Marcel Hulst

Lelystad virus, the cause of porcine epidemic abortion and respiratory syndrome: a review of mystery swine disease research at Lelystad

This paper reviews the laboratory investigations that led us to isolate the Lelystad virus and demonstrate that this virus causes mystery swine disease. We describe: 1) isolating the virus from the disease; 2) characterizing the virus as a new enveloped RNA virus; 3) reproducing the disease experimentally with the isolated Lelystad virus; 4) isolating the virus from the experimentally induced disease.

Passage of Classical Swine Fever Virus in Cultured Swine Kidney Cells Selects Virus Variants That Bind to Heparan Sulfate due to a Single Amino Acid Change in Envelope Protein Erns

ABSTRACT Infection of cells with Classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoprotein Ernsand E2 with the cell surface. In this report we studied the role of the cell surface glycoaminoglycans (GAGs), chondroitin sulfates A, B, and C (CS-A, -B, and -C), and heparan sulfate (HS) in the initial binding of CSFV strain Brescia to cells. Removal of HS from the surface of swine kidney cells (SK6) by heparinase I treatment almost completely abolished infection of these cells with virus that was extensively passaged in swine kidney cells before it was cloned (clone C1.1.1). Infection with C1.1.1 was inhibited completely by heparin (a GAG chemically related to HS but sulfated to a higher extent) and by dextran sulfate (an artificial highly sulfated polysaccharide), whereas HS and CS-A, -B, and -C were unable to inhibit infection. Bound C1.1.1 virus particles were released from the cell surface by treatment with heparin. Furthermore, C1.1.1 virus particles and CSFV Ernspurified from insect cells bound to immobilized heparin, whereas purified CSFV E2 did not. These results indicate that initial binding of this virus clone is accomplished by the interaction of Erns with cell surface HS. In contrast, infection of SK6 cells with virus clones isolated from the blood of an infected pig and minimally passaged in SK6 cells was not affected by heparinase I treatment of cells and the addition of heparin to the medium. However, after one additional round of amplification in SK6 cells, infection with these virus clones was affected by heparinase I treatment and heparin. Sequence analysis of the Erns genes of these virus clones before and after amplification in SK6 cells showed that passage in SK6 cells resulted in a change of an Ser residue to an Arg residue in the C terminus of Erns (amino acid 476 in the polyprotein of CSFV). Replacement of the Erns gene of an infectious DNA copy of C1.1.1 with the Erns genes of these virus variants proved that acquisition of this Arg was sufficient to alter an HS-independent virus to a virus that uses HS as an Erns receptor.

Development of a virus neutralisation test to detect antibodies against Schmallenberg virus and serological results in suspect and infected herds.

BackgroundAt the end of 2011, a new orthobunyavirus, tentatively named Schmallenberg virus (SBV), was discovered in Germany. This virus has since been associated with clinical signs of decreased milk production, watery diarrhoea and fever in dairy cows, and subsequently also with congenital malformations in calves, lambs and goat kids. In affected countries, initial surveillance for the infection was based on examination of malformed progeny. These suspicions were followed up by real-time reverse transcription polymerase chain reaction (RT-PCR) on brain tissue. For epidemiological purposes, a serological assay was, however, needed.ResultsA virus neutralisation test (VNT) was developed and optimized, and subsequently evaluated. This VNT has a specificity of >99% and the sensitivity is likely also very close to 100%. The assay is highly repeatable and reproducible. The final assay was used to test for antibodies in cows, ewes and does from herds known to be infected or suspected to be so. Targets for sampling in these herds were the mothers of malformed offspring. In herds with an RT-PCR confirmed SBV infection, more than 94% (190 out of 201) of the ewes and 99% (145 out of 146) of the cows were seropositive. In herds with suspicion of SBV infection based on birth of malformed offspring only (no or negative RT-PCR), more than 90% (231 out of 255) of the ewes and 95% (795 out of 834) of the cows were seropositive. In goats, on the other hand, only a low number of seropositives was found: overall 36.4%, being 16 out of 44 goats tested.ConclusionsGiven the characteristics of this VNT, it can be used at a relative high throughput for testing of animals for export, surveillance, screening and research purposes, but can also be used as a confirmation test for commercially available enzyme-linked immunosorbent assays (ELISA’s) and for (relative) quantification of antibodies.Suspicions of SBV infections that were confirmed by RT-PCR were almost always confirmed by serology in cows. Due to individual registration and identification of cows and calves, affected offspring could almost always be traced back to the mother. Ewes on the other hand were not always the mothers of affected lambs, but were in many cases herd mates with unaffected lambs. This indicated a high within-herd seroprevalence of antibodies against SBV.

Interaction of Classical Swine Fever Virus with Membrane-Associated Heparan Sulfate: Role for Virus Replication In Vivo and Virulence

ABSTRACT Passage of native classical swine fever virus (CSFV) in cultured swine kidney cells (SK6 cells) selects virus variants that attach to the surface of cells by interaction with membrane-associated heparan sulfate (HS). A Ser-to-Arg change in the C terminus of envelope glycoprotein Erns (amino acid 476 in the open reading frame of CSFV) is responsible for selection of these HS-binding virus variants (M. M. Hulst, H. G. P. van Gennip, and R. J. M. Moormann, J. Virol. 74:9553–9561, 2000). In this investigation we studied the role of binding of CSFV to HS in vivo. Using reverse genetics, an HS-independent recombinant virus (S-ST virus) with Ser476 and an HS-dependent recombinant virus (S-RT virus) with Arg476 were constructed. Animal experiments indicated that this adaptive Ser-to-Arg mutation had no effect on the virulence of CSFV. Analysis of viruses reisolated from pigs infected with these recombinant viruses indicated that replication in vivo introduced no mutations in the genes of the envelope proteins Erns, E1, and E2. However, the blood of one of the three pigs infected with the S-RT virus contained also a low level of virus particles that, when grown under a methylcellulose overlay, produced relative large plaques, characteristic of an HS-independent virus. Sequence analysis of such a large-plaque phenotype showed that Arg476 was mutated back to Ser476. Removal of HS from the cell surface and addition of heparin to the medium inhibited infection of cultured (SK6) and primary swine kidney cells with S-ST virus reisolated from pigs by about 70% whereas infection with the administered S-ST recombinant virus produced in SK6 cells was not affected. Furthermore, Erns S-ST protein, produced in insect cells, could bind to immobilized heparin and to HS chains on the surface of SK6 cells. These results indicated that S-ST virus generated in pigs is able to infect cells by an HS-dependent mechanism. Binding of concanavalin A (ConA) to virus particles stimulated the infection of SK6 cells with S-ST virus produced in these cells by 12-fold; in contrast, ConA stimulated infection with S-ST virus generated in pigs no more than 3-fold. This suggests that the surface properties of S-ST virus reisolated from pigs are distinct from those of S-ST virus produced in cell culture. We postulate that due to these surface properties, in vivo-generated CSFV is able to infect cells by an HS-dependent mechanism. Infection studies with the HS-dependent S-RT virus, however, indicated that interaction with HS did not mediate infection of lung macrophages, indicating that alternative receptors are also involved in the attachment of CSFV to cells.

Determinants of Virulence of Classical Swine Fever Virus Strain Brescia

ABSTRACT Two related classical swine fever virus (CSFV) strain Brescia clones were isolated from blood samples from an infected pig. Virus C1.1.1 is a cell-adapted avirulent variant, whereas CoBrB is a virulent variant. Sequence analysis revealed 29 nucleic acid mutations in C1.1.1, resulting in 9 amino acid substitutions compared to the sequence of CoBrB 476R. Using reverse genetics, parts of the genomes of these viruses, which contain differences that lead to amino acid changes, were exchanged. Animal experiments with chimeric viruses derived from C1.1.1 and CoBrB 476R showed that a combination of amino acid changes in the structural and nonstructural regions reduced the virulence of CSFV in pigs. Moreover, the presence of a Leu at position 710 in structural envelope protein E2 seemed to be an important factor in the virulence of the virus. Changing the Leu at position 710 in the CoBrB 476S variant into a His residue did not affect virulence. However, the 710His in the C1.1.1/CoBrB virus, together with adaptive mutations 276R, 476R, and 477I in Erns, resulted in reduced virulence in pigs. These results indicated that mutations in Erns and E2 alone do not determine virulence in pigs. The results of in vitro experiments suggested that a high affinity for heparan sulfate of C1.1.1 Erns may reduce the spread of the C1.1.1/CoBrB virus in pigs and together with the altered surface structure of E2 caused by the 710L→H mutation may result in a less efficient infection of specific target cells in pigs. Both these features contributed to the attenuation of the C1.1.1/CoBrB virus in vivo.

Mannose-specific interaction of Lactobacillus plantarum with porcine jejunal epithelium

Host-microorganism interactions in the intestinal tract are complex, and little is known about specific nonpathogenic microbial factors triggering host responses in the gut. In this study, mannose-specific interactions of Lactobacillus plantarum 299v with jejunal epithelium were investigated using an in situ pig Small Intestinal Segment Perfusion model. The effects of L. plantarum 299v wild-type strain were compared with those of two corresponding mutant strains either lacking the gene encoding for the mannose-specific adhesin (msa) or sortase (srtA; responsible for anchoring of cell surface proteins like Msa to the cell wall). A slight enrichment of the wild-type strain associated with the intestinal surface could be observed after 8 h of perfusion when a mixture of wild-type and msa-mutant strain had been applied. In contrast to the mutant strains, the L. plantarum wild-type strain tended to induce a decrease in jejunal net fluid absorption compared with control conditions. Furthermore, after 8 h of perfusion expression of the host gene encoding pancreatitis-associated protein, a protein with proposed bactericidal properties, was found to be upregulated by the wild-type strain only. These observations suggest a role of Msa in the induction of host responses in the pig intestine.

Nucleotide sequence of hog cholera virus RNA: properties of the polyprotein encoded by the open reading frame spanning the viral genomic RNA

Hog cholera virus RNA was cloned and sequenced. A single major open reading frame (ORF), encoding an amino acid sequence of 3898 residues, was found in the second reading frame of the sequence of one of the cDNA strands. We demonstrated that the ORF spans the length of the viral sense RNA, which implies that it is translated into a precursor polyprotein. Several properties of this polyprotein, like hydrophobicity, position of putative protease cleavage sites, distribution of N-linked glycosylation sites, distribution of cysteines and distribution of acidic and basic residues are described and discussed.

Effects of chronic stress: A Comparison between tethered and loose sows

The present study aimed to investigate whether long-lasting, recurrent tethering of sows leads to enduring effects on measures that may be indicative of chronic stress. Sows that had experienced tethering for about 1.5 or 4.5years and age-matched sows kept in a social housing system (loose sows) were compared. Immediately after slaughter, blood samples were taken to measure plasma cortisol levels, and the brain, spleen, and adrenals were dissected and weighed. Gene expression in the frontal cortex and hippocampus was analyzed. Plasma cortisol levels were higher in the tethered sows than in the loose sows. The older, but not the younger, tethered sows had heavier adrenal glands than their loose counterparts. The weight of the spleen was not affected by the housing conditions, but the pituitary gland was lighter in tethered sows than in loose sows. Microarray analyses revealed an increased expression of beta-globin mRNA in the hippocampus and to a lesser extent in the frontal cortex of the older tethered sows, compared with the older loose sows. Taken together, the findings indicate that chronically stressed pigs develop depression-like symptoms. However, it can be questioned whether the pig subjected to repeated, long-term stress can be regarded an animal model of major depression.

Erns Protein of Pestiviruses

Publisher Summary Together with flaviviruses and hepatitis C virus (HCV), an important human pathogen, the pestiviruses are classified as a genus within the family Flaviviridae. Viruses in this family are small, enveloped, positive-strand RNA viruses. Compared with HCV, the pestivirus genome encodes two additional proteins, an N-terminal autoprotease, N pro , and the envelope protein E rns . Comparison of the amino acid sequences of E rns of pestiviruses with amino acid sequences in databases identified two short stretches, both eight amino acids in length, that are homologous to the active site domains of ribonucleases of the RNase T2 family. Enzymatic tests of purified E rns proved that these stretches are involved in ribonuclease activity. This chapter reviews the specific properties of this RNase activity and its function in relation to the life cycle of pestiviruses. The properties of the RNase activity of CSFV E rns were determined with purified native E rns , and E rns purified from insect cells. For ribonucleases of the RNase T2 family, it has been shown that the histidine residues in the two conserved domains are essential for RNase catalysis. Mutational studies showed that this is also true for E rns . Besides the possible cytotoxic action of E rns toward the host immune system there is also a possible role for E rns RNase activity in regulation of RNA synthesis in infected cells.

Transcription networks responsible for early regulation of Salmonella -induced inflammation in the jejunum of pigs

BackgroundThe aim of this study was to identify transcription factors/regulators that play a crucial role in steering the (innate) immune response shortly (within a few hours) after the first contact of the intestinal mucosa with an inflammatory mediator, and to test whether the processes regulated by these factors/regulators can be modulated by chemical substances of natural origin.MethodsWe experimentally induced inflammation by perfusion of surgically applied jejunal loops with Salmonella enterica subspecies enterica serovar Typhimurium DT104 in three pigs. Segments of mock and Salmonella treated loops were dissected after 2, 4 and 8 hours of perfusion. IL8 and IL1-beta mRNA expression levels were measured in mucosal scrapings of all segments. Furthermore, intra-animal microarray comparisons (isogenic) between Salmonella and mock treated segments after 8 hours, and inter-animal comparisons between similar Salmonella-treated loops of each pig at 2 and 4 hours, were performed.ResultsIL-1beta and IL8 mRNA levels, and intra-animal microarray comparisons at 8 hours between Salmonella and mock treated segments showed that the response-time and type of response to Salmonella was different in all three pigs. This plasticity allowed us to extract a comprehensive set of differentially expressed genes from inter-animal comparisons at 2 and 4 hours. Pathway analysis indicated that many of these genes play a role in induction and/or tempering the inflammatory response in the intestine. Among them a set of transcription factors/regulators known to be involved in regulation of inflammation, but also factors/regulators for which involvement was not expected. Nine out of twenty compounds of natural origin, which according to literature had the potential to modulate the activity of these factors/regulators, were able to stimulate or inhibit a Salmonella-induced mRNA response of inflammatory-reporter genes IL8 and/or nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha in cultured intestinal porcine epithelial cells.ConclusionsWe describe a set of transcription factors/regulators possibly involved in regulation of “very early” immune mechanism which determines the inflammatory status of the intestine later on. In addition, we show that these mechanisms may be modulated by chemical substances of natural origin.