Porcine Circovirus 2 Activates the PERK-Reactive Oxygen Species Axis To Induce p53 Phosphorylation with Subsequent Cell Cycle Arrest at S Phase in Favor of Its Replication.

Porcine circovirus type 2 (PCV2), the causative agent of porcine circovirus-associated diseases (PCVAD), is known to induce oxidative stress, activate p53 with induction of cell cycle arrest, and trigger the PERK (protein kinase R-like endoplasmic reticulum kinase) branch of the endoplasmic reticulum (ER) stress pathway. All these cellular responses could enhance PCV2 replication. However, it remains unknown whether PERK activation by PCV2 is involved in p53 signaling with subsequent changes of cell cycle. Here, we demonstrate that PCV2 infection induced cell cycle arrest at S phase to favor its replication via the PERK-reactive oxygen species (ROS)-p53 nexus. PCV2 infection promoted phosphorylation of p53 (p-p53) at Ser15 in porcine alveolar macrophages. Inhibition of PERK by RNA silencing downregulated total p53 (t-p53) and p-p53. Treatment with the MDM2 inhibitor nutlin-3 led to partial recovery of t-p53 in perk-silenced and PCV2-infected cells. perk silencing markedly downregulated ROS production. Scavenging of ROS with N-acetylcysteine (NAC) of PCV2-infected cells downregulated t-p53 and p-p53. Increased accumulation of p-p53 in the nuclei during PCV2 infection could be downregulated by silencing of perk or NAC treatment. Further studies showed that perk silencing or NAC treatment alleviated S phase accumulation and downregulated cyclins E1 and A2 in PCV2-infected cells. These findings indicate that the PCV2-activated PERK-ROS axis promotes p-p53 and contributes to cell cycle accumulation at S phase when more cellular enzymes are available to favor viral DNA synthesis. Overall, our study provides a novel insight into the mechanism how PCV2 manipulates the host PERK-ROS-p53 signaling nexus to benefit its own replication via cell cycle arrest. IMPORTANCE Coinfections or noninfectious triggers have long been considered to potentiate PCV2 infection, leading to manifestation of PCVAD. The triggering mechanisms remain largely unknown. Recent studies have revealed that PERK-mediated ER stress, oxidative stress, and cell cycle arrest during PCV2 infection are conducive to viral replication. However, how PCV2 employs such host cell responses requires further research. Here, we provide a novel mechanism of PCV2-induced ER stress and enhanced viral replication: the PCV2-activated PERK-ROS-p53 nexus increases S phase cell population, a cell cycle period of DNA synthesis favorable for PCV2 replication. The fact that PCV2 deploys the simple ROS molecules to activate p53 to benefit its replication provides novel insights into the triggering factors, that is, certain stimuli or management measures that induce ER stress with subsequent generation of ROS would exacerbate PCVAD. Use of antioxidants is justified on farms where PCVAD is severe.

Kinetic analysis of pathogenicity and tissue tropism of gyrovirus 3 in experimentally infected chickens.

Gyrovirus 3 (GyV3), the third novel emerging species of the genus Gyrovirus of the Anelloviridae family, has been described in multiple hosts. Epidemiologically, there are suggestions that GyV3 is associated with diarrhea/proventriculitis, however, no direct causal evidence exists between GyV3 infection and specific clinical diseases. Herein, we infected special pathogen-free (SPF) chickens with GyV3, and then assessed the pathogenicity and tissue tropism. The results revealed that GyV3 induced persistent infection characterized by diarrhea, aplastic anemia, immunosuppression, and persistent systemic lymphocytic inflammation. Clinically, the infected chickens presented ruffled feathers, diarrhea, anemia, and weight loss. Aplastic anemia was characterized by progressive depletion of hematopoietic cells in the bone marrow, immunosuppression was associated with atrophy of the thymus, spleen, and bursa of Fabricious, progressive lymphocytic inflammations were characterized by proventriculitis, adrenalitis, pancreatitis, hepatitis, nephritis, and bronchitis. Viral loads of GyV3 in tissues exhibited "M", "N", "W" or "V" type dynamic changes. The highest level of viral loads was reported in bone marrow at 7dpi, followed by the adrenal gland at 2 dpi, the sciatic nerve at 7 dpi, and bile at 35 dpi. The bone marrow and kidney demonstrate the strongest immunostaining of GyV3-VP1 antigen and were suggested as the target tissues of GyV3. Collectively, GyV3 is an immunosuppressive pathogenic virus that targets the bone marrow and kidney in chickens. Exploring the pathogenicity and tissue tropism of GyV3 will guide the basic understanding of the biology of GyV3 and its pathogenesis in chickens.

Viruses Infecting the European Catfish (Silurus glanis).

In aquaculture, disease management and pathogen control are key for a successful fish farming industry. In past years, European catfish farming has been flourishing. However, devastating fish pathogens including limiting fish viruses are considered a big threat to further expanding of the industry. Even though mainly the ranavirus (Iridoviridea) and circovirus (Circoviridea) infections are considered well- described in European catfish, more other agents including herpes-, rhabdo or papillomaviruses are also observed in the tissues of catfish with or without any symptoms. The etiological role of these viruses has been unclear until now. Hence, there is a requisite for more detailed information about the latter and the development of preventive and therapeutic approaches to complete them. In this review, we summarize recent knowledge about viruses that affect the European catfish and describe their origin, distribution, molecular characterisation, and phylogenetic classification. We also highlight the knowledge gaps, which need more in-depth investigations in the future.

Macrophage Polarization Modulated by Porcine Circovirus Type 2 Facilitates Bacterial Coinfection.

Polarization of macrophages to different functional states is important for mounting responses against pathogen infections. Macrophages are the major target cells of porcine circovirus type 2 (PCV2), which is the primary causative agent of porcine circovirus-associated disease (PCVAD) leading to immense economic losses in the global swine industry. Clinically, PCV2 is often found to increase risk of other pathogenic infections yet the underlying mechanisms remain to be elusive. Here we found that PCV2 infection skewed macrophages toward a M1 status through reprogramming expression of a subset of M1-associated genes and M2-associated genes. Mechanistically, induction of M1-associated genes by PCV2 infection is dependent on activation of nuclear factor kappa B (NF-κB) and c-jun N-terminal kinase (JNK) signaling pathways whereas suppression of M2-associated genes by PCV2 is via inhibiting expression of jumonji domain containing-3 (JMJD3), a histone 3 Lys27 (H3K27) demethylase that regulates M2 activation of macrophages. Finally, we identified that PCV2 capsid protein (Cap) directly inhibits JMJD3 transcription to restrain expression of interferon regulatory factor (IRF4) that controls M2 macrophage polarization. Consequently, sustained infection of PCV2 facilitates bacterial infection in vitro. In summary, these findings showed that PCV2 infection functionally modulated M1 macrophage polarization via targeting canonical signals and epigenetic histone modification, which contributes to bacterial coinfection and virial pathogenesis.

Detection and molecular characterization of two canine circovirus genotypes co-circulating in Vietnam.

BACKGROUND: Canine circovirus is reported in dogs in many countries, including the USA, China and Thailand. It has been detected in healthy dogs and dogs with diarrhea, hemorrhagic gastroenteritis, and vasculitis. It comprises five genotypes and is frequently found as a coinfection with canine parvovirus-2 (CPV-2). AIM: To characterize canine circovirus genotypes co-circulating with CPV-2 in Vietnam. METHOD: PCR assessment of 81 CPV-2-positive fecal samples from Vietnamese diarrheic dogs up to seven months of age for other viral enteric pathogens, including canine bocavirus, canine adenovirus, paramyxovirus, canine coronavirus, porcine circovirus-3 and canine circovirus. In addition, eight selected full genome sequences of Vietnamese canine circovirus were analyzed and used for phylogeny. RESULTS: In total 19.8% of samples were found to be positive for canine circovirus. Phylogeny revealed that the Vietnamese canine circovirus strains were clustered in two different genotypes (genotype-1 and -3). The genetic diversity among Vietnamese canine circovirus was 86.0-87.2%. The nucleotide discrepancy among both genotypes altered the deduced amino acid sequence in 14 and ten residues of the replicase and capsid proteins, respectively. Genetic recombination analysis revealed that the Vietnamese canine circovirus-6 strain has the American and Chinese canine circovirus as its major and minor parents, respectively. Only a single dog revealed triple detections of CPV-2c, Canine circovirus and canine adenovirus (1.2%). CONCLUSION: The co-circulation of two different genotypes of canine circovirus and CPV-2c in dogs in Vietnam has been illustrated. CLINICAL RELEVANCE: The mortality rate with CPV-2 only (22%) doubled in dogs with canine circovirus and CPV-2 co-infection.

Coinfection with Porcine Circovirus Type 2 (PCV2) and Streptococcus suis Serotype 2 (SS2) Enhances the Survival of SS2 in Swine Tracheal Epithelial Cells by Decreasing Reactive Oxygen Species Production.

Porcine circovirus type 2 (PCV2) and Streptococcus suis serotype 2 (SS2) clinical coinfection cases have been frequently detected. The respiratory epithelium plays a crucial role in host defense against a variety of inhaled pathogens. Reactive oxygen species (ROS) are involved in killing of bacteria and host immune response. The aim of this study is to assess whether PCV2 and SS2 coinfection in swine tracheal epithelial cells (STEC) affects ROS production and investigate the roles of ROS in bacterial survival and the inflammatory response. Compared to SS2 infection, PCV2/SS2 coinfection inhibited the activity of NADPH oxidase, resulting in lower ROS levels. Bacterial intracellular survival experiments showed that coinfection with PCV2 and SS2 enhanced SS2 survival in STEC. Pretreatment of STEC with N-acetylcysteine (NAC) also helps SS2 intracellular survival, indicating that PCV2/SS2 coinfection enhances the survival of SS2 in STEC through a decrease in ROS production. In addition, compared to SS2-infected STEC, PCV2/SS2 coinfection and pretreatment of STEC with NAC prior to SS2 infection both downregulated the expression of the inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-1ß. Further research found that activation of p38/MAPK promoted the expression of inflammatory cytokines in SS2-infected STEC; however, PCV2/SS2 coinfection or NAC pretreatment of STEC inhibited p38 phosphorylation, suggesting that coinfection of STEC with PCV2 and SS2 weakens the inflammatory response to SS2 infection through reduced ROS production. Collectively, coinfection of STEC with PCV2 and SS2 enhances the intracellular survival of SS2 and weakens the inflammatory response through decreased ROS production, which might exacerbate SS2 infection in the host.

Research Note: Molecular and pathologic characterization of avian adenovirus isolated from the oviducts of laying hens in eastern Japan.

Cases of poor egg production were investigated in 2 layer farms from Ibaraki Prefecture in eastern Japan. To identify any microbial agents that may have caused the problem, necropsy, bacterial isolation, histopathology, and virus detection were performed. Members of the avian adenoviruses was detected by PCR in oviduct samples from both farms; chicken anemia virus coinfection was also confirmed in one of the farms. Avian adenovirus was isolated from the oviducts of the affected chickens on each farm. Inoculation into chick embryos showed tropism for the chorio-allantoic membrane. Stunting and hemorrhaging was observed in all infected embryos, as well as death in a few. Inoculation of 1-day-old specific pathogen-free chicks, and 400-day-old commercial hens, did not result in any significant findings. The isolated viruses were analyzed by sequencing of the hexon gene and were confirmed as fowl adenovirus type-c serotype-4 (FAdV-4). The 2 virus strains were found to be 99.29% similar to each other. One of the strains, Japan/Ibaraki/Y-H6/2016, was 99.15% similar to the KR5 strain. The other, Japan/Ibaraki/M-HB2/2016, was 99.57% similar to the KR5 strain. Fiber-2 gene analysis confirmed the identity as FAdV-4 that is closely related to nonpathogenic strains. Although nonpathogenic to chicks and laying hens, this infection can possibly cause economic damage. Perhaps the bigger concern is the effect on infected breeder operations. Because the virus is fatal to 9.09% of infected embryos, this could translate to a considerable loss in chick production owing to embryonic death. This is the first report of detection and isolation of FAdV-4 from the chicken oviduct; however, further studies are needed to elucidate its impact on both layer and breeder flocks. Indeed, FAdV-4 has negative effects on the avian reproductive tract as well.

Detection of cyprinid herpesvirus 1 (CyHV-1) in barbel (Barbus barbus): First molecular evidence for the presence of CyHV-1 in fish other than carp (Cyprinus carpio).

Two adult barbels (Barbus barbus) with visible skin tumours were subjected to histopathological and molecular examinations. The fish were caught in the River Danube near Budapest. Papillomas were found around their oral cavity, at the operculum and at the pectoral fins, while epidermal hyperplasias were seen on the body surface. Cyprinid herpesvirus 1 (CyHV-1) was detected in the kidney of the specimens by polymerase chain reaction (PCR), and barbel circovirus 1 (BaCV1) was found in all internal organs and in the tissues of the tumours. The whole genome of BaCV1 and three conserved genes from the genome of CyHV-1 were sequenced. Previously, BaCV1 had been reported only once from a mass mortality event among barbel fry. The whole genome sequence of our circovirus shared 99.9% nucleotide identity with that of the formerly reported BaCV1. CyHV-1 is known to infect common carp and coloured carp (Cyprinus carpio), and has been assumed to infect other cyprinid fish species as well. We found the nucleotide sequences of the genes of CyHV-1 to be identical in 98.7% to those of the previous isolates from carp. To the best of our knowledge, this is the first molecular confirmation of the presence of CyHV-1 DNA in cyprinid fish species other than carp.

PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication.

Porcine circovirus type 2 (PCV2) is an important swine pathogen that causes significant economic losses to the pig industry. PCV2 interacts with host cellular factors to regulate its replication. High-mobility-group box 1 (HMGB1) protein, a major nonhistone protein in the nucleus, was recently discovered to participate in viral infections. Here, we demonstrate that nuclear HMGB1 negatively regulated PCV2 replication as shown by overexpression of HMGB1 or blockage of its nucleocytoplasmic translocation with ethyl pyruvate. The B box domain was essential in restricting PCV2 replication. Nuclear HMGB1 restricted PCV2 replication by sequestering the viral genome via binding to the Ori region. However, PCV2 infection induced translocation of HMGB1 from cell nuclei to the cytoplasmic compartment. Elevation of reactive oxygen species (ROS) induced by PCV2 infection was closely associated with cytosolic translocation of nuclear HMGB1. Treatment of PCV2-infected cells with ethyl pyruvate or N-acetylcysteine downregulated PCV2-induced ROS production, suppressed nucleocytoplasmic HMGB1 translocation, and decreased PCV2 replication. Collectively, these findings offer new insight into the mechanism of the PCV2 evasion strategy: PCV2 manages to escape restriction of its replication by nuclear HMGB1 by inducing ROS to trigger the nuclear-to-cytoplasmic translocation of HMGB1.IMPORTANCE Porcine circovirus type 2 (PCV2) is a small DNA virus that depends heavily on host cells for its infection. This study reports the close relationship between subcellular localization of host high-mobility-group box 1 (HMGB1) protein and viral replication during PCV2 infection. Restriction of PCV2 replication by nuclear HMGB1 is the early step of host defense at the host-pathogen interface. PCV2 then upregulates host reactive oxygen species (ROS) to prevent sequestration of its genome by expelling nuclear HMGB1 into the cytosol. It will be interesting to study if a similar evasion strategy is employed by other circoviruses such as beak and feather disease virus, recently discovered PCV3, and geminiviruses in plants. This study also provides insight into the justification and pharmacological basis of antioxidants as an adjunct therapy in PCV2 infection or possibly other diseases caused by the viruses that deploy the ROS-HMGB1 interaction favoring their replication.

Porcine Circovirus 2 Induction of ROS Is Responsible for Mitophagy in PK-15 Cells via Activation of Drp1 Phosphorylation.

Mitochondrial dynamics is essential for the maintenance of cell homeostasis. Previous studies have shown that porcine circovirus 2 (PCV2) infection decreases the mitochondrial membrane potential and causes the elevation of reactive oxygen species (ROS), which may ultimately lead to mitochondrial apoptosis. However, whether PCV2 induce mitophagy remains unknown. Here we show that PCV2-induced mitophagy in PK-15 cells via Drp1 phosphorylation and PINK1/Parkin activation. PCV2 infection enhanced the phosphorylation of Drp1 and its subsequent translocation to mitochondria. PCV2-induced Drp1 phosphorylation could be suppressed by specific CDK1 inhibitor RO-3306, suggesting CDK1 as its possible upstream molecule. PCV2 infection increased the amount of ROS, up-regulated PINK1 expression, and stimulated recruitment of Parkin to mitochondria. N-acetyl-L-cysteine (NAC) markedly decreased PCV2-induced ROS, down-regulated Drp1 phosphorylation, and lessened PINK1 expression and mitochondrial accumulation of Parkin. Inhibition of Drp1 by mitochondrial division inhibitor-1 Mdivi-1 or RNA silencing not only resulted in the reduction of ROS and PINK1, improved mitochondrial mass and mitochondrial membrane potential, and decreased mitochondrial translocation of Parkin, but also led to reduced apoptotic responses. Together, our study shows that ROS induction due to PCV2 infection is responsible for the activation of Drp1 and the subsequent mitophagic and mitochondrial apoptotic responses.

Pathological and virological analysis of concurrent disease of chicken anemia virus infection and infectious bronchitis in Japanese native chicks.

A concurrent infection of chicken anemia virus (CAV) and infectious bronchitis virus (IBV) was detected in Japanese native chicks in 2017, in which a high mortality rate (97.7%) was recorded in a small flock of 130 chicks exhibiting poor growth. Histological examination revealed that the affected chicks exhibited two different pathological entities: one was severe hematopoietic and lymphocytic depletion with abnormally large cells containing intranuclear inclusion bodies of CAV, whereas the other was renal tubular necrosis due to IBV infection. Immunohistochemistry detected CAV antigens in the bone marrow, liver, and spleen as well as IBV antigens in the kidneys, trachea, and air sacs. CAV was isolated from the liver sample of the chicks, and the isolated strain was designated as CAV/Japan/HS1/17. A phylogenetic analysis of the CAV VP1 gene revealed that CAV/Japan/HS1/17 is genetically similar to Chinese strains collected from 2014 to 2016. An experimental infection was performed using CAV/Japan/HS1/17 and specific-pathogen-free chicks to determine the pathogenicity of CAV/Japan/HS1/17. The isolate caused 100% anemia and 70% mortality to chicks inoculated at one day old, 80% of chicks inoculated at seven days old also developed anemia, and 10% died from CAV infection. These results suggest that the unusually high mortality in Japanese native chicks can be attributed to dual infection with both CAV and IBV. The results of the experimental infection suggest that CAV/Japan/HS1/17 has a pathogenic potential to specific-pathogen-free chicks and a relatively higher pathogenicity than previous Japanese CAV strains.

Porcine circovirus type 2 exploits JNK-mediated disruption of tight junctions to facilitate Streptococcus suis translocation across the tracheal epithelium.

Porcine circovirus type 2 (PCV2) is considered as the primary pathogen of porcine circovirus-associated disease (PCVAD), which results in significant economic losses worldwide. Clinically, PCV2 often causes disease through coinfection with other bacterial pathogens, including Streptococcus suis (S. suis), and especially the highly prevalent S. suis serotype 2 (SS2). The present study determined that continuous PCV2 infection in piglets down-regulates tight junction proteins (TJ) ZO-1 and occludin in the lungs. Swine tracheal epithelial cells (STEC) were used to explore the mechanisms and consequences of disruption of TJ, and an in vitro tracheal epithelial barrier model was established. Our results show that PCV2 infection in STEC decreases the expression levels of ZO-1 and occludin and increases the permeability of the tracheal epithelial barrier, resulting in easier translocation of SS2. Moreover, Western blot analysis indicates that PCV2 infection activates the JNK/MAPK pathway. The disruption of TJ in SETC and increased permeability of the epithelial barrier induced by PCV2 could be alleviated by inhibition of JNK phosphorylation, which indicates that the JNK/MAPK pathway regulates the expression of ZO-1 and occludin during PCV2 infection. This study allows us to better understand the mechanisms of PCV2 coinfection with bacterial pathogens and provides new insight into controlling the occurrence of PCVAD.

Monitoring of free-ranging and captive Psittacula populations in Western Europe for avian bornaviruses, circoviruses and polyomaviruses.

Avian pathogens such as bornaviruses, circoviruses and polyomaviruses are widely distributed in captive collections of psittacine birds worldwide and can cause fatal diseases. In contrast, only little is known about their presence in free-ranging psittacines and their impact on these populations. Rose-ringed parakeets (Psittacula krameri) and Alexandrine parakeets (Psittacula eupatria) are non-native to Europe, but have established stable populations in parts of Western Europe. From 2012-2017, we surveyed free-ranging populations in Germany and France as well as captive Psittacula individuals from Germany and Spain for avian bornavirus, circovirus and polyomavirus infections. Samples from two out of 469 tested free-ranging birds (0.4%; 95% confidence interval [CI-95]: 0.1-1.5%) were positive for beak and feather disease virus (BeFDV), whereas avian bornaviruses and polyomaviruses were not detected in the free-ranging populations. In contrast, avian bornaviruses and polyomaviruses, but not circoviruses were detected in captive populations. Parrot bornavirus 4 (PaBV-4) infection was detected by RT-PCR in four out of 210 captive parakeets (1.9%; CI-95: 0.7-4.8%) from four different holdings in Germany and Spain and confirmed by detection of bornavirus-reactive antibodies in two of these birds. Three out of 160 tested birds (1.9%; CI-95: 0.5-5.4%) possessed serum antibodies directed against budgerigar fledgling disease virus (BuFDV). PaBV-4 and BuFDV were also detected in several psittacines of a mixed holding in Germany, which had been in contact with free-ranging parakeets. Our results demonstrate that Psittacula parakeets are susceptible to common psittacine pathogens and their populations in Western Europe are exposed to these viruses. Nevertheless, the prevalence of avian bornaviruses, circoviruses and polyomaviruses in those populations is very low.RESEARCH HIGHLIGHTS Psittacula parakeets are susceptible to bornavirus, circovirus and polyomavirus infection.Introduced Psittacula populations in Europe have been exposed to these viruses.Nevertheless, they may be absent or present at only low levels in free-ranging Psittacula populations.Free-ranging populations in Europe pose a minor threat of transmitting these viruses to captive Psittaciformes.

Development and use of a triplex real-time PCR assay for detection of three DNA viruses in psittacine birds.

Aves polyomavirus 1, psittacine beak and feather disease virus, and psittacid herpesvirus 1 are important pathogens of psittacine birds with the potential to cause substantial morbidity and mortality. Using publically available nucleotide sequences, we developed and validated a triplex real-time PCR (rtPCR) assay to rapidly detect these 3 viruses. The assay had high analytical sensitivity, detecting <6 copies of viral DNA per reaction, and 100% analytical specificity, showing no cross-reactivity with 59 other animal pathogens. Archived formalin-fixed, paraffin-embedded tissues from psittacine birds diagnosed at postmortem as infected with each of the viruses as well as virus-negative birds were used to validate the utility of the assay. Birds were selected for the positive cohort if they showed histologic evidence of infection (i.e., characteristic inclusion bodies in tissues); birds in the negative cohort had final diagnoses unrelated to the pathogens of interest. The triplex rtPCR assay confirmed 98% of histopathology-positive cases, and also identified subclinical infections that were not observed by histologic examination, including coinfections. Birds that tested positive only by rtPCR had significantly higher cycle threshold values compared to those with histologic evidence of infection. Positive, negative, and overall percentage agreements as well as the kappa statistic between the results of the assay and histopathology were high, demonstrating the usefulness of the assay as a tool to confirm disease diagnoses, and to improve detection of subclinical infections.

Enhanced protective immune response to PCV2 adenovirus vaccine by fusion expression of Cap protein with InvC in pigs.

The major immunogenic protein capsid (Cap) of porcine circovirus type 2 (PCV2) is critical to induce neutralizing antibodies and protective immune response against PCV2 infection. This study was conducted to investigate the immune response of recombinant adenovirus expressing PCV2b Cap and C-terminal domain of Yersinia pseudotuberculosis invasin (Cap-InvC) fusion protein in pigs. The recombinant adenovirus rAd-Cap-InvC, rAd-Cap and rAd were generated and used to immunize pigs. The phosphate-buffered saline was used as negative control. The specific antibodies levels in rAd-Cap-InvC and ZJ/C-strain vaccine groups were higher than that of rAd-Cap group (p < 0.05), and the neutralization antibody titer in rAd-Cap-InvC group was significantly higher than those of other groups during 21-42 days post-immunization (DPI). Moreover, lymphocyte proliferative level, interferon-γ and interleukin-13 levels in rAd-Cap-InvC group were increased compared to rAd-Cap group (p < 0.05). After virulent challenge, viruses were not detected from the blood samples in rAd-Cap-InvC and ZJ/C-strain vaccine groups after 49 DPI. And the respiratory symptom, rectal temperature, lung lesion and lymph node lesion were minimal and similar in the ZJ/C-strain and rAd-Cap-InVC groups. In conclusion, our results demonstrated that rAd-Cap-InvC was more efficiently to stimulate the production of antibody and protect pigs from PCV2 infection. We inferred that InvC is a good candidate gene for further development and application of PCV2 genetic engineering vaccine.

Prevalence of Aves Polyomavirus 1 and Beak and Feather Disease Virus From Exotic Captive Psittacine Birds in Chile.

Avian polyomavirus disease and psittacine beak and feather disease (PBFD) are both contagious viral diseases in psittacine birds with similar clinical manifestations and characterized by abnormal feathers. To determine the prevalence of Aves polyomavirus 1 (APyV) and beak and feather disease virus (BFDV) in captive, exotic psittacine birds in Chile, feathers from 250 psittacine birds, representing 17 genera, were collected and stored during the period 2013-2016. Polymerase chain reaction testing was used to detect APyV and BFDV were detected in feather bulb samples. The results indicated that 1.6% (4/250) of the samples were positive for APyV, 23.2% (58/250) were positive to BFDV, and 0.8% (2/250) were positive to both APyV and BFDV. This is the first report, to our knowledge, of APyV and BFDV prevalence in captive, exotic psittacine birds in South America. Analysis of 2 Chilean partial sequences of the gene encoding agnoprotein 1a (APyV) and the replication-associated protein (BFDV) extends the knowledge of genomic variability for both APyV and BFDV isolates and their spectrum of hosts. No geographical marker was detected for the local isolates.

Establishment and Characterization of an Immortalized Porcine Oral Mucosal Epithelial Cell Line as a Cytopathogenic Model for Porcine Circovirus 2 Infection.

Porcine circovirus 2 (PCV2) is a major etiological agent for porcine circovirus-associated diseases and causes enormous economic losses in domestic and overseas swine production. However, there are currently no suitable cell models to study the cytopathic effects (CPE) of PCV2 in vitro, which severely restricts the study of PCV2 pathogenesis. In the present study, we established an immortalized porcine oral mucosal epithelial cell line (hTERT-POMEC) by introducing the hTERT gene into primary porcine oral mucosal epithelial cells (POMECs) derived from a neonatal, unsuckled piglet. The hTERT-POMEC cells have a homogeneous cobblestone-like morphology and retain the basic physiological properties of primary POMECs. No chromosome abnormality and tumorigenicity transformation was observed in immortalized hTERT-POMECs. Viral infection assays demonstrated that PCV2 propagated and caused CPE in hTERT-POMECs. We conclude that the immortalized cell line hTERT-POMEC is a crucial tool for further research into the pathogenesis of PCV2.

Characterization of the endonuclease activity of the replication-associated protein of beak and feather disease virus.

Beak and feather disease virus (BFDV) belongs to the family Circoviridae. A rolling-circle replication strategy based on a replication-associated protein (Rep) has been proposed for BFDV. The Rep gene of BFDV was expressed and purified, and it was shown to cleave short oligonucleotides containing the conserved nonanucleotide sequence found in the replication origin of circoviruses. This endonuclease activity was most efficient in the presence of the divalent metal ions Mg2+ and Mn2+. Rep proteins containing mutation in the ATPase/GTPase motifs and the 14FTLNN18, 61KKRLS65, 89YCSK92, and 170GKS172 motifs lacked endonuclease activity. The endonuclease activity was not affected by ATPase inhibitors, with the exception of N-ethylmaleimide (NEM), or by GTPase inhibitors, but it was decreased by treatment with the endonuclease inhibitor L-742001. Both the ATPase and GTPase activities were decreased by site-directed mutagenesis and deletion of the ATPase/GTPase and endonuclease motifs. The Rep protein was able to bind a double-stranded DNA fragment of P36 (dsP36) containing the stem-loop structure of the replication origin of BFDV. All of the Rep mutant proteins showed reduced ability to bind this fragment, suggesting that all the ATPase/GTPase and endonuclease motifs are involved in the binding. Other than NEM, all ATPase, GTPase, and endonuclease inhibitors inhibited the binding of the Rep protein to the dsP36 fragment. This is the first report describing the endonuclease activity of the Rep protein of BFDV.

Identification of four serotypes of fowl adenovirus in clinically affected commercial poultry co-infected with chicken infectious anaemia virus in Trinidad and Tobago.

Fowl adenovirus (FAdV), which causes the high-impact diseases such as inclusion body hepatitis and hepatitis-hydropericardium syndrome, is of major concern to the poultry industry internationally. This study was carried out in direct response to mortality rates of up to 75% in commercial broiler flocks in Trinidad, West Indies. Symptoms in 3- to 8-week-old broilers and 13- to 18-week-old pullets pointed to infection with an immunosuppressive viral pathogen. The objectives of the study were to determine whether the infectious agent FAdV, along with other viral pathogens, was responsible for the clinical disease, and to obtain information on the serotypes of FAdV that were infecting the birds. Tissue samples from clinically affected birds from eight different farms were tested for chicken infectious anaemia virus (CIAV) and infectious bursal disease virus (IBDV) by real-time reverse transcription polymerase chain reaction (PCR) and for FAdV by conventional PCR. The birds tested positive for FAdV and CIAV, but negative for IBDV. The gene corresponding to the L1 loop of the hexon protein for FAdV was amplified and sequenced. Phylogenetic analysis of seven FAdV strains inferred that four serotypes were likely to be circulating in the chickens. Well supported genetic relatedness was observed for serotype 8a (97.8%), 8b (97.8%), 9 (95.8%) and 11 (98.8%-99.5%). This is the first published report from Trinidad and Tobago on the presence and circulation of pathogenic FAdV strains, in combination with CIAV, in poultry. The data demonstrate a possible need for the introduction of serotype-specific vaccines against FAdV, as well as vaccines against CIAV, in broilers in the region and emphasize the importance of maintaining high levels of biosecurity on farms to prevent the spread of these potentially devastating viruses between farms.