Sebastiaan Theuns

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.

Complete Genome Characterization of Recent and Ancient Belgian Pig Group A Rotaviruses and Assessment of Their Evolutionary Relationship with Human Rotaviruses

ABSTRACT 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.

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.

Porcine group A rotaviruses with heterogeneous VP7 and VP4 genotype combinations can be found together with enteric bacteria on Belgian swine farms.

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.

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.

Genetic Characterization of the Belgian Nephropathogenic Infectious Bronchitis Virus (NIBV) Reference Strain B1648

The virulent nephropathogenic infectious bronchitis virus (NIBV) strain B1648 was first isolated in 1984, in Flanders, Belgium. Despite intensive vaccination, B1648 and its variants are still circulating in Europe and North Africa. Here, the full-length genome of this Belgian NIBV reference strain was determined by next generation sequencing (NGS) to understand its evolutionary relationship with other IBV strains, and to identify possible genetic factors that may be associated with the nephropathogenicity. Thirteen open reading frames (ORFs) were predicted in the B1648 strain (5′UTR-1a-1b-S-3a-3b-E-M-4b-4c-5a-5b-N-6b-3′UTR). ORFs 4b, 4c and 6b, which have been rarely reported in literature, were present in B1648 and most of the other IBV complete genomes. According to phylogenetic analysis of the full-length genome, replicase transcriptase complex, spike protein, partial S1 gene and M protein, B1648 strain clustered with the non-Massachusetts type strains NGA/A116E7/2006, UKr 27-11, QX-like ITA/90254/2005, QX-like CK/SWE/0658946/10, TN20/00, RF-27/99, RF/06/2007 and SLO/266/05. Based on the partial S1 fragment, B1648 clustered with the strains TN20/00, RF-27/99, RF/06/2007 and SLO/266/05 and, further designated as B1648 genotype. The full-length genome of B1648 shared the highest sequence homology with UKr 27-11, Gray, JMK, and NGA/A116E7/2006 (91.2% to 91.6%) and was least related with the reference Beaudette and Massachusetts strains (89.7%). Nucleotide and amino acid sequence analyses indicated that B1648 strain may have played an important role in the evolution of IBV in Europe and North Africa. Further, the nephropathogenicity determinants might be located on the 1a, spike, M and accessory proteins (3a, 3b, 4b, 4c, 5a, 5b and 6b). Overall, strain B1648 is distinct from all the strains reported so far in Europe and other parts of the world.

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 24 h post-reseeding, and this monolayer could be maintained for 10 days with a viability of 90 %. Binding of WSSV to cells reached a maximum (73 ± 3 % of cells and 4.84 ± 0.2 virus particles per virus-binding cell) at 120 min at 4 °C. WSSV entered cells by endocytosis. The co-localization of WSSV and early endosomes was observed starting from 30 min post-inoculation (p.i.). Double indirect immunofluorescence staining showed that all cell-bound WSSV particles entered these cells in the period between 0 and 60 min p.i. and that the uncoating of WSSV occurred in the same period. After 1 h inoculation at 27 °C, the WSSV nucleocapsid protein VP664 and envelope protein VP28 started to be synthesized in the cytoplasm from 1 and 3 h p.i., and were transported into nuclei from 3 and 6 h p.i., respectively. The percentage of cells that were VP664- and VP28-positive in their nuclei peaked (50 ± 4 %) at 12 h p.i. Quantitative PCR showed that WSSV DNA started to be synthesized from 6 h p.i. In vivo titration of the supernatants showed that the progeny WSSV were released from 12 h p.i. and peaked at 18 h 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

Abstract 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.

Identification of an enterovirus recombinant with a torovirus-like gene insertion during a diarrhea outbreak in fattening pigs

Abstract Diarrhea outbreaks in pig farms have raised major concerns in Europe and USA, as they can lead to dramatic pig losses. During a suspected outbreak in Belgium of porcine epidemic diarrhea virus (PEDV), we performed viral metagenomics to assess other potential viral pathogens. Although PEDV was detected, its low abundance indicated that other viruses were involved in the outbreak. Interestingly, a porcine bocavirus and several enteroviruses were most abundant in the sample. We also observed the presence of a porcine enterovirus genome with a gene insertion, resembling a C28 peptidase gene found in toroviruses, which was confirmed using re-sequencing, bioinformatics, and proteomics approaches. Moreover, the predicted cleavage sites for the insertion suggest that this gene was being expressed as a single protein, rather than a fused protein. Recombination in enteroviruses has been reported as a major mechanism to generate genetic diversity, but gene insertions across viral families are rather uncommon. Although such inter-family recombinations are rare, our finding suggests that these events may significantly contribute to viral evolution.