Coxsackievirus B3 Strains Research Articles

Mechanisms of Coxsackievirus-Induced Damage to Human Pancreaticβ -Cells1

Enteroviruses may be involved in the pathogenesis of insulin-dependent diabetes mellitus, either through direct beta-cell infection or as triggers of autoimmunity. In the present study we investigated the patterns of infection in adult human islet cell preparations (consisting of 56+/-14% beta-cells) by several coxsackieviruses. The cells were infected with prototype strains of coxsackievirus B (CBV) 3, 4, and 5 as well as coxsackievirus A9 (CAV-9). The previously characterized diabetogenic strain of coxsackievirus B4 (CBV-4-E2) was used as a reference. All viruses replicated well in beta-cells, but only CBVs caused cell death. One week after infection, the insulin response of the beta-cells to glucose or glucose plus theophylline was most severely impaired by CBV-3 and CBV-5 infections. CBV-4 also caused significant functional impairment, whereas CAV-9-infected cells responded like uninfected controls. After 2 days of infection, about 40% of CBV-5-infected cells had undergone morphological changes characteristic of pyknosis, i.e. highly distorted nuclei with condensed but intact chromatin. Both mitochondria and plasma membrane were intact in these cells. DNA fragmentation was found in 5.9+/-1.1% of CBV-5-infected beta-cell nuclei (2.1+/-0.3% in controls; P<0.01). CAV-9 infection did not induce DNA fragmentation. One week after infection the majority of infected cells showed characteristics of secondary necrosis. Medium nitrite and inducible nitric oxide synthase messenger ribonucleic acid levels were not significantly up-regulated by CBV infection. These results suggest that several enteroviruses may infect human beta-cells. The infection may result in functional impairment or death of the beta-cell or may have no apparent immediate adverse effects, as shown here for CAV-9. Coxsackie B viruses cause functional impairment and beta-cell death characterized by nuclear pyknosis. Apoptosis appears to play a minor role during a productive CBV infection in beta-cells.

The role of oxidative stress in viral infections.

Oxidative stress is implicated as a pathogenic factor in a number of viral infections. Our work has shown that nutritionally induced oxidative stress exacerbates the pathogenesis of coxsackievirus B3 (CVB3) infection in mice. Of particular note, mice fed on a diet deficient in antioxidants developed myocarditis when infected with a normally benign strain of CVB3. This change in virulence was found to be due to changes in the viral genome. Immune functions of the oxidatively stressed mice were also altered. Another example of the effect of oxidative stress on a viral pathogen took place in Cuba in the 1990s. An epidemic of optic and peripheral neuropathy in the population occurred that was associated with a lack of dietary antioxidants and with smoking (a pro-oxidant). A coxsackie-like virus was isolated from the cerebrospinal fluid from 84% of patients cultured. Thus, oxidative stress can have profound effects, not only on the host, but on the pathogen as well.

A glyconutritional mixture (Ambrotose®) provides some amelioration to mice with coxsackievirus-induced pancreatitis.

Challenge of adolescent male CD-1 mice with a coxsackievirus B3 (CVB3) strain (CVB3m) induces mild to severe destruction of pancreatic acinar cells, but causes no deaths and does not induce hyperglycemia. A weekly parenteral (intraperitoneal) administration of a glyconutritional mixture (Ambrotose® to virus-challenged mice was assessed to determine if there were any benefits to recovery over an eight month period. Virus-challenged mice showed a significant weight loss over the initial five weeks of the experiment, but injection of Ambrotose® to similar virus-challenged mice restored the total body weight to levels found in normal mice. Normal mice given Ambrotose® exhibited a small weight gain. Mice given Ambrotose® showed reduced severity of pancreatitis, as evidenced by significant reductions in percentages of pancreatic acinar cells destroyed and proportion of sections of pancreata with destroyed acinar cells, compared to virus control-mice not injected with Ambrotose®. Statistical analyses of the extent of acinar cell pathology in all virus-challenged mice showed that Ambrotose® contributed significantly to recovery of the acinar cell population in virus-inoculated mice. Anti-viral antibody titers were not affected by Ambrotose® injections. One potential mechanism to explain the benefits derived from Ambrotose® injections came from studies of antioxidant levels of glutathione in splenic macrophages/monocytes. Whereas CVB3 challenge of mice reduced glutathione levels in the latter cells, Ambrotose® injections to virus-challenged mice restored glutathione levels to those found in normal mice. In summary, most but not all mice derived benefits from Ambrotose® injections, i.e. a reduction in pathology in the pancreas and restored levels of the antioxidant glutathione in macrophages/monocytes. Higher doses of Ambrotose® could provide greater benefits for more mice, a study for the future.

Selenium and host defence towards viruses.

The association between viral disease and nutrition has long been thought to be due to effects on the host immune system. This theory suggests that when a host is malnourished, the immune system is compromised, and thus increased susceptibility to viral infection will occur. However, the virus itself may also be affected by the nutritional status of the host. We have demonstrated that a normally-benign strain of coxsackievirus B3 (CVB3/0) becomes virulent in either Se-deficient or vitamin E-deficient mice. Although the deficient animals are immunosuppressed, the virus itself is also altered. Six nucleotide changes were found in the virus that replicated in the deficient mice, and once these mutations occurred, even mice with normal nutrition became susceptible to disease. Thus, the nutritional status of the host was able to transform an avirulent virus into a virulent one due to genomic changes in the virus. We believe that a common mechanism of oxidative stress is the underlying cause of the genetic changes. Both vitamin E and Se act as antioxidants, and benign virus inoculated into GSH peroxidase (EC 1.11.1.9)-knockout mice will also convert to virulence due to genomic changes. Our work points to the importance of host nutrition during a viral disease, not only from the perspective of the host, but from the perspective of the viral pathogen as well.

Cleavage of RasGAP and phosphorylation of mitogen-activated protein kinase in the course of coxsackievirus B3 replication.

Recently, we reported on tyrosine phosphorylation of distinct cellular proteins in the course of enterovirus infections (M. Huber, H.-C. Selinka, and R. Kandolf, J. Virol. 71:595-600, 1997). These phosphorylation events were mediated by Src-like kinases and were shown to be necessary for effective virus replication. That study is now extended by examination of the interaction of the adapter protein Sam68, a cellular target of Src-like kinases which has been shown to interact with the poliovirus 3D polypeptide, with cellular signaling proteins as well as the function of the latter during infection. Here, we report that the RNA-binding and protein-binding protein Sam68 associates with the p21(ras) GTPase-activating protein RasGAP. Remarkably, RasGAP is cleaved during infections with different strains of coxsackievirus B3 as well as with echovirus 11 and echovirus 12, yielding a 104-kDa protein fragment. This cleavage event, which cannot be prevented by the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, may promote the activation of the Ras pathway, as shown by the activating dual phosphorylation of the mitogen-activated protein kinases Erk-1 and Erk-2 in the late phase of infection. Moreover, downstream targets of the mitogen-activated protein kinases, i.e., the p21(ras) exchange factor Sos-1 and cytoplasmic phospholipase A2, are phosphorylated with parallel time courses during infection. Activation or inhibition of cellular signaling pathways may play a general role in regulating effective enterovirus replication and pathogenesis, and the results of this study begin to unravel the molecular cross talk between enterovirus infection and key cellular signaling networks.

Viral myocarditis: identification of five differentially expressed genes in coxsackievirus B3-infected mouse heart.

Differences in host susceptibility to viral myocarditis caused by a given strain of coxsackievirus B3 (CVB3) are known to be largely related to host genetic factors. Little is known, however, about the key genes that encode determinants (mediators) of myocarditis development or the nature of injury. To identify these genes and further understand the molecular mechanisms of the disease process, we have used a murine model and the differential display technique to fingerprint mRNAs from CVB3-infected mouse hearts. Total RNA was extracted from hearts of 4- and 10-week-old A/J(H-2(a)) mice at day 4 after CVB3 infection, and mRNAs were detected by reverse transcriptase-polymerase chain reaction and subsequently analyzed on polyacrylamide DNA sequencing gels. The differentially displayed bands were confirmed by Northern hybridization using the bands as cDNA probes. Twenty-eight upregulated or downregulated bands were selected from the sequencing gels; among these, 2 upregulated and 3 downregulated cDNA fragments were confirmed by Northern hybridization. DNA sequence analysis and GenBank searching have determined that 4 of the 5 candidate genes are homologous to genes encoding Mus musculus inducible GTPase, mouse mitochondrial hydrophobic peptide (a subunit of NADH dehydrogenase), mouse beta-globin, and Homo sapiens cAMP-regulated response element binding protein (CREB) binding protein (CBP), respectively. The remaining candidate gene matches an unpublished cDNA clone, M musculus Nip21 mRNA (GenBank accession number, AF035207), which is homologous to human Nip2, a Bcl-2 binding protein. Our data suggest preliminarily that both structural and nonstructural genes are involved in myocarditis development. For the structural gene, beta-globin, we further confirmed its downregulation at the protein level by measuring the mean cell volume of red blood cells and found it was marginally reduced in the CVB3-infected group (P<0.06), with no change in hemoglobin concentration. Cardiac myoglobin concentration was also measured and found to be decreased (P<0.005), with a parallel decrease in total soluble protein in the CVB3-infected mouse myocardium (P<0.01). We also noted that the ratio of myoglobin to total protein was not significantly changed; this may be due to the downregulation of additional genes in the host heart, a number being observed on the differential display gels. The significant downregulation of beta-globin major gene expression in the heart may be relevant to impaired cardiac function in both the early and late postinfection period. The other identified nonstructural genes are known to be involved in regulation of gene expression, signal transduction pathways, and apoptotic cell death. The altered expression of structural and nonstructural genes may play important roles in the mediation of myocarditis development and perhaps other pathological processes in the heart.

Selenium and viral virulence.

A mouse model of coxsackievirus-induced myocarditis is being used to investigate nutritional determinants of viral virulence. This approach was suggested by research carried out in China which showed that mice fed diets composed of low selenium ingredients from a Keshan disease area suffered more extensive heart damage when infected with a coxsackie B4 virus than infected mice fed the same diet but supplemented with selenium by esophageal intubation. Selenium deficiency in our mice increased the virulence of an already virulent strain of coxsackievirus B3 (CVB3/20) and also allowed conversion of a non-virulent strain (CVB3/0) to virulence. Such conversion of CVB3/0 was accompanied by a change in the viral genome to more closely match that of the virulent virus, CVB3/20. As far as the authors are aware, this is the first report of host nutrition influencing the genetic make-up of an invading pathogen. Nutritionists may need to consider this mechanism of increased viral virulence in order to gain a better understanding of diet/infection relationships.

Glutathione peroxidase protects mice from viral-induced myocarditis.

Glutathione peroxidase 1 (GPX-1) is a selenium-dependent enzyme with antioxidant properties. Previous investigations determined that mice deficient in selenium developed myocarditis when infected with a benign strain of coxsackievirus B3 (CVB3/0). To determine whether this effect was mediated by GPX-1, mice with a disrupted Gpx1 gene (Gpx1-/-) were infected with CVB3/0. Gpx1-/- mice developed myocarditis after CVB3/0 infection, whereas infected wild-type mice (Gpx1+/+) were resistant. Sequencing of viruses recovered from Gpx1(-/-)-infected mice demonstrated seven nucleotide changes in the viral genome, of which three occurred at the G residue, the most easily oxidized base. No changes were found in virus isolated from Gpx1+/+ mice. These results demonstrate that GPX-1 provides protection against viral-induced damage in vivo due to mutations in the viral genome of a benign virus.

Dietary oxidative stress and the potentiation of viral infection.

Oxidative stress is implicated in the pathogenesis of several viral infections, including hepatitis, influenza, and AIDS. Dietary oxidative stress due to either selenium or vitamin E deficiency increases cardiac damage in mice infected with a myocarditic strain of coxsackievirus B3. Such dietary oxidative stress also allows a normally benign (i.e., amyocarditic) coxsackievirus B3 to convert to virulence and cause heart damage. This conversion to virulence is due to a nucleotide sequence change in the genome of the benign virus, which then resembles more closely the nucleotide sequence of virulent strains. Although it has been known for many years that poor nutrition can affect host response to infection, this is the first report of host nutrition affecting the genetic sequence of a pathogen. Further research is needed to determine whether poor host nutrition plays any role in the emergence of new viral diseases via alterations in he genotype of an infectious agent.

Nucleotide sequence of an attenuated mutant of coxsackievirus B3 compared with the cardiovirulent wildtype: assessment of candidate mutations by analysis of a revertant to cardiovirulence

Background: Coxsackievirus B3 (CVB3) causes myocarditis in the SWR (H 2q) mouse model and persistence of CVB3 in myocardium disposes to the development of dilated cardiomyopathy. An attenuated strain of CVB3 has been isolated, sequenced and several candidate mutations for attenuation identified. Derivation of a revertant to cardiovirulence allows the significance of these mutations to be assessed. Objectives: To ascertain which candidate mutation(s) determine(s) the attenuated phenotype. Study design: A revertant to cardiovirulence was isolated following passage through severe combined immunodeficient disease (SCID) mouse heart. The 5′-non-translated region (NTR) and region coding for capsid proteins were sequenced and compared to the wildtype and attenuant. Results: There are five candidates for attenuation: (1) A–G at base 580 in the 5′-NTR; (2) A–T at base 690 in the 5′-NTR; (3) CG–GC at bases 1401/2 (Thr to Ser at amino acid 151 in VP2); (4) AA–GT at bases 2691/2 (Lys to Ser at amino acid 80 in VP1); (5) A–G at base 2916 (Asp to Gly at amino acid 155 in VP1). It was shown previously that mutations at 580, 690 and 2691/2 are not important in attenuation. Additionally, there are three novel mutations in the coding region of the revertant and one in the 5′-NTR which are unlikely to be relevant for attenuation as they are not present in the attenuant. Of nucleotide changes seen at 1401/2 and 2916 in the attenuant, only 2916 reverts to the wildtype sequence and so is a strong candidate for a determinant of attenuation. Conclusions: The A–G mutation at 2916 (Asp to Gly at amino acid 155 in VP1) is a strong candidate for attenuation. It is located at the top of the receptor binding cleft and mutation of the Asp to a Gly may destabilise the receptor binding site.

A decay-accelerating factor-binding strain of coxsackievirus B3 requires the coxsackievirus-adenovirus receptor protein to mediate lytic infection of rhabdomyosarcoma cells.

The composition of the cellular receptor complex for coxsackievirus B3 (CVB3) has been an area of much contention for the last 30 years. Recently, two individual components of a putative CVB3 cellular receptor complex have been identified as (i) decay-accelerating factor (DAF) and (ii) the coxsackievirus-adenovirus receptor protein (CAR). The present study elucidates the individual roles of DAF and CAR in cell entry of CVB3 Nancy. First, we confirm that the DAF-binding phenotype of CVB3 correlates to the presence of key amino acids located in the viral capsid protein, VP2. Second, using antibody blockade, we show that complete protection of permissive cells from infection by high input multiplicities of CVB3 requires a combination of both anti-DAF and anti-CAR antibodies. Finally, it is shown that expression of the CAR protein on the surface of nonpermissive DAF-expressing RD cells renders them highly susceptible to CVB3-mediated lytic infection. Therefore, although the majority of CVB3 Nancy attaches to the cell via DAF, only virus directly interacting with the CAR protein mediates lytic infection. The role of DAF in CVB3 cell infection may be analogous to that recently described for coxsackievirus A21 (D. R. Shafren, D. J. Dorahy, R. A. Ingham, G. F. Burns, and R. D. Barry, J. Virol. 71:4736-4743, 1997), in that DAF may act as a CVB3 sequestration site, enhancing viral presentation to the functional CAR protein.

Sites other than nucleotide 234 determine cardiovirulence in natural isolates of coxsackievirus B3

The genetic site(s) that naturally determine the cardiovirulence phenotype of coxsackievirus B3 (CVB3) have yet to be mapped. Using two closely related CVB3 strains that differed in terms of cardiovirulence phenotype in mice, we previously reported the difference in phenotype mapped to a single site, nucleotide 234 (nt234) in the 5' non-translated region (NTR) of the CVB3 genome. When nt234 was C, the virus was attenuated and when U, the virus was cardiovirulent. To determine whether this finding was applicable to other strains of CVB3, we examined 13 different naturally occurring CVB3 strains isolated in different years in the United States. We determined that only two isolates induced severe inflammatory heart muscle disease in C3H/HeJ male mice. Using PCR products as sequencing templates, we determined the 5' NTR sequence from each viral genome. Alignment of these sequences and other published CVB3 5' NTR sequences suggests as many as four separate lineages, with commonly used laboratory strains clustering closely in one branch. An examination of the sequences showed that regardless of cardiovirulence phenotype, nt234 was invariably uridine. Thus, the previously reported cytidine at nt234 is most likely the result of a rare mutation and is not a naturally occurring variation and other sites must account for the variance in virulence seen in natural isolates of CVB3.

Expression and distribution of the receptors for coxsackievirus B3 during fetal development of the Balb/c mouse and of their brain cells in culture

This study was designed mainly to determine the relationships between the expression and distribution of the cellular receptor proteins for coxsackievirus B3 (CVB3) and susceptibility of mouse brain cells during fetal development of Balb/c mice. Immunoblot analysis of fetal extracts demonstrated that the CVB3 receptor proteins were first expressed at day 14 of the fetal stage, and that maximal expression of the cellular receptor occurred at near term or newborn stage. Results also suggested that newborn mouse brain tissue expressed much larger quantities of viral receptor proteins, compared to other tissues. In vitro studies showed that both mouse neurons and astrocytes could be infected by two CVB3 strains, pantropic CVB3 Nancy strain (CVB3N) and myocardiotropic CVB3 Woodruff strain (CVB3W). CVB3N, however, replicated and grew to high titer in primary astrocyte cultures and in primary neuron cultures, whereas, primary astrocyte cultures were relatively resistant to CVB3W. Virus binding assays revealed that CVB3N bound faster and in greater amounts to mouse brain cells than CVBW. These two virus strains, however, were found to share the same receptor specificity by virus competition assays. The number of virus binding sites for CVB3 on newborn mouse brain cells was approximately 1.8 × 10 4 per cell. The data suggested that preferential expression of the cellular receptors on newborn mouse brain cells may be related to their high susceptibilities to CVB3 infection.

A non-cardiovirulent strain of coxsackievirus B3 causes myocarditis in mice with severe combined immunodeficiency syndrome.

The most extensively studied animal model of coxsackievirus B3 (CVB3)-induced inflammatory heart muscle disease is the murine model. In the acute and chronic phase of the disease, it has been suggested that autoimmune mechanisms play a major role in the pathogenesis of the disease. In this study, C3H mice without functional T- and B-lymphocytes (C3H SCID) were inoculated either with a cardiovirulent (CVB3/20) or a non-cardiovirulent (CVB3/0) strain of coxsackievirus B3. Both viruses caused myocarditis in SCID mice. Furthermore, it could be demonstrated, that CVB3/0 had mutated to a cardiovirulent phenotype, able to cause myocarditis in immunocompetent mice.

The cardiovirulent phenotype of coxsackievirus B3 is determined at a single site in the genomic 5' nontranslated region.

We report the construction of chimeric coxsackievirus B3 (CVB3) strains in which sequences of an infectious cDNA copy of a noncardiovirulent CVB3 genome were replaced by the homologous sequences from a cardiovirulent CVB3 genome to identify which of 10 predicted genetic sites determine cardiovirulence. Cardiovirulent phenotype expression was consistently linked to nucleotide 234 (U in cardiovirulent CVB3 and C in avirulent CVB3) in the 5' nontranslated region. Reconstructions of the parental noncardiovirulent CVB3 genome from chimeras restored the noncardiovirulent phenotype when tested in mice. Inoculation of severe combined immunodeficient (scid) mice with the noncardiovirulent CVB3 strain resulted in massive cardiomyocyte necrosis in all animals. Sequence analysis of viral genomes isolated from twelve scid mouse hearts showed that only nucleotide position 234 was different (a C-->U transition) from that in the input parental noncardiovirulent CVB3 genome. Higher-order RNA structures predicted by two different algorithms did not demonstrate an obvious local effect caused by the C-->U change at nucleotide 234. Initial studies of parental and chimeric CVB3 replication in primary cultures of fetal murine heart fibroblasts and in adult murine cardiac myocytes demonstrated that viral RNA transcriptional efficiency is approximately 10-fold lower for noncardiovirulent CVB3 than for cardiovirulent CVB3. CVB3 did not shut off protein synthesis in murine cardiac fibroblasts, nor were levels of viral protein synthesis significantly different as a function of viral phenotype. Taken together, these data support a significant role for determination of the CVB3 cardiovirulence phenotype by nucleotide 234 in the 5' nontranslated region, possibly via a transcriptional mechanism.

Pathogenesis of Virus-Induced Diabetes in Mice

Male CD-1 mice were challenged with a diabetogenic strain (E2) of coxsackievirus B4 (CVB4). At 7 weeks, 6 months, and 1 year after inoculation, mean histopathologic scores, postprandial blood glucose levels, and serum levels of antibody to islet cells were significantly elevated and mean fasting serum insulin levels significantly reduced in infected mice versus uninfected controls (P < .001 for each). At 7 weeks after infection, viral RNA, but not protein or infectious virus, was demonstrated in the pancreases of most infected mice. The pancreases of 4 of 12 and 0 of 10 infected animals were positive for viral RNA at 6 months and at 1 year after infection, respectively. Interferon-gamma-stimulated peritoneal macrophages were cytotoxic to islet cells with and without sera with high titers of islet cell autoantibody (ICA). Thus, islet cell destruction in mice infected with CVB4 strain E2 is associated with chronic islet cell inflammation, elevation of islet cell antibody, and prolonged presence of viral RNA in the pancreas. Stimulated peritoneal macrophages lyse islet cells directly and by an antibody-dependent mechanism.

Replication and persistence of Coxsackieviruses B3 in human fibroblasts

Although in some cases a carrier state of Coxsackieviruses in human fibroblasts was described, the persistence mechanism has remained unknown. Our results demonstrate a replication of Coxsackievirus B3 (CVB3) in diploid human fibroblasts that is dependent on the virus strain as well as on the cell line involved. Two CVB3 Nancy strains could be multiplied over more than 10 passages in cell line H, whereas CVB3 strains SH"C" and SH"W" did not longer form infectious virus following several passages in these fibroblasts. None of the CVB3 strains replicated in cell lines J and K after a few passages. These results were not influenced by variations of culture conditions such as duration of incubation (3 or 7 days), temperature (33 or 37 degrees C) or application of trypsin. In line H, only 3-5 per cent of human fibroblasts were virus-infected. This was demonstrated by 1. antigen evidence by immunofluorescence, 2. determination of infectious centres, and 3. virus reproduction in dependence on MOI of the virus. We observed a carrier state of CVB3 Nancy strains over 16 cell passages in line H fibroblasts infected once. As shown in virus passage experiments, only few cells were productively infected. An addition of specific anti-CVB3 antiserum terminated this persistent infection. Generally, no cytopathic effect was observed. However, in one case a cell destruction by CVB3 Nancy "P" at the end of the life-time of the carrier cells could be found. This virus caused a complete cytopathic effect during more than 10 passages in line H and could still be neutralized by CVB3-specific antiserum.

Coxsackievirus B4 Alters Pancreatic Glutamate Decarboxylase Expression in Mice Soon After Infection

The 64,000-M(r) (64K) islet autoantigen, which is considered to be a target protein of beta cell destruction in insulin-dependent diabetes mellitus (IDDM), has recently been identified as the enzyme glutamic acid decarboxylase (GAD). We reported a two- to three-fold increased expression of the antigen in islets of diabetes-susceptible mice following infection with a diabetogenic strain of Coxsackievirus B4 (CB4) at 72-h postinfection (p.i.), a time point of active virus replication in the islets. Most of the infected animals subsequently developed 64K autoantibodies and hyperglycemia. Since the infection increases 64K expression, we have analysed immunoreactive GAD expression with a panel of peptide antisera and two widely-used polyclonal antisera against GAD, and measured GAD activity in the brain, pancreas and islets of these mice. Two isoforms, GAD65 and GAD67, are detected in these tissues from non-infected mice. Both GADs are also present in the infected mice brain at 72 h p.i.; however, their islets contain about three-fold more GAD65, and essentially no detectable GAD67. GAD activity is significantly higher in the brain compared with whole pancreas or islets, and islet GAD activity is higher than pancreas GAD activity. The infection significantly reduces islet GAD activity, but not brain GAD activity. CB4-induced abnormalities in islet GAD expression may play a role in virus-induced diabetes.

Antigenic analysis of coxsackievirus B3 with monoclonal antibodies.

All the 16 mouse monoclonal antibodies (McAbs) raised against Nancy strain of coxsackievirus B3, CoxB3(N), were type 3-specific. The reactivity of CoxB3(N) to this McAb panel during the serial passages showed a variation. Three field isolates of CoxB3, all neutralizable with the reference Anti-CoxB3 hyperimmune serum, were checked for the reactivities with the McAb panel. The isolates from the patients in Fujiang reacted with 12 McAbs and variably with the remaining four McAbs. On the basis of the reactivities with different virus strains, the McAbs were found to fall into four groups possibly corresponding to at least four epitopes. A considerable antigenic variation of CoxB3 was thus suggested.

Mapping of the RD phenotype of the Nancy strain of coxsackievirus B3

The RD variants of group B coxsackieviruses differ from their parental strains in having the ability to replicate in a human rhabdomyosarcoma cell line, RD. The nucleotide sequence of the P1region of the RD variant of coxsackievirus B3 strain Nancy (CB3NRD) was determined by sequencing cloned cDNAs, obtained by PCR amplification. A comparison between the established nucleotide sequence and that of the P1 region from the parental virus revealed 12 point mutations which corresponded to six amino acid replacements. To identify if the P1 region is responsible for the phenotype of CB3NRD, a chimeric virus was constructed, using an infectious cDNA clone of CB3. The P1 region of the infectious cDNA was replaced by cDNA fragments from CB3N (parental strain Nancy) or CB3NRD and the resulting recombinants were assayed for their ability to infect and replicate in RD cells. The results showed that the RD phenotype of CB3NRD maps in the P1 region. Furthermore, a chimera which only contained the 5' part of the P1 region derived from CB3NRD and the remaining P1 sequence from CB3N was able to replicate in RD cells, suggesting that the VP2 polypeptide contains at least one determinant for the RD phenotype.