Multiple Viral Variants Research Articles

847 ACH-2684 DEMONSTRATES POTENT VIRAL SUPPRESSION IN GENOTYPE 1 HEPATITIS C PATIENTS WITH AND WITHOUT CIRRHOSIS: SAFETY, PHARMACOKINETIC, AND VIRAL KINETIC ANALYSIS

Background: ACH-2684 is a second-generation HCV NS3 protease inhibitor in Phase Ib development for the treatment of chronic HCV infection. ACH-2684 demonstrates potent activity in vitro against genotype-1 (GT1) HCV and, as compared to other protease inhibitors, has improved activity against multiple viral variants. Presented here are phase I study results in cirrhotic and non-cirrhotic patients with chronic HCV GT1 infection. Methods: ACH-2684 or placebo was administered to non-cirrhotic (n = 30) and cirrhotic (n = 8) patients with chronic HCV GT1 infection. Pharmacokinetic, (PK) viral load and safety assessments were collected on Days 1 through 35. Serial blood samples were collected on Day 1 and Day 3, and plasma non-compartmental pharmacokinetic parameters were determined. Drug efficiency was estimated by applying a single population viral dynamics model (Neumann et al, Science 1998, 282,103). Results: ACH-2684 was safe and well tolerated in non-cirrhotic and cirrhotic patients. There were no serious adverse events, and no discontinuations for safety concerns. Mean maximum reductions in plasma HCV RNA were 3.36, 3.73 and 4.16 log10 (IU/ml) at doses of 100 mg daily, 400 mg daily and 400 mg twice daily, respectively. In cirrhotic patients administered 400 mg daily, mean maximum reduction was 3.67 log10 (IU/ml). The systemic exposures were generally greater than doseproportional for the dose range studied in non-cirrhotics, and approximately 2-fold higher in cirrhotic versus non-cirrhotic patients. The drug efficiency of the 400 mg daily dose in non-cirrhotic and cirrhotic subjects was similar at 0.9993 ± 0.0005 and 0.9991 ± 0.0007, respectively. Conclusions: ACH-2684 was safe and well-tolerated, and produced more than a 3 log10 (IU/ml) mean maximum reduction in plasma HCV RNA levels in all groups. A 400 mg dose of ACH-2684 yielded approximately a 2-fold higher exposure in cirrhotics than non-cirrhotics, but was still safe and well-tolerated, and was equally effective in reducing HCV RNA levels. Viral kinetics parameters were robust and demonstrated high drug efficiency. These data provide evidence that ACH-2684 is a safe and well-tolerated protease inhibitor with potent anti-viral activity in both cirrhotic and non-cirrhotic patients with chronic GT-1 HCV infection, making it a promising candidate for future treatment of chronic HCV infection.

Impact of Mucosal Inflammation on Oral Simian Immunodeficiency Virus Transmission

Mucosal tissues are the primary route of transmission for most respiratory and sexually transmitted diseases, including human immunodeficiency virus (HIV). There is epidemiological evidence that genital mucosal inflammation leads to enhanced HIV type 1 (HIV-1) transmission. The objective of this study was to assess the influence of periodontal inflammation on oral HIV transmission using a nonhuman primate model of teeth ligature-induced periodontitis. Simian immunodeficiency virus (SIV) was nontraumatically applied to the gingiva after moderate gingivitis was identified through clinical and immunologic analyses (presence of inflammatory cytokines). Overall oral SIV infection rates were similar in the gingivitis-induced and control groups (5 infections following 12 SIV administrations for each), although more macaques were infected with multiple viral variants in the gingivitis group. SIV infection also affected the levels of antiviral and inflammatory cytokines in the gingival crevicular fluid, and a synergistic effect was observed, with alpha interferon and interferon-inducible protein 10 undergoing significant elevations following SIV infection in macaques with gingivitis compared to controls. These increases in antiviral and inflammatory immune modulators in the SIV-infected gingivitis macaques could also be observed in blood plasma, although the effects at both compartments were generally restricted to the acute phase of the infection. In conclusion, while moderate gingivitis was not associated with increased susceptibility to oral SIV infection, it resulted in elevated levels of cytokines in the oral mucosa and plasma of the SIV-infected macaques. These findings suggest a synergy between mucosal inflammation and SIV infection, creating an immune milieu that impacts the early stages of the SIV infection with potential implications for long-term pathogenesis.

HIV-1 Subtypes and Recombinants in Northern Tanzania: Distribution of Viral Quasispecies

This study analyzed the distribution and prevalence of HIV-1 subtypes, multiplicity of HIV-1 infection, and frequency of inter-subtype recombination among HIV-1-infected female bar and hotel workers in Moshi, Kilimanjaro Region, Tanzania, from 2004 to 2007. The HIV-1 viral sequences spanning the V1-C5 region of HIV-1 env gp120 were analyzed from 50 subjects by single genome amplification and sequencing (SGA/S) technique. A total of 1740 sequences were amplified and sequenced from the HIV-1 proviral DNA template. The median env sequences analyzed per subject per two time points was 38 (IQR 28–50) over one year of HIV infection. In a subset of 14 subjects, a total of 239 sequences were obtained from HIV-1 RNA template at the baseline visit. The most prevalent HIV-1 subtypes were A1 (56%) and C (30%), while HIV-1 subtype D and inter-subtype recombinant viruses were found in 6% and 8% of subjects respectively. Transmission of multiple HIV-1 variants was evident in 27% of the subjects infected with pure HIV-1 subtypes A1, C, or D. The HIV-1 inter-subtype recombinants were found in 8% including HIV-1 C/A, D/A, and complex mosaic recombinants. Multiple viral variants were found in two subjects infected with inter-subtype recombinants. One subject harbored quasispecies of both pure HIV-1 A1 and C/A recombinant. The other subject was infected with two complex mosaic inter-subtype recombinant variants belonging to subtype D. HIV-1 multiple infections and ongoing recombination contribute significantly to the genetic diversity of circulating HIV-1 in Tanzania and have important implications for vaccine design and the development of therapeutic strategies.

Detection of Minority Resistance during Early HIV-1 Infection: Natural Variation and Spurious Detection rather than Transmission and Evolution of Multiple Viral Variants

Reports of a high frequency of the transmission of minority viral populations with drug-resistant mutations (DRM) are inconsistent with evidence that HIV-1 infections usually arise from mono- or oligoclonal transmission. We performed ultradeep sequencing (UDS) of partial HIV-1 gag, pol, and env genes from 32 recently infected individuals. We then evaluated overall and per-site diversity levels, selective pressure, sequence reproducibility, and presence of DRM and accessory mutations (AM). To differentiate biologically meaningful mutations from those caused by methodological errors, we obtained multinomial confidence intervals (CI) for the proportion of DRM at each site and fitted a binomial mixture model to determine background error rates for each sample. We then examined the association between detected minority DRM and the virologic failure of first-line antiretroviral therapy (ART). Similar to other studies, we observed increased detection of DRM at low frequencies (average, 0.56%; 95% CI, 0.43 to 0.69; expected UDS error, 0.21 ± 0.08% mutations/site). For 8 duplicate runs, there was variability in the proportions of minority DRM. There was no indication of increased diversity or selection at DRM sites compared to other sites and no association between minority DRM and AM. There was no correlation between detected minority DRM and clinical failure of first-line ART. It is unlikely that minority viral variants harboring DRM are transmitted and maintained in the recipient host. The majority of low-frequency DRM detected using UDS are likely errors inherent to UDS methodology or a consequence of error-prone HIV-1 replication.

Hepatitis B virus (HBV) X gene diversity and evidence of recombination in HBV/HIV co‐infected persons

The high frequency of mutation during hepatitis B virus (HBV) infection has resulted in 8 genotypes (A-H) with varying effects on disease severity and treatment efficacy. However, analysis of intrapatient HBV diversity is limited, especially during HIV co-infection. Therefore, a preliminary study was performed to analyze HBV X gene diversity in 17 HBV/HIV co-infected individuals. Phylogenetic analysis revealed HBV genotype A in 13 individuals (76.5%) or genotype E in 1 individual (5.9%). Additionally, 3 individuals were dually infected with HBV genotypes A and G (17.6%). Overall, higher genetic distance and entropy were observed in the X region and overlapping polymerase (Pol(X)) regions when compared to the PreS, S, and overlapping polymerase (Pol(PS) and Pol(S)) regions analyzed in the same patients as part of a previous study. In addition, multiple viral variants from 2 individuals with dual HBV infection did not group with either genotype A or G by phylogenetic analysis, indicating possible recombination. SimPlot bootscan analysis confirmed recombination breakpoints within the X gene in both individuals. Recombination between HBV genotypes may represent an important evolutionary strategy that enhances overall pathogenic potential and/or alters the downstream effects of the HBV X protein.

Transmission of Single and Multiple Viral Variants in Primary HIV-1 Subtype C Infection

To address whether sequences of viral gag and env quasispecies collected during the early post-acute period can be utilized to determine multiplicity of transmitted HIV's, recently developed approaches for analysis of viral evolution in acute HIV-1 infection [1], [2] were applied. Specifically, phylogenetic reconstruction, inter- and intra-patient distribution of maximum and mean genetic distances, analysis of Poisson fitness, shape of highlighter plots, recombination analysis, and estimation of time to the most recent common ancestor (tMRCA) were utilized for resolving multiplicity of HIV-1 transmission in a set of viral quasispecies collected within 50 days post-seroconversion (p/s) in 25 HIV-infected individuals with estimated time of seroconversion. The decision on multiplicity of HIV infection was made based on the model's fit with, or failure to explain, the observed extent of viral sequence heterogeneity. The initial analysis was based on phylogeny, inter-patient distribution of maximum and mean distances, and Poisson fitness, and was able to resolve multiplicity of HIV transmission in 20 of 25 (80%) cases. Additional analysis involved distribution of individual viral distances, highlighter plots, recombination analysis, and estimation of tMRCA, and resolved 4 of the 5 remaining cases. Overall, transmission of a single viral variant was identified in 16 of 25 (64%) cases, and transmission of multiple variants was evident in 8 of 25 (32%) cases. In one case multiplicity of HIV-1 transmission could not be determined. In primary HIV-1 subtype C infection, samples collected within 50 days p/s and analyzed by a single-genome amplification/sequencing technique can provide reliable identification of transmission multiplicity in 24 of 25 (96%) cases. Observed transmission frequency of a single viral variant and multiple viral variants were within the ranges of 64% to 68%, and 32% to 36%, respectively.

Long-distance airborne transport of infectious PRRSV and Mycoplasma hyopneumoniae from a swine population infected with multiple viral variants

Airborne transport of porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae ( M hyo) has been reported out to 4.7 km. This study attempted to determine whether this event could occur over longer distances and across multiple viral variants. To accomplish this goal, a mixed infection of 3 PRRSV variants (1-8-4, 1-18-2 and 1-26-2) and M hyo 232 was established in a source population of growing pigs. Over 21-day period, air samples were collected from the source population and at designated distances from the herd. Samples were tested for PRRSV RNA and M hyo DNA by PCR and if positive, further characterized. In exhaust air from the source population, PRRSV and M hyo were detected in 21 of 21 and 8 of 21 air samples, respectively. Five of 114 (4.4%) long-distance air samples were positive for PRRSV and 6 of 114 (5.2%) were positive for M hyo. The 5 PRRSV-positive samples were collected at 2.3, 4.6, 6.6 and 9.1 km from the herd. All contained infectious virus and were >99.2% homologous to PRRSV 1-8-4. No evidence of PRRSV 1-18-2 or 1-26-2 was detected in long-distance samples. All 6 M hyo-positive samples were 99.9% homologous to M hyo 232 and 3 samples (collected at 3.5, 6.8 and 9.2 km from the herd) were infectious. These results indicate that airborne transport of PRRSV 1-8-4 and M hyo 232 occurs over longer distances than previously reported and that both pathogens remained infectious.

Lack of viral selection in human immunodeficiency virus type 1 mother-to-child transmission with primary infection during late pregnancy and/or breastfeeding

Mother-to-child transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1) as described for women with an established infection is, in most cases, associated with the transmission of few maternal variants. This study analysed virus variability in four cases of maternal primary infection occurring during pregnancy and/or breastfeeding. Estimated time of seroconversion was at 4 months of pregnancy for one woman (early seroconversion) and during the last months of pregnancy and/or breastfeeding for the remaining three (late seroconversion). The C2V3 envelope region was analysed in samples of mother-child pairs by molecular cloning and sequencing. Comparisons of nucleotide and amino acid sequences as well as phylogenetic analysis were performed. The results showed low variability in the virus population of both mother and child. Maximum-likelihood analysis showed that, in the early pregnancy seroconversion case, a minor viral variant with further evolution in the child was transmitted, which could indicate a selection event in MTCT or a stochastic event, whereas in the late seroconversion cases, the mother's and child's sequences were intermingled, which is compatible with the transmission of multiple viral variants from the mother's major population. These results could be explained by the less pronounced selective pressure exerted by the immune system in the early stages of the mother's infection, which could play a role in MTCT of HIV-1.

Evolution of proviral gp120 over the first year of HIV-1 subtype C infection

The evolution of proviral gp120 during the first year after seroconversion in HIV-1 subtype C infection was addressed in a case series of eight subjects. Multiple viral variants were found in two out of eight cases. Slow rate of viral RNA decline and high early viral RNA set point were associated with a higher level of proviral diversity from 0 to 200 days after seroconversion. Proviral divergence from MRCA over the same period also differed between subjects with slow and fast decline of viral RNA, suggesting that evolution of proviral gp120 early in infection may be linked to the level of viral RNA replication. Changes in the length of variable loops were minimal, and length reduction was more common than length increase. Potential N-linked glycosylation sites ranged ±one site, showing common fluctuations in the V4 and V5 loops. These results highlight the role of proviral gp120 diversity and diversification in the pathogenesis of acute HIV-1 subtype C infection.

Compendium of Animal Rabies Prevention and Control, 2008

sive encephalitis caused by a Lyssavirus. Although the United States has been declared free of canine rabies virus variant transmission, multiple viral variants are maintained in wild mammal populations, and there is always a risk of reintroduction of canine rabies. 2 All mammals are believed to be susceptible to the disease, and for purposes of this document, use of the term animal refers to mammals. The recommendations in this compendium serve as a basis for animal rabies-prevention and -control programs throughout the United States and facilitate standardization of procedures among jurisdictions, thereby contributing to an effective national rabies-control program. This document is reviewed annually and revised as necessary, and the most current version replaces all previous versions. These recommendations do not supersede state and local laws or requirements. Principles of rabies prevention and control are detailed in Part I; Part II contains recommendations for parenteral vaccination procedures; all animal rabies vaccines licensed by the USDA and marketed in the United States are listed in Part III.

Molecular and functional analysis of occult hepatitis B virus isolates from patients with hepatocellular carcinoma

Occult HBV infection is characterized by the persistence of HBV DNA in the liver of individuals negative for HBV surface antigen (HBsAg). Occult HBV may exist in the hepatocytes as a free genome, although the factors responsible for the very low viral replication and gene expression usually observed in this peculiar kind of infection are mostly unknown. Aims of this study were to investigate whether the viral genomic variability might account for the HBsAg negativity and the inhibition of the viral replication in occult HBV carriers, and to verify in vitro the replication capability of occult HBV strains. We studied liver viral isolates from 17 HBV patients, 13 with occult infection and 4 HBsAg-positive. Full-length HBV genomes from each case were amplified and directly sequenced. Additionally, full-length HBV DNA from eight occult-HBV and two HBsAg-positive cases were cloned and sequenced. Finally, three entire, linear HBV genomes from occult cases were transiently transfected in HuH7 cells. Direct sequencing showed the absence of mutations capable of interfering with viral replication and gene expression in the major viral population of each case. Cloning experiments showed highly divergent HBV strains both in HBsAg-positive and HBsAg-negative individual cases (range of divergence 1.4%-7.1%). All of the 3 transfected full-length HBV isolates showed normal patterns of replication in vitro. Multiple viral variants accumulate in the liver of occult HBV-infected patients. Occult HBV strains are replication-competent in vitro, suggesting that host, rather than viral factors are responsible for cryptic HBV infection.

Wide Range of Quasispecies Diversity during Primary Hepatitis C Virus Infection

Hepatitis C virus (HCV) infections may be initiated by multiple infectious particles, resulting in a genetically heterogeneous viral population, or by a single particle, leading to a clonal population in the initial stage of infection. To determine which of these scenarios is most common, we evaluated the genetic diversity of HCV quasispecies in 12 seronegative subjects with primary infection following community exposures, six acutely infected recipients of HCV-seropositive blood transfusions and six seropositive individuals with infections of undetermined durations. RNA isolated from plasma and a region of the HCV envelope gene including the first hypervariable region (HVR-1) was reverse transcription-PCR amplified and subcloned, and multiple plasmid clones were sequenced. Phylogenetic analysis indicated that all HCV variants clustered by individuals. Genetic distances among HCV variants within recently infected subjects ranged from 1 to 7.8%. On the basis of the estimated mutation rate of HCV in vivo and the Taq polymerase error rate, primary infection viral quasispecies were classified as genetically heterogeneous when the maximum sequence divergence between genetic variants in the same person was >3%. Heterogeneous quasispecies were detected in 4 of 12 preseroconversion subjects, 1 of 6 transfusion recipients, and 4 of 6 seropositive subjects. The high level of viral quasispecies genetic diversity found in at least a third of recently infected individuals is consistent with the transmission of multiple infectious particles. Community-acquired HCV infection, predominantly the result of needle sharing by injection drug users, therefore appears to be frequently initiated by the successful transmission of multiple viral variants.

HIV vaccine development by computer assisted design: the GAIA vaccine

The design of epitope-driven vaccines that address the global variability of HIV has been significantly hampered by concerns about conservation of the vaccine epitopes across clades of HIV. We developed two computer-driven methods for improving epitope-driven HIV vaccines: the Epi-Assembler, which derives representative or “immunogenic consensus sequence” (ICS) epitopes from multiple viral variants, and VaccineCAD, which reduces junctional immunogenicity when epitopes are aligned in a string-of-beads format for insertion in a DNA expression vector. In this study, we report on 20 ICS HIV-1 peptides. The core 9-mer contained in these consensus peptides was conserved in 105–2250 individual HIV-1 strains. Nineteen of the 20 ICS epitopes (95%) evaluated in this study were confirmed in ELISpot assays using peripheral blood monocytes obtained from 13 healthy HIV-1 infected subjects. Twenty-five ICS peptides (all 20 of the peptides evaluated in this study and 5 additional ICS epitopes) were then aligned in a pseudoprotein string using “VaccineCAD”, an epitope alignment tool that eliminates immunogenicity created by the junctions between the epitopes. Reordering the construct reduced the immunogenicity of the junctions between epitopes as measured by EpiMatrix, an epitope mapping algorithm. The reordered construct was also a more effective immunogen in vivo when tested in HLA-DR transgenic mice. These data confirm the utility of bioinformatics tools to design novel vaccines containing “immunogenic consensus sequence” T cell epitopes for a globally relevant vaccine against HIV.

Identification of modifiable factors that affect the genetic diversity of the transmitted HIV-1 population.

Our previous studies have shown that the majority of African women were infected with multiple HIV-1 genetic variants, while in the remaining women only a single viral genotype was detected early in infection. Infection with multiple viral variants was associated with higher plasma HIV-1 RNA levels and faster CD4 T-cell decline. Socio-behavioral characteristics, use of hormonal contraceptives, and the presence of sexually transmitted diseases were prospectively assessed at approximately monthly intervals around the time of HIV-1 acquisition in female sex workers in Kenya. We assessed the relationship between these factors and HIV-1 genetic complexity early in infection. One hundred and fifty-six women were included in this analysis, of whom 89 had multiple viral genotypes and 67 had a single genotype at primary infection. Women with multiple variants were more likely to have a genital tract infection [odds ratio (OR), 4.7; 95% confidence interval (CI), 1.4-18.1] or to be using hormonal contraceptives (OR, 2.7; 95% CI, 1.3-5.6) at the time of their infection than those with a single variant. In multivariate analyses, these factors were independent predictors of early HIV-1 genetic complexity, and the presence of multiple viral variants early in infection remained significantly associated with a higher steady state plasma HIV-1 RNA level. The presence of genital tract infections and hormonal contraceptive use at the time of transmission were associated with the acquisition of multiple HIV-1 variants.

HIV type 1 chemokine receptor usage in mother-to-child transmission.

To investigate the role of the HIV-1 phenotype in mother-to-child HIV-1 transmission, we evaluated coreceptor usage and replication kinetics in chemokine receptor-expressing U87MG.CD4 cells of primary isolates from 32 HIV-1-infected mothers of Italian origin, none under preventive antiretroviral therapy, and from their infected infants. Five of 15 mothers of infected children and 2 of 17 mothers of uninfected children harbored viruses able to use CXCR4 as coreceptor. However, all isolates used CCR5, alone or in association with CXCR4. The replicative capacity in coreceptor-expressing cells of the viral isolates did not differ between the two groups of mothers. All mothers with an R5 virus transmitted a virus with the same coreceptor usage, whereas those four with a multitropic virus transmitted such a virus in one case. Although the presence of a mixed viral population was documented in the mothers, we did not observe transmission solely of X4 viruses. Interestingly, the only child infected with a multitropic virus carried a defective CCR5 allele. Analysis of the env V3 region of the provirus from this child revealed infection with multiple viral variants with a predominance of R5-type over X4-type sequences. These findings show that CCR5 usage of a viral isolate is not a discriminating risk factor for vertical transmission. Furthermore, X4 viruses can be transmitted to the newborn, although less frequently. In particular, we document the transmission of multiple viral variants with different coreceptor usage in a Delta32 CCR5 heterozygous child, and demonstrate that the heterozygous genotype per se does not contribute to the restriction of R5-type virus spread.

V3 sequence diversity of HIV-1 subtype E in infected mothers and their infants.

To elucidate genetic characteristics of HIV-1 subtype E involved in vertical transmission, V3 regions of HIV-1 subtype E isolated from 17 infected mothers (M1-M17) and their infants (I1-I17) at 1 month after birth were sequenced after cloned into pCRII vectors. At least three clones of each sample were collected. All mothers were asymptomatic and had been infected through a heterosexual route. Nine infants (I9-I17) showed mild symptomatic and immunosuppression within the first year of life. The interpatient nucleotide distance of mothers and infants in this group (0.065+/-0.008) were of greater diversity than those of a nonimmunosuppression group (0.039+/-0.006) by a significant amount (Fischer's exact test, p = .003). The substitution with asparagine (N) at threonine (T) at position 13 and aspartic acid (D) at position 29 of the V3 sequence were significantly associated with nonimmunosuppression in the first year of life (F-test, p = 0.003). Either a single or multiple viral variants could transmit from mothers to their infants.

Long terminal repeat and nef gene variants of human immunodeficiency virus type 1 in perinatally infected long-term survivors and rapid progressors.

HIV-1 sequences from perinatally infected children were analyzed in the long terminal repeat (LTR) region and nef in order to investigate associations of viral variation and disease progression. Four long-term survivors who reached 10 years of age or older, and four rapid progressors who survived less than 2 years, participated in this study. LTR sequences of multiple independent viral variants from each individual were compared. No sequence pattern within the LTR consistently distinguished long-term survivors from rapid progressors or vice versa. Deletions and insertions within transcription factor binding sites of the LTR and nef ranging from 8 to 341 bp were found in viral variants from the eldest long-term survivor (LTS047). These deletions and duplications may be associated with the survival of LTS047 via an unknown mechanism. Among all children in this study, the sites in the untranslated region (NF-kappaB, SP1, and TATA box) were more conserved than the sites in the nef/LTR overlap region (NFAT, purine-rich region, USF, TCF1alpha), reflecting the importance of the sites in the untranslated region for viral replication. A mutation in the E box motif within the USF site among the sequences from a long-term survivor (LTS113) is predicted to disrupt protein binding and may be associated with slow disease progression. Mutations of the SP1-III site in a rapid progressor (RP056) indicate that this site is not necessary for rapid disease progression.

Coinfection with multiple strains of the Epstein-Barr virus in human immunodeficiency virus-associated hairy leukoplakia.

Epstein-Barr virus DNA was analyzed from specimens of hairy leukoplakia, an oral lesion that occurs in patients infected with the human immunodeficiency virus. The simultaneous presence of both type 1 and type 2 Epstein-Barr virus was demonstrated by Southern blot analysis and polymerase chain reaction assay. Restriction fragment length polymorphisms in the BamHI WYH region and in clones of the EcoRI C region suggested the presence of multiple strains of type 1 and type 2 viruses. The demonstration of multiple variably sized BamHI H fragments on Southern blot analysis and cloning of the EBNA-2 gene coding region also suggested the presence of multiple viral strains or variants coinfecting hairy leukoplakia. Recombination of the viral genome in and around the EBNA-2 gene apparently generated viral variants that replicated efficiently, one of which appeared to increase in abundance in a lesion over time. These data indicate that hairy leukoplakia involves coinfection with multiple strains of replicating Epstein-Barr virus and the endogenous generation of viral variants, some of which have mutations of the EBNA-2 gene.