, respectively (P < 0.05). HPV6 L1 seropositivity was not significantly associated with CIN lesions (P = 0.11). Presence of viral DNA for either HPV16, 52, or 58 correlated with a significant antibody response against the homologous L1 capsids but not heterologous L1 capsids. Furthermore, seropositivity to multiple types of HPV16, 52, and 58 was more strongly associated with an increased risk of CIN development than seropositivity to a single type (P for trend <0.001). These findings indicate that IgG antibodies to L1 capsids of HPV16, 52, and 58 represent an increased risk of CIN development, with antibodies to multiple types being indicative of a further increase in risk. The presence of CIN lesions in women with seropositivity to multiple types suggests that viral exposure to a given type may not be protective against infections by other types and subsequent CIN development.

Adult↗
PubMed2002

Baculovirus expression of erythrovirus V9 capsids and screening by ELISA: serologic cross-reactivity with erythrovirus B19.

Diagnosis of erythrovirus B19 (B19) relies on serology and the detection of viral DNA. Recently, a distinct erythrovirus isolate termed V9, markedly different from erythrovirus B19 (> 11% nucleotide disparity), was isolated. Standard B19 PCR assays were inconclusive and serologic tests failed to categorize V9 as an acute B19-like infection. Sequencing, combined with PCR studies, have since demonstrated the need for specific and differentiated techniques when examining samples for possible B19 or V9 viremia. The antigenic properties of the V9 capsid proteins have not been characterized previously. To address this question, V9 VP1 and VP2 open reading frames were cloned and expressed in insect cells using a baculovirus vector. Large quantities of purified recombinant V9 capsid protein were produced and electron micrographs revealed self-assembly of V9 VP1/VP2 and VP2 capsids into empty icosahedral erythrovirus-like particles with a diameter of approximately 23 nm. Screening of a panel of 270 clinical samples for the presence of V9 IgM and IgG antibodies in ELISA showed 100% serologic cross-reactivity between B19 and V9 when comparing V9 VP2 capsids to a commercial B19 VP2 assay. This suggests that both a V9 and a B19 antibody response may be diagnosed equally well by ELISA using either V9 or B19 recombinant capsids as antigen source. Retrospectively, translation of the V9 sequence indicates that despite a significant genetic variation on the DNA level, the majority of the discrepant DNA sequence represents silent mutations leading to an amino acid sequence very similar to the known B19 strains (96-97% homology).

Adolescent↗