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Next-generation sequencing (NGS) technologies are becoming increasingly accessible, leading to an expanded interest in the composition of the porcine enteric virome. In the present study, the fecal virome of a non-diarrheic Belgian piglet was determined. Although the virome of only a single piglet was analyzed, some interesting data were obtained, including the second complete genome of a pig group C rotavirus (RVC). This Belgian strain was only distantly related to the only other completely characterized pig RVC strain, Cowden. Its relatedness to RVC strains from other host species was also analyzed and the porcine strain found in our study was only distantly related to RVCs detected in humans and cows. The gene encoding the outer capsid protein VP7 belonged to the rare porcine G3 genotype, which might be serologically distinct from most other pig RVC strains. A putative novel RVC VP6 genotype was identified as well. A group A rotavirus strain also present in this fecal sample contained the rare pig genotype combination G11P[27], but was only partially characterized. Typical pig RVA genotypes I5, A8, and T7 were found for the viral proteins VP6, NSP1, and NSP3, respectively. Interestingly, the fecal virome of the piglet also contained an astrovirus and an enterovirus, of which the complete genomes were characterized. Results of the current study indicate that many viruses may be present simultaneously in fecal samples of non-diarrheic piglets. In this study, these viruses could not be directly associated with any disease, but still they might have had a potential subclinical impact on pig growth performance. The fast evolution of NGS will be a powerful tool for future diagnostics in veterinary practice. Its application will certainly lead to better insights into the relevance of many (sub)clinical enteric viral infections, that may have remained unnoticed using traditional diagnostic techniques. This will stimulate the development of new and durable prophylactic measures to improve pig health and production., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In the present study, the replication kinetics of nephropathogenic (B1648) and respiratory (Massachusetts-M41) IBV strains were compared in vitro in respiratory mucosa explants and blood monocytes (KUL01(+) cells), and in vivo in chickens to understand why some IBV strains have a kidney tropism. B1648 was replicating somewhat better than M41 in the epithelium of the respiratory mucosa explants and used more KUL01(+) cells to penetrate the deeper layers of the respiratory tract. B1648 was productively replicating in KUL01(+) monocytic cells in contrast with M41. In B1648 inoculated animals, 10(2.7-6.8) viral RNA copies/100 mg were detected in tracheal secretions at 2, 4, 6, 8, 10 and 12 days post inoculation (dpi), 10(2.4-4.5) viral RNA copies/mL in plasma at 2, 4, 6, 8, 10 and 12 dpi and 10(1.8-4.4) viral RNA copies/10(6) mononuclear cells in blood at 2, 4, 6 and 8 dpi. In M41 inoculated animals, 10(2.6-7.0) viral RNA copies/100 mg were detected in tracheal secretions at 2, 4, 6, 8 and 10 dpi, but viral RNA was not demonstrated in plasma and mononuclear cells (except in one chicken at 6 dpi). Infectious virus was detected only in plasma and mononuclear cells of the B1648 group. At euthanasia (12 dpi), viral RNA and antigen positive cells were detected in lungs, liver, spleen and kidneys of only the B1648 group and in tracheas of both the B1648 and M41 group. In conclusion, only B1648 can easily disseminate to internal organs via a cell-free and -associated viremia with KUL01(+) cells as important carrier cells.

Murine cytomegalovirus (MCMV) infection in mice is a commonly used animal model for studying human cytomegalovirus (HCMV) infections. In our previous studies, a mouse model based on an oronasal MCMV infection was set up for mimicking a natural infection, and the spleen was hypothesized to regulate viremia and virus dissemination to distal organs such as submandibular glands. Here, the role of the spleen during an MCMV infection was investigated by the comparison of intact and splenectomized Balb/c mice. Both highly passaged MCMV Smith and low passaged MCMV HaNa1 were used. Various samples were collected at 7, 14, and 21 days post inoculation (dpi) for analyses by virus isolation/titration, co-cultivation and qPCR. The results showed that for both virus strains, 1) cell-associated virus in PBMC (determined by co-cultivation) was detected in intact mice but not in splenectomized mice; 2) the mean viral DNA load in PBMC of splenectomized mice was 4.4-(HaNa1)/2.7-(Smith) fold lower at the peak viremia (7dpi) in contrast to that of intact mice; and 3) infectious virus in the submandibular glands was detected later in splenectomized mice (14dpi) than in intact mice (7dpi). Moreover, the average virus titers in submandibular glands of splenectomized mice were 10-(HaNa1)/7.9-(Smith) fold lower at 14dpi and 1.7-(HaNa1)/2.1-(Smith) fold lower at 21dpi compared with that of intact mice. Upon inoculation with MCMV Smith, infectious virus was found in the kidneys and liver of intact mice, but not in splenectomized mice. Taken together, all these data clearly demonstrate that virus dissemination to distant organs is reduced in splenectomized mice, further confirming the importance of the spleen as a viremia booming site for a natural MCMV infection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Cytomegaloviruses may infect mammals via oronasal route. However, up till now it remains unclear how this exposure leads to a general infection and shedding. To address this issue, BALB/c female mice were oronasally inoculated with either the highly passaged murine cytomegalovirus (MCMV) Smith or the low passaged MCMV HaNa1. Virus titration showed a productive virus replication of both strains in the nasal mucosa from 1 dpi until the end of the experiment (14 dpi), in lungs from 5 until 14 dpi, and in submandibular glands from 7 until 14 dpi. In contrast to MCMV HaNa1, MCMV Smith also established a low level productive infection in abdominal organs (spleen, liver and kidneys) from 5 dpi (spleen), 7 dpi (liver), and 10 dpi (kidneys) until the end of the experiment. Co-culture showed that for both strains, cell-associated virus was detected in a non-infectious form in nasopharynx-associated lymphoid tissues (NALT) from 1 until 14 dpi, in submandibular lymph nodes from 3 until 5 dpi, in deep cervical lymph nodes from 3 until 14 dpi, in mediastinal lymph nodes from 7 until 14 dpi, in spleen from 5 until at least 10 dpi and in the peripheral blood mononuclear cells (PBMC) at 7 and 10 dpi. The present study shows that upon oronasal exposure, MCMV first enters the nasal mucosa and NALT, from where the virus disseminates to the spleen possibly via the draining lymphatic system and blood; a subsequent cell-associated viremia transports MCMV to submandibular glands and for MCMV Smith also to liver and kidneys, where a second productive replication starts., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Feline infectious peritonitis (FIP) results from mutations in the viral genome during a common feline enteric coronavirus (FECV) infection. Since many virological and immunological data on FECV infections are lacking, the present study investigated these missing links during experimental infection of three SPF cats with FECV strain UCD. Two cats showed mild clinical signs, faecal shedding of infectious virus from 4 dpi, a cell-associated viraemia at inconsistent time points from 5 dpi, a highly neutralising antibody response from 9 dpi, and no major abnormalities in leukocyte numbers. Faecal shedding lasted for 28-56 days, but virus shed during this stage was less infectious in enterocyte cultures and affected by mutations. Remarkably, in the other cat neither clinical signs nor acute shedding were seen, but virus was detected in blood cells from 3 dpi, and shedding of non-enterotropic, mutated viruses suddenly occurred from 14 dpi onwards. Neutralising antibodies arose from 21 dpi. Leukocyte numbers were not different compared to the other cats, except for the CD8(+) regulatory T cells. These data indicate that FECV can infect immune cells even in the absence of intestinal replication and raise the hypothesis that the gradual adaptation to these cells can allow non-enterotropic mutants to arise.

Porcine epidemic diarrhea virus (PEDV) is a member of the family Coronaviridae and can cause severe outbreaks of diarrhea in piglets from different age groups. Here, we report the complete genome sequence (28,028 nt) of a PEDV strain isolated during a novel outbreak in Belgium., (Copyright © 2015 Theuns et al.)

Unlabelled: Group A rotaviruses (RVAs) are an important cause of diarrhea in young pigs and children. An evolutionary relationship has been suggested to exist between pig and human RVAs. This hypothesis was further investigated by phylogenetic analysis of the complete genomes of six recent (G2P[27], G3P[6], G4P[7], G5P[7], G9P[13], and G9P[23]) and one historic (G1P[7]) Belgian pig RVA strains and of all completely characterized pig RVAs from around the globe. In contrast to the large diversity of genotypes found for the outer capsid proteins VP4 and VP7, a relatively conserved genotype constellation (I5-R1-C1-M1-A8-N1-T7-E1-H1) was found for the other 9 genes in most pig RVA strains. VP1, VP2, VP3, NSP2, NSP4, and NSP5 genes of porcine RVAs belonged to genotype 1, which is shared with human Wa-like RVAs. However, for most of these gene segments, pig strains clustered distantly from human Wa-like RVAs, indicating that viruses from both species have entered different evolutionary paths. However, VP1, VP2, and NSP3 genes of some archival human strains were moderately related to pig strains. Phylogenetic analysis of the VP6, NSP1, and NSP3 genes, as well as amino acid analysis of the antigenic regions of VP7, further confirmed this evolutionary segregation. The present results also indicate that the species barrier is less strict for pig P[6] strains but that chances for successful spread of these strains in the human population are hampered by the better adaptation of pig RVAs to pig enterocytes. However, future surveillance of pig and human RVA strains is warranted., Importance: Rotaviruses are an important cause of diarrhea in many species, including pigs and humans. Our understanding of the evolutionary relationship between rotaviruses from both species is limited by the lack of genomic data on pig strains. In this study, recent and ancient Belgian pig rotavirus isolates were sequenced, and their evolutionary relationship with human Wa-like strains was investigated. Our data show that Wa-like human and pig strains have entered different evolutionary paths. Our data indicate that pig P[6] strains form the most considerable risk for interspecies transmission to humans. However, efficient spread of pig strains in the human population is most likely hampered by the adaptation of some crucial viral proteins to the cellular machinery of pig enterocytes. These data allow a better understanding of the risk for direct interspecies transmission events and the emergence of pig rotaviruses or pig-human reassortants in the human population., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Group A rotaviruses (RVA) are an important cause of diarrhea in young piglets, resulting in significant economic losses. However, the role of RVA in the etiology of piglet diarrhea on Belgian swine farms was previously unreported. In the present study, different techniques, including fast antigen detection tests, virus isolation, RT-PCR and RT-qPCR have been applied for detection of RVA in diarrheic (n=28) and asymptomatic (n=6) fecal samples collected on Belgian pig farms. RT-qPCR was shown to be most sensitive. Routine bacteriological analysis of the fecal samples showed that most diarrheic RVA positive samples were also co-infected with one or more bacterial species, such as Escherichia coli, Clostridium perfringens, Salmonella sp. and/or Brachyspira sp. Further genetic characterization of the VP7 and VP4 genes of 26 RVA strains resulted in the detection of six different G-genotypes (G2, G3, G4, G5, G9 and G11), and five different P-genotypes (P[6], P[7], P[13], P[23], P[27]), in a total of 12 different G/P combinations. A large intra-genotypic diversity was also apparent. In conclusion, results of the present study help us better understand the role of RVA in the pathogenesis of piglet diarrhea, and provide better insights into the vast genetic diversity present among circulating porcine group A rotaviruses., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Monocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV.

Feline infectious peritonitis (FIP) is a fatal, coronavirus-induced systemic disease in domestic and wild felids. The pathology associated with FIP (multifocal granulomatous vasculitis) is considered to be elicited by exaggerated activation and subsequent extravasation of leukocytes. As changes in the expression of adhesion molecules on circulating leukocytes precede their margination and emigration, we reasoned that the expression of leukocyte adhesion molecules may be altered in FIP. In present study, the expression of principal adhesion molecules involved in leukocyte transmigration (CD15s, CD11a, CD11b, CD18, CD49d, and CD54) on peripheral blood leukocytes from cats with naturally occurring FIP (n=15) and controls (n=12) was quantified by flow cytometry using a formaldehyde-based rapid leukocyte preparation technique. T- and B-lymphocytes from FIP patients exhibit higher expression of both subunits (CD11a and CD18) composing the β2 integrin lymphocyte function-associated antigen (LFA)-1. In addition, the expression of the α4 subunit (CD49d) of the β1 integrin very late antigen (VLA)-4 was elevated on B-lymphocytes from FIP patients. The expression of CD11b and CD18, that combine to form the β2 integrin macrophage-1 antigen (Mac-1), was elevated on monocytes, whereas the density of CD49d was reduced on this population in FIP. Granulocytes of FIP cats displayed an increased expression of the α chain of Mac-1 (CD11b). These observations suggest that leukocytes from FIP patients show signs of systemic activation causing them to extravasate into surrounding tissues and ultimately contribute to pyogranuloma formation seen in FIP., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: The in vitro culture of endothelial cells (ECs) is an indispensable tool for studying the role of the endothelium in physical and pathological conditions. Primary ECs, however, have a restricted proliferative lifespan which hampers their use in long-term studies. The need for standardized experimental conditions to obtain relevant and reproducible results has increased the demand for well-characterized, continuous EC lines that retain the phenotypic and functional characteristics of their non-transformed counterparts., Results: Primary feline ECs from aorta and vena cava were successfully immortalized through the successive introduction of simian virus 40 large T (SV40LT) antigen and the catalytic subunit of human telomerase (hTERT). In contrast to the parental ECs, the transformed cells were able to proliferate continuously in culture. Established cell lines exhibited several inherent endothelial properties, including typical cobblestone morphology, binding of endothelial cell-specific lectins and internalization of acetylated low-density lipoprotein. In addition, the immortalization did not affect the functional phenotype as demonstrated by their capacity to rapidly form cord-like structures on matrigel and to express cell adhesion molecules following cytokine stimulation., Conclusion: The ability to immortalize feline ECs, and the fact that these cells maintain the EC phenotype will enable a greater understanding of fundamental mechanisms of EC biology and endothelial-related diseases. Furthermore, the use of cell lines is an effective implementation of the 3-R principles formulated by Russel and Burch.

Feline infectious peritonitis (FIP) is the most feared infectious cause of death in cats, induced by feline infectious peritonitis virus (FIPV). This coronavirus is a virulent mutant of the harmless, ubiquitous feline enteric coronavirus (FECV). To date, feline coronavirus (FCoV) research has been hampered by the lack of susceptible cell lines for the propagation of serotype I FCoVs. In this study, long-term feline intestinal epithelial cell cultures were established from primary ileocytes and colonocytes by simian virus 40 (SV40) T-antigen- and human Telomerase Reverse Transcriptase (hTERT)-induced immortalization. Subsequently, these cultures were evaluated for their usability in FCoV research. Firstly, the replication capacity of the serotype II strains WSU 79-1683 and WSU 79-1146 was studied in the continuous cultures as was done for the primary cultures. In accordance with the results obtained in primary cultures, FCoV WSU 79-1683 still replicated significantly more efficient compared to FCoV WSU 79-1146 in both continuous cultures. In addition, the cultures were inoculated with faecal suspensions from healthy cats and with faecal or tissue suspensions from FIP cats. The cultures were susceptible to infection with different serotype I enteric strains and two of these strains were further propagated. No infection was seen in cultures inoculated with FIPV tissue homogenates. In conclusion, a new reliable model for FCoV investigation and growth of enteric field strains was established. In contrast to FIPV strains, FECVs showed a clear tropism for intestinal epithelial cells, giving an explanation for the observation that FECV is the main pathotype circulating among cats.

A strong cell-mediated immunity (CMI) is thought to be indispensable for protection against infection with feline infectious peritonitis virus (FIPV) in cats. In this study, the role of natural killer (NK) cells and regulatory T cells (Tregs), central players in the innate and adaptive CMI respectively, was examined during natural FIPV infection. When quantified, both NK cells and Tregs were drastically depleted from the peripheral blood, mesenteric lymph node (LN) and spleen in FIP cats. In contrast, mesentery and kidney from FIP cats did not show any difference when compared to healthy non-infected control animals. In addition, other regulatory lymphocytes (CD4+CD25-Foxp3+ and CD3+CD8+Foxp3+) were found to be depleted from blood and LN as well. Phenotypic analysis of blood-derived NK cells in FIP cats revealed an upregulation of activation markers (CD16 and CD25) and migration markers (CD11b and CD62L) while LN-derived NK cells showed upregulation of only CD16 and CD62L. LN-derived NK cells from FIPV-infected cats were also significantly less cytotoxic when compared with healthy cats. This study reveals for the first time that FIPV infection is associated with severe suppression of NK cells and Tregs, which is reflected by cell depletion and lowered cell functionality (only NK cells). This will un-doubtfully lead to a reduced capacity of the innate immune system (NK cells) to battle FIPV infection and a decreased capacity (Tregs) to suppress the immunopathology typical for FIP. However, these results will also open possibilities for new therapies targeting specifically NK cells and Tregs to enhance their numbers and/or functionality during FIPV infection., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The ability to productively infect monocytes/macrophages is the most important difference between the low virulent feline enteric coronavirus (FECV) and the lethal feline infectious peritonitis virus (FIPV). In vitro, the replication of FECV in peripheral blood monocytes always drops after 12h post inoculation, while FIPV sustains its replication in the monocytes from 45% of the cats. The accessory proteins of feline coronaviruses have been speculated to play a prominent role in virulence as deletions were found to be associated with attenuated viruses. Still, no functions have been ascribed to them. In order to investigate if the accessory proteins of FIPV are important for sustaining its replication in monocytes, replication kinetics were determined for FIPV 79-1146 and its deletion mutants, lacking either accessory protein open reading frame 3abc (FIPV-Δ3), 7ab (FIPV-Δ7) or both (FIPV-Δ3Δ7). Results showed that the deletion mutants FIPV-Δ7 and FIPV-Δ3Δ7 could not maintain their replication, which was in sharp contrast to wt-FIPV. FIPV-Δ3 could still sustain its replication, but the percentage of infected monocytes was always lower compared to wt-FIPV. In conclusion, this study showed that ORF7 is crucial for FIPV replication in monocytes/macrophages, giving an explanation for its importance in vivo, its role in the development of FIP and its conservation in field strains. The effect of an ORF3 deletion was less pronounced, indicating only a supportive role of ORF3 encoded proteins during the infection of the in vivo target cell by FIPVs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Natural killer (NK) cells play a central role in innate immunity and have been shown to influence adaptive immune responses as well. This study aimed to provide a general NK cell quantification and phenotyping in several compartments of healthy cats and assess their functional properties. The results indicated that NK numbers, both absolute and relative, and phenotype mostly correspond with those found in bovine, ovine, human and murine immunology. However, there were also distinct differences, especially with regard to the expression of the integrin CD11b and the selectin CD62L (between 10 and 30% of feline NK cells stain positive for these markers) and the relative frequencies in lymph nodes (6.7%), which stand central in NK cell development. Caution should be taken when extrapolating findings on NK cell properties over species, notwithstanding the generally accepted evolutionary conservation of NK cells and their subtypes. It was also shown that K562 cells, the 'golden' target cell line for NK functionality tests did not work for feline cells. The feline kidney cell line CRFK proved to be very responsive to NK- and NKT-mediated lysis and therefore, represents an ideal alternative target. This study is a good reference for NK cell numbers, both absolute and relative, phenotype and function in several anatomical compartments of healthy cats and for cat-specific cytotoxic assays involving both NK and NKT cells., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In this study an in vitro assay was optimized to detect feline proliferating lymphocytes as an assessment for the cell-mediated immune response. For this purpose, 5-bromo-2'-deoxyuridine (BrdU) labeling was chosen because of its sensitivity and the possibility of further characterization of proliferating cells. The assay was optimized by selecting the best batch and concentration of fetal bovine serum, β-mercaptoethanol concentration, cell density, BrdU incubation time and antigen presenting cell type. Cats were vaccinated with the attenuated Nobivac vaccine Tricat and the peripheral blood lymphocyte proliferation responses were quantified upon in vitro restimulation with inactivated and infectious feline panleukopenia virus (FPV), feline calicivirus (FCV) and felid herpesvirus 1 (FeHV-1). Proliferation signals were detected with inactivated FeHV-1 in the CD8(+) but not in the CD8(-) T lymphocyte population, with inactivated FCV and FPV in both CD8(-) and CD8(+) T lymphocyte populations. Restimulation with infectious FCV caused significant proliferation in the CD8(-) T lymphocyte population only while infectious FPV and FeHV-1 seemed to suppress lymphocyte proliferation in both T cell populations. Additional IFN-γ quantification in the culture supernatant revealed a large correlation between the proliferation signals and IFN-γ production, indicating that BrdU labeling is a very reliable technique to assess and characterize feline lymphoproliferative responses to viral antigens in vitro., (Copyright © 2012 Elsevier B.V. All rights reserved.)