Aberrant Lymphocyte Activation Precedes Delayed Virus-Specific T-Cell Response after both Primary Infection and Secondary Exposure to Hepadnavirus in the Woodchuck Model of Hepatitis B Virus Infection^▿

Animals and WHV inoculations.

Ten healthy, adult woodchucks housed in the Woodchuck Hepatitis Research Facility at Memorial University, St. John's, Canada were infected with large, liver-pathogenic doses of WHV. Prior exposure to WHV was excluded based on negative serological markers for WHV infection and the absence of WHV DNA in randomly selected serum, peripheral blood mononuclear cell (PBMC), and liver biopsy samples assessed by nested PCR-nucleic acid hybridization (PCR-NAH) assays (sensitivity, <10 virus genome equivalents [vge]/ml) (9, 42). The animals were divided into three groups. Four animals (group A) were investigated in the first phase to determine the overall features of T-cell proliferative responses accompanying primary infection and subsequent exposures to hepadnavirus. The animals were intravenously injected with 1.9 × 10¹¹ DNase-protected vge of WHV/tm4 inoculum carrying the wild-type WHV, as determined by a whole-genome sequence analysis (P. M. Mulrooney-Cousins and T. I. Michalak, unpublished data). They were followed for 65 weeks post-primary infection (w.p.p.i.) and then challenged with the same dose of WHV/tm4 and rechallenged 15 weeks later with either 1.9 × 10¹¹ vge (animals 1/F and 2/F) or 1.9 × 10² vge (animals 3/F and 4/M) of the inoculum (Fig. 1A). Four other woodchucks constituted study group B. These animals were injected once with WHV, followed for 16 w.p.p.i., and investigated to investigate a possible interdependence between the heightened T-lymphocyte proliferation capacity occurring immediately after WHV exposure identified in the first study and the lymphocyte susceptibility to apoptotic death, the delayed WHV-specific T-cell response, and the expression of selected proinflammatory and antiviral cytokines in lymphoid cells. In this group, two animals (5/M and 6/M) were infected with 1.9 × 10¹¹ vge of WHV/tm4, while two others (7/M and 8/M) were infected with a WHV/tm3 inoculum (GenBank accession number AY334075) (41) at 1.1 × 10¹⁰ DNase-protected vge per dose (Fig. 1B). In addition, two woodchucks, each injected with a single dose of WHV/tm4 at 1.9 × 10¹¹ vge, were examined for 112 weeks postinfection (w.p.i.) as controls. The study protocol was approved by the Institutional President's Committee on Animal Bioethics and Care.

Sample collection.

Animals were bled at the time points indicated in Fig. 1. Sera were collected and preserved. PBMC were isolated as described before (23). Their viability normally exceeded 98%, as determined by trypan blue dye exclusion. Liver biopsies were obtained by surgical laparotomy at the time points showed in Fig. 1 and examined for WHV DNA load and histological lesions using the scoring criteria reported before (40-42).

Serological and WHV DNA detection assays.

Serial serum samples were tested for WHV surface antigen (WHsAg), antibodies to WHV core antigen (anti-WHc), and antibodies to WHsAg (anti-WHs) by using in-house specific-enzyme-linked immunoassays reported before (9, 41, 42). The levels of WHV DNA in sera and liver biopsies were quantified by real-time PCR (41) and, when negative, by PCR-NAH assay, as reported previously (42).

WHV antigens and mitogens for T-cell proliferation assays.

Recombinant WHV core, e, and X proteins, designated rWHc, rWHe, and rWHx, respectively, and used to measure WHV-specific T-cell proliferation in vitro, were produced in the pET41b(+) Escherichia coli expression system (Novagen, Darmstadt, Germany) and affinity purified as recently reported (62). They were extensively tested for nonspecific T-cell proliferation against potential bacterial contaminants and found to be entirely devoid of such activity (62). In addition, a synthetic WHV peptide, containing the immunodominant epitope corresponding to amino acids 97 to 110 (WHc_97-110) of the virus nucleocapsid protein (33), was synthesized. WHV envelope particles carrying WHsAg specificity were purified from pooled sera of a chronic WHV carrier, as described previously (62). As inducers of generalized, nonspecific, polyclonal lymphocyte responses, concanavalin A (ConA; Pharmacia Fine Chemicals, Uppsala, Sweden), pokeweed mitogen (PWM, Phytolacca americana agglutinin; ICN Biochemicals, Inc., Aurora, OH), and phytohemagglutinin (PHA; ICN Biochemicals) were used (see below).

Adenine incorporation T-cell proliferation assay.

A [³H]adenine incorporation T-lymphocyte proliferation assay was applied to examine WHV-specific and mitogen-induced T-cell responsiveness and performed as described elsewhere (23, 30). Briefly, freshly isolated PBMC were cultured in 96-well flat-bottomed plates (Becton Dickinson Labware, Franklin Lakes, NJ) at a density of 1 × 10⁵ cells/well in complete AIM-V lymphocyte culture medium (Gibco-Invitrogen Corp., Auckland, New Zealand) with 10% fetal calf serum (Gibco-Invitrogen). Recombinant WHV proteins and WHsAg were added in triplicate at 1 μg/ml and 2 μg/ml, whereas the WHc_97-110 peptide was used at five twofold dilutions from 1.25 μg/ml to 20 μg/ml. Similarly, to assess the generalized lymphocyte response, each PBMC sample was exposed in triplicate to five twofold dilutions of ConA or PHA from 1.25 μg/ml to 20 μg/ml and PWM from 0.6 μg/ml to 10 μg/ml or to medium alone as a control. The cultures were incubated for 96 h at 37°C, pulsed with 0.5 μCi of [2-³H]adenine (Amersham Pharmacia Biotech, AB, Uppsala, Sweden) per well, and incubated for an additional 12 to 18 h. Then, the cells were harvested and the radioactivity counts per minute (cpm) in each well were determined (23). The mean cpm values for either stimulated or control cells were calculated by averaging the counts in the respective triplicate wells. The stimulation index (SI) was calculated by dividing the mean cpm obtained either after WHV antigen or mitogen stimulation by the mean cpm detected in control, unstimulated wells. SI values of ≥3.1 for rWHc, rWHe, and rWHx and ≥2.1 for WHc_97-110 and WHsAg were considered as positive based on the cutoff values given by 3 standard deviations above the mean values obtained from control wells. The mean mitogenic SI (MMSI) was determined by averaging the SI values obtained for all five concentrations of a given mitogen, as described previously (23). For each time point, the data were presented as the MMSI, which was calculated by averaging the SI values obtained after stimulation with five different concentrations of a given mitogen.

CFSE flow cytometry T-cell proliferation assay.

Since WHV-specific T-cell responses examined by using a [³H]adenine incorporation assay gave readings at the detection limit of the assay in samples collected after the third injection with WHV, i.e., 80 w.p.p.i., and to confirm the observations made for study group A in group B animals, a more-sensitive flow cytometric assay with 5- and-6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, Oregon) was employed as recently reported (23). Briefly, woodchuck PBMC were labeled with 1 μM of CFSE and either stimulated with WHV antigens or the WHc_97-110 peptide at the same concentrations as those in the adenine incorporation assay or left unstimulated as controls. The plates were incubated for 5 days. The cells were harvested and analyzed in a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). The halved CFSE fluorescence in dividing PBMC was determined by using CellQuest Pro software (Becton Dickinson). The cell division index (CDI) was defined by dividing the percentage of cells showing halved CFSE fluorescence after stimulation with WHV antigens by that of cells with halved CFSE fluorescence cultured in the absence of stimulation, as we described before (23). The highest CDI value found at a given time point for any concentration of WHV protein/peptide tested was taken as a measure of WHV-specific T-cell response. The cutoff CDI values of ≥3.1 were considered to represent a positive WHV-specific response (23).

CFSE-annexin V-PE-7-AAD assay for simultaneous detection of T-cell proliferation and apoptosis.

An assay simultaneously measuring the proliferation of lymphocytes and their apoptotic death was adopted, using ConA and rWHe as stimulators and three-color CFSE-annexin V-phycoerythrin (PE) -7-actinomycin-D (7-AAD) labeling. Briefly, freshly isolated PBMC were labeled with 1 μM of CFSE, cultured at a density of 5 × 10⁵ cells per well in a 48-well tissue culture plate, and stimulated with either ConA (2.5 and 5 μg/ml) or rWHe (1 and 2 μg/ml) as indicated above. The cells were harvested, washed with annexin-labeling buffer (5 mM NaCl, 5 mM KCl, and 2 mM CaCl₂ in 10 mM HEPES), stained for 30 min on ice with 50 μg/ml of 7-AAD (Molecular Probes) and 50 μl/ml of R-PE-conjugated recombinant human annexin V (annexin V-PE; Caltag Laboratories, Burlingame, CA), and analyzed by flow cytometry. In addition, to monitor the death of lymphoid cells potentially occurring in vivo, freshly isolated PBMC were directly labeled with annexin V-7-AAD without stimulation with ConA or rWHe and analyzed. The authenticity of specific binding of annexin V and 7-AAD was verified by visualizing the intracellular fluorescence distribution (see Fig. 6A) using an Olympus BX 50WI confocal microscopy system (Olympus America, Inc., Center Valley, PA).

The CDI was defined by dividing the percentage of CFSE^dim cells after stimulation with rWHe or ConA by the percentage of CFSE^dim cells without any stimulation (medium only), as indicated above. A CDI of ≥3.1 after rWHe or ConA stimulation was accepted as a positive T-cell response. The level of cell apoptosis was assessed by using CellQuestPro software (Becton Dickinson) and by separating test cells into four quadrants as follows: live cells, i.e., annexin V-7-AAD negative (lower-left quadrant); early apoptotic, i.e., annexin V positive only (lower-right quadrant); late apoptotic, i.e., annexin V and 7-AAD positive (upper-right quadrant); and necrotic, i.e., 7-AAD positive only (upper-left quadrant), as illustrated in Fig. 6A. The total percentage of apoptotic cells was determined by adding the percentages of early apoptotic, late apoptotic, and necrotic cells.

Real-time RT-PCR.

PBMC and liver biopsy samples collected prior to and during WHV infection were analyzed for the expression of selected cytokines and cell marker genes by real-time reverse transcription-PCR (RT-PCR) by using a LightCycler (Roche Diagnostics, Mannheim, Germany). For this purpose, 5 × 10⁵ to 1 × 10⁶ PBMC were resuspended immediately after isolation in 1 ml of Trizol reagent (Invitrogen, Auckland, New Zealand) and stored at −20°C. After all experimental samples were collected, RNA was extracted, and 2-μg samples of RNA were reverse transcribed to cDNA, as reported previously (9). The levels of expression of woodchuck IFN-α, IFN-γ, TNF-α, interleukin-2 (IL-2), IL-4, IL-10, IL-12, CD3, CD4, CD14, and CD56 were quantified by using the equivalent of 50 ng of total RNA and the gene-specific primer pairs shown in Table 1, and their levels of transcription normalized against that of woodchuck β-actin (C. S. Guy, P. M. Mulrooney-Cousins, N. D. Churchill, and T. I. Michalak, unpublished data).

To quantify WHV mRNA, cDNA derived from the equivalent of 50 ng RNA, along with serial 10-fold dilutions of complete recombinant WHV DNA as standards (42), was amplified by real-time PCR using WHVc gene-specific primers (Table 1). When WHV RNA was undetectable, 2 μg of cDNA was analyzed by nested PCR-NAH using WHV-specific primers and conditions described before (41).

Statistical analysis.

The two-tailed, unpaired Student t test, with a 95% confidence interval, was applied to compare the means of the results for the sample groups investigated, and P values of <0.05 were considered to be statistically significant.

Serological and WHV DNA profiles after primary and multiple exposures to WHV.

Among animals belonging to study group A, three woodchucks developed classical self-limited AH with WHsAg detectable in their sera between 3 and 7 w.p.p.i. (Fig. 1A). Woodchucks 1/F, 2/F, and 3/F seroconverted to anti-WHs between 22 and 37 w.p.p.i., while woodchuck 4/M became transiently anti-WHs reactive 1 week after challenge with WHV, i.e., at 66 w.p.p.i. (Fig. 1A). Anti-WHc appeared at 6 w.p.p.i. and remained detectable during the entire follow-up period in all animals. The level of anti-WHc did not fluctuate noticeably after challenge or rechallenge with WHV. Serum WHV DNA became detectable from 1 w.p.p.i. and persisted until the end of follow-up in all four woodchucks. After clearance of serum WHsAg, the level of circulating WHV DNA declined and usually did not exceed 10² vge/ml thereafter, as has been previously reported for animals with secondary occult HBV infection continuing after resolution of AH (10, 41, 42). Serum WHV DNA remained at a similar low level after the second and third injections with WHV (Fig. 1A). Hepatic WHV loads were at 2 × 10³ to 8 × 10³ vge/μg of total liver DNA during AH, and then subsided to about 3 × 10² vge/μg and persisted at an approximately similar level until the end of follow-up (Fig. 1A), as previously observed (10, 41, 42).

In study group B, three animals infected with WHV/tm3 or WHV/tm4 developed immunovirological and WHV DNA profiles indicative of a self-limiting episode of AH which were closely comparable to those seen in animals in group A (Fig. 1B), while woodchuck 8/M developed histologically evident mild, protracted hepatitis. In this animal, serum WHsAg appeared at 3 w.p.i. and persisted until the end of follow-up, i.e., 16 w.p.i. Anti-WHs became detectable in animals 6/M and 7/M after clearance of WHsAg from serum around 13 and 10 w.p.i., respectively. Liver biopsies collected at 7 w.p.i. contained WHV DNA loads ranging from 2.4 × 10⁶ to 2.5 × 10⁷ vge/μg of total DNA (mean ± standard deviation, 1.1 × 10⁷ ± 4.9 × 10⁶ vge/μg) and WHV mRNA at 3.1 × 10⁵ to 5.5 × 10⁶ copies/μg of total RNA (mean ± standard deviation, 2.7 × 10⁶ ± 1.1 × 10⁶ copies/μg).

Both primary infection and challenge with WHV induce delayed virus-specific T-cell response.

In samples from animals from group A, the WHV-specific T-cell proliferation against WHV antigens or the WHc_97-110 peptide measured by the [³H]adenine incorporation assay was multispecific and of relatively high magnitude between 10 and 14 w.p.p.i. (Fig. 2). Persistently detectable virus-specific proliferative responses to rWHe, rWHx, and the WHc_97-110 peptide first appeared around 6 to 8 w.p.p.i. and preceded those against rWHc and WHsAg, which became detectable at 10 to 14 w.p.p.i. The strong WHV-specific response was evident for up to 30 w.p.p.i. and then subsided and became intermittently detectable with the [³H]adenine incorporation assay until challenge at 65 w.p.p.i. (Fig. 2). In two control animals, the pattern of WHV-specific responses during the acute phase of infection was similar to that described above, and the reactivity remained detectable until termination of the experiment at 112 w.p.i. (data not shown). Overall, the virus-specific T-cell response was strong and multispecific during the acute phase of primary infection, with the greatest magnitudes after ex vivo stimulation with rWHe, followed by rWHc or rWHx, and the lowest after stimulation with WHc_97-110 or WHsAg.

Challenge of the convalescent woodchucks with the same 1.9 × 10¹¹ vge dose of WHV/tm4 did not induce the expected immediate, virus-specific memory T-cell response (Fig. 2). However, a strong T-cell response to WHV antigens was found in all animals between 5 and 10 weeks after challenge, i.e., 70 to 75 w.p.p.i. The magnitudes of the secondary responses to rWHe (P = 0.01), rWHc (P = 0.03), and rWHx (P = 0.001) were significantly greater than those during the primary infection (Fig. 2). In contrast, the levels of secondary T-cell response against the WHc_97-110 peptide (P = 0.6) and WHsAg (P = 0.9) were comparable to those detected in the primary infection. After rechallenge with either a high (animals 1/F and 2/F) or low (animals 3/F and 4/M) dose of WHV/tm4, samples from all animals demonstrated minuscule or undetectable virus-specific T-cell reactivity when tested with the [³H]adenine incorporation assay (Fig. 2). However, a transient increase in the magnitude of WHV-specific T-cell proliferation was evident when the more-sensitive CFSE flow cytometric assay was applied (Fig. 2, insets).

The generalized proliferative capacity of lymphocytes increases immediately after primary and subsequent exposures to WHV.

Almost immediately after the primary injection with WHV, i.e., 1 to 3 w.p.p.i., the lymphocytes acquired from animals in study group A showed a strongly heightened capacity to proliferate in response to ConA, PWM, and PHA (Fig. 3). Then, the response subsided between 6 and 30 w.p.p.i., at the time when the WHV-specific T-cell response appeared, and endured at high levels (Fig. 4). The exception was animal 4/M, with a serum WHsAg-negative but anti-WHc- and WHV DNA-positive infection in which the nonspecific lymphocyte reactivity after the initial lowering increased between 10 and 14 w.p.p.i. During the phase of lowered responsiveness to mitogenic stimuli, temporal increases in this T-cell response occurred. Interestingly, these periodic increases appeared to be synchronized in all animals, as recapitulated in Fig. 4. It also is of note that ConA, PHA, and PWM induced similar profiles of the proliferative response (Fig. 3), suggesting that no particular lymphocyte subset was predominantly involved in the periodic increases.

After challenge with WHV at 65 w.p.p.i., this nonspecific T-cell proliferative reactivity was again augmented in all animals, with the peak occurring between 1 and 3 weeks following challenge, i.e., 66 and 68 w.p.p.i. (Fig. 3 and 4). However, the magnitudes of the cell proliferation responses to ConA (P = 0.2), PWM (P = 0.5), and PHA (P = 0.1) were not significantly different from those detected after the first injection with WHV (Fig. 3). This heightened response to mitogens subsided between 68 and 75 w.p.p.i., which coincided again with the rise of the secondary WHV-specific T-cell response (Fig. 3 and 4). Subsequently, when the virus-specific T-cell reactivity progressively decreased, the T-cell proliferative capacity in response to mitogens rebounded and remained at a high level until 80 w.p.p.i. when the animals were rechallenged with WHV (Fig. 4). The third injection with either a high or low WHV dose induced another, although lower in magnitude, immediate increase in the T-cell generalized proliferative response (Fig. 3 and 4).

Immediate augmented lymphocyte generalized proliferative response and delayed virus-specific T-cell response are consistently induced by WHV infection.

To confirm findings made in the first study by assessing the WHV-specific response using a CFSE flow cytometric assay, as well as to test if another WHV inoculum will induce the same effects in T-cell responses and to systematically evaluate lymphoid cell predisposition to apoptotic death after exposure to WHV, weekly PBMC samples collected from animals constituting study group B were investigated. Thus, the results of the CFSE-based lymphocyte proliferation assay with rWHe confirmed that the primary exposure to WHV induced a delayed virus-specific T-cell response which appeared from 7 w.p.i. and persisted until the end of the 16-week follow-up (Fig. 5A). Further, the capacity of the lymphoid cells to proliferate in response to stimulation with a mitogenic stimulus (ConA) was evident again immediately after exposure to WHV, i.e., as early as 1 w.p.i. This response rapidly subsided between 4 and 6 w.p.i., then transiently increased between 7 and 8 w.p.i., and receded to approximately preinfection levels thereafter (Fig. 5B). All four animals belonging to study group B displayed closely comparable patterns of both WHV-specific and ConA-induced T-cell proliferative responses, including animal 8/M, which developed a protracted infection suggesting progression to chronic hepatitis.

Considering the distinct and, for the most part, opposing kinetics of the generalized, mitogen-induced, and WHV-specific T-cell proliferative responses, the progression of acute WHV infection was divided into phases (Fig. 5A and B). Thus, phase A corresponded to the time period prior to WHV infection; phase B, or the preacute phase, occurred between 1 and 3 w.p.i. and was characterized by heightened nonspecific and essentially absent WHV-specific T-cell responses; phase C, or the early acute phase between 4 and 6 w.p.i., was accompanied by subsiding nonspecific and absent virus-specific T-cell responses, and phase D, or the acute phase (7 w.p.i. onwards), was associated with the initially slightly increasing but then normalized mitogen-induced proliferative capacity and with significantly augmented WHV-specific T-cell responses. As shown by the results in Fig. 5C, the magnitude of WHV-specific T-cell reactivity during acute infection (phase D) was significantly (P < 0.0001) greater than those detected during the preacute (phase B) and early acute (phase C) periods of WHV infection. In contrast, PBMC displayed significantly (P < 0.0001) increased magnitudes of mitogen-induced proliferation immediately after exposure to WHV (Fig. 5D), i.e., during the preacute period (phase B). This reactivity declined during the early acute period (phase C), displaying a mean response lower (P = 0.02) than that observed prior to infection (phase A), and subsequently rebounded in the acute phase (phase D) to a mean level greater (P = 0.03) than that prior to infection (phase A), but still significantly (P < 0.005) lower than that characterizing phase B.

Decline in the generalized proliferation capacity of T cells coincides with increased activation-induced cell death.

To recognize a possible reason behind the observed rapid decline in the T-cell proliferative response to mitogenic stimuli seen during the early acute period (phase C) of WHV infection, the rate of apoptotic death of lymphomononuclear cells in weekly PBMC samples from group B animals was evaluated by using the assay employing staining with annexin V-PE, 7-AAD, and CFSE. In preliminary experiments, the authenticity of staining of woodchuck lymphoid cells with recombinant human annexin V-PE and 7-AAD was verified by visualizing appropriate subcellular compartments with confocal microscopy. As illustrated in Fig. 6A, annexin V, 7-AAD, and CFSE produced cell surface, nuclear, and cytoplasmic staining, respectively, in woodchuck lymphocytes undergoing apoptosis.

To assess whether naturally circulating lymphocytes from different phases of acute WHV infection (Fig. 5) might be differentially predisposed to apoptosis, freshly isolated, unmanipulated, weekly PBMC samples were stained with annexin V and 7-AAD. The results showed that the fresh cells from different phases did not display any noticeable variations in mean percentage of apoptotic cells in comparison to the rate of apoptosis of the cells collected prior to infection (Fig. 6B). However, when the rate of apoptosis was measured in the same cell samples after stimulation with ConA or rWHe, significantly greater rates and infection phase-dependent changes in apoptotic death were identified (Fig. 6B). Overall, the results indicated that lymphocytes derived from the early acute period (phase C) of WHV infection, when nonspecific T-cell responsiveness profoundly declined but the WHV-specific response had not yet risen, were significantly more prone to activation-induced death than the cells collected soon after infection (phase B) or during AH (phase D).

Cytokine and cell marker gene expression profiles in unmanipulated circulating lymphoid cells.

Since the expression patterns of antiviral and proinflammatory cytokines in peripheral lymphoid cells after primary hepadnaviral infection have been described previously (20, 60), we initially (study group A) examined the expression kinetics after reexposure to WHV. We found that following challenge of animals convalescent from AH with WHV, the heightened expression of IFN-α became immediately evident in sequential PBMC samples collected after challenge, i.e., between 66 and 69 w.p.p.i. This cytokine transcription then subsided and increased again, but to a lower level, around the time of rechallenge with the same virus inoculum (Fig. 7A). This significantly increased (P < 0.0001) expression of IFN-α coincided with the phase of augmented proliferative responsiveness of T-cells to mitogen stimuli (Fig. 7A and Fig. 3, respectively). The transcription of TNF-α (P < 0.0001) and, to some extent, IFN-γ (P = 0.015) was transiently enhanced between 68 and 69 w.p.p.i. The mRNA levels of IFN-α, IFN-γ, TNF-α, and, to a lesser degree, CD3 subsided 5 weeks later, i.e., around 70 w.p.p.i. In general, the enhanced expression of IFN-α, IFN-γ, and TNF-α coincided with the highly augmented T-cell nonspecific proliferative capacity and clearly preceded the reappearance of the WHV-specific T-cell proliferative response, which occurred between weeks 70 and 75 postinfection (Fig. 7A and 2, respectively). The expression profiles of IL-2 and IL-4 did not correlate with the magnitudes of either specific or nonspecific T-cell responses, except for animal 4/M, which displayed increased expression of both these cytokines at the time of greater expression of IFN-γ and TNF-α. The CD14 and CD56 transcription levels measured in serial PBMC samples showed unremarkable profiles (data not shown). Overall, the data revealed a close association between the augmented expression of IFN-α, TNF-α, and, to a lesser extent, IFN-γ in circulating unmanipulated lymphoid cells and their heightened proliferative capacity in response to mitogenic, but not virus-specific, stimuli after challenge with WHV.

To further recognize events accompanying distinctive phases of WHV-specific and nonspecific T-cell proliferative responses accompanying acute WHV infection, the transcriptional activities of genes encoding selected cytokines and immune-cell subtype-specific markers were quantified by real-time RT-PCR in unmanipulated PBMC collected weekly from animals belonging to study group B. As shown by the results in Fig. 7B, the significantly augmented expression of IFN-α (P = 0.0001), IL-12 (P = 0.005), IL-2 (P = 0.005), and IL-4 (P = 0.01) relative to the levels detected prior to infection (phase A) was evident almost immediately after primary exposure to WHV (phase B). This coincided with the elevated expression of CD3 (P = 0.04) and CD56 (P = 0.01). This selective upregulation of progrowth lymphoid cell cytokines, along with natural killer (NK)- and T-cell-specific markers, was associated with the significantly heightened lymphocyte proliferative responsiveness to stimulation with ConA, but not with WHV (Fig. 5C and D). However, these elevated levels of expression of the cytokines and cell markers indicated above became drastically reduced during the early acute period (phase C), which coincided with the augmented susceptibility of lymphoid cells to undergo activation-induced apoptosis (Fig. 6B). It is important to note that WHV infection failed to induce the expression of either TNF-α or IFN-γ in lymphoid cells during both preacute (phase B) and early acute (phase C) periods (Fig. 7B). Finally, during acute WHV infection (phase D), the levels of expression of IFN-α, TNF-α, IFN-γ, IL-12, IL-2, IL-10, and IL-4 were synchronously upregulated, along with the significantly greater expression of CD3 (P < 0.0001) and CD4 (P < 0.0001) which coincided with the appearance of the strong WHV-specific T-cell response (Fig. 5A).

WHV replication peaks in lymphoid cells after their increased susceptibility to activation-induced death subsides.

As shown by the results for animals from study group B in presented Fig. 8, the WHV mRNA was detectable in serial PBMC samples beginning from the first week postinfection at levels not exceeding 200 copies/μg of total RNA. From 5 w.p.i., i.e., at the time of termination of the increased susceptibility of the cells to activation-induced apoptosis, WHV mRNA levels progressively increased in circulating lymphoid cells, peaked at around 11 w.p.i., and then subsided until the end of follow-up. Overall, the results revealed that WHV replication in peripheral lymphoid cells is established as early as 1 week after exposure to a large, liver-pathogenic dose of WHV and progresses at a low level until acute infection becomes serologically and histologically evident (phase D).