Antigenic comparison of feline coronavirus isolates: evidence for markedly different peplomer glycoproteins.

The antigenic relationships among seven feline coronavirus isolates were investigated by using a panel of 26 monoclonal antibodies (MAbs). The MAbs were categorized into five immunoreactive groups which were used to delineate two antigenic types of feline coronaviruses. One antigenic type included the more virulent feline infectious peritonitis virus (FIPV) isolates (FIPV-UCD-1, FIPV-UCD-4, FIPV-TN406, FIPV-DF2, and FIPV-79-1146), whereas the second antigenic type was composed of the avirulent isolate FIPV-UCD-2. The feline enteric coronavirus isolate FECV-79-1683 shared some characteristics of both of the major antigenic groups. Epitopes on the nucleocapsid and envelope polypeptides were in general highly conserved among both antigenic types, although a few type-specific antigenic sites were discriminated. The most striking finding was the marked antigenic difference in the peplomer (E2) glycoproteins between the two antigenic types. Seven anti-E2 MAbs reacted with one antigenic type of E2, whereas seven other anti-E2 MAbs recognized a different antigenic form of E2. None of the 14 anti-E2 MAbs reacted with all of the isolates.     

Acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein

In feline coronavirus (FCoV) pathogenesis, the ability to infect macrophages is an essential virulence factor. Whereas the low-virulence feline enteric coronavirus (FECV) isolates primarily replicate in the epithelial cells of the enteric tract, highly virulent feline infectious peritonitis virus (FIPV) isolates have acquired the ability to replicate efficiently in macrophages, which allows rapid dissemination of the virulent virus throughout the body. FIPV 79-1146 and FECV 79-1683 are two genetically closely related representatives of the two pathotypes. Whereas FECV 79-1683 causes at the most a mild enteritis in young kittens, FIPV 79-1146 almost invariably induces a lethal peritonitis. The virulence phenotypes correlate with the abilities of these viruses to infect and replicate in macrophages, a feature of FIPV 79-1146 but not of FECV 79-1683. To identify the genetic determinants of the FIPV 79-1146 macrophage tropism, we exchanged regions of its genome with the corresponding parts of FECV 79-1683, after which the ability of the FIPV/FECV hybrid viruses to infect macrophages was tested. Thus, we established that the FIPV spike protein is the determinant for efficient macrophage infection. Interestingly, this property mapped to the C-terminal domain of the protein, implying that the difference in infection efficiency between the two viruses is not determined at the level of receptor usage, which we confirmed by showing that infection by both viruses was equally blocked by antibodies directed against the feline aminopeptidase N receptor. The implications of these findings are discussed.     

Intrinsic resistance of feline peritoneal macrophages to coronavirus infection correlates with in vivo virulence.

Cats infected with virulent feline coronavirus strains develop feline infectious peritonitis, an invariably fatal, immunologically mediated disease; avirulent strains cause either clinically inapparent infection or mild enteritis. Four virulent coronavirus isolates and five avirulent isolates were assessed by immunofluorescence and virus titration for their ability to infect and replicate in feline peritoneal macrophages in vitro. The avirulent coronaviruses infected fewer macrophages, produced lower virus titers, were less able to sustain viral replication, and spread less efficiently to other susceptible macrophages than the virulent coronaviruses. Thus, the intrinsic resistance of feline macrophages may play a pivotal role in the outcome of coronavirus infection in vivo.     

Enhancement and Neutralization of Feline Infectious Peritonitis Virus Infection in Feline Macrophages by Neutralizing Monoclonal Antibodies Recognizing Different Epitopes

The interaction between the enhancing and neutralizing activities of three monoclonal antibodies (MAbs) (5-6-2, 6-4-2 and 7-4-1) to the spike protein of feline infectious peritonitis virus (FIPV) strain 79-1146 was determined using feline macrophages. At a high MAb concentration, all of the three MAbs completely inhibited the FIPV infection at 37 C. However, two of them (6-4-2 and 7-4-1) enhanced FIPV infection when either the MAb concentration or reaction temperature was lowered. These MAbs also exerted an immediate infectivity-enhancing activity for up to 10 min of reaction and by 20 min, neutralizing activities were observed. Only MAb 5-6-2 consistently showed neutralizing activity regardless of the reaction conditions. Competition with sera from cats experimentally infected with FIPV strain 79-1146 or feline enteric coronavirus strain 79-1683 showed that the two epitopes recognized by MAb 5-6-2 and MAb 6-4-2, respectively, are also recognized by the natural host.     

Comparison of the Amino Acid Sequence and Phylogenetic Analysis of the Peplomer,Integral Membrane and Nucleocapsid Proteins of Feline,Canine and Porcine Coronaviruses

Complete nucleotide sequences were determined by cDNA cloning of peplomer (S), integral membrane (M) and nucleocapsid (N) genes of feline infectious peritonitis virus (FIPV) type I strain KU-2, UCD1 and Black, and feline enteric coronavirus (FECV) type II strain 79–1683. Only M and N genes were analyzed in strain KU-2 and strain 79–1683, which still had unknown nucleotide sequences. Deduced amino acid sequences of S, M and N proteins were compared in a total of 7 strains of coronaviruses, which included FIPV type II strain 79–1146, canine coronavirus (CCV) strain Insavc-1 and transmissible gastroenteritis virus of swine (TGEV) strain Purdue. Comparison of deduced amino acid sequences of M and N proteins revealed that both M and N proteins had an identity of at least 90% between FIPV type I and type II. The phylogenetic tree of the M and N protein-deduced amino acid sequences showed that FIPV type I and type II form a group with FECV type II, and that these viruses were evolutionarily distant from CCV and TGEV. On the other hand, when the S protein-deduced amino acid sequences was compared, identity of only about 45% was found between FIPV type I and type II. The phylogenetic tree of the S protein-deduced amino acid sequences indicated that three strains of FIPV type I form a group, and that it is a very long distance from the FIPV type II, FECV type II, CCV and TGEV groups.     

Feline aminopeptidase N serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup I.

Two members of coronavirus serogroup I, human respiratory coronavirus HCV-229E and porcine transmissible gastroenteritis virus (TGEV), use aminopeptidase N (APN) as their cellular receptors. These viruses show marked species specificity in receptor utilization, as HCV-229E can utilize human but not porcine APN, while TGEV can utilize porcine but not human APN. To determine whether feline APN could serve as a receptor for two feline coronaviruses in serogroup I, feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FeCV), we cloned the cDNA encoding feline APN (fAPN) by PCR from cDNA isolated from a feline cell line and stably expressed it in FIPV- and FeCV-resistant mouse and hamster cells. The predicted amino acid sequence of fAPN shows 78 and 77% identity with human and porcine APN, respectively. When inoculated with either of two biologically different strains of FIPV or with FeCV, fAPN-transfected mouse and hamster cells became infected and viral antigens developed in the cytoplasm. Infectious FIPV was released from hamster cells stably transfected with fAPN. The fAPN-transfected mouse and hamster cells were challenged with other coronaviruses in serogroup I including canine coronavirus, porcine coronavirus TGEV, and human coronavirus HCV-229E. In addition to serving as a receptor for the feline coronaviruses, fAPN also served as a functional receptor for each of these serogroup I coronaviruses as shown by development of viral antigens in the cytoplasm of infected mouse or hamster cells stably transfected with fAPN. In contrast, fAPN did not serve as a functional receptor for mouse hepatitis virus (MHV-A59), which is in serogroup II and utilizes mouse biliary glycoprotein receptors unrelated to APN. Thus, fAPN serves as a receptor for a much broader range of group I coronaviruses than human and porcine APNs. The human, porcine, and canine coronaviruses in serogroup I that are able to use fAPN as a receptor have previously been shown to infect cats without causing disease. Therefore, host factors in addition to receptor specificity apparently affect the virulence and transmissibility of nonfeline serogroup I coronaviruses in the cat.     

Feline Coronavirus Type II Strains 79-1683 and 79-1146 Originate from a Double Recombination between Feline Coronavirus Type I and Canine Coronavirus

Recent evidence suggests that the type II feline coronavirus (FCoV) strains 79-1146 and 79-1683 have arisen from a homologous RNA recombination event between FCoV type I and canine coronavirus (CCV). In both cases, the template switch apparently took place between the S and M genes, giving rise to recombinant viruses which encode a CCV-like S protein and the M, N, 7a, and 7b proteins of FCoV type I (K. Motowaka, T. Hoh- datsu, H. Hashimoto, and H. Koyama, Microbiol. Immunol. 40:425–433, 1996; H. Vennema, A. Poland, K. Floyd Hawkins, and N. C. Pedersen, Feline Pract. 23:40–44, 1995). In the present study, we have looked for additional FCoV-CCV recombination sites. Four regions in the pol gene were selected for comparative sequence analysis of the type II FCoV strains 79-1683 and 79-1146, the type I FCoV strains TN406 and UCD1, the CCV strain K378, and the TGEV strain Purdue. Our data show that the type II FCoVs have arisen from double recombination events: additional crossover sites were mapped in the ORF1ab frameshifting region of strain 79-1683 and in the 5′ half of ORF1b of strain 79-1146.     

A reverse genetics approach to study feline infectious peritonitis

Feline infectious peritonitis (FIP) is a lethal immunopathological disease caused by feline coronaviruses (FCoVs). Here, we describe a reverse genetics approach to study FIP by assessing the pathogenicity of recombinant type I and type II and chimeric type I/type II FCoVs. All recombinant FCoVs established productive infection in cats, and recombinant type II FCoV (strain 79-1146) induced FIP. Virus sequence analyses from FIP-diseased cats revealed that the 3c gene stop codon of strain 79-1146 has changed to restore a full-length open reading frame (ORF).     

Immunogenicity of a bovine rotavirus glycoprotein fragment.

Previous experiments demonstrated that an antigenic site responsible for virus neutralization and cell attachment was located on a 14,000-molecular-weight fragment of the major bovine rotavirus (BRV) glycoprotein (M. Sabara, J. E. Gilchrist, G. R. Hudson, and L. A. Babiuk, J. Virol. 53:58-66, 1985). However, it was necessary to investigate whether this fragment also had the ability to induce the production of neutralizing antibodies. Upon immunization of mice, the bovine serum albumin-conjugated 14,000-molecular-weight fragment, the unconjugated 14,000-molecular-weight fragment, and the native glycoprotein all induced a similar neutralizing antibody response, albeit to a lesser extent than did the infectious, whole virus. In addition, immuno-blot enzyme-linked immunosorbent assay analysis of the reactivity of anti-peptide serum versus anti-glycoprotein serum with the glycoprotein was very comparable. These results suggest that the 14,000-molecular-weight fragment may represent not only a biologically active region but also an immunodominant area of the glycoprotein.     

Isofemale Line Analysis of Meloidogyne incognita Virulence to Cowpea Resistance Gene Rk

Isofemale lines (IFL) from single egg masses were studied for genetic variation in Meloidogyne incognita isolates avirulent and virulent to the resistance gene Rk in cowpea (Vigna unguiculata). In parental isolates cultured on susceptible and resistant cowpea, the virulent isolate contained 100% and the avirulent isolate 7% virulent lineages. Virulence was selected from the avirulent isolate within eight generations on resistant cowpea (lineage selection). In addition, virulence was selected from avirulent females (individual selection). Virulence differed (P ≤ 0.05) both within and between cohorts of IFL cultured for up to 27 generations on susceptible or resistant cowpea. Distinct virulence profiles were observed among IFL. Some remained avirulent on susceptible plants and became extinct on resistant plants; some remained virulent on resistant and susceptible plants; some changed from avirulent to virulent on resistant plants; and others changed from virulent to avirulent on susceptible plants. Also, some IFL increased in virulence on susceptible plants. Single descent lines from IFL showed similar patterns of virulence for up to six generations. These results revealed considerable genetic variation in virulence in a mitotic parthenogenetic nematode population. The frequencies of lineages with stable or changeable virulence and avirulence phenotypes determined the overall virulence potential of the population.     

Formalin Inactivation of Japanese Encephalitis Virus Vaccine Alters the Antigenicity and Immunogenicity of a Neutralization Epitope in Envelope Protein Domain III

Formalin-inactivated Japanese encephalitis virus (JEV) vaccines are widely available, but the effects of formalin inactivation on the antigenic structure of JEV and the profile of antibodies elicited after vaccination are not well understood. We used a panel of monoclonal antibodies (MAbs) to map the antigenic structure of live JEV virus, untreated control virus (UCV), formalin-inactivated commercial vaccine (FICV), and formalin-inactivated virus (FIV). The binding activity of T16 MAb against Nakayama-derived FICV and several strains of FIV was significantly lower compared to live virus and UCV. T16 MAb, a weakly neutralizing JEV serocomplex antibody, was found to inhibit JEV infection at the post-attachment step. The T16 epitope was mapped to amino acids 329, 331, and 389 within domain III (EDIII) of the envelope (E) glycoprotein. When we explored the effect of formalin inactivation on the immunogenicity of JEV, we found that Nakayama-derived FICV, FIV, and UCV all exhibited similar immunogenicity in a mouse model, inducing anti-JEV and anti-EDII 101/106/107 epitope-specific antibodies. However, the EDIII 329/331/389 epitope-specific IgG antibody and neutralizing antibody titers were significantly lower for FICV-immunized and FIV-immunized mouse serum than for UCV-immunized. Formalin inactivation seems to alter the antigenic structure of the E protein, which may reduce the potency of commercially available JEV vaccines. Virus inactivation by H₂O₂, but not by UV or by short-duration and higher temperature formalin treatment, is able to maintain the antigenic structure of the JEV E protein. Thus, an alternative inactivation method, such as H₂O₂, which is able to maintain the integrity of the E protein may be essential to improving the potency of inactivated JEV vaccines.     

A human coronavirus evolves antigenically to escape antibody immunity

There is intense interest in antibody immunity to coronaviruses. However, it is unknown if coronaviruses evolve to escape such immunity, and if so, how rapidly. Here we address this question by characterizing the historical evolution of human coronavirus 229E. We identify human sera from the 1980s and 1990s that have neutralizing titers against contemporaneous 229E that are comparable to the anti-SARS-CoV-2 titers induced by SARS-CoV-2 infection or vaccination. We test these sera against 229E strains isolated after sera collection, and find that neutralizing titers are lower against these “future” viruses. In some cases, sera that neutralize contemporaneous 229E viral strains with titers >1:100 do not detectably neutralize strains isolated 8–17 years later. The decreased neutralization of “future” viruses is due to antigenic evolution of the viral spike, especially in the receptor-binding domain. If these results extrapolate to other coronaviruses, then it may be advisable to periodically update SARS-CoV-2 vaccines.     

Characterization of Immune Responses Induced by Immunization with the HA DNA Vaccines of Two Antigenically Distinctive H5N1 HPAIV Isolates

The evolution of the H5N1 highly pathogenic avian influenza virus (HPAIV) has resulted in high sequence variations and diverse antigenic properties in circulating viral isolates. We investigated immune responses induced by HA DNA vaccines of two contemporary H5N1 HPAIV isolates, A/bar-headed goose/Qinghai/3/2005 (QH) and A/chicken/Shanxi/2/2006 (SX) respectively, against the homologous as well as the heterologous virus isolate for comparison. Characterization of antibody responses induced by immunization with QH-HA and SX-HA DNA vaccines showed that the two isolates are antigenically distinctive. Interestingly, after immunization with the QH-HA DNA vaccine, subsequent boosting with the SX-HA DNA vaccine significantly augmented antibody responses against the QH isolate but only induced low levels of antibody responses against the SX isolate. Conversely, after immunization with the SX-HA DNA vaccine, subsequent boosting with the QH-HA DNA vaccine significantly augmented antibody responses against the SX isolate but only induced low levels of antibody responses against the QH isolate. In contrast to the antibody responses, cross-reactive T cell responses are readily detected between these two isolates at similar levels. These results indicate the existence of original antigenic sin (OAS) between concurrently circulating H5N1 HPAIV strains, which may need to be taken into consideration in vaccine development against the potential H5N1 HPAIV pandemic.     

Infection Profiles of Selected Aquabirnavirus Isolates in CHSE Cells

The wide host range and antigenic diversity of aquabirnaviruses are reflected by the presence of a collection of isolates with different sero- and genotypic properties that have previously been classified as such. Differences in cytopathogenic mechanisms and host responses induced by these isolates have not been previously examined. In the present study, we investigated infection profiles induced by genetically and serologically closely related as well as distant isolates in-vitro. CHSE-214 cells were infected with either E1S (serotype A3, genogroup 3), VR-299 (serotype A1, genogroup 1), highly virulent Sp (TA) or avirulent Sp (PT) (serotype A2, genogroup 5). The experiments were performed at temperatures most optimum for each of the isolates namely 15°C for VR-299, TA and PT strains and 20°C for E1S. Differences in virus loads and ability to induce cytopathic effect, inhibition of protein synthesis, apoptosis, and induction of IFNa, Mx1, PKR or TNFα gene expression at different times post infection were examined. The results showed on one hand, E1S with the highest ability to replicate, induce apoptosis and IFNa gene expression while VR-299 inhibited protein synthesis and induced Mx1 and PKR gene expression the most. The two Sp isolates induced the highest TNFα gene expression but differed in their ability to replicate, inhibit protein synthesis, and induce gene expression, with TA being more superior. Collectively, these findings point towards the adaptation by different virus isolates to suit environments and hosts that they patronize. Furthermore, the results also suggest that genetic identity is not prerequisite to functional similarities thus results of one aquabirnavirus isolate cannot necessarily be extrapolated to another.     

Utilization of DC-SIGN for entry of feline coronaviruses into host cells

The entry and dissemination of viruses in several families can be mediated by C-type lectins such as DC-SIGN. We showed that entry of the serotype II feline coronavirus strains feline infectious peritonitis virus (FIPV) WSU 79-1146 and DF2 into nonpermissive mouse 3T3 cells can be rescued by the expression of human DC-SIGN (hDC-SIGN) and that infection of a permissive feline cell line (Crandall-Reese feline kidney) was markedly enhanced by the overexpression of hDC-SIGN. Treatment with mannan considerably reduced infection of feline monocyte-derived cells expressing DC-SIGN, indicating a role for FIPV infection in vivo.     

Novel swine virulence determinant in the left variable region of the African swine fever virus genome

Previously we have shown that the African swine fever virus (ASFV) NL gene deletion mutant E70DeltaNL is attenuated in pigs. Our recent observations that NL gene deletion mutants of two additional pathogenic ASFV isolates, Malawi Lil-20/1 and Pr4, remained highly virulent in swine (100% mortality) suggested that these isolates encoded an additional virulence determinant(s) that was absent from E70. To map this putative virulence determinant, in vivo marker rescue experiments were performed by inoculating swine with infection-transfection lysates containing E70 NL deletion mutant virus (E70DeltaNL) and cosmid DNA clones from the Malawi NL gene deletion mutant (MalDeltaNL). A cosmid clone representing the left-hand 38-kb region (map units 0.05 to 0.26) of the MalDeltaNL genome was capable of restoring full virulence to E70DeltaNL. Southern blot analysis of recovered virulent viruses confirmed that they were recombinant E70DeltaNL genomes containing a 23- to 28-kb DNA fragment of the Malawi genome. These recombinants exhibited an unaltered MalDeltaNL disease and virulence phenotype when inoculated into swine. Additional in vivo marker rescue experiments identified a 20-kb fragment, encoding members of multigene families (MGF) 360 and 530, as being capable of fully restoring virulence to E70DeltaNL. Comparative nucleotide sequence analysis of the left variable region of the E70DeltaNL and Malawi Lil-20/1 genomes identified an 8-kb deletion in the E70DeltaNL isolate which resulted in the deletion and/or truncation of three MGF 360 genes and four MGF 530 genes. A recombinant MalDeltaNL deletion mutant lacking three members of each MGF gene family was constructed and evaluated for virulence in swine. The mutant virus replicated normally in macrophage cell culture but was avirulent in swine. Together, these results indicate that a region within the left variable region of the ASFV genome containing the MGF 360 and 530 genes represents a previously unrecognized virulence determinant for domestic swine.     

Switching species tropism: an effective way to manipulate the feline coronavirus genome

Feline infectious peritonitis virus (FIPV), a coronavirus, is the causative agent of an invariably lethal infection in cats. Like other coronaviruses, FIPV contains an extremely large positive-strand RNA genome of ca. 30 kb. We describe here the development and use of a reverse genetics strategy for FIPV based on targeted RNA recombination that is analogous to what has been described for the mouse hepatitis virus (MHV) (L. Kuo et al., J. Virol. 74:1393-1406, 2000). In this two-step process, we first constructed by targeted recombination a mutant of FIPV, designated mFIPV, in which the ectodomain of the spike glycoprotein was replaced by that of MHV. This switch allowed for the selection of the recombinant virus in murine cells: mFIPV grows to high titers in these cells but has lost the ability to grow in feline cells. In a second, reverse process, mFIPV was used as the recipient, and the reintroduction of the FIPV spike now allowed for selection of candidate recombinants by their regained ability to grow in feline cells. In this fashion, we reconstructed a wild-type recombinant virus (r-wtFIPV) and generated a directed mutant FIPV in which the initiation codon of the nonstructural gene 7b had been disrupted (FIPV Delta 7b). The r-wtFIPV was indistinguishable from its parental virus FIPV 79-1146 not only for its growth characteristics in tissue culture but also in cats, exhibiting a highly lethal phenotype. FIPV Delta 7b had lost the expression of its 7b gene but grew unimpaired in cell culture, confirming that the 7b glycoprotein is not required in vitro. We establish the second targeted RNA recombination system for coronaviruses and provide a powerful tool for the genetic engineering of the FIPV genome.     

Oxidative burst elicited by Bacillus mycoides isolate Bac J, a biological control agent, occurs independently of hypersensitive cell death in sugar beet

Response of sugar beet cultivars C40 and USH11 to syringe infiltration of live and dead Bacillus mycoides isolate Bac J, a biological control agent, and virulent and avirulent isolates of Erwinia carotovora pv. betavasculorum was measured by monitoring systemic acquired resistance control of Cercospora beticola, specific activity of chitinase and beta-glucanase, the oxidative burst, and hypersensitive cell death at the infiltration site. Priming sugar beet with B. mycoides Bac J (1 x 10(8) cells/ml) and avirulent isolates of E. carotovora pv. betavasculorum (1 x 10(6) cells/ml) reduced C. beticola symptoms by nearly 70% on distal, untreated leaves. Systemic resistance responses elicited by live B. mycoides Bac J and avirulent E. carotovora pv. betavasculorum isolates, measured by assays for chitinase and beta-glucanase, were statistically equivalent, and biphasic hydrogen peroxide production was observed. Although similar in timing, the second hydrogen peroxide burst was twofold lower for B. mycoides Bac J than for avirulent E. carotovora pv. betavasculorum. Hypersensitive cell death was elicited by avirulent E. carotovora pv. betavasculorum but not B. mycoides Bac J. An oxidative burst was elicited by spray-applied B. mycoides Bac J under both light and green light conditions, indicating that the signal produced by B. mycoides Bac J was not reliant on the stomata for entry into sugar beet. A working model for signal delivery and systemic resistance induction by B. mycoides Bac J in sugar beet is proposed.     

Antibody-selected variation and reversion in Sindbis virus neutralization epitopes.

Sindbis virus variants evidencing a complex and bidirectional tendency toward spontaneous antigenic change were isolated and characterized. Variants were selected on the basis of their escape from neutralization by individual monoclonal antibodies to either of the two envelope glycoproteins, E2 and E1. Multisite variants, including one altered in three neutralization sites, were obtained by selecting mutants consecutively in the presence of different neutralizing monoclonal antibodies. Two phenotypic revertants, each of which reacquired prototype antigenicity, were back-selected on the basis of their reactivity with a neutralizing monoclonal antibody. An incidental oligonucleotide marker distinguished these and the variant from which they arose from parental Sindbis virus and other mutants, thereby confirming that the revertants were true progeny of the antigenic variant. Prototype Sindbis virus and variants derived from it were compared on the basis of their reactivities with each of a panel of monoclonal antibodies; patterns revealed a minimum of five independently mutable Sindbis virus neutralization epitopes, segregating as three antigenic sites (two E2 and one E1).     

Localization of neutralizing regions of the envelope gene of feline leukemia virus by using anti-synthetic peptide antibodies.

We synthesized 27 synthetic peptides corresponding to approximately 80% of the sequences encoding gp70 and p15E of Gardner-Arnstein feline leukemia virus (FeLV) subtype B. The peptides were conjugated to keyhole limpet hemocyanin and injected into rabbits for preparation of antipeptide antisera. These sera were then tested for their ability to neutralize a broad range of FeLV isolates in vitro. Eight peptides elicited neutralizing responses against subtype B isolates. Five of these peptides corresponded to sequences of gp70 and three to p15E. The ability of these antipeptide antisera to neutralize FeLV subtypes A and C varied. In certain circumstances, failure to neutralize a particular isolate corresponded to sequence changes within the corresponding peptide region. However, four antibodies which preferentially neutralized the subtype B viruses were directed to epitopes in common with Sarma subtype C virus. These results suggest that distal changes in certain subtypes (possibly glycosylation differences) alter the availability of certain epitopes in one virus isolate relative to another. We prepared a "nest" of overlapping peptides corresponding to one of the neutralizing regions of gp70 and performed slot blot analyses with both antipeptide antibodies and a monoclonal antibody which recognized this epitope. We were able to define a five-amino-acid sequence required for reactivity. Comparisons were made between an anti-synthetic peptide antibody and a monoclonal antibody reactive to this epitope for the ability to bind both peptide and virus, as well as to neutralize virus in vitro. Both the anti-synthetic peptide and the monoclonal antibodies bound peptide and virus to high titers. However, the monoclonal antibody had a 4-fold-higher titer against virus and a 10-fold-higher neutralizing titer than did the anti-synthetic peptide antibody. Competition assays were performed with these two antibodies adjusted to equivalent antivirus titers against intact virions affixed to tissue culture plates. The monoclonal antibody had a greater ability to compete for virus binding, which suggested that differences in neutralizing titers may relate to the relative affinities of these antisera for the peptide conformation in the native structure.