Lowiese M.B. Desmarets

Complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in belgium, january 2015.

ABSTRACT 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.

ORF7-encoded accessory protein 7a of feline infectious peritonitis virus as a counteragent against IFN-α-induced antiviral response

The type I IFN-mediated immune response is the first line of antiviral defence. Coronaviruses, like many other viruses, have evolved mechanisms to evade this innate response, ensuring their survival. Several coronavirus accessory genes play a central role in these pathways, but for feline coronaviruses this has never to our knowledge been studied. As it has been demonstrated previously that ORF7 is essential for efficient replication in vitro and virulence in vivo of feline infectious peritonitis virus (FIPV), the role of this ORF in the evasion of the IFN-α antiviral response was investigated. Deletion of ORF7 from FIPV strain 79-1146 (FIPV-Δ7) rendered the virus more susceptible to IFN-α treatment. Given that ORF7 encodes two proteins, 7a and 7b, it was further explored which of these proteins is active in this mechanism. Providing 7a protein in trans rescued the mutant FIPV-Δ7 from IFN sensitivity, which was not achieved by addition of 7b protein. Nevertheless, addition of protein 7a to FIPV-Δ3Δ7, a FIPV mutant deleted in both ORF3 and ORF7, could no longer increase the replication capacity of this mutant in the presence of IFN. These results indicate that FIPV 7a protein is a type I IFN antagonist and protects the virus from the antiviral state induced by IFN, but it needs the presence of ORF3-encoded proteins to exert its antagonistic function.

Presence and characterization of pig group A and C rotaviruses in feces of Belgian diarrheic suckling piglets.

The importance of group A and C rotaviruses (RVA and RVC) in the pathogenesis of diarrhea in Belgian suckling pigs is poorly investigated, and it is not known which strains are circulating in the Belgian suckling pig population. Obtaining better insights in the occurrence of both viral species in the swine population is essential in order to develop accurate diagnostic, therapeutic and prophylactic strategies to protect suckling pigs against diarrhea in a durable manner. In the present study, viral loads of RVA and RVC were quantified in diarrhea samples of suckling piglets less than 2 weeks old, collected on 36 different Belgian farms. On 22 of 36 farms tested (61%), high viral loads of RVA (6.96-11.95 log10 copies/g feces) and/or RVC (5.40-11.63 log10 copies/g feces) were detected. Seventeen RVA isolates were genotyped for their outer capsid proteins VP7 and VP4. Four different G-genotypes (G3, G4, G5 and G9) for VP7 were found together with 4 different P-genotypes (P[6], P[7], P[13] and P[23]) for VP4, in 8 different G/P combinations. All characterized RVC strains belonged to genotype G6 (VP7), except for one strain possessing the G1 genotype. VP4 genes of Belgian RVC strains were genetically heterogeneous, but were classified in the genotype P5. Most rotavirus positive samples also contained Escherichia coli, whereas Clostridium perfringens infections were mainly detected in rotavirus negative samples. Results of the present study offer better insights in the occurrence of RVA and RVC infections in Belgian diarrheic suckling piglets. As a conclusion, routine diagnostic testing for both viral species in cases of diarrhea in suckling pigs is highly recommended. Furthermore, the present findings also offer valuable information for the development of new prophylactic measures against rotavirus. Finally, the relatedness between RVC strains from pigs and other host species is described, and their possible implications in interspecies transmission events are discussed.

Experimental feline enteric coronavirus infection reveals an aberrant infection pattern and shedding of mutants with impaired infectivity in enterocyte cultures.

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.

Virus replication cycle of white spot syndrome virus in secondary cell cultures from the lymphoid organ of Litopenaeus vannamei.

The replication cycle of white spot syndrome virus (WSSV) was investigated in secondary cell cultures from the lymphoid organ of Litopenaeus vannamei. The secondary cells formed a confluent monolayer at 24u200ah post-reseeding, and this monolayer could be maintained for 10u200adays with a viability of 90u2009%. Binding of WSSV to cells reached a maximum (73u2009±u20093u2009% of cells and 4.84u2009±u20090.2 virus particles per virus-binding cell) at 120u200amin at 4u2009°C. WSSV entered cells by endocytosis. The co-localization of WSSV and early endosomes was observed starting from 30u200amin post-inoculation (p.i.). Double indirect immunofluorescence staining showed that all cell-bound WSSV particles entered these cells in the period between 0 and 60u200amin p.i. and that the uncoating of WSSV occurred in the same period. After 1u200ah inoculation at 27u2009°C, the WSSV nucleocapsid protein VP664 and envelope protein VP28 started to be synthesized in the cytoplasm from 1 and 3u200ah p.i., and were transported into nuclei from 3 and 6u200ah p.i., respectively. The percentage of cells that were VP664- and VP28-positive in their nuclei peaked (50u2009±u20094u2009%) at 12u200ah p.i. Quantitative PCR showed that WSSV DNA started to be synthesized from 6u200ah p.i. In vivo titration of the supernatants showed that the progeny WSSV were released from 12u200ah p.i. and peaked at 18u200ah p.i. In conclusion, the secondary cell cultures from the lymphoid organ were proven to be ideal for examination of the replication cycle of WSSV.

Characterization of a genetically heterogeneous porcine rotavirus C, and other viruses present in the fecal virome of a non-diarrheic Belgian piglet

n Abstractn n 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.n n

Productive replication of nephropathogenic infectious bronchitis virus in peripheral blood monocytic cells, a strategy for viral dissemination and kidney infection in chickens

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, 102.7–6.8 viral RNA copies/100xa0mg were detected in tracheal secretions at 2, 4, 6, 8, 10 and 12xa0days post inoculation (dpi), 102.4–4.5 viral RNA copies/mL in plasma at 2, 4, 6, 8, 10 and 12xa0dpi and 101.8–4.4 viral RNA copies/106 mononuclear cells in blood at 2, 4, 6 and 8xa0dpi. In M41 inoculated animals, 102.6–7.0 viral RNA copies/100xa0mg 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 6xa0dpi). Infectious virus was detected only in plasma and mononuclear cells of the B1648 group. At euthanasia (12xa0dpi), 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.

Pattern of circulation of MCMV mimicking natural infection upon oronasal inoculation

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.

Role of sialic acids in feline enteric coronavirus infections

To initiate infections, many coronaviruses use sialic acids, either as receptor determinants or as attachment factors helping the virus find its receptor underneath the heavily glycosylated mucus layer. In the present study, the role of sialic acids in serotype I feline enteric coronavirus (FECV) infections was studied in feline intestinal epithelial cell cultures. Treatment of cells with neuraminidase (NA) enhanced infection efficiency, showing that terminal sialic acid residues on the cell surface were not receptor determinants and even hampered efficient virus-receptor engagement. Knowing that NA treatment of coronaviruses can unmask viral sialic acid binding activity, replication of untreated and NA-treated viruses was compared, showing that NA treatment of the virus enhanced infectivity in untreated cells, but was detrimental in NA-treated cells. By using sialylated compounds as competitive inhibitors, it was demonstrated that sialyllactose (2,6-α-linked over 2,3-α-linked) notably reduced infectivity of NA-treated viruses, whereas bovine submaxillary mucin inhibited both treated and untreated viruses. In desialylated cells, however, viruses were less prone to competitive inhibition with sialylated compounds. In conclusion, this study demonstrated that FECV had a sialic acid binding capacity, which was partially masked by virus-associated sialic acids, and that attachment to sialylated compounds could facilitate enterocyte infections. However, sialic acid binding was not a prerequisite for the initiation of infection and virus-receptor engagement was even more efficient after desialylation of cells, indicating that FECV requires sialidases for efficient enterocyte infections.

Differences in Env and Gag protein expression patterns and epitope availability in feline immunodeficiency virus infected PBMC compared to infected and transfected feline model cell lines

Env and Gag are key components of the FIV virion that are targeted to the plasma membrane for virion assembly. They are both important stimulators and targets of anti-FIV immunity. To investigate and compare the expression pattern and antigenic changes of Gag and Env in various research models, infected PBMC (the natural FIV host cells) and GFox, and transfected CrFK were stained over time with various Env and Gag specific MAbs. In FIV infected GFox and PBMC, Env showed changes in epitope availability for antibody binding during processing and trafficking, which was not seen in transfected CrFK. Interestingly, epitopes exposed on intracellular Env and Env present on the plasma membrane of CrFK and GFox seem to be hidden on plasma membrane expressed Env of FIV infected PBMC. A kinetic follow up of Gag and Env expression showed a polarization of both Gag and Env expression to specific sites at the plasma membrane of PBMC, but not in other cell lines. In conclusion, mature trimeric cell surface expressed Env might be antigenically distinct from intracellular monomeric Env in PBMC and might possibly be unrecognizable by feline humoral immunity. In addition, Env expression is restricted to a small area on the plasma membrane and co-localizes with a large moiety of Gag, which may represent a preferred FIV budding site, or initiation of virological synapses with direct cell-to-cell virus transmission.