The p17 nonstructural protein of avian reovirus triggers autophagy enhancing virus replication via activation of phosphatase and tensin deleted on chromosome 10 (PTEN) and AMP-activated protein kinase (AMPK), as well as dsRNA-dependent protein kinase (PKR)/eIF2α signaling pathways.

Autophagy has been shown to facilitate replication or production of avian reovirus (ARV); nevertheless, how ARV induces autophagy remains largely unknown. Here, we demonstrate that the nonstructural protein p17 of ARV functions as an activator of autophagy. ARV-infected or p17-transfected cells present a fast and strong induction of autophagy, resulting in an increased level of autophagic proteins Beclin 1 and LC3-II. Although autophagy was suppressed by 3-methyladenine or shRNAs targeting autophagic proteins (Beclin 1, ATG7, and LC3) as well as by overexpression of Bcl-2, viral transcription, σC protein synthesis, and virus yield were all significantly reduced, suggesting a key role of autophagosomes in supporting ARV replication. Furthermore, we revealed for the first time that p17 positively regulates phosphatase and tensin deleted on chromosome 10 (PTEN), AMP-activated protein kinase (AMPK), and dsRNA dependent protein kinase RNA (PKR)/eIF2α signaling pathways, accompanied by down-regulation of Akt and mammalian target of rapamycin complex 1, thereby triggering autophagy. By using p53, PTEN, PKR, AMPK, and p17 short hairpin RNA (shRNA), activation of signaling pathways and LC3-II levels was significantly suppressed, suggesting that p17 triggers autophagy through activation of p53/PTEN, AMPK, and PKR signaling pathways. Furthermore, colocalization of LC3 with viral proteins (p17 and σC), p62 with LAMP2 and LC3 with Rab7 was observed under a fluorescence microscope. The expression level of p62 was increased at 18 h postinfection and then slightly decreased 24 h postinfection compared with mock infection and thapsigargin treatment. Furthermore, disruption of autophagosome-lysosome fusion by shRNAs targeting LAMP2 or Rab7a resulted in inhibition of viral protein synthesis and virus yield, suggesting that formation of autolysosome benefits virus replication. Taken together, our results suggest that ARV induces formation of autolysosome but does not induce complete autophagic flux.

Bovine ephemeral fever virus uses a clathrin-mediated and dynamin 2-dependent endocytosis pathway that requires Rab5 and Rab7 as well as microtubules.

The specific cell pathways involved in bovine ephemeral fever virus (BEFV) cell entry have not been determined. In this work, colocalization of the M protein of BEFV with clathrin or dynamin 2 was observed under a fluorescence microscope. To better understand BEFV entry, we carried out internalization studies with a fluorescently labeled BEFV by using a lipophilic dye, 3,30-dilinoleyloxacarbocyanine perchlorate (DiO), further suggesting that BEFV uses a clathrin-mediated endocytosis pathway. Our results suggest that clathrin-mediated and dynamin 2-dependent endocytosis is an important avenue of BEFV entry. Suppression of Rab5 or Rab7a through the use of a Rab5 dominant negative mutant and Rab7a short hairpin RNA (shRNA) demonstrated that BEFV requires both early and late endosomes for endocytosis and subsequent infection in MDBK and Vero cells. Treatment of BEFV-infected cells with nocodazole significantly decreased the M protein synthesis and viral yield, indicating that microtubules play an important role in BEFV productive infection, likely by mediating trafficking of BEFV-containing endosomes. Furthermore, BEFV infection was strongly blocked by different inhibitors of endosomal acidification, suggesting that virus enters host cells by clathrin-mediated and dynamin 2-dependent endocytosis in a pH-dependent manner.

Cell entry of avian reovirus follows a caveolin-1-mediated and dynamin-2-dependent endocytic pathway that requires activation of p38 mitogen-activated protein kinase (MAPK) and Src signaling pathways as well as microtubules and small GTPase Rab5 protein.

Very little is known about the mechanism of cell entry of avian reovirus (ARV). The aim of this study was to explore the mechanism of ARV entry and subsequent infection. Cholesterol mainly affected the early steps of the ARV life cycle, because the presence of cholesterol before and during viral adsorption greatly blocked ARV infectivity. Although we have demonstrated that ARV facilitating p38 MAPK is beneficial for virus replication, its mechanism remains unknown. Here, we show that ARV-induced phosphorylation of caveolin-1 (Tyr(14)), dynamin-2 expression, and Rac1 activation through activation of p38 MAPK and Src in the early stage of the virus life cycle is beneficial for virus entry and productive infection. The strong inhibition by dynasore, a specific inhibitor of dynamin-2, and depletion of endogenous caveolin-1 or dynamin-2 by siRNAs as well as the caveolin-1 colocalization study implicate caveolin-1-mediated and dynamin-2-dependent endocytosis as a significant avenue of ARV entry. By means of pharmacological inhibitors, dominant negative mutants, and siRNA of various cellular proteins and signaling molecules, phosphorylation of caveolin-1, dynamin-2 expression, and Rac1 activation were suppressed, suggesting that by orchestrating p38 MAPK, Src, and Rac1 signaling cascade in the target cells, ARV creates an appropriate intracellular environment facilitating virus entry and productive infection. Furthermore, disruption of microtubules, Rab5, or endosome acidification all inhibited ARV infection, suggesting that microtubules and small GTPase Rab5, which regulate transport to early endosome, are crucial for survival of ARV and that exposure of the virus to acidic pH is required for productive infection.

Inhibitors of phosphatidylinositol 3-kinase and mTOR but not Akt enhance replication of bovine ephemeral fever virus.

Non-segmented, negative-sense RNA viruses (NNSVs) depend on Akt (protein kinase B) for efficient replication. Infection with bovine ephemeral fever virus (BEFV) increases Akt phosphorylation. This study examined the effect of inhibition of phosphatidylinositol 3-kinase (PI3K)-Akt signalling on BEFV replication, since PI3K is the major upstream regulator of Akt. Treatment of BEFV-infected cells with two specific PI3K inhibitors (wortmannin and LY294002) enhanced replication of BEFV when compared to the effects of Akt inhibitors III and IV. BEFV antagonised the effects of PI3K inhibitors on Akt dephosphorylation. Inhibition of mTOR by rapamycin also enhanced replication of BEFV. The results provide evidences that inhibition of PI3K and mTOR has positive effects on replication of BEFV.

Advanced glycation end products down-regulate gap junctions in human hepatoma SKHep 1 cells via the activation of Src-dependent ERK1/2 and JNK/SAPK/AP1 signaling pathways.

Hyperglycemia and advanced glycation end products (AGEs) are associated with an elevated risk of developing several cancers in diabetic patients. However, the detailed mechanisms remain to be elucidated. The mechanism of AGE-bovine serum albumin (BSA) on gap junction intercellular communication in human hepatoma cell line, SKHep 1, was investigated. Both Cx32 and Cx43 are major gap junction forming proteins in the liver, the loss of which has been shown to facilitate tumorigenesis. Although the MTT assay results showed that AGE-BSA significantly increased cell growth by 31%, AGE-BSA down-regulated Cx32 and Cx43 expression in a dose- and time-dependent manner. The present study also demonstrated that ERK1/2 and JNK/SAPK were significantly activated by AGE-BSA and that Src, ERK1/2, and JNK/SAPK inhibitors significantly reversed the reduction of Cx32 and Cx43 proteins by AGE-BSA. Taken together, these results strongly support the hypothesis that Src-dependent ERK1/2 and JNK/SAPK/AP1 signaling pathways play a key role in AGE-BSA-mediated down-regulation of Cx32 and Cx43 protein expression in SKHep 1 cells.

Analysis of sequence and haemagglutinin activity of the HN glycoprotein of Newcastle disease virus.

Reverse transcription-polymerase chain reaction (RT-PCR) was used to generate sequence data for recent Taiwanese strains of Newcastle disease virus (NDV) isolated from 1999 to 2003, covering the full length of the haemagglutinin-neuraminidase (HN) gene and protein. Nucleotide sequence analysis of the HN gene of these recent isolates revealed that the whole HN gene carries an open reading frame encoding 571 amino acids and possesses a shorter C-terminal extension. Six amino acid substitutions in epitopes on the HN glycoprotein of the recent Taiwanese NDV isolates were also found. All the recent Taiwanese NDV isolates have the amino acid sequence (112)RRQKRF(117) for the F protein. A phylogenetic tree analysis based on the nucleotide sequences of the F gene revealed that all recent Taiwanese isolates were related to genotype VII viruses. Since the recent Taiwanese NDV isolates exhibited a low level of haemagglutination (HA) activity, we generated two sets of mutants to elucidate whether mutations in the heptad repeat region of the HN protein could affect the HA activity. To demonstrate the presence of the viruses used in the HA test, a real-time RT-PCR was established to determine the copy number of NDV isolates. From sequence analysis, site-directed mutagenesis, and haemadsorption assays, it was found that the HN glycoprotein of recent Taiwanese NDV isolates carrying a substitution at the amino acid residue 81 (I to M) in the heptad repeat region in the stalk domain showed a dramatic decrease in the activity of HA. We infer from these results that a specific amino acid sequence within the heptad repeat region of the stalk is important for the HA activity of the HN glycoprotein.

Avian reovirus nonstructural protein p17-induced G(2)/M cell cycle arrest and host cellular protein translation shutoff involve activation of p53-dependent pathways.

The effects of avian reovirus (ARV) p17 protein on cell cycle progression and host cellular protein translation were studied. ARV infection and ARV p17 transfection resulted in the accumulation of infected and/or transfected cells in the G(2)/M phase of the cell cycle. The accumulation of cells in the G(2)/M phase was accompanied by upregulation and phosphorylation of the G(2)/M-phase proteins ATM, p53, p21(cip1/waf1), Cdc2, cyclin B1, Chk1, Chk2, and Cdc25C, suggesting that p17 induces a G(2)/M cell cycle arrest through activation of the ATM/p53/p21(cip1/waf1)/Cdc2/cyclin B1 and ATM/Chk1/Chk2/Cdc25C pathways. The G(2)/M cell cycle arrest resulted in increased virus replication. In the present study, we also provide evidence demonstrating that p17 protein is responsible for ARV-induced host cellular protein translation shutoff. Increased phosphorylation levels of the eukaryotic translation elongation factor 2 (eEF2) and initiation factor eIF2alpha and reduced phosphorylation levels of the eukaryotic translation initiation factors eIF4E, eIF4B, and eIF4G, as well as 4E-BP1 and Mnk-1 in p17-transfected cells, demonstrated that ARV p17 suppresses translation initiation factors and translation elongation factors to induce host cellular protein translation shutoff. Inhibition of mTOR by rapamycin resulted in a decrease in the levels of phosphorylated 4E-BP1, eIF4B, and eIF4G and an increase in the levels eEF2 but did not affect ARV replication, suggesting that ARV replication was not hindered by inhibition of cap-dependent translation. Taken together, our data indicate that ARV p17-induced G(2)/M arrest and host cellular translation shutoff resulted in increased ARV replication.

Intracellular cleavage of sigmaA protein of avian reovirus.

By Western blot analyzes of expression of avian reovirus proteins, one unknown fragment was detected by an anti-sigmaA monoclonal antibody in virus-infected cells lysate. It was interesting to note that RNA interference against sigmaA resulted in the suppression of the unknown fragment. Using various lengths of sigmaA constructs conjugated with different tags, we present evidences to demonstrate that the fragment comes from the cleavage of sigmaA and is the larger carboxyl-terminus, termed sigmaAC. Cleavage of sigmaA simultaneously produces a smaller amino-terminus, named sigmaAN. sigmaAC could be seen early in viral infection and accumulated with time and dose of infection, indicating that the derived products are not just transient intermediates of protein degradation. The same type of cleaved products were also observed in different genotypes and serotypes of ARV as well as in different cell lines, suggesting that this intracellular modification of sigmaA is common to all ARVs. Similar localization of sigmaAC in both cytosol and nucleus with sigmaA suggested that further modification of sigmaA may be important for its function. Our evidences suggest that besides the outer capsid protein muB, sigmaA may also have post-translational cleavage which has never been reported before even in related mammalian reovirus.

AMP-activated protein kinase facilitates avian reovirus to induce mitogen-activated protein kinase (MAPK) p38 and MAPK kinase 3/6 signalling that is beneficial for virus replication.

Stimulated by energetic stress, AMP-activated protein kinase (AMPK) controls several cellular functions. It was discovered here that infection of Vero cells with avian reovirus (ARV) upregulated AMPK and mitogen-activated protein kinase (MAPK) p38 phosphorylation in a time- and dose-dependent manner. Being an energy status sensor, AMPK is potentially an upstream regulator of MAPK p38. Treatment with 5-amino-4-imidazolecarboxamide ribose (AICAR), a well-known activator of AMPK, induced phosphorylation of MAPK p38. Unlike AICAR, wortmannin or rapamycin did not induce phosphorylation of MAPK p38, suggesting that mTOR inhibition is not a determining factor in MAPK p38 phosphorylation. Inhibition of AMPK by compound C antagonized the effect of AICAR on MAPK p38 in Vero cells. Specific inhibition of AMPK by small interfering RNA or compound C also suppressed ARV-induced phosphorylation of MAPK kinase (MKK) 3/6 and MAPK p38 in Vero and DF-1 cells, thereby providing a link between AMPK signalling and the MAPK p38 pathway. The mechanism of ARV-enhanced phosphorylation of MKK 3/6 and MAPK p38 in cells was not merely due to glucose deprivation, a probable activator of AMPK. In the current study, direct inhibition of MAPK p38 by SB202190 decreased the level of ARV-induced syncytium formation in Vero and DF-1 cells, and decreased the protein levels of ARV sigma A and sigma C and the progeny titre of ARV, suggesting that activation of MAPK p38 is beneficial for ARV replication. Taken together, these results suggested that AMPK could facilitate MKK 3/6 and MAPK p38 signalling that is beneficial for ARV replication. Although well studied in energy metabolism, this study provides evidence for the first time that AMPK plays a role in modulating ARV and host-cell interaction.

Avian reovirus influences phosphorylation of several factors involved in host protein translation including eukaryotic translation elongation factor 2 (eEF2) in Vero cells.

Viral infection usually influences cellular protein synthesis either actively or passively via modification of various translation initiation factors. Here we demonstrated that infection with avian reovirus (ARV) interfered with cellular protein synthesis. This study demonstrated for the first time that ARV influenced the phosphorylation of translation initiation factors including eIF4E and eIF-4G. Interestingly, ARV also induced phosphorylation of eukaryotic translation elongation factor (eEF2) in a time- and dose-dependent manner. Inhibition of mTOR by rapamycin notably increased the level of phosphorylated eEF2 in infected cells. However, rapamycin did not show any negative effects on ARV replication, suggesting that phosphorylation of eEF2 in infected cells did not reduce ARV propagation. These results demonstrated for the first time that ARV promotes phosphorylation of eEF2 which in turn influenced host protein production not simply by modulating the function of translation initiation factors but also by regulating elongation factor eEF2.

Recent patents on cell cycle regulatory proteins.

Cell cycle progression and cell division are driven by the sequential activation of a group of serine-threonine kinases called cyclin-dependent kinases (Cdks). Multiple Cdks control the cell cycle in mammals and have been long considered essential for normal proliferation, development and homeostasis. The importance of the Cdk-cyclin complexes in cell proliferation is underscored by the fact that deregulation of the Cdk activity is found in virtually the whole spectrum of human tumors. Advances in the cell cycle proteins in the last 25 years, since the discovery of cyclins, have been discussed and have shed even more light on this essential life sustaining process. Recent information from different models for the various cyclins and Cdks have made some of the generally accepted concepts of cell cycle regulation to be revised and new and exciting questions to be investigated. There is also increasing evidence that suggests that Cdks such as Cdc2 are also commonly targeted by viral proteins, which modulate host cell cycle machinery to benefit viral survival or replication. This review, describes some of the most recent and important US patents related to cell cycle regulation and those on viral proteins involved in cell cycle modulation particularly the G2/M phase transition and cancer therapy.

PI3K-Akt signaling and viral infection.

PI3K-Akt pathway is an important mechanism through which viral infection influences various cell functions. Activating PI3K-Akt signaling is a strategy employed by viruses to slow down apoptosis and prolong viral replication in both acute and persistent infection. It is also probable that prevention of cell death facilitates virus-induced carcinogenesis. Accumulating evidence suggests that the activity of PI3K or Akt is critical for survival of a few viruses. Adenovirus relies on PI3K-mediated organization of actin filament for active internalization. Non-segmented negative-sense RNA viruses require Akt to enhance synthesis of viral RNAs. On the other hand, PI3K-Akt signaling is associated with up-regulating interferon response. Higher PI3K-Akt activity might impede viral propagation due to activation of cellular defenses. Influenza A virus is an interesting case which requires active PI3K for penetration despite the negative effects of inducing immune response. Unlike most viruses, it was reported that VP1 protein of foot-and-mouth disease virus inhibits Akt to promote cell death. These reports confirm the multiple roles of PI3K-Akt pathway in viral infection. Here, more new information on the interaction between PI3K-Akt signaling and viral infection is discussed.

Baculovirus surface display of sigmaC and sigmaB proteins of avian reovirus and immunogenicity of the displayed proteins in a mouse model.

Avian reovirus (ARV), an important pathogen in poultry, causes arthritis, chronic respiratory disease, and malabsorption syndrome that cause considerable economic losses to the poultry industry. In present study, we have succeeded in construction of a universal baculovirus surface display system (UBSDS) that can display different foreign proteins on the envelope of baculovirus. Sequences encoding the signal peptide (SS), transmembrane domain (TM), and cytoplasmic domain (CTD) derived from the gp64 protein of baculovirus and histidine tag, respectively were inserted into the pBacCE vector. Four restriction enzyme sites between the histidine tag and gp64 transmembrane domain were established for expression of different foreign proteins. The transmembrane domain and CTD of gp64 in the platform were designed in order to improve stability and quantity of foreign proteins on the envelope of baculovirus. The sigmaC and sigmaB proteins of ARV are known to elicit neutralizing antibodies against ARV. The UBSDS was therefore used to express sigmaC and sigmaB proteins on the envelope of baculovirus. Two recombinant baculoviruses BacSC-sigmaC and BacSC-sigmaB have been successfully constructed. After infection, both His6-tagged recombinant sigmaC (rsigmaC) and sigmaB (rsigmaB) proteins were displayed on the envelope of recombinant baculoviruses and the recombinant viral proteins were anchored on the plasma membrane of Sf-9 cells, as revealed by immunofluorescence staining (IFS) and confocal microscopy. The antigenicity of rsigmaC and rsigmaB proteins was demonstrated by Western blotting assay. Immunogold electron microscopy demonstrated that both recombinant viruses displayed rsigmaC and rsigmaB proteins on the viral surface. Immunization of BALB/c mice with recombinant viruses, demonstrated that serum from the BacSC-sigmaC and BacSC-sigmaB treated models had significant higher levels of virus neutralization activities than the control groups. This demonstrates that the recombinant baculoviruses BacSC-sigmaC and BacSC-sigmaB can be a potential vaccine against ARV infections.

Proteasome inhibition reduces avian reovirus replication and apoptosis induction in cultured cells.

The interplay between avian reovirus (ARV) replication and apoptosis and proteasome pathway was studied in cultured cells. It is shown that inhibition of the proteasome did not affect viral entry and host cell translation but had influence on ARV replication and ARV-induced apoptosis. Evidence is provided to demonstrate that ubiquitin-proteasome blocked ARV replication at an early step in viral life cycle. However, viral transcription and protein translation were also reduced markedly after addition of proteasome inhibitor MG132. Treatment of BHK-21 cells with the MG132 markedly decreased virus titer as well as prevented virus-induced apoptosis. The expression of ARV proteins sigmaC, sigmaA, and sigmaNS was also reduced markedly, suggesting that suppression of virus replication is due to down-regulation of these ARV proteins by ubiquitin-proteasome system. MG132 was also shown to suppress ARV sigmaC-induced phosphrylation of p53 on serine 46, caspase 3 activities, and DNA fragmentation leading to complete inhibition of ARV-induced apoptosis.

Development of a reliable assay protocol for identification of diseases (RAPID)-bioactive amplification with probing for detection of avian reovirus.

Avian reovirus (ARV) causes several disease syndromes in poultry including arthritis, malabsorption syndrome and chronic respiratory disease that result in major economic losses. Early detection is very important for the control of the ARV-induced infections. This study was therefore aimed at developing a reliable assay protocol for identification of diseases (RAPID)-bioactive amplification with probing (BAP) assay for detection of ARV. This assay combines nested polymerase chain reaction (PCR) and magnetic bead-based DNA probing systems greatly increasing its sensitivity and specificity. Alignment of ARV S2 gene from different ARV genotypes and serotypes was done to find the highly conserved regions for primer and probe design. Two reverse transcription (RT)-PCR primer pairs, six nested PCR primer pairs, and one magnetic probe were tested to find the most specific ones for ARV detection. The optimal conditions for RT-PCR, nested PCR, and hybridization of magnetic probe were established. The optimal annealing temperatures for RT-PCR and nested PCR were 62.1 and 54.8 degrees C, respectively. The optimal hybridization temperature was 51.2 degrees C using hybridization buffer (5x SSC and 0.5% SDS). The sensitivity of the kit was 5 copies/microl of ARV genomic RNA. The kit was very specific as all negative controls failed to show any positive reactions. The kit shows good reproducibility with intra- and inter-assay coefficient of variation (CV) of 1.3 and 1.7%, respectively. In addition, different serotypes and genotypes of ARV were tested by RAPID-BAP assay to estimate the practicability of the kit in clinical samples. All of ARV serotypes and genotypes tested could be detected by this kit proving that the kit is suitable for clinical application.

Suppression of protein expression of three avian reovirus S-class genome segments by RNA interference.

RNA interference was used to suppress protein expression of three S-class genome segments of avian reovirus (ARV). Viral progeny titer was successfully down-regulated by RNA interference. Suppression of S1 genome segment, which has three open reading frames, not only decreased the expression level of the structural protein sigmaC but also reduced cell fusion and the level of Ser(15)-phosphorylated p53 protein caused by the nonstructural proteins p10 and p17, respectively. Suppression of S2 or S4 genome segment by RNA interference could also reduce the expression level of sigmaA or sigmaNS. Interestingly, suppression of sigmaNS resulted in down regulation of the expression of other viral products. In terms of variability of different genes among viral strains and of the impact after their suppression, it seems that the viral products involved in construction of viroplasm or core particles, like sigmaNS, are considerable choices to efficiently inhibit ARV multiplication by RNA interference. Using a GFP reporter system, it was discovered that ARV could not inhibit activated RNA interference, suggesting that RNA interference may be used in the suppression of ARV infection.

The cleavage of the hemagglutinin protein of H5N2 avian influenza virus in yeast.

Influenza viruses belonging to the Orthomyxoviridae family are enveloped viruses with segmented negative sense RNA genome surrounded by a helical symmetry shell. Influenza viruses, especially the highly pathogenic avian influenza virus (HPAI) such as H5 or H7 subtype are important pathogens for the poultry industry. Due to genetic reassortments between avian and human influenza viruses, global pandemics may emerge and the naive human immunity could not be ready for them. The full-length HA-encoding gene of H5N2 AIV was inserted into a secretory pPICZalphaA vector and integrated into the genome of Pichia pastoris by heterologous recombination. The HA protein secretion into the medium was induced with methanol. Besides the expected 69kDa protein, another smaller fragment about 47kDa was recognized by an anti-AIV-HA monoclonal antibody in Western blot assay. This is the first report on the cleavage of HA(0) into HA(1) and HA(2) in the methylotrophic yeast P. pastoris. This possibly was due to digestion by proteases from P. pastoris based on the amino acid sequences at the predicted cleavage site, (326)R-X-K-R(329). With similar modifications to the eukaryotes, large quantity, proper antigenicity, and low cost, this expression system may provide a simple tool to produce HA proteins for further use in preparation of ELISA kits and subunit vaccines.

Suppression of bovine ephemeral fever virus by RNA interference.

RNA interference (RNAi) was used to suppress bovine ephemeral fever virus (BEFV). Plasmids expressing continuously shRNAs were used against G gene of BEFV to induce RNA interference in cultured cells. A GFP reporter assay was established to determine the efficiency and specificity of siRNA and the potential of BEFV to hamper RNAi. Two of five small interfering RNAs (siRNAs) were shown to suppress BEFV. Suppression of the G gene of BEFV corresponded with reduction of viral plaques and progeny titer. The results suggest that RNAi has the potential for use in suppression of BEFV infection with possible therapeutic implications.

Avian reovirus-induced apoptosis related to tissue injury.

Apoptosis plays an important role in pathogenesis of many viral infections. Infection of chicken with avian reovirus S1133 causes tissue injury related to virus-induced apoptosis. To determine whether avian reovirus (ARV) induced apoptosis in chicken tissues, six 3-week-old specific pathogen free White Leghorn chicks were inoculated with ARV S1133. Tissues were dual-labelled for the simultaneous detection of viral antigen containing and apoptotic cells. DNA laddering was detected in ARV-infected but not mock-infected chicken tissues. Dual-labelling assay revealed that the majority of antigen-expressing cells were not apoptotic. Surprisingly, some apoptotic but non-antigen-expressing cells were frequently located in the vicinity of antigen-expressing cells. Syncytium formation in ARV-infected chicken tissues undergoing apoptosis was apparent, suggesting a correlation between virus replication and apoptosis in chicken tissues.