Biomarkers for early effects of carcinogenic dual-acting PPAR agonists in rat urinary bladder urothelium in vivo.

Small-molecule agonists of the peroxisome proliferator-activated receptor (PPAR) alpha and gamma isoforms (dual-acting PPAR agonists) can cause urothelial cancers in rodents. Rats were dosed orally for 16 days with bladder carcinogenic (ragaglitazar) as well as non-bladder carcinogenic (fenofibrate and rosiglitazone) PPAR agonists and protein changes were assayed in the urinary bladder urothelium by Western blotting. Dose levels reflected 10-20 x human exposure, and the ragaglitazar dose was in the carcinogenic range. Ragaglitazar induced expression of the transcription factor Egr-1, phosphorylation of the c-Jun transcription factor and phosphorylation of the ribosomal S6 protein were observed. These changes were also observed in rats dosed with either rosiglitazone or fenofibrate. However, the protein changes were stronger (Egr-1 induction) or of a longer duration (S6 phosphorylation) in ragaglitazar-treated animals. Animals co-administered fenofibrate (a specific PPARalpha agonist) and rosiglitazone (a specific PPARgamma agonist) exhibited Egr-1 and S6 protein changes more similar to those induced by ragaglitazar (a dual-acting PPARalpha/gamma agonist) than either fenofibrate or rosiglitazone alone. The findings suggest that ragaglitazar causes Egr-1, c-Jun and S6 protein changes in the urothelium by a mechanism involving PPARalpha as well as PPARgamma, and that the Egr-1, c-Jun and S6 protein changes might have potential biomarker value.

Development of a novel quantitative real-time RT-PCR assay for the simultaneous detection of all serotypes of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) spreads extremely fast and the need for rapid and robust diagnostic virus detection systems was obvious during the recent European epidemic. Using a novel real-time RT-PCR system based on primer-probe energy transfer (PriProET) we present here an assay targeting the 3D gene of FMDV. The assay was validated for the efficacy to detect all known FMDV serotypes. The test method was linear over a range of at least 7 orders of magnitude and the detection limit was below the equivalent of 10 genomic copies. Analysing recent African probang samples the method was able to detect FMDV in materials from both cattle and buffalo. When compared to traditional virus cultivation the virus detection sensitivity was similar but the RT-PCR method can provide a laboratory result much faster than virus cultivation. The real-time PCR method confirms the identity of the amplicon by melting point analysis for added specificity and at the same time allows the detection of mutations in the probe region. As such, the described new method is suitable for the robust real-time detection of index cases caused by any serotype of FMDV.

Generation of an infectious clone of VR-2332, a highly virulent North American-type isolate of porcine reproductive and respiratory syndrome virus.

A full-length cDNA clone of the prototypical North American porcine reproductive and respiratory syndrome virus (PRRSV) isolate VR-2332 was assembled in the plasmid vector pOK(12). To rescue infectious virus, capped RNA was transcribed in vitro from the pOK(12) clone and transfected into BHK-21C cells. The supernatant from transfected monolayers were serially passaged on Marc-145 cells and porcine pulmonary alveolar macrophages. Infectious PRRSV was recovered on Marc-145 cells as well as porcine pulmonary macrophages; thus, the cloned virus exhibited the same cell tropism as the parental VR-2332 strain. However, the cloned virus was clearly distinguishable from the parental VR-2332 strain by an engineered marker, a BstZ17I restriction site. The full-length cDNA clone had 11 nucleotide changes, 2 of which affected coding, compared to the parental VR-2332 strain. Additionally, the transcribed RNA had an extra G at the 5' end. To examine whether these changes influenced viral replication, we examined the growth kinetics of the cloned virus in vitro. In Marc-145 cells, the growth kinetics of the cloned virus reflected those of the parental isolate, even though the titers of the cloned virus were consistently slightly lower. In experimentally infected 5.5-week-old pigs, the cloned virus produced blue discoloration of the ears, a classical clinical symptom of PRRSV. Also, the seroconversion kinetics of pigs infected with the cloned virus and VR-2332 were very similar. Hence, virus derived from the full-length cDNA clone appeared to recapitulate the biological properties of the highly virulent parental VR-2332 strain. This is the first report of an infectious cDNA clone based on American-type PRRSV. The availability of this cDNA clone will allow examination of the molecular mechanisms behind PRRSV virulence and attenuation, which might in turn allow the production of second-generation, genetically engineered PRRSV vaccines.

Reproduction of postweaning multisystemic wasting syndrome (PMWS) in immunostimulated and non-immunostimulated 3-week-old piglets experimentally infected with porcine circovirus type 2 (PCV2).

Postweaning multisystemic wasting syndrome (PMWS) in swine is causally associated with the newly recognised pathogen, porcine circovirus type 2 (PCV2). In this study, 3-week-old SPF PCV2-seronegative piglets were inoculated intranasally with PCV2. The effect of immunostimulation on the induction of PMWS was investigated by immunisation with keyhole limpet hemocyanin (KLH) emulsified in incomplete Freunds adjuvant. The study was terminated 5 weeks after inoculation. While disease was not observed in the age-matched controls, two out of five non-immunised PCV2-infected piglets died on postinoculation day (PID) 21, and one was euthanized on PID 25 in moribund condition. These animals had appeared lethargic with persistent fever from PID 12 onwards. The euthanized pig appeared smaller than littermates and suffered from jaundice. At postmortem examination, gastric ulceration, icterus, and liver and thymus atrophy were observed. Furthermore, histological lesions of degenerating hepatocytes and hepatitis in combination with lymphoid depletion and syncytial cells in lymph nodes were consistent with the diagnosis of PMWS. One out of five immunostimulated PCV2-infected piglets was euthanized on PID 22 with convulsions after a period with wasting. This pig was lethargic from PID 14 onwards with persistent fever from PID 8 and transient dyspnoea. No differences in clinical signs, gross pathologic or histological findings were observed for the remaining non-immunostimulated and immunostimulated PCV2-infected piglets. All 10 PCV2-inoculated piglets seroconverted to PCV2 within 14 days after inoculation. By virus isolation, quantitative polymerase chain reaction (Q-PCR), and immunostaining of cryostat sections, it was demonstrated that lymphoid tissue contained abundant PCV2 antigen. Viral DNA load in serum samples was assessed by Q-PCR. All four PMWS-affected piglets had high levels of PCV2 DNA in serum, suggesting that there was a correlation between high levels of viral DNA in serum and the development of PMWS. In conclusion, infection with PCV2 caused PMWS in SPF piglets, however, the immunostimulation did not seem to play a critical role.

Identification of radically different variants of porcine reproductive and respiratory syndrome virus in Eastern Europe: towards a common ancestor for European and American viruses.

We determined 22 partial porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 sequences, representing pathogenic field strains mainly from Poland and Lithuania, and two currently available European-type live PRRSV vaccines. Also, the complete ORF7 of two Lithuanian and two Polish strains was sequenced. We found that Polish, and in particular Lithuanian, PRRSV sequences were exceptionally different from the European prototype, the Lelystad virus, and in addition showed a very high national diversity. The most diverse present-day European-type PRRSV sequences were from Poland (2000) and Lithuania (2000), and exhibited only 72.2% nucleotide identity in the investigated ORF5 sequence. While all sequences determined in the present study were clearly of European type, inclusion of the new Lithuanian sequences in the genealogy resulted in a common ancestor for the European type virus significantly closer to the American-type PRRSV than previously seen. In addition, the length of the ORF7 of the Lithuanian strains was 378 nucleotides, and thus intermediate between the sizes of the prototypical EU-type (387 nucleotides) and US-type (372 nucleotides) ORF7 lengths. These findings for the Lithuanian PRRSV sequences provide support for the hypothesis that the EU and US genotypes of PRRSV evolved from a common ancestor. Also, this is the first report of ORF7 protein size polymorphism in field isolates of EU-type PRRSV.

Experimental inoculation of late term pregnant sows with a field isolate of porcine reproductive and respiratory syndrome vaccine-derived virus.

The use of a live attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in piglets has been associated with reproductive disorders in non-vaccinated sows. Vaccine-derived virus (VDV) has been isolated from foetuses, stillborn pigs, and dead piglets, indicating that the live vaccine spread from vaccinated piglets to non-vaccinated sows, and that the virus might be implicated in the severe reproductive problems observed. In the present study, one such VDV isolate was used to experimentally infect pregnant sows in the last trimester. The chosen isolate, which had more than 99.6% identity to the attenuated vaccine virus, originated from the lungs of a stillborn pig from a swine herd with a sudden high level of stillborn pigs and increased piglet mortality in the nursing period. Intranasal inoculation of sows with the virus isolate resulted in congenital infection, foetal death, and preweaning pig mortality. As such, the present study showed that vaccine-derived PRRSV can cause disease in swine consistent with PRRS.

A molecular clock dates the common ancestor of European-type porcine reproductive and respiratory syndrome virus at more than 10 years before the emergence of disease.

The disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) emerged independently and almost simultaneously in Europe (1990) and North America (1987). The original reservoir of the virus and the date it entered the pig populations is not known. In this study, we demonstrate an accurate molecular clock for the European PRRSV ORF 3 gene, place the root in the genealogy, estimate the rate of nucleotide substitution, and date the most recent common viral ancestor of the data set to 1979; more than 10 years before the onset of the European epidemic. Based on these findings, we conclude that PRRSV virus most likely entered the pig population some time before the epidemic emergence of the virus, and hence, that emergence of European-type PRRSV is not the result of a recent species transmission event. Together, our results show that ORF3 sequencing is a valuable epidemiologic tool for examining the emergence and spread of PRRSV in Europe. As such, the panel of well-characterized and highly divergent ORF3 sequences described in this study provides a reference point for future molecular epidemiologic studies.

Phylogenetic characterisation of the G(L) sequences of equine arteritis virus isolated from semen of asymptomatic stallions and fatal cases of equine viral arteritis in Denmark.

The study describes for the first time the phylogenetic relationship between equine arteritis virus (EAV) isolated from asymptomatic virus-shedding stallions and fatal cases of equine viral arteritis (EVA) in an European country. EAV was isolated from three dead foals and an aborted foetus during three different outbreaks of EVA. From these fatalities, the complete open reading frame 5, encoding the EAV G(L) protein, was amplified by reverse transcription-polymerase chain reaction and subjected to nucleotide sequence analysis. Furthermore, DNA sequences were obtained from virus isolated from semen samples of seven virus-shedding, but clinically healthy, Danish stallions. DNA sequence alignment revealed an overall divergence of 0-14 and 0-10% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis including 24 previously published sequences revealed that European as well as North American "types" of EAV were present in the semen of asymptomatic carrier stallions and in fatal cases of EVA. Our results reveal that the presence of EAV-shedding stallions in Denmark represents a potential source of severe EVA.

Epitope mapping porcine reproductive and respiratory syndrome virus by phage display: the nsp2 fragment of the replicase polyprotein contains a cluster of B-cell epitopes.

We screened phage display libraries of porcine reproductive and respiratory syndrome virus (PRRSV) protein fragments with sera from experimentally infected pigs to identify linear B-cell epitopes that are commonly recognized during infection in vivo. We identified 10 linear epitope sites (ES) 11 to 53 amino acids in length. In the replicase polyprotein, a total of eight ES were identified, six of which localized to the Nsp2 replicase polyprotein processing end product. In the structural proteins, a total of two ES were identified, in the ORF3 and ORF4 minor envelope glycoproteins. The ORF4 ES was previously identified by monoclonal antibody mapping (J. J. M. Meulenberg, A. P. van Nieuwstadt, A. van Essen-Zandenbergen, and J. P. M. Langeveld, J. Virol. 71:6061-6067, 1997), but its immunogenicity had not been examined in pigs. We found that six experimentally PRRSV-infected pigs consistently had very high antibody titers against the ORF4 ES. In some animals, sera diluted 1:62,500 still gave weak positive enzyme immunoassay reactivity against the ORF4 ES. This hitherto unrecognized immunodominance likely caused phages displaying the ORF4 ES to outcompete phages displaying other ES during library screening with porcine sera and accounted for our failure to identify more than two ES in the structural genes of PRRSV. Genetic analysis showed that variable ES were also the most immunogenic in vivo. Serological analysis indicated differences in the immunoglobulin A responses between short-term and longer-term viremic pigs towards some ES. The implications of these findings for PRRSV diagnostics and immunopathogenesis are discussed.

Analysis of ORF 1 in European porcine reproductive and respiratory syndrome virus by long RT-PCR and restriction fragment length polymorphism (RFLP) analysis.

A rapid method was developed for partial characterization of the replicase-encoding open reading frame 1 (ORF 1) of porcine reproductive and respiratory syndrome virus (PRRSV). It comprised long RT-PCR amplification of 11.1 kb (94%) of ORF 1, followed by restriction fragment length polymorphism analysis. The method was used to compare ORF 1 sequences of two divergent European-type PRRSV strains. Our results indicated that the structural and replicase parts of these two strains had evolved at overall similar rates.

Emergence of porcine reproductive and respiratory syndrome virus deletion mutants: correlation with the porcine antibody response to a hypervariable site in the ORF 3 structural glycoprotein.

By using porcine immune sera to select a library of phage-displayed random peptides, we identified an antigenic sequence (RKASLSTS) in the C-terminus of the ORF 3 structural glycoprotein of European-type porcine reproductive and respiratory syndrome virus (PRRSV). Through the use of overlapping reading frames, the same PRRSV genetic locus codes for the ORF 3 "RKASLSTS" sequence, and a previously described ORF 4 epitope (Meulenberg, J. J. M., Van Nieuwstadt, A. P., Van Essen-Zandbergen, A., and Langeveld, J. P. M., 1997, J. Virol. 71, 6061-6067). Sequence analysis identified naturally occurring deletion mutants at this ORF 34 site. Phylogenetic analysis showed the presence of a highly accurate ORF 3 molecular clock, according to which deletion mutants and nondeleted viruses evolved at differing speeds. Furthermore, deletion mutants and nondeleted viruses evolved as separate lineages. These distinctions suggested that deletion mutants were a hitherto unrecognized subtype of European-type PRRSV. Currently, deletion mutants appear to be outcompeting nondeleted viruses in the field, highlighting the importance of the porcine antibody response against the minor structural glycoproteins of European-type PRRSV for viral evolution.

Heterologous challenge with porcine reproductive and respiratory syndrome (PRRS) vaccine virus: no evidence of reactivation of previous European-type PRRS virus infection.

In Denmark, a porcine reproductive and respiratory syndrome virus (PRRSV) control programme, comprising vaccination of seropositive herds with a live American type PRRSV vaccine, was started in 1996. In several of these herds, spread of vaccine virus from vaccinated 3-18 week old pigs to non-vaccinated sows was demonstrated by the isolation of vaccine virus from fetuses and stillborn piglets. Surprisingly, sows infected with the American type vaccine strain consistently exhibited significantly stronger serological responses towards European type PRRSV than American type PRRSV. In order to elucidate whether the unexpectedly strong serological reaction towards European-type PRRSV in American type PRRSV infected sows was due to a booster reaction, or reactivation of an unrecognized, latent infection in the sows with European type PRRSV, a challenge study with the vaccine was carried out. In this study, the stronger serological response towards European type PRRSV than towards American type PRRSV was reproduced, and reactivation of the previous natural infection with European PRRSV could neither be demonstrated by virus isolation nor by RT-PCR. So, the increase in antibody titers towards European PRRSV in previously European PRRSV infected pigs after challenge with the vaccine strain seems to be the result of a boosting effect on the immune system, induced by the heterologous vaccine PRRSV strain.

Determination of 5'-leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs.

We determined the untranslated 5'-leader sequence for three different isolates of porcine reproductive and respiratory syndrome virus (PRRSV): pathogenic European- and American-types, as well as an American-type vaccine strain. 5'-leader from European- and American-type PRRSV differed in length (220 and 190 nt, respectively), and exhibited only approximately 50% nucleotide homology. Nevertheless, highly conserved areas were identified in the leader of all 3 PRRSV isolates, which constitute candidate motifs for binding of protein(s) involved in viral replication. These comparative data provide a priori knowledge for mutational identification of virulence determinants in the 5' nontranslated part of the PRRSV genome.

Examination of the selective pressures on a live PRRS vaccine virus.

We determined the ORF5 and 7 sequences of 20 pathogenic revertants of a live PRRSV vaccine. The sequence analysis confirmed all 20 isolates to be of vaccine origin. Having established that clonal introduction of American (vaccine) PRRS virus had occurred in Denmark, we could perform analysis of the selective pressure this attenuated virus had experienced during reversion. An analysis of nucleotide mutations showed a similar rate of mutations in the two genes (ORF5 and 7). However, non-synonymous mutations in ORF7 were eliminated by purifying selection. In contrast, non-synonymous mutations in ORF5 were tolerated or even selected for. The cDNA sequencing of the 20 vaccine virus revertants identified two single nucleotide mutations located in ORF5 and in ORF6 that we suggest are involved or at least linked to the attenuation of the vaccine virus and to the subsequent reversion to virulence.

Sensitive detection and typing of porcine reproductive and respiratory syndrome virus by RT-PCR amplification of whole viral genes.

Following the recent use of a live vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) in Denmark, both American (vaccine) and European-type PRRSV now coexist in Danish herds. This situation highlighted a requirement for supplementary tests for precise virus-typing. As a result, we developed a RT-PCR assay able to detect as well as type PRRSV. To provide maximal sequence information, complete viral open reading frames (ORFs 5 and 7) were targeted for amplification. The RT-PCR test was able to amplify complete PRRSV ORFs from complex materials such as boar semen containing as little as 1 TCID50 ml(-1) of PRRSV. Typing of viruses was accomplished by any one of three strategies: (i) use of type-specific PCR primers, (ii) size determination of ORF 7 amplicons, (iii) DNA sequencing. All three typing strategies showed complete concordance with the currently used method of typing with monoclonal antibodies (MAbs) when used on a panel of PRRSV field isolates covering the period 1992-1997. The ORF 7-based test had particularly desirable characteristics, namely, highly sensitive detection of PRRSV without apparent type bias, typing of the detected virus, discrimination between pure and mixed virus populations, and semi-quantitative assessment of type ratios in mixed populations, all in a single PCR reaction. In addition, the obtained sequence data were used to predict two simple and rapid strategies (single-enzyme restriction length polymorphy analysis and oligonucleotide hybridization) for confirmation of the specificity of ORF 7 RT-PCR reactions. As such, the RT-PCR assay provides a new, powerful diagnostic tool to study the population dynamics between present and emerging PRRSV-types.

Two parvoviruses that cause different diseases in mink have different transcription patterns: transcription analysis of mink enteritis virus and Aleutian mink disease parvovirus in the same cell line.

The two parvoviruses of mink cause very different diseases. Mink enteritis virus (MEV) is associated with rapid, high-level viral replication and acute disease. In contrast, infection with Aleutian mink disease parvovirus (ADV) is associated with persistent, low-level viral replication and chronic severe immune dysregulation. In the present report, we have compared viral transcription in synchronized CRFK cells infected with either MEV or ADV using a nonradioactive RNase protection assay. The overall level of viral transcription was 20-fold higher in MEV- than in ADV-infected cells. Furthermore, MEV mRNA encoding structural proteins (MEV mRNA R3) was dominant throughout the infectious cycle, comprising approximately 80% of the total viral transcription products. In marked contrast, in ADV-infected cells, transcripts encoding nonstructural proteins (ADV mRNA R1 and R2) comprised more than 84% of the total transcripts at all times after infection, whereas ADV mRNA R3 comprised less than 16%. Thus, the ADV mRNA coding for structural proteins (ADV mRNA R3) was present at a level at least 100-fold lower than the corresponding MEV mRNA R3. These findings paralleled previous biochemical studies analyzing in vitro activities of the ADV and MEV promoters (J. Christensen, T. Storgaard, B. Viuff, B. Aasted, and S. Alexandersen, J. Virol. 67:1877-1886, 1993). The overall low levels of ADV mRNA and the paucity of the mRNA coding for ADV structural proteins may reflect an adaptation of the virus for low-level restricted infection.

Prejunctional modulation by angiotensins of noradrenaline release from sympathetic neurons in isolated rabbit aorta.

The aims of the present work were to compare the modulating effect of angiotensins I, II, III, IV and (1-7) [AI, AII, AIII, AIV and A(1-7) respectively] on stimulation-evoked noradrenaline release from postganglionic sympathetic nerves in rabbit isolated aorta; to examine the influence of inhibiting the neuronal and extraneuronal uptake of noradrenaline on the modulating effect of AII and thirdly, to determine the role of angiotensin converting enzyme (ACE) in the modulating effects of AI and AII and the role of aminopeptidases A and M in the effects of AII and AIII. Rings of aorta were preloaded with (-)-3H-noradrenaline and then subjected to electrical field stimulation. Cumulative addition of AI (10(-8)-10(-6) M), AII (3 x 10(-11)-10(-8) M) and AIII (3 x 10(-10)-10(-6) M) enhanced the stimulation-evoked 3H-overflow up to 142, 165 and 188% respectively. The order of potency was AII > AIII > AI. AIV (10(-10)-10(-7) M) and A(1-7) (10(-10)-10(-7) M) caused no change. Single concentrations (10(-9)-10(-7) M) of AI, AII and AIII caused initial enhancement which subsequently decreased, i.e. development of tachyphylaxis. The effect of AII was independent of stimulation frequency at 1-10 Hz, but absent at 30 Hz. Cocaine (3 x 10(-5) M) plus corticosterone (4 x 10(-5) M) did not alter the enhancing effect of AII. CaNa2EDTA (3 x 10(-5) M) did not alter the enhancing effect of AI. Captopril (10(-6) and 10(-5) M) and lisinopril (10(-6) M) attenuated the enhancing effect of AI. Captopril and lisinopril (both 10(-6) and 10(-5) M) did not alter the enhancing effect of AII. Captopril (10(-7)-10(-4) M) and lisinopril (10(-7)-10(-4) M) themselves did not alter the stimulation-evoked 3H-overflow. Amastatin (10(-5) M) increased the enhancement seen with AIII (3 x 10(-11)-10(-9) M) but did not alter the enhancing effect of AII (10(-9)-10(-8) M). Amastatin (10(-9)-10(-5) M) had no effect on the stimulation-evoked 3H-overflow. It is concluded that AI, AII and AIII facilitate the stimulation-evoked 3H-noradrenaline release to various degrees (relative order of potency: AII > AIII > AI and of efficacy: AIII > AII > AI). The estimates may be compromised by the development of tachyphylaxis. The facilitation by AII was independent of the neuronal and extraneuronal uptake mechanisms. The action of AI is in part due to its conversion to AII. The effect of AIII was probably underestimated due to its degradation to AIV. AII is apparently not a substrate for aminopeptidase M.

Structure of mink immunoglobulin gamma chain cDNA.

Two cDNA clones, encoding mink Ig gamma chains were characterized. The pIGG47 clone contains a part of the leader segment, VDJ and C regions, and pIGG14 contains a part of the J and a complete C region. The clones differ by only four nucleotides in the C region, and they most probably represent allelic variants of the same gene. The V gene segment of pIGG47 was found to be highly similar to human VHIII subgroup sequences; there was 86-87% similarity for the whole V gene segment and 91% for the VHIII specific regions (codons 65-87). Southern blot analysis demonstrated that a high proportion of mink VH genes is VHIII related. The V gene segment used as a probe revealed 19-23 bands in mink DNA under stringent conditions. This is in agreement with our previous data showing that a high proportion of mink Ig contains an 'alternative' binding site for protein A, a feature common to VHIII-related molecules. According to Southern blot analysis there may be 5-7 C gamma genes at the mink IgH locus.

Pathogenesis of Aleutian mink disease parvovirus infection: effects of suppression of antibody response on viral mRNA levels and on development of acute disease.

We suppressed the B-cell development and antibody response in mink by using treatment with polyclonal anti-immunoglobulin M (anti-IgM) to study the effects of antiviral antibodies on development of Aleutian mink disease parvovirus (ADV)-induced disease in more detail. Newborn mink kits were injected intraperitoneally with 1 mg of either anti-IgM or a control preparation three times a week for 30 to 34 days. At 21 days after birth, groups of mink kits were infected with the highly virulent United isolate of ADV. At selected time points, i.e., postinfection days 9, 13, 29, and 200, randomly chosen mink kits were sacrificed, and blood and tissues were collected for analyses. The efficacy of immunosuppressive treatment was monitored by electrophoretic techniques and flow cytometry. Effects of treatment on viral replication, on viral mRNA levels, and on development of acute or chronic disease were determined by histopathological, immunoelectrophoretic, and molecular hybridization techniques. Several interesting findings emerged from these studies. First, antiviral antibodies decreased ADV mRNA levels more than DNA replication. Second, suppression of B-cell development and antibody response in mink kits infected at 21 days of age resulted in production of viral inclusion bodies in alveolar type II cells. Some of these kits showed mild clinical signs of respiratory disease, and one kit died of respiratory distress; however, clinical signs were seen only after release of immunosuppression, suggesting that the production of antiviral antibodies, in combination with the massive amounts of free viral antigen present, somehow is involved in the induction of respiratory distress. It is suggested that the antiviral antibody response observed in mink older than approximately 14 days primarily, by a yet unknown mechanism, decreases ADV mRNA levels which, if severe enough, results in restricted levels of DNA replication and virion production. Furthermore, such a restricted ADV infection at low levels paves the way for a persistent infection leading to immunologically mediated disease. The potential mechanisms of antibody-mediated restriction of viral mRNA levels and mechanisms of disease induction are discussed.