Can You Mono Again From Another Strain

Multiple Epstein-Barr Virus Strains in Patients with Infectious Mononucleosis: Comparison of Ex Vivo Samples with In Vitro Isolates by Use of Heteroduplex Tracking Assays

Rosemary J. Tierney,

1Cancer Inquiry Uk Institute for Cancer Studies, University of Birmingham, Birmingham, U.k.;

Reprints or correspondence: Prof. Alan Rickinson, CRUK Constitute for Cancer Studies, Academy of Birmingham, Edgbaston, Birmingham, B15 2TT, Uk (a.b.rickinson@bham.ac.uk)

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Rachel Hood Edwards,

2Lineberger Comprehensive Cancer Center,

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Diane Sitki-Dark-green,

twoLineberger Comprehensive Cancer Centre,

3School of Dentistry, and

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Deborah Croom-Carter,

1Cancer Research United kingdom Institute for Cancer Studies, University of Birmingham, Birmingham, United Kingdom;

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Sushmita Roy,

1Cancer Inquiry Britain Institute for Cancer Studies, University of Birmingham, Birmingham, United Kingdom;

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Qing-Yun Yao,

iCancer Research Uk Institute for Cancer Studies, University of Birmingham, Birmingham, United Kingdom;

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Nancy Raab-Traub,

twoLineberger Comprehensive Cancer Center,

4Section of Microbiology, Academy of Northward Carolina at Chapel Hill, Chapel Hill

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Alan B. Rickinson

iCancer Research UK Plant for Cancer Studies, University of Birmingham, Birmingham, Uk;

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a

Present affiliation: Department of Oral Pathology, School of Clinical Dentistry, Sheffield, United kingdom

Author Notes

Published:

fifteen January 2006

Abstract

Recent work using a heteroduplex tracking assay (HTA) to identify resident viral sequences has suggested that patients with infectious mononucleosis (IM) who are undergoing master Epstein-Barr virus (EBV) infection oft harbor different EBV strains. Here, we examine samples from patients with IM by use of a new Epstein-Barr nuclear antigen ii HTA aslope the established latent membrane protein 1 HTA. Coresident allelic sequences were detected in ex vivo blood and pharynx wash samples from xiii of 14 patients with IM; almost patients carried 2 or more type 1 strains, one patient carried 2 blazon ii strains, and one patient carried both virus types. In contrast, coresident strains were detected in only 2 of fourteen patients by in vitro B prison cell transformation, despite screening >20 isolates/patient. Nosotros infer that coacquisition of multiple strains is mutual in patients with IM, although only i strain tends to be rescued in vitro; whether nonrescued strains are present in low abundance or are transformation defective remains to be adamant

Epstein-Barr virus (EBV), a γ-herpesvirus that is widespread in all man populations, can be isolated in vitro via its ability to transform resting human being B cells into permanent lymphoblastoid cell lines (LCLs) expressing the virus-coded antigens EBNA1, 2, 3A, 3B, 3C, and LP and the latent membrane proteins (LMPs) 1, 2A, and 2B. EBV isolates can exist categorized as type one or type 2 on the ground of marked allelic polymorphisms within the EBNA2, 3A, 3B, and 3C genes [1, 2] and into distinct strains on the basis of more-subtle sequence variations within the EBNA1, EBNA2, and LMP1 genes and certain lytic bicycle genes [3–ix]. Studies in which multiple LCLs were established from claret and throat launder (TW) samples suggested that, in European populations, most people carried a single dominant strain—usually of type one merely, in ∼10% of cases, of type 2 [10, eleven]. In dissimilarity, immunocompromised patients were oft found to bear multiple virus strains—once again, usually of blazon one [10, 12]. The exceptions were male homosexual patients with AIDS who, like HIV-negative homosexual men [xiii], ofttimes harbored both virus types [14, 15]

Because LCL outgrowth may select for some only not all resident EBV strains [xvi], other studies have used direct amplification of viral Dna sequences from ex vivo samples, either to screen for type 1/type 2 coinfection [17, 18] or for the presence of coresident strains with different LMP1 alleles, distinguished either by the number of copies of a 33-bp repeat sequence or by the presence or absence of a 30-bp deletion [19, twenty]. Some of these studies indicated that use of in vitro isolation may have led to an underestimation of the incidence of multiple infection in long-term virus carriers. More surprisingly, withal, use of a heteroduplex tracking analysis (HTA) to map coresident LMP1 gene sequences has suggested that well-nigh patients with infectious mononucleosis (IM) who are undergoing main EBV infection frequently harbor different EBV strains [21, 22]. This was unexpected considering, autonomously from 1 unusual case [23], previous in vitro studies had given no indication of the presence of coresident strains. Here, we further examine the situation in patients with IM by employ of both the LMP1 HTA and a newly developed HTA that detects polymorphisms at the EBNA2 gene locus, comparison the results for ex vivo samples with those for in vitro isolates

Patients, Materials, and Methods

Patients Patients with acute IM were identified on clinical grounds, and diagnoses were confirmed by a positive heterophile antibody (monospot) examination and by the presence of EBV-specific CD8⁺ T cell expansion in the blood [24]. Unfractionated blood mononuclear (UM) cells and TW samples were obtained during astute IM. These were analyzed directly ex vivo in polymerase chain reaction (PCR) assays and were as well used (as described elsewhere [xv]) to rescue resident virus strains equally UM jail cell–derived spontaneous LCLs (sp-LCLs) or as TW–derived LCLs (TW-LCLs)

Standard PCR analysis for EBNA2 type and LMP1 echo and deletion status All LCLs were screened using the EBNA2 blazon–specific primer/probe combination described past Sample et al. [ii]. The type ane/blazon 2 status of the ex vivo UM cell and TW samples was determined using a more sensitive nested PCR approach with first-round E2C and E2SEQ4 primers and 2nd-round E2C and 2A.2 primers; products were then Southern blotted and hybridized with type-specific radiolabeled probes (table i). Standard PCR assays directed against strain-specific polymorphic markers in the LMP1 gene were also used to place the presence or absenteeism of a xxx-bp deletion and to determine the number of copies of the 33-bp echo present [five, 15]

Table i

Oligonucleotide primer/probe combinations for standard polymerase concatenation reaction (PCR) and heteroduplex tracking assay (HTA) analysis

Table one

Oligonucleotide primer/probe combinations for standard polymerase chain reaction (PCR) and heteroduplex tracking analysis (HTA) analysis

HTA at the EBNA2 and LMP1 factor loci The LMP1 HTA was performed as described elsewhere, using probes specific for two selected variants, Ch2 and Med+ [25]. An EBNA2 HTA was developed that was based on a polymorphic region (B95.eight coordinates 48959–49208) in the EBNA2 gene. Starting time, EBNA2 type-specific HTA probes were generated by PCR amplification of the prototypic type ane strain B95.viii with the E2SEQ7.1/E2SEQ8.1 primers and from the prototypic type 2 strain Ag876 with the E2SEQ7.2/E2SEQ8.2 primers (table 1). The resulting PCR products were then digested with EcoRI and BglTwo, gel purified, and ligated into EcoRI/BglII-digested pSG5, to generate the E2.1HTA-pSG5 and E2.2HTA-pSG5 plasmids, respectively. Radiolabeled HTA probes were generated from these plasmids every bit described elsewhere [25]. For EBNA2 HTA analysis, aliquots of DNA from ex vivo samples or LCLs were PCR amplified with first-circular E2C and E2SEQ4 primers, so separate aliquots of the offset-circular products were reamplified with type-specific second-round combinations—namely, E2SEQ7.1 and E2SEQ8.1, which are type 1 specific, and E2SEQ7.two and E2SEQ8.ii, which are type two specific. The subsequent production/probe binding reactions and heteroduplex analysis were performed equally described elsewhere [25]

Results

Development of an EBNA2 HTA

Sequencing >50 geographically diverse EBV isolates within a region of the EBNA2 factor (B95.eight coordinates 48810–49374) identified 5 major variants of the type 1 sequence. To maintain consistency with a previously published nomenclature [6], these are referred to as EBNA2 alleles one.ane, 1.two, 1.3A, one.3B, and i.3E; allele-specific changes relative to the B95.8 sequence (defined as allele ane.ane) inside the most polymorphic region (B95.8 coordinates 48959–49208) are shown in figure 1A. In contrast, all type 2–carrying LCLs analyzed were identical to the Ag876 prototype sequence in this region. To distinguish between these different EBNA2 gene sequences, we developed an HTA based on PCR amplification of this most-polymorphic region followed by heteroduplex formation with the corresponding one.1 allele–specific (B95.8) and type two–specific (Ag876) probes (effigy 1B). Mobility of the complexes depends on the degree of sequence homology with the probe, with homoduplexes having the fastest mobility. Results of HTA analysis on ten representative type 1 LCL isolates (A–Thou) and on 2 type two LCL isolates (50 and 1000) are illustrated in figure 2A. The 2 virus types could be conspicuously distinguished from one some other, because there was no detectable complex formation betwixt the resulting PCR products and probes of the incorrect type. More chiefly, by heteroduplex mobility the blazon 1 isolates could be placed in 1 of the 5 subsets (i.i, 1.2, 1.3A, i.3B, or ane.3E) on the basis of the identity of their EBNA2 allelic sequence

Figure 1

Classification of EBNA2 and latent membrane protein (LMP) 1 variant sequences. Panel A shows the position of nucleotide substitutions within the i.ii, i.3A, 1.3B, and 1.3E EBNA2 alleles, relative to the 1.1 (B95.8) allelic sequence between coordinates 48959 and 49208. Note that the 1.iii family unit of EBNA2 alleles comprise a CTC insertion not present in the 1.1 and ane.two alleles (shaded squares). Panel B shows a sequence alignment of the B95.8-derived (EBNA2 1.1 allelic) probe and the respective Ag876-derived (EBNA2 blazon 2 allelic) probe used in the heteroduplex tracking analysis (HTA). The genome coordinates of the nucleotide changes found in the type one allelic variants are shown to a higher place the B95.eight sequence. The arrow betwixt coordinates 49136 and 49137 indicates the site of the CTC insertion establish in the 1.3 family of EBNA2 alleles. Console C shows the positions of nucleotide substitutions in the 7 LMP1 allelic sequence variants, relative to the B95.eight LMP1 allele; the shaded squares indicate nucleotides that are removed by the xxx-bp deletion. Console D shows the sequence of the B95.8 LMP1 allele from coordinate 169163 to 169427, the region analyzed by the LMP1 HTA. The genome coordinates for each of the potential nucleotide changes found in the LMP1 allelic variants are shown above the sequence. The position of the 30-bp deletion is indicated past the shaded box

Figure 1

Nomenclature of EBNA2 and latent membrane protein (LMP) 1 variant sequences. Panel A shows the position of nucleotide substitutions inside the one.2, one.3A, 1.3B, and ane.3E EBNA2 alleles, relative to the one.i (B95.8) allelic sequence betwixt coordinates 48959 and 49208. Note that the i.3 family of EBNA2 alleles contain a CTC insertion not present in the 1.1 and 1.2 alleles (shaded squares). Panel B shows a sequence alignment of the B95.eight-derived (EBNA2 1.1 allelic) probe and the corresponding Ag876-derived (EBNA2 blazon 2 allelic) probe used in the heteroduplex tracking assay (HTA). The genome coordinates of the nucleotide changes found in the blazon i allelic variants are shown above the B95.8 sequence. The arrow betwixt coordinates 49136 and 49137 indicates the site of the CTC insertion found in the i.3 family of EBNA2 alleles. Panel C shows the positions of nucleotide substitutions in the 7 LMP1 allelic sequence variants, relative to the B95.viii LMP1 allele; the shaded squares bespeak nucleotides that are removed by the xxx-bp deletion. Panel D shows the sequence of the B95.eight LMP1 allele from coordinate 169163 to 169427, the region analyzed past the LMP1 HTA. The genome coordinates for each of the potential nucleotide changes plant in the LMP1 allelic variants are shown above the sequence. The position of the thirty-bp deletion is indicated past the shaded box

Effigy 2

Analysis of a console of reference lymphoblastoid prison cell lines (LCLs) that carry unique Epstein-Barr virus isolates, using the EBNA2 and latent membrane protein (LMP) 1 heteroduplex tracking assays (HTAs). Type 1–specific and blazon 2–specific EBNA2 polymerase chain reactions (PCRs) were performed on Deoxyribonucleic acid from x type 1 LCL isolates (A–M) and 2 type 2 LCL isolates (L and Grand). The resulting PCR products were subjected to HTA assay with a one.one EBNA2 allele–specific probe and a blazon 2 EBNA2–specific probe, respectively (A). The same samples were too subjected to LMP1-specific PCR amplification followed by HTA analysis with a Ch2 allele–specific and a Med+ allele–specific probe (B). The results of these assays are summarized in panel C and demonstrate that the EBNA2 and LMP1 polymorphisms are not linked

Figure 2

Analysis of a panel of reference lymphoblastoid cell lines (LCLs) that acquit unique Epstein-Barr virus isolates, using the EBNA2 and latent membrane protein (LMP) ane heteroduplex tracking assays (HTAs). Type ane–specific and type 2–specific EBNA2 polymerase chain reactions (PCRs) were performed on DNA from 10 type ane LCL isolates (A–K) and 2 type 2 LCL isolates (L and Thou). The resulting PCR products were subjected to HTA analysis with a 1.1 EBNA2 allele–specific probe and a type ii EBNA2–specific probe, respectively (A). The aforementioned samples were also subjected to LMP1-specific PCR amplification followed by HTA analysis with a Ch2 allele–specific and a Med+ allele–specific probe (B). The results of these assays are summarized in panel C and demonstrate that the EBNA2 and LMP1 polymorphisms are not linked

The same representative panel of LCL isolates were examined for LMP1 sequence identity using the previously described LMP1 HTA [25]. This assay spans the 30-bp deletion locus and can distinguish the 7 different LMP1 variants shown in figure 1C—B95.8, Ch2, AL, NC, and Med−, none of which accept the xxx-bp deletion, and Med+ and Ch1, both of which have the deletion. Analysis of PCR products with a combination of Ch2 and Med+ probes allowed each of the LCLs to be identified as carrying a particular LMP1 sequence (effigy 2B). Importantly, this showed that private LMP1 alleles were not linked to item EBNA2 alleles. Thus, combining the two HTAs produced an EBNA2/LMP1 allele signature for each LCL (figure 2C) that provided much better discrimination between private strains than did either assay alone

Characterization of Virus Strains Carried past Patients with IM

With these assays in place, we then turned to the analysis of the EBV strains present in 14 patients with IM. The ex vivo UM cell and TW samples and the in vitro LCL isolates derived from them (a mean of 13 sp-LCLs and x TW-LCLs per patient) were analyzed (1) at the EBNA2 factor locus by standard Deoxyribonucleic acid typing PCR analysis and so by allele-specific HTA and (two) at the LMP1 gene locus by standard PCRs spanning the 33-bp repeats and the xxx-bp deletion and so past allele-specific HTA. Four dissimilar patterns of results were obtained

Detection of a single allelic sequence in a blazon 1 virus–infected patient Assays for one patient, IM87, detected a single EBNA2 allele (i.3B) and a single LMP1 allele (Ch1) in the ex vivo UM cell and TW samples too equally in all 12 sp-LCLs and nine TW-LCLs rescued in vitro. This suggested that this patient carried merely a unmarried type 1 strain

Detection of multiple allelic sequences in type one virus–infected patients A farther 11 of the 14 patients were carrying type 1 sequences just (in the absence of type two) at the EBNA2 gene locus; yet, in all 11 patients in that location was evidence of the presence of multiple EBNA2 and/or LMP1 allelic sequences in ex vivo samples. Figure 3 shows data from the ex vivo samples and from some LCL isolates (representative of 16 sp-LCLs and ten TW-LCLs) from patient IM75. Standard EBNA2 typing PCR analysis (figure 3A) detected type one signals throughout. Nonetheless, the more discriminatory EBNA2 HTA (figure 3B) detected both one.1 and i.3B allelic sequences in the ex vivo UM cell sample, whereas only the one.3B sequence was detected in the ex vivo TW sample. Interestingly, just the 1.3B allele was rescued in vitro—and not just in the TW-LCLs merely also in every sp-LCL, even though the 1.1 allele had given the stronger HTA indicate in the ex vivo UM cell sample. This was the start indication that not all resident viral sequences were represented in the in vitro–isolated LCLs

Figure 3

Analysis of Epstein-Barr virus sequences nowadays in ex vivo unfractionated blood mononuclear (UM) cell and pharynx wash (TW) samples, in up to 4 (representative of 16) UM prison cell–derived spontaneous lymphoblastoid cell lines (sp-LCLs), and in upwards to four (representative of 10) TW–derived LCLs (TW-LCLs) from patient IM75. Panel A shows the results of an EBNA2 typing polymerase chain reaction (PCR) analysis, with B95.8 and Ag876 serving as type i and type 2 reference controls, respectively. Only EBNA2 type i sequences are detectable in the ex vivo UM cell and TW samples and in all of the sp-LCLs and TW-LCLs. Console B shows the results of the EBNA2 heteroduplex tracking assay (HTA), which indicates the presence of a one.3B allele and a 1.i allele (both indicated past an pointer) in the ex vivo UM cell sample and the presence of a ane.3B allele only in the ex vivo TW sample and in all 4 representative LCL isolates. The gel also shows results obtained in the same experiment from reference control isolates known to comport a 1.1, one.ii, one.3A, i.3B, or 1.3E allele. The faint band running beneath the ane.ane heteroduplex represents probe homoduplex. Panel C shows the results of the latent membrane poly peptide (LMP) 1 repeat and deletion PCRs performed on the aforementioned samples and reference controls as for console A. LMP1 sequences with 4.5 and 5 copies of the repeat (both indicated by an arrow) are detectable in both the ex vivo UM prison cell and the TW sample, whereas all in vitro isolates have 5 copies of the repeat. Screening at the LMP1 deletion locus showed that all ex vivo samples and LCL isolates contained sequences that were nondeleted (in the figure, "WT" indicates nondeletion, and "DEL" indicates deletion). The B95.eight and Ag876 virus strains are known to have a iv.5-echo, nondeleted LMP1 allele and a 4-echo, deleted LMP1 allele, respectively. Console D shows the results of the LMP1 HTA; in this instance, only the data for the ex vivo samples are shown, alongside reference controls containing B95.eight and Ch2 allelic sequences in one track; Med+, Med−, and AL allelic sequences in a 2nd track; and Ch1 and NC allelic sequences in a third runway. The left panel shows an HTA performed with a Med+ allelic probe, and the correct panel shows an HTA performed with a Ch2 allelic probe. The results show that the ex vivo UM cell sample from IM75 contains detectable Ch1 and Med− sequences (both indicated by an arrow) and that the ex vivo TW sample from IM75 contains Med− sequences only

Figure iii

Assay of Epstein-Barr virus sequences nowadays in ex vivo unfractionated blood mononuclear (UM) jail cell and throat wash (TW) samples, in upward to four (representative of 16) UM cell–derived spontaneous lymphoblastoid cell lines (sp-LCLs), and in up to iv (representative of 10) TW–derived LCLs (TW-LCLs) from patient IM75. Panel A shows the results of an EBNA2 typing polymerase chain reaction (PCR) analysis, with B95.8 and Ag876 serving every bit type 1 and type ii reference controls, respectively. Only EBNA2 type 1 sequences are detectable in the ex vivo UM cell and TW samples and in all of the sp-LCLs and TW-LCLs. Panel B shows the results of the EBNA2 heteroduplex tracking assay (HTA), which indicates the presence of a one.3B allele and a 1.i allele (both indicated by an pointer) in the ex vivo UM cell sample and the presence of a 1.3B allele only in the ex vivo TW sample and in all 4 representative LCL isolates. The gel also shows results obtained in the same experiment from reference control isolates known to carry a 1.1, i.2, 1.3A, one.3B, or one.3E allele. The faint band running below the ane.1 heteroduplex represents probe homoduplex. Panel C shows the results of the latent membrane protein (LMP) 1 repeat and deletion PCRs performed on the aforementioned samples and reference controls as for console A. LMP1 sequences with four.v and 5 copies of the repeat (both indicated by an arrow) are detectable in both the ex vivo UM cell and the TW sample, whereas all in vitro isolates have 5 copies of the echo. Screening at the LMP1 deletion locus showed that all ex vivo samples and LCL isolates contained sequences that were nondeleted (in the figure, "WT" indicates nondeletion, and "DEL" indicates deletion). The B95.eight and Ag876 virus strains are known to have a 4.v-echo, nondeleted LMP1 allele and a four-repeat, deleted LMP1 allele, respectively. Panel D shows the results of the LMP1 HTA; in this example, only the information for the ex vivo samples are shown, alongside reference controls containing B95.8 and Ch2 allelic sequences in one track; Med+, Med−, and AL allelic sequences in a second runway; and Ch1 and NC allelic sequences in a third track. The left panel shows an HTA performed with a Med+ allelic probe, and the right console shows an HTA performed with a Ch2 allelic probe. The results show that the ex vivo UM cell sample from IM75 contains detectable Ch1 and Med− sequences (both indicated by an arrow) and that the ex vivo TW sample from IM75 contains Med− sequences only

When the aforementioned samples were analyzed at the LMP1 gene locus, the standard LMP1 xxx-bp deletion PCR was uninformative, because all of the samples were constitute to contain a nondeleted LMP1 sequence. However, distension across the LMP1 33-bp repeats detected 2 dissimilar products, with 4.five and 5 copies of the repeat in the ex vivo UM prison cell and TW samples, respectively, whereas just the sequence with 5 copies of the repeat had been rescued in vitro (figure 3C). LMP1 HTA assay detected the presence of 2 LMP1 variant sequences, Med− and Ch1, in the blood but only 1 sequence, Med−, in the throat; again, only 1 of these sequences, Med−, had been rescued in the LCLs (figure 3D). The results suggest that this patient was infected with 2 blazon one strains. One strain, preferentially rescued in vitro, had a 1.3B EBNA2 allele and a Med− LMP1 allele with 5 copies of the 33-bp repeat; the nonrescued strain is predicted to accept a 1.one EBNA2 allele and a Ch1 LMP1 allele with iv.five copies of the repeat

In a second such patient (IM81) found to be carrying only type 1 virus sequences past standard EBNA2 typing PCR analysis (figure 4A), the EBNA2 HTA detected stiff 1.i and weak 1.3B allelic signals in the ex vivo UM cell sample but only i.3B sequences in the pharynx. Again, all 12 sp-LCLs and 12 TW-LCLs from this patient carried simply 1 of the sequences, one.3B (figure 4B and data not shown). Standard PCR assays at the LMP1 factor locus reinforced the testify indicating that unlike virus strains were dominant in the blood (4.five copies of the echo, nondeleted) versus the throat (5 copies of the repeat, deleted) and that just the latter virus had been isolated in vitro (figure 4C). However, the more discriminatory LMP1 HTA revealed iii different LMP1 allelic sequences in the blood (Ch1, B95.viii, and Ch2), of which ii (Ch1 and B95.8) were besides detectable in the throat. Again simply 1 of these, the Ch1 allele, had been rescued in vitro (figure 4D). This suggested that patient IM81 carried 3 type i virus strains. One strain, preferentially rescued in vitro, carried a i.3B EBNA2 allele and a Ch1 LMP1 allele. The other 2 predicted strains appear to have different alleles at the LMP1 gene locus (B95.8 and Ch2); their identity at the EBNA2 cistron locus could not be determined unequivocally, merely at least 1 must carry a 1.1 EBNA2 allele

Figure 4

Analysis of Epstein-Barr virus sequences present in ex vivo unfractionated claret mononuclear (UM) prison cell and throat wash (TW) samples, in up to 4 (representative of 12) UM prison cell–derived spontaneous lymphoblastoid cell lines (sp-LCLs), and in up to four (representative of 12) TW–derived LCLs (TW-LCLs) from patient IM81. Results are presented substantially as described in figure 3. Panel A (EBNA2 typing polymerase chain reaction [PCR] analysis) shows that only EBNA2 type 1 sequences are detectable in the ex vivo UM cell and TW samples and in all sp-LCLs and TW-LCLs. Console B (EBNA2 heteroduplex tracking assay [HTA]) shows that ane.i and 1.3B EBNA2 allelic sequences (both indicated by an arrow) are nowadays in the ex vivo UM prison cell sample but that simply the 1.3B allelic sequence is detectable in the TW sample and in all of the sp-LCLs and TW-LCLs. Panel C (latent membrane protein [LMP] 1 repeat and deletion PCRs) shows the presence in the ex vivo UM cell sample of an LMP1 allele that has 4.5 copies of the echo and that is nondeleted (in the figure, "WT" indicates nondeletion, and "DEL" indicates deletion) at the 30-bp deletion locus, whereas the ex vivo TW sample and all of the LCLs carried an LMP1 allele with 5 repeats and a xxx-bp deletion. Console D (LMP1 HTA) shows the presence of iii LMP1 allelic sequences (Ch1, B95.viii, and Ch2) (all indicated by an arrow) in the ex vivo UM prison cell sample and of 2 alleles (Ch1 and B95.viii) in the ex vivo TW sample; all sp-LCLs and TW-LCLs carried only the Ch1 allele

Figure 4

Assay of Epstein-Barr virus sequences present in ex vivo unfractionated blood mononuclear (UM) cell and throat wash (TW) samples, in up to 4 (representative of 12) UM cell–derived spontaneous lymphoblastoid cell lines (sp-LCLs), and in upwardly to 4 (representative of 12) TW–derived LCLs (TW-LCLs) from patient IM81. Results are presented essentially as described in effigy 3. Panel A (EBNA2 typing polymerase chain reaction [PCR] assay) shows that only EBNA2 type i sequences are detectable in the ex vivo UM jail cell and TW samples and in all sp-LCLs and TW-LCLs. Panel B (EBNA2 heteroduplex tracking assay [HTA]) shows that 1.1 and 1.3B EBNA2 allelic sequences (both indicated by an arrow) are present in the ex vivo UM jail cell sample but that only the i.3B allelic sequence is detectable in the TW sample and in all of the sp-LCLs and TW-LCLs. Panel C (latent membrane poly peptide [LMP] i repeat and deletion PCRs) shows the presence in the ex vivo UM prison cell sample of an LMP1 allele that has 4.five copies of the repeat and that is nondeleted (in the figure, "WT" indicates nondeletion, and "DEL" indicates deletion) at the thirty-bp deletion locus, whereas the ex vivo TW sample and all of the LCLs carried an LMP1 allele with 5 repeats and a 30-bp deletion. Console D (LMP1 HTA) shows the presence of 3 LMP1 allelic sequences (Ch1, B95.8, and Ch2) (all indicated by an arrow) in the ex vivo UM cell sample and of 2 alleles (Ch1 and B95.8) in the ex vivo TW sample; all sp-LCLs and TW-LCLs carried only the Ch1 allele

Of the 11 patients with IM who were obviously coinfected with 2 or more than type one viruses, different allelic sequences were detected in both the ex vivo UM cell samples and the TW samples from 7 patients (IM72, IM75, IM79, IM80, IM81, IM82, and IM84); in the TW samples only not in the UM jail cell samples from ii patients (IM85 and IM86); and in the UM cell samples but non (where tested) in the TW samples from 2 patients (IM73 and IM78). Virtually importantly, from all simply one of these 11 patients just a single virus strain was always rescued in vitro, despite the analysis of multiple LCLs in each case

Detection of type 1 and type 2 allelic sequences Ane patient, IM76, showed evidence of both blazon 1 and blazon 2 EBNA2 allelic sequences. These were initially detected in the ex vivo TW sample past standard EBNA2 typing PCR analysis, although the same assay detected merely type 2 sequences in the blood (effigy 5A). EBNA2 HTA screening of the same ex vivo samples confirmed these results, with the TW sample carrying both a 1.3A allele and a type 2 allele and the UM cell sample carrying but a blazon 2 allele (figure 5B). However, a blazon i strain was preferentially rescued in vitro, with just one of 10 sp-LCLs (sp-LCL7) and 1 of 10 TW-LCLs (TW-LCL8) with detectable type 2 too every bit type 1 signals in standard EBNA2 typing PCR analysis (effigy 5A). Surprisingly, however, EBNA2 HTA analysis showed that the type 1 virus beingness rescued in vitro carried a 1.3B allelic sequence, not the 1.3A allele that had been detected ex vivo (effigy 5B). Furthermore, assays at the LMP1 cistron locus (data non shown) showed that a virus strain with a Med− allele and v copies of the 33-bp repeat was preferentially rescued in vitro, whereas a Ch1 allele with 6 copies of the 33-bp repeat was the dominant sequence in the ex vivo samples. This suggests that this patient harbored 3 virus strains. I type 1 strain carrying a ane.3B EBNA2 allele and a Med− LMP1 allele was preferentially rescued in vitro, and another predicted type 1 strain apparently conveying a 1.3A allele was non rescued. The coresident blazon ii strain, conveying a type 2 EBNA2 allele and a Ch1 LMP1 allele, was rescued in vitro, but with low efficiency

Figure 5

Analysis of Epstein-Barr virus sequences present in ex vivo unfractionated blood mononuclear (UM) cell and pharynx wash (TW) samples, in upward to 4 (representative of x) UM jail cell–derived spontaneous lymphoblastoid cell lines (sp-LCLs), and in up to four (representative of 10) TW–derived LCLs (TW-LCLs) from patient IM76. Panel A (EBNA2 typing polymerase chain reaction [PCR] assay) shows that just type 2 EBNA2 allelic sequences are detectable in the ex vivo UM cell sample, whereas both type one and type ii sequences are detectable in the ex vivo TW sample. All of the LCLs carried blazon 1 alleles, but type 2 allelic sequences were too weakly detected in certain LCLs—namely, sp-LCL7 and TW-LCL8. Console B (EBNA2 heteroduplex tracking assay [HTA]) shows information for ex vivo samples and some of the to a higher place LCLs, alongside reference control isolates known to carry a type 1 EBNA2 allele or a blazon 2 allele. The upper panel shows an HTA performed with a 1.i allelic probe, and the lower panel shows an HTA performed with a type 2 allelic probe. The ex vivo UM cell sample contains only detectable blazon two sequences, whereas the ex vivo TW sample contains both type 2 and 1.3A allelic sequences (indicated past an arrow). However, all of the LCLs carried a one.3B allelic sequence (indicated past an arrow) either alone or, in the case of sp-LCL7 and TW-LCL8, together with a type 2 allele

Figure 5

Analysis of Epstein-Barr virus sequences present in ex vivo unfractionated blood mononuclear (UM) jail cell and throat wash (TW) samples, in upwards to 4 (representative of 10) UM jail cell–derived spontaneous lymphoblastoid prison cell lines (sp-LCLs), and in upward to 4 (representative of 10) TW–derived LCLs (TW-LCLs) from patient IM76. Panel A (EBNA2 typing polymerase concatenation reaction [PCR] analysis) shows that but blazon 2 EBNA2 allelic sequences are detectable in the ex vivo UM cell sample, whereas both type 1 and type 2 sequences are detectable in the ex vivo TW sample. All of the LCLs carried type 1 alleles, but type 2 allelic sequences were also weakly detected in sure LCLs—namely, sp-LCL7 and TW-LCL8. Panel B (EBNA2 heteroduplex tracking assay [HTA]) shows data for ex vivo samples and some of the in a higher place LCLs, alongside reference control isolates known to conduct a type 1 EBNA2 allele or a type ii allele. The upper panel shows an HTA performed with a one.1 allelic probe, and the lower console shows an HTA performed with a type 2 allelic probe. The ex vivo UM cell sample contains simply detectable type 2 sequences, whereas the ex vivo TW sample contains both type 2 and 1.3A allelic sequences (indicated by an arrow). Nevertheless, all of the LCLs carried a 1.3B allelic sequence (indicated by an arrow) either lone or, in the case of sp-LCL7 and TW-LCL8, together with a type 2 allele

Detection of multiple allelic sequences in a blazon 2 virus– infected patient A final patient, IM 43, was found to be carrying only type 2 sequences (in the absence of type 1) at the EBNA2 factor locus, both by standard EBNA2 typing PCR analysis and by HTA. Results for the ex vivo UM prison cell sample and for 8 representative LCLs are shown in figure 6A and 6B. However, the corresponding assays at the LMP1 gene locus detected in the ex vivo TW sample the presence of a dominant B95.eight allelic sequence, nondeleted and with 4.five copies of the repeat, and of an additional sequence that had the 30-bp deletion. Parallel assays of the ex vivo UM cell sample identified a dominant Ch1 allelic sequence, deleted and with half-dozen copies of the repeat, and an additional B95.8 allelic sequence. The type 2 strain carrying the Ch1 LMP1 allele was preferentially rescued in vitro not simply from the blood, where it was dominant, but as well from the throat, where, ex vivo assays suggested, it was the modest strain (figure 6C and 6D)

Figure 6

Analysis of Epstein-Barr virus sequences present in ex vivo unfractionated blood mononuclear (UM) cell and pharynx wash (TW) samples, in up to iv (representative of 12) UM cell–derived spontaneous lymphoblastoid jail cell lines (sp-LCLs), and in up to all 4 available TW–derived LCLs (TW-LCLs) from patient IM43. Results are presented essentially as described in figure iii. Panel A (EBNA2 typing polymerase chain reaction [PCR] assay) shows that only type 2 EBNA2 allelic sequences are detectable in the ex vivo UM cell and TW samples and in all sp-LCLs and TW-LCLs. Console B (EBNA2 heteroduplex tracking analysis [HTA]) confirms the presence of type 2 sequences (indicated by an arrow) in all samples, with the exception of the ex vivo TW sample, for which no signal was obtained; note that no blazon i allelic sequences were detected in whatsoever sample by EBNA2 HTA with the 1.1 probe (data not shown). Panel C (latent membrane poly peptide [LMP] 1 repeat and deletion PCRs) shows the presence of an LMP1 allele with 6 repeats and a xxx-bp deletion in the ex vivo UM cell sample also as weak signals (indicated by arrows) for a 4.5-repeat sequence and for both deleted and nondeleted (in the figure, "WT" indicates nondeletion, and "DEL" indicates deletion) sequences in the ex vivo TW sample. All of the sp-LCLs and TW-LCLs carried the 6-echo, thirty-bp deleted LMP1 allele. Console D (LMP1 HTA) revealed the presence of a dominant Ch1 allele and a weak B95.eight allele (seen on longer gel exposures) in the ex vivo UM prison cell sample and the presence of a B95.8 allele only in the ex vivo TW sample. However, all of the sp-LCLs and TW-LCLs carried a Ch1 LMP1 allele

Figure six

Assay of Epstein-Barr virus sequences present in ex vivo unfractionated blood mononuclear (UM) jail cell and throat wash (TW) samples, in upwards to four (representative of 12) UM cell–derived spontaneous lymphoblastoid cell lines (sp-LCLs), and in upward to all four available TW–derived LCLs (TW-LCLs) from patient IM43. Results are presented essentially as described in figure iii. Console A (EBNA2 typing polymerase concatenation reaction [PCR] analysis) shows that only type 2 EBNA2 allelic sequences are detectable in the ex vivo UM cell and TW samples and in all sp-LCLs and TW-LCLs. Panel B (EBNA2 heteroduplex tracking assay [HTA]) confirms the presence of blazon two sequences (indicated by an pointer) in all samples, with the exception of the ex vivo TW sample, for which no bespeak was obtained; note that no type 1 allelic sequences were detected in whatever sample past EBNA2 HTA with the 1.i probe (data non shown). Console C (latent membrane protein [LMP] ane repeat and deletion PCRs) shows the presence of an LMP1 allele with six repeats and a 30-bp deletion in the ex vivo UM cell sample as well as weak signals (indicated by arrows) for a 4.5-echo sequence and for both deleted and nondeleted (in the figure, "WT" indicates nondeletion, and "DEL" indicates deletion) sequences in the ex vivo TW sample. All of the sp-LCLs and TW-LCLs carried the 6-echo, 30-bp deleted LMP1 allele. Panel D (LMP1 HTA) revealed the presence of a dominant Ch1 allele and a weak B95.8 allele (seen on longer gel exposures) in the ex vivo UM prison cell sample and the presence of a B95.8 allele just in the ex vivo TW sample. However, all of the sp-LCLs and TW-LCLs carried a Ch1 LMP1 allele

The detailed results for all fourteen patients with IM analyzed are summarized in effigy vii. This figure shows, for each patient, the EBNA2 and LMP1 sequences detected in ex vivo samples and in their derived in vitro isolates. The patients are arranged by shading into the iv categories described in a higher place, and in each case we give our interpretation of the number of resident virus strains present

Figure 7

Summary of results for all 14 patients with infectious mononucleosis (IM) analyzed, showing the outcome of standard polymerase concatenation reaction (PCR) and heteroduplex tracking assay (HTA) analysis at the EBNA2 and latent membrane protein (LMP) ane gene loci on ex vivo unfractionated blood mononuclear (UM) cell and throat wash (TW) samples and on UM jail cell–derived spontaneous lymphoblastoid cell lines (sp-LCLs) and TW–derived LCLs (TW-LCLs) (no. of available LCLs are shown in brackets). The right-paw column shows our interpretation of the data in terms of the no. of resident Epstein-Barr virus strains in the blood and the throat. Patients are bundled into 4 master categories, identified by shading, to match the 4 patterns described in Results. DEL, deletion; WT, nondeletion

Effigy seven

Summary of results for all 14 patients with infectious mononucleosis (IM) analyzed, showing the consequence of standard polymerase chain reaction (PCR) and heteroduplex tracking analysis (HTA) analysis at the EBNA2 and latent membrane protein (LMP) 1 factor loci on ex vivo unfractionated claret mononuclear (UM) cell and throat wash (TW) samples and on UM prison cell–derived spontaneous lymphoblastoid cell lines (sp-LCLs) and TW–derived LCLs (TW-LCLs) (no. of bachelor LCLs are shown in brackets). The correct-hand column shows our interpretation of the data in terms of the no. of resident Epstein-Barr virus strains in the blood and the throat. Patients are arranged into iv main categories, identified by shading, to lucifer the 4 patterns described in Results. DEL, deletion; WT, nondeletion

Word

Recent evidence suggesting that patients with IM are often infected with multiple EBV strains [21, 22] was entirely based on HTA analysis of ex vivo samples at a single locus, the LMP1 gene locus. Given the credible discordance between these findings and the results of earlier in vitro isolation studies, we extended the HTA analysis to a second polymorphic locus, the EBNA2 gene locus [3, half dozen, 26], and to ex vivo samples from patients from whom a large number of in vitro LCL isolates had already been recovered. The type and strain specificity of the newly developed EBNA2 HTA was validated using a panel of type i and blazon 2 EBV reference isolates. These experiments showed that EBNA2 and LMP1 allelic polymorphisms are not linked. This strengthened a conclusion from earlier work with standard PCR assays, in which the EBNA2 blazon of virus strains did non correlate with the xxx-bp deletion status of the LMP1 allele [27]. Information technology was therefore clear that combining the 2 HTAs would provide greater discriminatory power than would either the EBNA2 or the LMP1 HTA lone

The relative prevalences of blazon 1 versus type 2 EBV strains in unlike human populations has remained contentious. Some studies have suggested that the majority of European and Southeast Asian donors carry type 1 strains, with depression incidences of detectable blazon 2 infection or blazon ane/type 2 coinfection [10, 11, 28–thirty], whereas studies of other white populations have reported a higher prevalence of type 2 strains [17, eighteen]. To some extent, these anomalies may reflect subtle differences in the composition of study cohorts, such as differences in precise geographic origin or fifty-fifty in social group. For instance, male homosexuals in Western societies have a much college incidence of blazon 2 virus infection than does the general population [12, 13, xv, 17, 18], thereby explaining the unexpectedly loftier frequency of blazon ii virus infection in early AIDS cohorts [14, 31, 32]. The present study focused on classic IM as it presents in young adults in the United Kingdom population. On the basis of EBNA2 gene amplification, it was found that, of our 14 patients, only type 1 sequences were detectable in 12 patients, only type 2 sequences were detectable in 1 patient, and both type 1 and type 2 sequences were detectable in one patient. Such a distribution is broadly in line with previous estimates of blazon 1 and type 2 virus infection prevalences in the full general United Kingdom population [eleven, 12]. Our data leads us to question earlier suggestions that type 1/type two coinfection is common in patients with IM [17]

More importantly, the combination of EBNA2 and LMP1 HTA analysis detected dissimilar coresident EBV sequences in all but 1 of the patients with IM studied, results that are in line with contempo findings obtained using only the LMP1 analysis [21, 22]. In 7 of 12 informative patients in the present study, the same sequences were nowadays in both the blood and the throat. Of the other patients, 2 had an additional sequence in the blood that was not detected in the throat, and iii had an boosted sequence in the throat that was not detected in the blood; such differences in the range of sequences found at dissimilar sites may reverberate limitations in analysis sensitivity rather than a genuine compartmentalization of infection in vivo. The reproducibility of the HTA information for the ex vivo samples (all were tested at to the lowest degree twice), also as the fact that standard PCR amplification assays at the LMP1 echo and deletion loci frequently detected coresident sequences in the same samples, strongly advise that the present results are not artifactual but genuinely reverberate the presence of 2 or more contained virus strains in patients with IM

Information technology was still surprising that, in many cases, only 1 virus strain was rescued in vitro despite the screening of multiple independent LCL isolates. Furthermore, the rescued strain'due south EBNA2 and LMP1 alleles were not necessarily those that gave the strongest signals in ex vivo assays. In that regard, the results of such assays, because they involve 2 rounds of PCR, may non accurately stand for the relative allele loads in ex vivo samples. It is therefore even so possible that in vitro isolation does, in fact, identify the dominant strain, at least when the contest for in vitro outgrowth involves coresident type 1 viruses. In contrast, type 1 strains will tend to be rescued preferentially above any coresident type 2 virus [16]. However, another possible explanation for the differences in results between the ex vivo samples and the in vitro isolates is that some viruses are not rescued in vitro because they are transformation defective. It will be important to written report this possibility farther, considering the presence of transformation-lacking strains in the claret of patients with IM would imply that transforming ability is not required for colonization of the B jail cell system

We believe that the presence of multiple strains in patients with IM can be explained only past coacquisition of these strains during chief infection. Although it has been postulated that EBV quasispeciation within the host occurs during virus persistence, leading to EBV-positive malignancies [33, 34], there is no house documentary evidence of such rapid evolution in vivo, nor is in that location any show indicating that EBV Deoxyribonucleic acid polymerase is particularly error prone [35]. Furthermore, it seems implausible that such quasispeciation would always produce the particular range of EBNA2 and LMP1 allelic sequences described here. The most likely scenario is that the multiple strains detected in many of the patients with IM in the present study were coacquired from a single source—namely, a virus carrier who was shedding multiple strains in saliva [22]. It is clear that infection with multiple strains is non unique to patients with IM, because many healthy carriers with no history of the disease show evidence of coinfection when analyzed by LMP1 HTA [21] and by other assays [eighteen, 28]. It is therefore possible that many subclinical primary infections, as well as cases of IM, involve the coacquisition of multiple strains. If and so, it could explain the apparent loftier frequency of multiple infection in the general population. Nonetheless, information technology does not rule out the possibility that at least some instances of multiple infection are the result of serial acquisition over time from independent sources. Prospective studies are required to resolve this important point. If serial acquisition does occur, it would hateful that infection with 1 or more chief strains does not return the host allowed to subsequent virus challenge. This would severely harm the prospects of designing an EBV vaccine that provided sterile amnesty confronting natural virus infection

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Presented in part: 11th biennial conference of the International Association for Research on Epstein-Barr Virus and Associated Diseases, Regensburg, Germany, twenty–25 September 2004 (abstract 03.03)

Potential conflicts of involvement: none reported

Financial support: Cancer Enquiry UK (grant C910/A3891); National Institutes of Health (grants CA32979 and DE11644)

Author notes

a

Nowadays affiliation: Department of Oral Pathology, School of Clinical Dentistry, Sheffield, United Kingdom

© 2005 past the Infectious Diseases Society of America

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