Antigenic and genetic stability of bovine immunodeficiency virus during long-term persistence in cattle experimentally infected with the BIVR29 isolate

Susan Carpenter1,2, Eric M. Vaughnb,1, Jun Yang1,2, Prasith Baccam1,3, James A. Roth1 and Yvonne Wannemuehler1

Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, 2Interdepartmental Genetics Program and 3Department of Mathematics, Iowa State University, Ames, IA 50011, USA

Author for correspondence: Susan Carpenter at College of Veterinary Medicine. Fax +1 515 294 8500. e-mail scarp{at}iastate.edu


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Experimental infection of cattle with bovine immunodeficiency virus (BIV) is characterized by persistent, low levels of virus replication in the absence of clinical disease. A virus neutralization (VN) assay was developed to examine the role of VN antibodies in controlling virus replication in cattle experimentally infected with the BIVR29 isolate of BIV. All animals developed VN antibody, but there was no correlation between VN titres and restriction of virus replication in vivo. BIV infection did not induce high-titred, cross-neutralizing antibody and there was no evidence for antigenic variation through more than 4 years in vivo. Genetic comparisons among the BIVR29 inoculum virus and viruses isolated from infected animals identified only limited genetic variation during 4 years in vivo. Moreover, there was no evidence that the observed variation was due to selection. Analyses of genetic diversity in the virus stock used for inoculation indicated a fairly homogeneous population. In the absence of high levels of virus replication and overt clinical disease, there appeared to be little selection of virus variants, resulting in antigenic and genetic stability of BIVR29 during long-term, persistent infection.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Bovine immunodeficiency virus (BIV), a member of the lentivirus subfamily of retroviruses, was originally isolated from a dairy cow with persistent lymphocytosis, lymphoid hyperplasia and perivascular cuffing in the brain (Van Der Maaten et al., 1972 ). Serological studies indicate that BIV is present worldwide (Cockerell et al., 1992 ; Forman et al., 1992 ; Horner, 1991 ; Amborski et al., 1989 ; McNab et al., 1994 ), with high seroprevalence among some herds in southeastern regions of the United States (St Cyr Coats et al., 1994 ; Snider et al.,1996 ). BIV seropositivity has been correlated with decreased milk production in dairy cattle (McNab et al., 1994 ), but has not been directly linked with clinical disease in naturally infected cattle. An increased incidence of encephalitis and secondary bacterial infections has been reported in herds with high BIV seroprevalence (Snider et al., 1996 ), and there is a report of atypical lymphosarcoma in a calf experimentally infected with BIV (Rovid et al., 1996 ). However, there has been no demonstration that BIV played a direct role in the aetiology of these syndromes. In many cases, such a demonstration is complicated by the presence of confounding factors, including co-infection with bovine leukaemia virus (BLV). Experimental infection of calves with BIV stocks derived from the original BIVR29 isolate resulted in a transient increase in lymphocytes and a lymphoid hyperplasia similar to that found early after infection with immunosuppressive lentiviruses (Carpenter et al., 1992 ; Suarez et al., 1993 ). Alterations in immune function and suppressed antibody response to other antigens have been observed (Zhang et al., 1997b ; Flaming et al.,1993 , 1997 ; Onuma et al., 1992 ). To date, however, there have been no reports of clinical disease or signs of immune suppression in BIVR29-infected cattle after more than 4 years post-infection (Flaming et al., 1997 ).

Lentivirus infections are typically characterized by the establishment of a persistent, lifelong infection and a slow, progressive disease course in the naturally infected host. There is wide variation in clinical disease both among and within the different members of the lentivirus subfamily. Two factors which influence the clinical outcome of infection are the virulence of the inoculum virus and the effectiveness of the host immune response. Reports of clinical disease syndromes in herds with a high incidence of BIV seroprevalence (Snider et al., 1996 ; St Cyr Coats et al., 1994 ) suggest that field isolates of BIV may be more pathogenic than the cell culture-adapted BIVR29 isolate. In addition, calves experimentally infected with the field-derived BIVFL112 isolate exhibited a more pronounced leukocytosis (Suarez et al., 1993 ) and higher levels of virus replication in vivo in comparison to BIVR29-infected calves (S. Carpenter & Y. Wannemuehler, unpublished observations). Attenuation of the BIVR29 isolate following extensive passage in cell culture may contribute to the lack of clinical disease observed in cattle experimentally infected with this isolate. In addition, the persistent low level of virus replication during long-term experimental infection with BIVR29 may be attributed to an effective host immune response. In the present study, we examined the role of virus-neutralizing (VN) antibody in controlling BIV replication in cattle experimentally infected with BIVR29.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Animal inoculation, serum collection and clinical monitoring.
Eight male Holstein calves, 2–4 months of age, were experimentally inoculated with 1·8x104 syncytia-forming units (SFU) of BIV propagated in foetal bovine lung cells (FBL) as previously described (Carpenter et al.,1992 ; Isaacson et al., 1995 ). Five of the eight animals, #344, 345, 347, 348 and 349, received 107 peripheral blood mononuclear cells (PBMC) from a lymphocytotic cow persistently infected with BLV as part of a separate study on the interactive effects of the two viruses. The other three calves, #340, 341 and 342, received 107 PBMC from a BLV-negative cow. The groups were housed in separate pens and monitored for signs of clinical disease. Blood was collected by jugular venipuncture on a weekly basis for the first 11 weeks post-inoculation (p.i.) and every 8–12 weeks thereafter through 4 years p.i. Haematological evaluation, including total and differential leukocyte analysis, clinical evaluation and evaluation of immune function have been reported previously (Flaming et al., 1993 , 1997 ; Rovid et al., 1995 ). Results from these in vivo studies indicated that animals co-infected with BIV and BLV did not differ from singly infected animals in any parameters of immune function, virus replication or pathological sequelae that have been examined. Therefore, the two groups are not considered separately in the analyses described herein.

{blacksquare} Cells and virus.
Primary FBL cells (Whetstone et al., 1991 ) were maintained in DMEM supplemented with 10% foetal calf serum (FCS) and antibiotics. A number of BIV stocks and/or isolates were derived from BIVR29 following in vivo and/or in vitro passage of the original virus (Van Der Maaten et al., 1972 ). The virus stock used for experimental inoculation of the eight animals was propagated in FBL cells (Carpenter et al., 1992 ) and is designated as BIVR29-4093. BIVR29-346M was derived by in vitro culture of monocyte-derived macrophages collected from a steer (#346) experimentally infected with BIVR29-4093 (Rovid et al., 1996 ). BIVFL112 (Suarez et al., 1993 ) was provided by D. Suarez. All virus stocks were maintained by passage in FBL cells. Additional in vivo isolates were obtained from experimentally infected cattle at 4 years p.i. by co-cultivation of PBMC with FBL as described below.

{blacksquare} Virus isolation.
Whole blood was collected by jugular venipuncture and PBMC were isolated by centrifugation, collection of the buffy coat, and flash lysis of erythrocytes (Roth et al., 1981 ). Approximately 107 PBMC were co-cultivated with FBL cells in the presence of 8 µg/ml polybrene (Carpenter et al., 1992 ). Cultures were passaged twice-weekly and visually monitored for syncytium formation. At each passage, replicate cultures were assayed for the presence of BIV, BLV, bovine spumavirus and bovine viral diarrhoea virus using immunofluorescence or immunoperoxidase assays as previously described (Carpenter et al., 1992 ; Wannemuehler et al., 1993 ). In most cases, cells were sub-cultured at least six times before they were considered virus-negative. Viruses isolated from experimentally infected cattle at 4 years p.i. which were used for antigenic and genetic characterization are referred to as BIVR29-341, BIVR29-342, BIVR29-345, BIVR29-347 and BIVR29-348.

{blacksquare} Virus neutralization assay.
Sera collected from BIV-infected cattle prior to or post-inoculation were heat-inactivated to destroy complement, serially diluted twofold in DMEM with 10% FCS, and used in a VN assay similar to that previously described (Carpenter et al., 1987 ). Virus stocks were prepared from clarified BIV-infected culture supernatant and were diluted to 200 SFU in a 250 µl volume. Virus was added to an equal volume of serially diluted serum, incubated for 15 min at 4 °C and inoculated in duplicate onto FBL cells seeded the previous day at a density of 2x104 cells/cm2 into 6-well tissue culture plates. Cultures were incubated at 37 °C with 5% CO2 for 24 h, at which time the BIV/serum inoculum was removed and fresh DMEM with 10% FCS was added. After 3 days, cells were fixed in methanol, and BIV-induced syncytia were detected by immunocytochemistry (ICC) using monoclonal antibody to BIV Gag as previously described (Wannemuehler et al., 1993 ). The number of BIV-specific SFU were counted and the serum dilution that resulted in at least 80% reduction in SFU as compared to negative control sera was considered to be the virus neutralization titre.

{blacksquare} Cloning and sequence analysis.
Total DNA was isolated from virus-infected FBL using SDS–proteinase K digestion. BIV env sequences were amplified by PCR using primers to conserved regions of the gene. The upstream primer sequence was 5' CTATGGATCAGGACCTAGAC (5' nt 5413) and the downstream primer sequence was 5' CAGCACAAGCAGGAATATTGC (5' nt 7092). The nucleotide sequence numbers of the primers are based on BIV127 (Garvey et al., 1990 ). Approximately 100 ng of total DNA was amplifed by PCR using the following conditions: 94 °C for 2 min, 30 cycles of 1 min denaturation at 94 °C, 1 min annealing at 50 °C, 2 min extension at 72 °C with a final 5 min extension at 72 °C. For direct sequencing of PCR products, amplified DNA was purified by column chromatography using a commercial kit (Promega) and sequenced using internal primers. For analysis of individual variants present in the BIVR29-4093 inoculum, 1 µl of amplified DNA was ligated to pCR2.1 (Invitrogen) and individual clones were isolated and sequenced. For amplification of viral sequences in the absence of selection for replication on FBL, DNA was isolated from PBMC collected from steer #348 at 4 years p.i. and a 656 nt fragment was amplified with the upstream primer 5' GTTGTCCCATATGTAGTTGGC (5' nt 5737), located in tat exon 1, and the downstream primer 5' GCAAACTTTGGAGGTATTTC (5' nt 6393), located in env. Amplification conditions were similar to those described above. The amplicon was cloned, and individual clones were picked and sequenced as above.

The program SITES, version 1.1 (Hey & Wakeley, 1997 ), was used to identify the number of synonymous (S) and non-synonymous (N) nucleotide changes, as well the number of synonymous sites and non-synonymous sites. These values were used to calculate the N/S and Kn/Ks ratios. SITES was also used to perform Tajima’s D test, which tests whether observed mutations are due to positive Darwinian selection or to neutral mutations (Tajima, 1989 ).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Development of VN antibodies in experimentally infected cattle
A VN assay was developed to examine the role of neutralizing antibody in controlling BIV replication in cattle experimentally infected with BIVR29. In initial studies, serially diluted sera samples were assayed for antibodies capable of neutralizing BIVR29-346M. This isolate, related to the BIVR29-4093 inoculum, replicates to sufficient titres in vitro for use in the VN assay. In all cases, sera collected from animals prior to experimental infection were negative for BIV-neutralizing antibody (Table 1). In seven of the eight animals, VN antibody titres were detectable by 17 weeks p.i. The single animal negative for VN antibody at 17 weeks had low but detectable titres by 22 months p.i. There was wide variability in VN antibody titres among the eight animals at all time-points examined, with maximum titres ranging from 16 (#344 and #340) to 256 (#342). In some cases, VN antibody titres increased throughout the 4 year period, with maximum titres observed at 44 months p.i. In two animals, #347 and #345, maximum titres were reached at 17 weeks p.i. and remained unchanged. Antibody titres in #348 peaked at 17 weeks p.i., declined by 22 months p.i., and increased until 44 months p.i., although titres at that time remained lower than those observed at 17 weeks p.i.


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Table 1. Neutralizing antibody titres and virus isolation from BIV-infected cattle

 
To determine the relationship between VN antibody titre and virus replication, we compared the VN titre with recovery of infectious virus from PBMC (Table 1). Results of virus isolation are expressed as the number of in vitro passages required for detection of BIV-specific syncytia. The highest VN titre was observed in #342, an animal from which BIV was consistently and readily isolated. In contrast, animals with the lowest VN titres, #340 and #344, were those which were most often negative for virus recovery. Over the 4 year period, we observed no evidence that VN antibody was effective in the clearance of BIV in vivo.

Limited antigenic variation during persistent BIV infection
Antigenic variation is a common feature of lentiviruses and the higher levels of virus replication observed in some animals (i.e. #342) may have been due to replication of virus antigenically distinct from the BIVR29-346M virus used in the neutralization assay. To explore this possibility, virus isolated from individual animals at 4 years p.i. was antigenically compared with the inoculum virus. BIVR29-342, BIVR29-345 and BIVR29-348 were used in a VN assay with sera collected from steers #342, #345 and #348 at 3 and 5 years p.i. In all three animals tested, sera collected at 3 years p.i. had neutralizing antibody to the inoculum virus, BIVR29-4093 (Table 2). Surprisingly, the same year three sera had equivalent or higher VN antibody titres to virus isolated at year 4 p.i. Moreover, VN titres to year 4 virus were essentially identical in sera collected at 3 and 5 years p.i. In all cases, VN titres to the inoculum virus and year 4 virus did not differ by more than twofold, indicating little or no antigenic variation between the inoculum virus and year 4 viruses.


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Table 2. Analysis of antigenic variation in BIV-infected cattle

 
Neutralizing antibody does not neutralize heterologous isolates of BIV
Studies with other lentiviruses have shown that later time-points after infection are characterized by a maturation of the immune response and development of broadly acting neutralizing antibody (Cole et al., 1997 ; Hammond et al., 1997 ). Therefore, the similarities in VN antibody titre to the inoculum virus and year 4 viruses may have been due to the presence of high-titred, cross-reacting neutralizing antibody. To test this possibility, sera collected from the experimentally infected animals were used in a VN assay against the heterologous BIVFL112 isolate (Suarez et al., 1993 ). No VN antibody to BIVFL112 was detected in year 5 sera samples from any of the animals inoculated with BIVR29-4093 (Table 3). To ensure that BIVFL112 was susceptible to in vitro neutralization in our assay system, serum from cattle experimentally infected with BIVFL112 or the related BIVFL491 were tested for type-specific and cross-neutralizing antibody. Sera from steer #1275, inoculated with BIVFL112, had low levels of VN antibody to BIVFL112, but no detectable VN antibody to BIVR29-4093. Partial neutralization of BIVFL112, but not BIVFL493, was observed at higher dilutions of #1275 sera. Sera from #1268, inoculated with BIVFL491, was unable to neutralize either the related BIVFL112 or the unrelated BIVR29-4093. The results indicated that BIV infection did not elicit high titres of cross-neutralizing antibody in either BIVR29-4093-inoculated steers or in calves inoculated with Florida isolates of BIV. Therefore, the comparable VN antibody titres to the BIVR29-4093 and the year 4 viruses (Table 2) reflect the antigenic similarity of these viruses rather than the presence of cross-neutralizing antibody.


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Table 3. Assessment of VN antibody to heterologous virus in sera from BIV-infected cattle

 
Limited genetic variation of BIV during long-term persistent infection
Our immunological analyses over the course of persistent infection suggested limited antigenic variation of BIV in vivo. To examine the extent of genetic variation over the same time-period, we compared the env sequence of BIVR29-4093 with the env sequences of virus isolated at 4–5 years p.i. from five of the experimentally infected steers. Virus was recovered from infected animals by co-cultivation of PBMC with FBL cells, and total DNA was isolated from virus-infected cells. Amplification of env sequences was done using primers conserved among heterologous isolates of BIV. Direct sequencing of the PCR products indicated limited sequence heterogeneity between the BIVR29-4093 inoculum and virus recovered from infected animals at 4 years p.i. (Fig. 1 and Table 4). Nucleotide diversity between BIVR29-4093 and in vivo virus ranged from 0·40% in steer #348 to 1·74% in #347. Over 75% of the nucleotide changes were non-synonymous, with the resulting amino acid variation between 1·19–5·23%. The ratio of non-synonymous to synonymous nucleotide changes (N/S) was greater than one, and the Kn/Ks ratio was greater than 0·33, suggesting that variation may have been due to positive Darwinian selection. Analysis of the five in vivo isolates using the Tajima test (Tajima, 1989 ), however, indicated that the observed variation was due to neutral mutations rather than positive selection.




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Fig. 1. Genetic heterogeneity among in vivo and in vitro isolates of BIV. The env sequences of BIVR29-4093, BIVR29-341, BIVR29-342, BIVR29-345, BIVR29-347 and BIVR29-348 were obtained from cell culture-propagated virus by direct sequencing of the PCR-amplified proviral DNA. The 348-A1, 348-A2, 348-A3, 348-A5, 348-B1 and 348-B2 env sequences are individual clones obtained from PCR amplification of proviral DNA isolated directly from PBMC collected from steer #348 at 4 years p.i. The 4093-E1, 4093-E5, 4093-E9, 4093-E10, 4093-E11 and 4093-E12 sequences are individual clones obtained following PCR amplification of proviral DNA isolated from BIVR29-4093-infected FBL cells. The sequence of BIV127 (Garvey et al., 1990 ), including the V2 insertion, was obtained from GenBank. Amino acid substitutions are indicated by the single letter designation. Dots represent amino acid identity and dashes indicate deletions. Previously described conserved and variable regions (Suarez & Whetstone, 1995 ) are indicated by double underlines and shaded boxes, respectively.

 

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Table 4. In vivo and in vitro variation in the BIV SU protein

 
The env sequences represent the predominant genotype of virus selected for in vitro replication in FBL cells and may not represent the predominant genotype in vivo. A more direct analysis of virus genotypes present in plasma or PBMC is difficult due to the low levels of BIV replication during persistent infection (Carpenter et al., 1992 ). However, it was important to determine if the env sequences obtained from virus propagated in FBL were reflective of virus sequences present in vivo. DNA was isolated from PBMC obtained from steers at 4 years p.i. and used to directly amplify env sequences present in vivo. In one of the animals, #348, we were able to amplify a 322 nt fragment from the 5' env gene. Although this region did not contain the size-variable V2 region of env, it did allow us to determine if sequences obtained directly from PBMC were similar to those amplified from the infected cell culture. The amplicon was cloned, and six individual clones were sequenced for comparison with the BIVR29-4093 and BIVR29-348 consensus sequences obtained by direct sequencing of the PCR-amplified product. The six sequences obtained directly from PBMC were similar to the consensus sequences of virus propagated in cell culture (Fig. 1 and Table 4). At the amino acid level, the percent divergence between the individual clones and BIVR29-4093 ranged from 0 to 3·4%, with the average divergence closely matching that observed for the entire BIVR29-348 consensus sequence. The individual clones differed from the BIVR29-348 consensus sequence by only one or two amino acids. The overall similarity among the consensus sequences and the individual clones indicates that the consensus sequences obtained following in vitro culture are reflective of sequences present in vivo. Moreover, the similarity between the in vivo-derived clones and the BIVR29-4093 inoculum supports our finding of genetic conservation of BIV during long-term persistent infection.

Analysis of genetic diversity in the R29-4093 inoculum
The extent of genetic diversity of env in the BIVR29-4093 stock was determined by PCR amplification, cloning and sequencing individual clones. Differences between individual clones and the BIVR29-4093 consensus sequence ranged from 0·26 to 0·72% divergence at the nucleotide level and 0·78 to 1·76% divergence at the amino acid level (Fig. 1 and Table 4). The total number of nucleotide substitutions/deletions found in BIVR29-4093 was only slightly less than that observed in the year 4 virus isolates (Table 4). It is possible that the observed nucleotide substitutions occurred during PCR amplification; however, the error rate of Taq polymerase using our standard laboratory conditions is 0·025%, about 20-fold less than the variation observed among the analysed sequences. Therefore, these findings suggest that BIV is genetically conserved during long-term persistent infection.

Previous studies of variation in the BIV surface (SU) protein have reported sequence divergence of up to 50% among unrelated BIV isolates (Suarez & Whetstone, 1995 ). In addition to nucleotide substitutions, insertions/duplications within the hypervariable V2 domain can result in a large amount of SU protein size variation (Garvey et al., 1990 ; Suarez & Whetstone, 1995 , 1997 ). Comparison of BIVR29-related sequences and sequences obtained from heterologous field isolates BIVFL112 and BIVOK40 of BIV demonstrate that BIVR29-related isolates are genetically quite diverse from the more recently described BIV genotypes (Fig. 2). In addition, we did not detect amino acid insertions in the V2 domain in any of the BIVR29-4093 variants or in vivo isolates (Figs 1 and 2). In fact, more than half of the observed amino acid differences among the BIVR29 variants were found to lie outside the previously described variable regions (Suarez & Whetstone, 1995 ). Therefore, both quantitative and qualitative differences in variation were observed in BIVR29-4093-infected cattle as compared to cattle infected with more recently described field isolates of BIV.



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Fig. 2. Genetic heterogeneity in BIV SU among characterized isolates of BIV. The SU sequences of BIVR29-4093, BIV127, BIVFL112 and BIVOK40 are represented by a horizontal bar. Each vertical line represents a single amino acid substitution from the BIVR29-4093 sequence. Stippled areas indicate deletions as compared to the larger genotype. BIV127, BIVFL112 and BIVOK40 sequences were obtained from GenBank.

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
Longitudinal studies of cattle experimentally infected with the BIVR29-related isolates have demonstrated virus persistence through 4 years post-infection with no progression to overt clinical disease (Flaming et al., 1997 ; Isaacson et al., 1995 ; Zhang et al., 1997a ). One possible reason why BIVR29-infected animals remain non-progressors is that BIVR29 is inherently apathogenic, with low levels of replication resulting in poor spread of the virus in vivo. In addition, an effective host immune response may control BIV replication and delay or prevent the onset of clinical disease. In the present study, a VN assay was developed in order to assess the role of BIV-neutralizing antibodies in controlling virus replication in vivo. During a 4 year period, eight of eight cattle experimentally infected with BIVR29-4093 developed neutralizing antibodies; however, differences were observed in neutralizing titre and rate at which neutralizing antibodies appeared. Higher-titred (>=128) neutralizing antibody appeared faster in the animals from which virus was most easily and consistently recovered. The appearance of neutralizing antibody was delayed, and peak titres were lower, in animals from which virus was not as readily recovered. Virus recovered from infected animals at 4 years p.i. was antigenically and genetically similar to the inoculum virus, and there was little evidence of immune selection by neutralizing antibody. Together, these results suggest that VN antibody does not play the major role in restriction of BIVR29 replication in vivo. It remains possible that an effective cell-mediated immune response is an important mechanism of immune control of BIVR29 replication in vivo.

Broadly reacting neutralizing antibodies are found in human immunodeficiency virus type 1-infected long-term non-progressors and may contribute to the lack of disease progression in these individuals (Cao et al., 1997 ). In the present study, the neutralizing antibody response in BIV-infected cattle exhibited a fairly narrow specificity and only low levels of cross-reacting neutralizing antibody were detected. Despite this narrow specificity, VN antibody did not appear to contribute to immune selection of antigenically variant virus. The twofold differences in VN titre to the inoculum virus and the year 4 viruses are within the range of experimental error and cannot be considered as evidence of antigenic variation. Antigenic variation in the absence of immune pressure by neutralizing antibody has been reported in other lentivirus infections (Carpenter et al., 1987 ; Cheevers et al., 1993 , 1999 ; Leroux et al., 1997 ). It is possible that other immune mechanisms exert selective pressure in vivo and that antigenic differences between BIVR29-4093 and year 4 viruses may be detectable by cytotoxic T lymphocytes and/or monoclonal antibodies. However, given the limited antigenic diversity observed, it was surprising that the host immune response failed to clear the virus after more than 4 years post-infection.

Previous studies of BIV variation in cattle experimentally infected with the Florida isolates of BIV identified nucleotide substitutions as well as insertions/duplications in the V2 region of the env gene (Suarez et al., 1995 ; Suarez & Whetstone, 1997 ). The BIVR29-4093 inoculum was derived from the same BIVR29 stock previously shown to contain at least two size variants, BIV106 and BIV127 (Garvey et al., 1990 ). BIV127 contains 29 additional amino acids in the V2 region of env as compared to BIV106 (Garvey et al., 1990 ). If the larger genotype has a selective advantage in vivo it might be expected to become the predominant genotype after 4 years in vivo. The only genotype detected in our studies was the smaller, BIV106-like, genotype. It is possible that the larger genotype was present in vivo, but was selected against during in vitro isolation in FBL cells. However, other studies have demonstrated selection of larger genotypes during in vitro replication of isolates containing mixed populations of larger and smaller genotypes (Suarez & Whetstone, 1997 ). Further studies are needed to clarify the role of the V2 region in BIV replication in vivo and in vitro.

Lentivirus infections are characterized by a high rate of genetic variation in vivo, and the genetic conservation in BIV env over a 4 year period in vivo was not expected. The percentage of non-synonymous nucleotide substitutions over 4 years in vivo was only slightly higher than that observed in the in vitro population. Although the ratios of N/S and Kn/Ks were higher than expected for random substitutions, the Tajima test indicated that the changes were due to neutral mutations. Moreover, the changes which did occur in vivo were not clustered in previously described hypervariable regions (Suarez & Whetstone, 1995 ), as might have been expected if the changes arose as a result of selective pressure. Together, these results strongly suggest that there was little selective pressure for virus variation in vivo. It is possible that the attenuation of BIVR29 following long-term culture in vitro, together with limited genetic diversity in the inoculum, contributed to the persistence of non-pathogenic virus genotypes in vivo. In the absence of high levels of virus replication and overt clinical disease, limited generation and selection of virus variants could result in antigenic and genetic stability of BIVR29 during long-term persistent infection. In vivo studies with BIVFL112 and BIVFL491 suggest that these isolates are somewhat more pathogenic than BIVR29-related isolates (Suarez et al., 1993 ), and it might be expected that other isolates of BIV are characterized by a higher rate of genetic and antigenic variation than observed in the present study. Delineating the factors which contribute to the genetic stability and persistence of BIV in vivo may provide insight into strategies that restrict lentivirus-associated disease in humans and animals.


   Acknowledgments
 
We thank Tom Skadow for excellent technical assistance and Janice Miller and David Suarez for providing serum samples. This work was partially supported by PHS grants CA50159 and CA59125.


   Footnotes
 
b Present address: Boehringer Ingelheim Laboratories, Inc., Ames, IA 50010, USA.


   References
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Introduction
Methods
Results
Discussion
References
 
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Received 8 November 1999; accepted 25 February 2000.



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