Human Rhinovirus Type Research Articles

Species-specific receptor recognition by a minor-group human rhinovirus (HRV): HRV serotype 1A distinguishes between the murine and the human low-density lipoprotein receptor.

Human rhinoviruses (HRV) of the minor receptor group use several members of the low-density lipoprotein receptor superfamily for cell entry. These proteins are evolutionarily highly conserved throughout species and are almost ubiquitously expressed. Their common building blocks, cysteine-rich ligand binding repeats about 40 amino acids in length, exhibit considerable sequence similarity. Various numbers of these repeats are present in the different receptors. We here demonstrate that HRV type 1A (HRV1A) replicates in mouse cells without adaptation. Furthermore, although closely related to HRV2, it fails to bind to the human low-density lipoprotein receptor but recognizes the murine protein, whereas HRV2 binds equally well to both homologues. This difference went unnoticed due to the presence of other receptors, such as the low-density lipoprotein receptor-related protein, which allow species-independent attachment. The species specificity of HRV1A reported here will aid in defining amino acid residues establishing the contact between the viral surface and the receptor.

Synergism between rhinovirus infection and oxidant pollutant exposure enhances airway epithelial cell cytokine production.

Of the several factors believed to exacerbate asthmatic symptoms, air pollution and viral infections are considered to be particularly important. Although evidence indicates that each of these respiratory insults individually can increase asthma severity in susceptible individuals, we know little about the extent to which exposure to environmental oxidant pollutants can influence the course of respiratory viral infection and its associated inflammation. To investigate the interaction of these two stimuli within their common epithelial cell targets in the upper and lower respiratory tracks, we infected primary human nasal epithelial cells and cells of the BEAS-2B line grown at the air-liquid interface with human rhinovirus type 16 (RV16) and exposed them to NO2 (2.0 ppm) or O3 (0.2 ppm) for 3 hr. Independently, RV16, NO2, and O3 rapidly increased release of the inflammatory cytokine interleukin-8 through oxidant-dependent mechanisms. The combined effect of RV16 and oxidant ranged from 42% to 250% greater than additive for NO2 and from 41% to 67% for O3. We abrogated these effects by treating the cells with the antioxidant N-acetylcysteine. Surface expression of intercellular adhesion molecule 1 (ICAM-1) underwent additive enhancement in response to combined stimulation. These data indicate that oxidant pollutants can amplify the generation of proinflammatory cytokines by RV16-infected cells and suggest that virus-induced inflammation in upper and lower airways may be exacerbated by concurrent exposure to ambient levels of oxidants commonly encountered the indoor and outdoor environments.

Virucidal activity and cytotoxicity of the liposomal formulation of povidone-iodine

Two drug formulations of povidone-iodine (PVP-I)—an aqueous PVP-I solution (Betaisodona®) and a liposomal PVP-I formulation—were tested for their virucidal activity and cytotoxicity in cell culture. As to the virudical activity against influenza A virus, herpes simplex virus type 1, adenovirus type 8 and human rhinovirus type 14, the liposomal formulation of PVP-I proved to be approximately as active as the aqueous one. Half maximum cytotoxic PVP-I concentrations were 0.01–0.07% for aqueous PVP-I and 0.03–0.27% for the liposomal PVP-I formulation (XTT reduction assay EZ4U). The detection of lactate dehydrogenase (LDH) release, DNA fragmentation (ELISA) and DNA strand breakage (TUNEL assay) after 24 h exposure of human embryonic lung fibroblasts to PVP-I revealed that necrosis predominates in cells treated with aqueous 0.08–0.32% PVP-I solutions, whereas apoptosis was the predominant type of cell death in cells treated with equivalent concentrations of liposomal PVP-I formulation. The favorable virucidal efficiency together with the preferred apoptotic route of cell death makes the liposomal PVP-I formulation a promising candidate for topical use in prevention and treatment of infections of the eye and the upper respiratory tract.

Antiviral Action of Euphorbium compositum® and Its Components

Introduction: Euphorbium compositum® SN (Biologische Heilmittel Heel GmbH, Baden-Baden, Germany, a homeopathic combination preparation available in form of drops, nasal spray, and injection solution), is prescribed for inflammation of the mucosae of the nose and sinuses. Infections in these areas are primarily of viral origin although bacterial superinfections are also common. Objective: The main question was whether or not this homeopathic remedy shows an activity against viruses responsible for infections of the respiratory tract. Methods:This in vitro study using virus plaque reduction assays examined the effect of Euphorbium compositum SN against pathogens causing various viral infections: influenza A virus, respiratory syncytial virus (RSV), human rhinovirus (HRV) and herpes simplex virus type 1 (HSV-1). Results: Analysis of virus production after treatment of the infected cells with the remedy showed an antiviral activity of Euphorbium compositum SN against RSV and HSV-1. In addition, an antiviral effect against influenza A virus and HRV, though minimal, was, also noted. Analyses of the plant-derived components of Euphorbium compositum SN, e.g. Euphorbium resinifera, Pulsatilla pratensis and Luffa operculata for their antiviral activity revealed a clear activity of Euphorbium resinifera and Pulsatilla pratensis against RSV. In contrast, no effect was detected using the same protocol with Luffa operculata. Conclusions: Euphorbium resinifera and Pulsatilla pratensis as components of Euphorbium compositum SN are responsible for its antiviral activity.

In vitro antiviral activity of the anthraquinone chrysophanic acid against poliovirus

Chrysophanic acid (1,8-dihydroxy-3-methylanthraquinone), isolated from the Australian Aboriginal medicinal plant Dianella longifolia, has been found to inhibit the replication of poliovirus types 2 and 3 ( Picornaviridae) in vitro. The compound inhibited poliovirus-induced cytopathic effects in BGM (Buffalo green monkey) kidney cells at a 50% effective concentration of 0.21 and 0.02 μg/ml for poliovirus types 2 and 3, respectively. The compound inhibited an early stage in the viral replication cycle, but did not have an irreversible virucidal effect on poliovirus particles. Chrysophanic acid did not have significant antiviral activity against five other viruses tested: Coxsackievirus types A21 and B4, human rhinovirus type 2 ( Picornaviridae), and the enveloped viruses Ross River virus ( Togaviridae) and herpes simplex virus type 1 ( Herpesviridae). Four structurally-related anthraquinones — rhein, 1,8-dihydroxyanthraquinone, emodin and aloe-emodin were also tested for activity against poliovirus type 3. None of the four compounds was as active as chrysophanic acid against the virus. The results suggested that two hydrophobic positions on the chrysophanic acid molecule (C-6 and the methyl group attached to C-3) were important for the compound's activity against poliovirus.

Membrane-Associated Respiratory Syncytial Virus F Protein Expressed from a Human Rhinovirus Type 14 Vector Is Immunogenic

Human rhinovirus (HRV) replicons have the potential to serve as respiratory vaccine vectors for mucosal immunization in humans. However, since many vaccine immunogens of interest are glycosylated, an important concern is whether HRV replicons are capable of expressing glycosylated proteins. The human respiratory syncytial virus (RSV) fusion (F) protein was chosen as a model glycoprotein and the HRV replicon ΔP1FVP3 was generated by inserting the F protein-coding sequence in frame and in lieu of the 5′ proximal 1489 nucleotides of the capsid-coding segment in the HRV-14 genome. When transfected into H1-HeLa cells, ΔP1FVP3 replicated and led to the expression of the F protein. Inhibition with guanidine demonstrated that F-protein expression was dependent on ΔP1FVP3 replication and did not result from translation of input RNA. Although most of the F protein remained as an immature, glycosylated precursor (F0), a readily detectable fraction of the protein was processed into the mature glycosylated subunit F1, an event known to occur within the Golgi apparatus. Packaged ΔP1FVP3 replicons were generated in transfected HeLa cells by coexpression of homologous HRV capsid proteins using the vaccinia virus/T7 RNA polymerase hybrid system. Packaged replicon RNAs were capable of infecting fresh cells, leading to accumulation of the F protein as in RNA-transfected cells. Mice immunized with HeLa cell lysates containing F protein expressed from ΔP1FVP3 produced neutralizing antibodies against RSV. These results indicate that an HRV-14 replicon can express a foreign glycosylated protein, providing further support for the potential of HRV replicons as a vaccine delivery system.

The C-terminal residues of poliovirus proteinase 2A(pro) are critical for viral RNA replication but not for cis- or trans-proteolytic cleavage.

Poliovirus proteinase 2A(pro) is an essential enzyme involved in cleavages of viral and cellular proteins during the infectious cycle. Evidence has been obtained that 2A(pro) is also involved in genome replication. All enteroviruses have a negatively charged cluster of amino acids at their C terminus (E(E)/(D)(E)/(D)AMEQ-NH(2)), a common motif suggesting function. When aligned with enterovirus sequences, the 2A(pro) proteinase of human rhinovirus type 2 (HRV2) has a shorter C terminus (EE.Q:-NH(2)) and, indeed, the HRV2 2A(pro) cannot substitute for poliovirus 2A(pro) to yield a viable chimeric virus. Here evidence is provided that the C-terminal cluster of amino acids plays an unknown role in poliovirus genome replication. Deletion of the EEAME sequence from poliovirus 2A(pro) is lethal without significantly influencing proteinase function. On the other hand, addition of EAME to HRV2 2A(pro), yielding a C terminus of this enzyme of EEEAMEQ:, stimulated RNA replication of a poliovirus/HRV2 chimera 100-fold. The novel role of the C-terminal sequence motif is manifested at the level of protein function, since silent mutations in its coding region had no effect on virus proliferation. Poliovirus type 1 Mahoney 2A(pro) could be provided in trans to rescue the lethal deletion EEAME in the poliovirus variant. Encapsidation studies left open the question of whether the C terminus of poliovirus 2A(pro) is involved in particle formation. It is concluded that the C terminus of poliovirus 2A(pro) is an essential domain for viral RNA replication but is not essential for proteolytic processing.

Membrane-Associated Respiratory Syncytial Virus F Protein Expressed from a Human Rhinovirus Type 14 Vector Is Immunogenic

Membrane-Associated Respiratory Syncytial Virus F Protein Expressed from a Human Rhinovirus Type 14 Vector Is Immunogenic

Translation and replication of human rhinovirus type 14 and mengovirus in Xenopus oocytes.

We have previously shown that Xenopus oocytes require coinjection of both poliovirus RNA and HeLa cell extracts to support a complete cycle of viral replication yielding high levels of infectious viral particles. This novel system provides a tool for identifying host factors and for biochemically dissect individual steps that lead to virus production. Here we demonstrate that Xenopus oocytes are able to support replication of other picornaviruses such as human rhinovirus 14 and mengovirus. Unlike poliovirus, microinjection of mengovirus RNA yields high viral titers (about 10(7) PFU/oocyte) without the need for coinjection of additional cell extracts. In contrast, formation of infectious rhinovirus particles requires coinjection of human cell extracts. We found that one of these human factors is required for efficient rhinovirus translation. Our findings uncover differences in the host factor requirements among members of the picornavirus family and provide the means to identify the human protein(s) involved in rhinovirus production.

Spectrum of virus inhibition by consensus interferon YM643.

The spectrum of viruses inhibited by a genetically engineered consensus interferon (IFN) YM643 (interferon alfacon-1) was evaluated using a cytopathic effect inhibition assay or plaque inhibition assay for five DNA viruses and 12 RNA viruses. This activity was compared to that of natural IFN-alpha derived from Namalwa lymphoblastoid cell line [IFN-alpha (Namalwa)]. The viruses inhibited by both IFNs were herpesvirus types 1 and 2, human cytomegalovirus, varicella-zoster virus, vesicular stomatitis virus, yellow fever virus, bovine viral diarrhoea virus, Semliki Forest virus, western equine encephalitis virus, encephalomyocarditis virus, rhinovirus type A, respiratory syncytial virus, Newcastle disease virus and influenza virus type A (H1N1). Neither IFN inhibited coxsackie virus B1, reovirus type 3 or vaccinia virus in the experimental conditions used. The specific activity of YM643 in human cells generally ranged from 3.6x10(7) to 2.1x10(9) IU/mg, which was greater than that of IFN-alpha (Namalwa), which ranged from 3.1x10(6) to 4.6x10(8) IU/mg against all sensitive viruses, except human cytomegalovirus and rhinovirus type 1A, which displayed approximately equal sensitivity to both IFNs. Significantly, the potency of YM643 against bovine viral diarrhoea virus and yellow fever virus, which were selected to serve as surrogates of hepatitis C virus, equalled or exceeded that of IFN-alpha (Namalwa). These results suggest that the genetically engineered YM643 is more potent than natural IFN-alpha.

Anti-rhinovirus activity of 3-methylthio-5-aryl-4-isothiazolecarbonitrile derivatives

A series of 3-methylthio-5-aryl-4-isothiazolecarbonitriles has been evaluated as anti rhinovirus agents against a panel of 17 representative human rhinovirus (HRV) serotypes, belonging to both A and B groups. No anti rhinovirus activity was detected for 3-methylthio-5-phenyl-4-isothiazolecarbonitrile ( IS-2). Isothiazole derivatives with bulky substituents (O-Bn or O-But groups) on the para position of the phenyl ring were the most effective compounds of this series. In fact, a reduction in virus-induced cytopathogenicity was demonstrated for the O-Bn substituted IS-50 compound against the majority (88%) of the rhinoviruses tested, whereas the compound with an O-Ts group ( IS-44) was found to be a specific inhibitor of group B serotypes, exhibiting the lowest IC 50 against HRVs type 2, 85 and 89. Our studies on the mechanism of action of IS-44 demonstrated that it prevents the thermal inactivation of HRV 2 infectivity, probably due to a conformational shift in the viral capsid and a decrease in affinity for the cellular receptor, resulting in an inhibition of attachment of the virions.

A coding RNA sequence acts as a replication signal in cardioviruses.

Theiler's virus and Mengo virus are representatives of the Cardiovirus genus within the picornavirus family. Their genome is an 8-kilobase long positive strand RNA molecule. This RNA molecule plays three roles in infected cells: It serves as a messenger RNA, acts as a template for genome replication, and is encapsidated to form progeny virions. We observed that a cis-acting signal required for replication of Theiler's virus was contained within a 130-nt stretch of the region encoding the capsid protein VP2. This RNA sequence does not influence internal ribosome entry site-mediated translation initiation and thus likely acts directly as a signal for the replication complex. We found a similar signal in the VP2-coding sequence of Mengo virus, and both signals could be functionally exchanged. Within the replication element, a 9-nt sequence that is highly conserved among cardioviruses was shown to be essential for replication. This conserved sequence was contained in mostly unpaired regions of the RNA secondary structure predicted for the replication elements of the various cardioviruses. Interestingly, a similar replication element has been reported to occur in the distantly related human rhinovirus type 14, suggesting that such elements could be conserved throughout the picornavirus family. However, the different location of the replication elements in rhinovirus and cardioviruses, and the fact that they were not functionally exchangeable, is raising intriguing questions about the evolution of such signals in picornaviruses.

Dual stem loops within the poliovirus internal ribosomal entry site control neurovirulence.

In the human central nervous system, susceptibility to poliovirus (PV) infection is largely confined to a specific subpopulation of neuronal cells. PV tropism is likely to be determined by cell-external components such as the PV receptor CD155, as well as cell-internal constraints such as the availability of a suitable microenvironment for virus propagation. We reported previously that the exchange of the cognate internal ribosomal entry site (IRES) within the 5' nontranslated region of PV with its counterpart from human rhinovirus type 2 (HRV2) can eliminate the neuropathogenic phenotype in a transgenic mouse model for poliomyelitis without diminishing the growth properties in HeLa cells. We now show that attenuation of neurovirulence of PV/HRV2 chimeras is not confined to CD155 transgenic mice but is evident also after intraspinal inoculation into Cynomolgus monkeys. We have dissected the PV and HRV2 IRES elements to determine those structures responsible for neurovirulence (or attenuation) of these chimeric viruses. We report that two adjacent stem loop structures within the IRES cooperatively determine neuropathogenicity.

The rhinovirus type 14 genome contains an internally located RNA structure that is required for viral replication.

Cis-acting RNA signals are required for replication of positive-strand viruses such as the picornaviruses. Although these generally have been mapped to the 5' and/or 3' termini of the viral genome, RNAs derived from human rhinovirus type 14 are unable to replicate unless they contain an internal cis-acting replication element (cre) located within the genome segment encoding the capsid proteins. Here, we show that the essential cre sequence is 83-96 nt in length and located between nt 2318-2413 of the genome. Using dicistronic RNAs in which translation of the P1 and P2-P3 segments of the polyprotein were functionally dissociated, we further demonstrate that translation of the cre sequence is not required for RNA replication. Thus, although it is located within a protein-coding segment of the genome, the cre functions as an RNA entity. Computer folds suggested that cre sequences could form a stable structure in either positive- or minus-strand RNA. However, an analysis of mutant RNAs containing multiple covariant and non-covariant nucleotide substitutions within these putative structures demonstrated that only the predicted positive-strand structure is essential for efficient RNA replication. The absence of detectable minus-strand synthesis from RNAs that lack the cre suggests that the cre is required for initiation of minus-strand RNA synthesis. Since a lethal 3' noncoding region mutation could be partially rescued by a compensating mutation within the cre, the cre appears to participate in a long-range RNA-RNA interaction required for this process. These data provide novel insight into the mechanisms of replication of a positive-strand RNA virus, as they define the involvement of an internally located RNA structure in the recognition of viral RNA by the viral replicase complex. Since internally located RNA replication signals have been shown to exist in several other positive-strand RNA virus families, these observations are potentially relevant to a wide array of related viruses.

Direct Cleavage of eIF4G by Poliovirus 2A Protease Is Inefficientin Vitro

Previously, the purified recombinant 2A proteases (2Apro) of coxsackievirus B4 (CVB4) and human rhinovirus type 2 (HRV2) were shown to cleave synthetic peptides derived from human or rabbit eIF4G as well as eIF4G protein purified from rabbit reticulocytes. These results were in contrast to previous evidence which supported the view that eIF4G cleavage activity in poliovirus-infected HeLa cells required a cellular factor(s) activated by poliovirus (PV) 2Apro. In the present study, recombinant PV 2Aprowas shown to cleave either rabbit or human eIF4G or their derived peptides in direct cleavage reactions, but cleaved the 4G-derived peptides with 100-fold lower efficiency than with a peptide derived from the poliovirus polyprotein. In these experiments, up to 25-fold molar excess of 2Aproover eIF4G protein was required to cause greater than 50% cleavage. CVB4 2Aprowas also tested in peptide cleavage assays under the same conditions as PV 2Aproand was found to cleave all eIF4G substrates with efficiencies similar to PV 2Apro. Finally, cleavage reactions utilizing recombinant eIF4G containing a G486E substitution at the cleavage site for CVB4 and HRV2 proteases resulted in drastically reduced cleavage by PV 2Apro, similar to the reduction previously seen with HRV2 and CVB4 2Apro, confirming that all three viral 2A proteases recognize the same cleavage site on eIF4G. These data show that PV 2Aprocan directly cleave eIF4Gin vitrowith efficiencies similar to those of CVB 2Apro, but cleavage efficiency of eIF4G is approximately 1000-fold lower than cleavage of a peptide derived from the authentic 2A cleavage site on the poliovirus polyprotein.

Eosinophils Bind Rhinovirus and Activate Virus-Specific T Cells

Episodes of virus-induced exacerbations of asthma are accompanied by increased eosinophils (EOS) in respiratory secretions and evidence of EOS degranulation. Although rhinoviruses (RV) are the viruses most often implicated in exacerbations of asthma in both children and adults, little is known about the immune response to this group of viruses and, in particular, EOS-RV interactions. To define such interactions, we incubated human rhinovirus type 16 (RV16), a serotype using ICAM-1 as a receptor, with EOS purified from PBMC, and measured EOS-RV binding, EOS-mediated Ag presentation and T cell activation, and EOS cell surface marker expression and superoxide production. Significant RV16 binding occurred to EOS that were pretreated with granulocyte-macrophage CSF, and this binding was inhibited by anti-ICAM-1 mAb. EOS also presented viral Ags to RV16-specific T cells, causing T cell proliferation and secretion of IFN-gamma. RV16 induced a significant shift from CD18dim to CD18bright, but did not affect EOS expression of CD54, CD69, or HLA-DR. Finally, RV16 did not induce superoxide production from peripheral blood EOS. These findings suggest that RV16 also binds to airway EOS, which resemble granulocyte-macrophage CSF-treated blood EOS in terms of high expression of ICAM-1. Furthermore, our findings suggest that EOS could participate in RV-induced immune responses through Ag presentation and T cell activation. By activating RV-specific T cells, EOS may play an important role in the initiation of antiviral T cell responses, and these effects could also contribute to enhanced airway inflammation and increased asthma symptoms in susceptible individuals.

Enzymatic characterization of refolded human rhinovirus type 14 2A protease expressed in Escherichia coli.

Reported here is the production of recombinant human rhinovirus 14 (HRV14) 2A protease from bacterial cells transformed with a heat-inducible plasmid containing the HRV14 2A cDNA sequence. Overexpressed 2A protein partitioned into the inclusion bodies was solubilized in urea and then refolded in the presence of Zn2+ Transition metals were required for the restoration of 2A protease activity as a structural component, but appeared to be inhibitory if added exogenously once the enzyme was refolded. Based on the cleavage specificity studies, a colorimetric assay was developed for the highly purified HRV14 2A protease. A peptide with the sequence RKGDIKSY-p-nitroanilide was found to be cleaved by the 2A protease with a k(cat)/Km ratio of approximately 335 M(-1)s(-1), which allows its activity to be measured continuously with a spectrophotometer or a microplate reader.

Human rhinovirus type 14:human immunodeficiency virus type 1 (HIV-1) V3 loop chimeras from a combinatorial library induce potent neutralizing antibody responses against HIV-1.

In an effort to develop a useful AIDS vaccine or vaccine component, we have generated a combinatorial library of chimeric viruses in which the sequence IGPGRAFYTTKN from the V3 loop of the MN strain of human immunodeficiency virus type 1 (HIV-1) is displayed in many conformations on the surface of human rhinovirus 14 (HRV14). The V3 loop sequence was inserted into a naturally immunogenic site of the cold-causing HRV14, bridged by linkers consisting of zero to three randomized amino acids on each side. The library of chimeric viruses obtained was subjected to a variety of immunoselection schemes to isolate viruses that provided the most useful presentations of the V3 loop sequence for potential use in a vaccine against HIV. The utility of the presentations was assessed by measures of antigenicity and immunogenicity. Most of the immunoselected chimeras examined were potently neutralized by each of the four different monoclonal anti-V3 loop antibodies tested. Seven of eight chimeric viruses were able to elicit neutralizing antibody responses in guinea pigs against the MN and ALA-1 strains of HIV-1. Three of the chimeras elicited HIV neutralization titers that exceeded those of all but a small number of previously described HIV immunogens. These results indicate that HRV14:HIV-1 chimeras may serve as useful immunogens for stimulating immunity against HIV-1. This method can be used to flexibly reconstruct varied immunogens on the surface of a safe and immunogenic vaccine vehicle.

Mutational Analyses Support a Model for the HRV2 2A Proteinase

The proteinase 2A of human rhinovirus 2 is a cysteine proteinase which contains a tightly bound Zn ion thought to be required for structural integrity. A three-dimensional model for human rhinovirus type 2 proteinase 2A (HRV2 2A) was established using sequence alignments with small trypsin-like Ser-proteinases and, for certain regions, elastase. The model was tested by expressing selected proteinase 2A mutants in bacteria and examining the effect on both intramolecular (“cis”) and intermolecular (“trans”) activities. The HRV2 proteinase 2A is proposed to have a two domain structure, with the catalytic site and substrate binding region on one face of the molecule and a Zn-binding motif on the opposite face. Residues Gly 123, Gly 124, Thr 121, and Cys 101 are proposed to be involved in the architecture of the substrate binding pocket and to provide the correct environment for the catalytic triad of His 18, Asp 35, and Cys 106. Residues Tyr 85 and Tyr 86 are thought to participate in substrate recognition. The presence of an extensive C-terminal helix, in which Asp 132, Arg 134, Phe 130, and Phe 136 play important roles, explains why mutations in this region are generally detrimental to proteinase activity. The proposed Zn-binding motif comprises Cys 52, Cys 54, Cys 112, and His 114. Exchange of these residues inactivates the enzyme. Furthermore, as measured by atom emission spectroscopy, Zn was absent from purified preparations of proteinase 2A in which His 114 had been replaced by Asn. The absence of disulphide bridges was confirmed by subjecting highly purified HRV2 proteinase 2A to one- and two-step alkylation procedures.

Hyper-antigenic variation occurs with human rhinovirus type 17.

Of 90 human rhinovirus (RV) serotypes tested, 50 can be placed into 16 groups according to antigenic relationships. It has been suggested that antigenic variants might arise in nature by immunologic pressure. To investigate this possibility, attempts were made to select variants by cloning 16 different plaque-purified RV serotypes in the presence of homologous, polyclonal antisera. Isolates were examined for evidence of variation in serum cross-neutralization tests using parental antisera and, in some cases, antisera prepared for the isolates. Only RV type 17 (RV-17) yielded major antigenic variants after cloning. With some variants, as much as a 500-fold difference in neutralizing titer was obtained with the parental antiserum. By using antisera for two of the variants, it was determined that they are prime strains of the parental RV-17. Continuing attempts at immunoselection by using antisera for one of these prime strains yielded additional antigenic variants. By using antisera prepared for three of these new variants, it was determined that one of them is a prime strain of the virus from which it was derived. Cross-neutralization tests with the two remaining isolates indicate that, according to conventional practice, they no longer would be classified as RV-17.