Reemerging Transmissible Gastroenteritis in Pigs - Simon Andersson

SZENT ISTVÁN UNIVERSITY FACULTY OF VETERINARY SCIENCE DEPARTMENT OF MICROBIOLOGY AND INFECTIOUS DISEASES RE-EMERGING TRANSMISSIBLE GASTROENTERITIS IN PIGS Written by: Simon Andersson Supervisors: Dr. Imre Biksi, PhD Dr. Márta Lőrincz, PhD student Dr. Tamás Tuboly, PhD Budapest 2010 TABLE OF CONTENTS INTRODUCTION 3 MATERIALS AND METHODS Samples RNA extraction Reverse transcriptase polymerase chain reaction Gel electrophoresis Cloning Sequence analysis 9 9 9 10 11 12 12 13 13 15 15 16 17 21 22 23 24 RESULTS Reverse transcriptase polymerase chain reaction Cloning Sequence analysis Serology DISCUSSION ACKNOWLEDGEMENTS SUMMARY ÖSSZEFOGLALÁS REFERENCES 2 INTRODUCTION Porcine transmissible gastroenteritis (TGE) is a disease in pigs caused by a coronavirus belonging into the Coronaviridae family of the order Nidovirales. Coronaviruses are enveloped (Figure 1.) and the genetic information is coded by a copy of a single stranded RNA genome of positive polarity, so far known as the largest such stable RNA genome of animal viruses, ranging in size between 26.4 – 31.7 kilobases (kb). Other families of Nidovirales, such as Arteriviridae and Roniviridae share some common characteristics with the Coronaviridae, besides the structural similarities and the nature of the genome. These virus families are all known for their nested set replication and transcription profile (de Vries et al., 1997). Peplomer (Spike, S) Nucleoprotein (N) Membrane (M) ssRNA Small membrane envelope (sM, E) Figure 1. Electron micrograph and a schematic drawing of the structure of a coronavirus. The family Coronaviridae is further divided into the coronavirus and the torovirus genera (Fauquet et al., 2005). The genus coronavirus comprises three groups, or most recently referred to as subgenera, namely Alpha-, Beta and Gamma-coronavirus. Coronaviruses have some rather prominent members that cause important diseases, both of human and veterinary interest such as, Severe Acute Respiratory Syndrome Virus (SARS-CoV), Infectious Bronchitis Virus (IBV), Feline Infectious Peritonitis Virus (FCoV), Porcine Hemagglutinating Encephalomyelitis (PHEV) and Transmissible Gastroenteritis (TGEV), just to mention a few of them. All in all the complete genome of some 26 coronaviruses have been described to this date. But there are probably more out there and this number is in no doubt going to change in 3 a not too distant future. After the SARS outbreak in 2002-2003 (Peiris et al., 2004), research into coronaviruses has really picked up the pace and new interesting things were found out, among them the zoonotic potential of some members of the genus (Shi and Hu, 2008; Hon et al., 2008). The ability to break the species barrier is also evident in case of the newly emerging enteric bovine coronaviruses that share a 98 % RNA sequence homology with the HECoV-4408 human enteric coronavirus, frequently isolated in diarrhea cases of children (Han et al., 2006). Also noteworthy in this respect is the fact that the recently emerged canine respiratory coronavirus (CRCoV), was originally a bovine coronavirus (Decaro et al., 2007). Coronaviruses are well known for their flexibility in adapting to new species or new environmental conditions in the host. Their plasticity has mainly been attributed to three factors, the high mutation rate due to the lack or limited proof reading activity of the RNA dependent RNA polymerase (Jenkins et al., 2002; Duffy et al., 2008); the polymerase may move to a different template during replication (Lai, 1992); and also there seems to be a negative correlation between genome size and mutation-rate. If the genome grows beyond a certain size the amount of accumulated deleterious mutations will prove fatal to the virus (Eigen, 1987). All these together make for a potentially fast evolving virus, which is able to adapt to new circumstances rather fast. There are three different coronavirus induced diseases of swine, namely Porcine Epidemic Diarrhea (PED), Porcine Hemagglutinating Encephalomyelitis (PHE) and TGE, with three different coronavirus species as the causative agents. PED was initially reported from Belgium and the United Kingdom in 1978 (Pensaert and de Bouck, 1978) and later from more countries in Europe and Korea (Debouck et al., 1982; Chae et al., 2000), and is very similar to the clinical picture observed in case of a TGEV infection, it has sometimes been referred to as a TGE-like disease. There is however a difference in the severity of the disease in the affected pigs, while the mortality in piglets bellow 2 weeks of age may rise to 100% in TGE cases, the losses induced by PEDV usually remain below 50% in the same age group. Although older pigs may also develop serious clinical signs. PHE was first observed causing disease in Ontario, Canada in 1957 (Alexander et al., 1959) and the virus was later isolated and classified by Greig et al. (1962). During the 1957 outbreak the disease mainly affected piglets younger than two weeks. Clinical signs are characterized by inappetance, shivering, loss of condition and vomiting. Apart from these clinical signs those of a severe encephalomyelitis can be observed. In these typically younger piglets the mortality may sometime reach 100%. Older piglets will most of the time only 4 show a slight posterior paralysis (Alexander et al., 1959). The disease seems to be limited to a single farrowing group, disappears by itself and does not recur (Werdin et al., 1976) as maternal immunity develops. Transmissible gastroenteritis was first identified in 1946 (Doyle and Hatchings, 1946) and caused problems in pig herds until the late 1980s when deletion mutants of the virus, the Porcine Respiratory Coronaviruses (PRCoV) emerged. TGE is characterized by vomiting, diarrhoea and dehydration (Hooper and Haelterman, 1969). Clinical signs can be experienced at 18 to 48 hours post exposure. Four days after infection neutralizing antibodies can be found in the circulation (Norman et al., 1973). During autopsy the stomach content is described as a solid caseous curd, which can be bile-stained and becomes more solid as the dehydration progresses. The most prominent lesions are detected in the intestines where a striking feature is the marked villous atrophy. The loss of villi is more or less uniform along the length of the small intestine, however there are some exceptions. The initial part of the duodenum seems to be unaffected, but villous atrophy can be complete in both the jejunum and the ileum These changes can be observed as early as 24 hours post infection (Hooper and Haelterman, 1969). Accompanying the villous atrophy is a failure of cells recruited from the crypt epithelium to differentiate to the normal columnar epithelium. The cells mainly affected by the TGE virus are those covering the villi and not the ones found in the crypts. At the microscopic level signs of recovery can be observed from day four but not until the seventh day post infection is there a complete regeneration of the villous epithelium. Virus in high titre can be found in cells of the intestinal mucosa. However, investigating other tissues, the virus can also be isolated from lung, liver and pancreas tissues. Pigs exposed to virus at 21 days or later do not have virus in the above mentioned organs (Norman et al., 1973; Underdahl, 1974). It has also been proven that the virus can replicate in alveolar macrophages in vitro (Laude et al, 1984). An important factor in predicting the severity of the infection is what age the pig is when it is exposed to the pathogen. The diarrhoea, vomiting and consequent electrolyte loss is more pronounced and has a longer duration in younger pigs. Pigs infected later in life may not even show clinical signs of an infection. As discussed earlier the hallmark of a TGEV infection on a microscopical level is villous atrophy and crypt cell hyperplasia. These changes are correlated with the severity of the clinical signs. Alas in younger pigs the histopathological alterations are more expressed (Moon et al., 1973). A remarkable thing happened in Belgium in 1984, there was a dramatic increase in animals being seropositive for TGEV. However, none of the animals displayed any of the 5 clinical signs associated with TGEV infection (Pensaert et al., 1986). On top of this TGEV could not be isolated from the seropositive animals, instead a new virus was found, a close relative to the virus causing TGE in pigs. Later it was found out that it most probably was a deletion mutant of TGEV, with deletions in the spike protein and a non-coding region (Rasschaert et al., 1990, Britton et al., 1991). PRCoV is pneumotropic, replicating mainly in alveolar cells, but also in epithelial cells of nasal mucosa, trachea, bronchi, bronchioli, in alveolar macrophages and in tonsils. It can also replicate in the gastrointestinal tract in cells located underneath the villi. To reach the gut the virus can either be swallowed or after primary replication in the respiratory tract there might be a viraemia, leading the virus to disseminate to the gastrointestinal tract. But virus is isolated at a much lower titre in the gastrointestinal tract compared to the respiratory tract (Cox et al., 1990). PRCoV infections are usually subclinical. What struck observers after the emergence of PRCoV was the relative absence of TGE outbreaks. The spreading of PRCoV in pig herds seemed to correlate with a decline in importance of TGE. The explanation for this was that the new virus was able to produce neutralizing antibodies against TGEV. Protective immunity on the antibody level against TGE is directed to the spike (S) structural protein of the virion. The spike is responsible for the attachment of the virus to cellular receptors. TGEV-S (Figure 2) uses two distinct receptor binding sites (RBS) and through them two different receptors. S protein trimer RBS-1 Anchor region RBS-2 Figure 2. Schematic representation of the Spike of TGEV with the approximate location of the receptor binding sites. (RBS-1: between residues 92 and 250, RBS-2: from amino acid 405 to 465). 6 RBS-2 is a motif, binding to the ubiquitous aminopeptidase-N molecules (Delmas et al., 1992) of cell membranes, whereas RBS-1 is the binding site of an approximately 200 KDa receptor limited to the small intestinal cells of newborn animals (Weingartl et al., 1994). One of the differences between TGEV and PRCoV is that TGEV carries both RBSs whereas PRCoV, the deletion mutant, lacks RBS-1, rendering it incapable to infect intestinal cells in the way TGEV does. The same deletion is suitable for the differentiation of TGEV and PRCoV by molecular methods targeting the S gene or by monoclonal antibody based serological methods both for antigen and antibody detection. There is a strong antigenic connection between TGEV and PRCoV. In vitro, the antibodies directed towards the structural proteins cross react. Sows infected with PRCoV secrete antibodies with the milk that are capable of decreasing the infection rate in the newborn gut with a dramatic reduction of the impact of TGEV on a litter, reducing clinical manifestation and consequently mortality (De Diego, 1994). Multiple exposures to PRCoV (Sestak et al., 1996) increase both the IgG and IgA titre in the milk. With the appearance and spread of PRCoV the incidence of TGE gradually decreased and from an OIE A list disease it became an almost forgotten disease throughout the world. Occasional reports (Elvander et al., 2000, Brendtsson et al., 2006) of TGEV-specific seropositivity (“singleton reactors”) indicated that the virus is still present in pig herds but without clinical manifestation. Recently, outbreaks of vomiting and diarrhea of 5-7 days old suckling piglets were observed on a large sow farm in Hungary. This PRRS-negative herd was newly established, so at first all sows were primiparous. Farrowing started in late August, and the problem did not reach significant magnitude until January the following year. Affected litters were mainly those of primiparous gilts, but litters of some multiparous sows showed similar clinical signs. These usually started with vomiting, then liquid, yellowish diarrhea was seen, and emaciation, dehydration of the piglets developed rapidly. In a given farrowing room signs usually started in 5-7 days old litters, but the disease spread rapidly to younger piglets, born later in the room. Morbidity in some groups was estimated at about 50%, mortality stayed low, at about 4-6%. Piglets treated with commercial electrolyte supplementation and antibiotic injections (Gentamox, Ceva) were likely to recover. In some instances, only electrolyte supplementation was used with the same result. Piglets routinely received preventive oral treatment against coccidiosis at four days of age (Baycox, Bayer), gilts were vaccinated twice during pregnancy with a commercial E. coli vaccine (Porcilis Coli, Schering-Plough, Intervet), and against PCV-2 infection (Circovac, Merial) when vaccines became available. Hypogalactia was 7 usually minimal or absent at the beginning of these outbreaks, sows were in good general condition, liveborn litter sizes and piglet birth weights were within acceptable limits. The farm was run according to strict hygienic standards and all-in/all-out was practiced in all production phases. Outbreaks of vomiting and diarrhea were not possible to relate to sow feed composition or to particular farrowing rooms. The problem was more frequently observed from late autumn to early spring. Other age groups on the farm did not experience a similar condition. Diagnostic investigations were initiated early in the course of this disease in several institutions. Initially no pathogen was detected that could be connected to the clinical signs in all the examined cases. Beta-haemolytic E. coli strains (not typed) were detected in a few instances, Clostridium perfringens A toxins (cpa, cpß2) were occasionally detected by multiplex polymerase chain reaction (PCR), but in the majority of the submitted cases no pathogens could be isolated. Fecal samples were consequently negative for coccidia (standard flotation), rotavirus (PCR), Clostridium difficile (commercial A/B toxin test) and PRRSV (PCR). Gross pathological lesions of piglets succumbed to the disease or euthanized due to terminal illness were restricted to signs of weight loss and dehydration, yellowish fluid filled, distended small intestinal loops and large intestines and to some mesocolonic edema. Histopathology of the small intestinal tract did not reveal major changes apart from mild shortening of the villi. Crypt hyperplasia was not evident in the examined cases and the colonic mucosa appeared normal. Parenchymal organs did not show pathognomonic alterations. Since the search for common causative agents was unsuccessful, more uncommon pathogens were also considered and investigations for their presence commenced. Although clinical signs and presentation of the condition would fit the description of enzootic TGE, detection of this agent was not attempted initially. This was because the widespread infection of Porcine Respiratory Coronavirus (PRCoV) thought to provide protective immunity to piglets, and therefore clinical TGE has not been detected in Hungary for the last 15 years. The purpose of the present study was to identify PRCoV and TGEV coronaviruses in affected animals, and to characterize the potential differences in the spike gene sequences of their respective genomes. 8 MATERIALS AND METHODS Samples In March, 2009, with the aim to detect if TGEV was present in piglets suffering clinical signs consistent with an enteral disease at the previously mentioned farm, four untreated piglets aged 4-6 days were sacrificed and dissected on the farm. Portions of their small and large intestines were placed into sterile plastic containers and transported on ice to the laboratory at the Department of Microbiology and Infectious Diseases within four hours of collection. Samples were also collected from the gastrointestinal tract and parenchymal organs for histopathology and group 1 coronavirus immunohistochemistry (IHC). Samples for aerobic bacteriological culture and detection of clostridial toxins were also collected from the small and large intestine. After the detection of the first TGEV positive cases a wider survey was initiated, where samples of small intestines, faeces and lung tissues were collected from approximately 150 animals of the most susceptible age group These samples were gathered at 14 different farms including the original farm, throughout the country. At the farm where the first TGEV positive piglets were found blood samples intended for serological examination were drawn from 15 primiparous and 15 multiparous sows nursing affected litters. These samples were tested for the presence of anti-TGEV antibodies with a TGEV/PRCoV differentiating commercial ELISA (Svanova) at the Large Animal Clinic of the Faculty of Veterinary Medicine located in Üllő. The technical details of these tests are not mentioned here, as they were performed by scientists other than the author of this thesis. The results however will be referred to, with permission of them, when needed RNA extraction About 0.1 g of tissue or faecal samples was used for extraction with the Viral Genespin™ Viral DNA/RNA Extraction Kit (iNtRON Biotechnology). Sterile distilled water up to 150 µl was added to the samples in a 1.5 ml microcentrifuge tube and homogenized with a mortar (Sigma-Aldrich) to break down the cell structures and with this release the nucleic acids, including the viral RNA, from the cells. 250 µl Lysis Buffer from the kit, heated to 80oC was added to the homogenate and mixed. Subsequently 5 µl 20 mg/ml Proteinase K (Fermentase) was added to digest proteins of the lysed cells and incubated at 55 oC for 10 9 min. To facilitate the binding of the nucleic acids to the matrix, 350 µl Binding buffer was added and the mixture loaded into the Spin Columns. The liquid phase was released by spinning through the cartridges at 12000 g for 1 min. The filter bound DNA/RNA mixture was washed with the appropriate Wash buffer from the kit twice, by letting 500 µl through at 12000 g, for 1 min each. The filter tubes were dried by centrifuging for an additional 2 min with the same force. The nucleic acids were then eluted into 30 µl of RNase free sterile distilled water preheated to 45 oC. The samples were stored at -80 oC until use. Reverese transcriptase polymerase chain reaction The following procedure was used for the reverse transcription of the RNA templates (using reagents from Fermentas, Lithuania). To 5 µl of the purified nucleic acids 1 µl random hexamer primer (100 µM), 10 U RNase Inhibitor and 6 µl sterile double distilled water were added, incubated at 65oC for 5 minutes. Following the incubation step the mixture was supplemented with 4 µl RevertAid™ Reverse Transcriptase buffer, 10 U RNase Inhibitor, 50 U M-MuLV RevertAid™ Reverse Transcriptase, together with 1 µl dNTP (10 mM) and incubated at 25 oC for 10 min and at 42 oC for 60 min. The PCR primers used for the specific amplification and differentiation of the TGEV/PRCoV S genes were designed by the Primer 3 program (Rozen and Skaletsky, 2000) based on the TGEV Purdue 115 full genome sequence (GenBank Accession Number: DQ811788), where the S gene is located at 20354-24697 nucleotides. The primers were TGE2: 5’-AAGGAAGGGTAAGTTGCTCA-3’ (binding at 20282-20301 nt) and TGE3: 5’GGTCCATCAGTTACGCCGAA-3’ (21538-21518 nt) flanking 1258 bases of the S gene, including the RBS-1 (aa: 92-219) and RBS-2 (aa: 405-465) coding sequences. The reactions were carried out with the TGradient Thermocycler (Biometra) with mixtures of a volume of 50 µl in microcentrifuge tubes. The components of the PCR mixture are listed in Table 1. As a negative control, 1 µl of water was used instead of cDNA. As a positive control a cloned TGEV-S gene (Tuboly et al., 1994) was used and it was mixed the same way as the samples. The Dream Taq Buffer contained KCl diluted in a PCR mix to provide optimal conditions for the polymerase, MgCl2 was used to increase hybridization strength during primer attachment. The steps of the PCR were as follows: 5 min at 95 oC, 40 cycles of denaturation at 95 oC, annealing at 55 oC (both for 30 sec) and elongation at 72 oC 1 min, and a final step at 72 oC for 7 min. 10 Table 1. Composition of the PCR reaction mixtures. Sample (cDNA) 10X DreamTaq Buffer (Fermentas) MgCl2 (25 mM) dNTP Mix (10 mM diluted 10 times) Primers (25 µM) Dream Taq DNA Polymerase (5U/µl) Double distilled water End volume 1 µl 5 µl 3 µl 1 µl 1 µl 0.2 µl 38.8 µl 50 µl Gel electrophoresis 2% agarose gels were prepared by mixing agarose (Q-Biogene) powder and TAE buffer (40 mM Tris acetate, 1 mM EDTA, containing 0.4 µg/ml ethidium bromide, later replaced by GR Safe DNA Stain I, Life Science Technologies). The mixture was boiled in a microwave until the agarose was completely dissolved and then poured into a casting box (closed with casting tape or with stockers) of appropriate size and sample combs according to how many samples were analyzed at the time. After the gel had solidified, the comb(s) was removed, the gel inserted into the electrophoresis tank and TAE was added as running buffer. 10 µl of each of the PCR products were thoroughly mixed with 2µl loading buffer and added to the wells in the agarose gel. For molecular mass standard 2µl of DNA ladder (Fermentas 1 kb and/or 50 bp Ladder) was added to flanking wells. The electrophoresis was done under constant voltage of 110 V for 20-60 min depending on gel size. The gel was visualized by ultraviolet light and photographed using the Kodak Electrophoretic Documentation and Analysis (EDAS 290) System. For GR Safe staining the detection was done with the Dark ReaderTM system between 420-500 nm wavelengths. 11 Cloning PCR fragments of sizes characteristic for TGEV or PRCoV S genes were cloned for long term storage and sequencing. The PCR products were cut off the agarose gel and purified with the QIAEX Gel Extraction Kit (Qiagen) according to the manufacturer’s instructions. Which briefly were as follows, the gel slice was dissolved by adding 3 volumes of binding buffer to 1 volume of gel kept at 50°C for 10 minutes or until completely dissolved. The DNA was adsorbed to the silica gel solution of the kit, incubated for 5 min at room temperature, and pelleted by spinning at 12000 g for 2 min. The pellet was washed with 500 µl of the Wash solution, pelleted again and air dried. The DNA was eluted from the silica gel with 10 µl of DNAse free distilled water. Cloning was done using the TOPO® TA Cloning® Kit (Invitrogen) following the instructions of the manufacturer. Which briefly were as follows: 4 μl of the purified PCR product was mixed with 1 μl of Salt Solution and 1 μl of the topoisomerase carrying TOPO® vector, incubated at room temperature for 5 min. The ligated product (1 μl) was used for the transformation of electro competent Escherichia coli bacterial cells (Electroporator 2510, Eppendorf) in 2 mm cuvettes at 2.5 kilo Volt. Bacteria then were plated onto Luria Broth (LB) plates containing 50-100 μg/ml ampicillin and grown overnight at 37 oC. Colonies were picked the next day and grown in liquid LB medium supplemented with ampicillin. The plasmid DNA was extracted using the Eppendorf mini plasmid isolation kit, according to the manufacturer’s instructions. The DNA was checked by electrophoresis as described earlier, after digestion with BamHI restriction endonuclease (Fermentas) with cleavage sites flanking the insert on the cloning vector. Sequence analysis The purified DNA clones were sequenced at the Biomi Ltd. (Gödöllő, Hungary) by the Big Dye terminator cycle sequencing, using the universal M13 reverse and forward primers, with an ABI310 automated sequencer. The sequence editing, analysis and prediction of amino acid sequences were conducted using the EditSeq program of the Lasergene package (DNASTAR Inc., Madison, USA). Sequences were compared to the TGEV Purdue 115 S gene and other TGEV S gene sequences of the GenBank. Sequence alignments were carried out with the MegAlign program (Lasergene) by Clustal multiple alignment algorithm. 12 RESULTS Upon the initial sampling of the first affected farm, TGEV was detected as the only significant pathogen in two of the four piglets that were sacrificed for the purpose and no other pathogen was isolated or detected from the two other piglets. Group 1 coronavirus immunohistochemistry on small intestinal samples gave dubious results, in the two TGEVpositive cases, mild villous atrophy was seen without any other significant gross or microscopic alteration in these. As the work done by the author of this thesis was limited to the molecular biological investigations, and to the evaluation of the serological results, none of the other results will be presented in details here. They will only be mentioned in the discussion to underline conclusions. Reverse transcriptase polymerase chain reaction The results of the rt-PCR reactions indicated that out of the close to 150 samples originating from 14 different swine herds only four herds were TGEV positive. The farms, based on their TGEV/PRCoV profiles could be divided into three groups. In the first group pigs that suffered from clinical signs associated with enzootic TGE usually had either PRCoV- or TGEV-S gene sequences in their samples. These gene fragments were estimated by the electrophoresis to be 550bp and 1250 bp for PRCoV- and TGEV-S genes respectively. Mixed infections were rarely detected. The PRCoV-S gene fragments were uniform in size. The typical picture seen after evaluating the rt-PCR by electrophoresis can be seen in figure 3. Figure 3. Agarose gel electrophoresis of porcine coronavirus S gene amplicons, samples are from piglets from a herd where clinical signs of enzootic TGEV infection was observed. 1-7: PRCoV-S amplicons with an estimated size of 550bp, 8: TGEV-S amplicon with an estimated size of 1250bp, M: 1kb DNA marker (Fermentas) P: positive control, full size 1285bp TGEV-S gene, N: negative control, sterile water. 13 The second group also consists of animals that were showing clinical signs of enzootic TGEV infection. Although in this group S genes of a variety of sizes were detected (panel A of Figure 4). Besides the full size fragment of the original TGEV-S (1258 bp, confirmed by sequencing) a variety of smaller sized fragments were also detected, dominating among them one with an approximate size of 600 bp and another with an approximate size of 250 bp, as judged by the agarose gel electrophoresis. These three fragments were selected for cloning and sequencing. Besides these three S gene variants, others of intermediate sizes were also detected, but cloning of these minor fragments was unsuccessful, or when successfully cloned and sequenced, the sequence did not show any homology with coronavirus genomes (data not shown). The original full size fragment of the TGEV-S was found together with the smaller fragments in the same animal, which sets this group apart from the first one. This so because in the first group mixed infections were rare. The third and last group consists of animals that did not show clinical signs of enzootic TGEV infection. In these farms the TGEV-S genes were not detected (panel B of Figure 4). The S gene amplicons were of approximately 550-600 bp in length, consistent with that what would be expected in PRCoV positive animals. In this group smaller fragments were also present. Figure 4. Electrophoresis of PCR amplicons of samples collected at a TGEV positive (panel A) and a TGEV negative but PRCoV positive (panel B) pig farm. Where fragment size has been indicated it is to be regarded as an estimate and refers to the strongest band(s). 1A: PRCoV-S amplicon 250bp, 2A: PRCoV-S amplicon 550-600bp 3A, 6A: TGEV-S amplicon 1250bp, PRCoV-S amplicon 600-550bp and PRCoV-S amplicon 250bp, 4A: PRCoV-S amplicons, 5A: negative sample, 1B-8B: PRCoV-S amplicons, 550-600bp, M: 1kb DNA marker (Fermentas) P: positive control, full size 1285bp TGEV-S gene, N: negative control, sterile water. When comparing the presence of TGEV and PRCoV in different organs, like the small intestine, lungs or lymph nodes, no difference could be observed. Usually lung and gut tissues or intestinal contents carried the virus. Hence organ preference or tropism could not be observed. 14 Cloning Cloning of the PCR fragments into the TOPO® TA Cloning vector was successful for the 1258 bp and the estimated 550-600 bp and 250 bp fragments of the TGEV/PRCoV cases. Figure 5 shows as an example the results of BamHI digested clones of the 550-600 bp inserts, together with an intact 1258 bp TGEV-S amplicon. Figure 5. BamHI digested cloned PCR fragments separated by agarose gel electrophoresis. M: 50 bp molecular mass ladder (Fermentas). The band indicated with a white circle is an intact 1285 bp long S gene amplicon. The bands indicated with black circles are successfully cloned 550-600 bp long fragments. The cloning of some other fragments was unsuccessful, probably due to the low amount of DNA amplicons produced by PCR. Where the intermediate sizes were successfully cloned the sequencing revealed that they were not of coronavirus origin but some unrelated, mostly bacterial nucleotides. Cloning of individual fragments of the mixed sized amplicons was necessary, as direct sequencing of the amplified products is not possible in such cases. Sequence analysis Sequence analysis determined the exact size of the three amplicon types that were successfully cloned. The largest fragment was 1258 bp as expected, the fragments originally estimated to be between 550 and 600 bp had a size of 586 bp. The shortest S gene amplicon with an estimated size of 250 bp was somewhat larger than expected, reaching 283 bp in length. The Genbank search of these nucleotide sequences showed that they were almost identical with the TGEV Miller M6 strain type viruses (Genbank accession Number: DQ811785) when considering the appropriate gene portion, with only a few nucleotide differences. When comparing the fragments to each other it was obvious that the same 15 coronavirus existed in at least three different forms if looking only at the S gene. Namely the full length TGEV-S type, a shorter one with a 672 nt deletion at the amino terminal half coding region of the S gene (characteristic to the European type of PRCoV sequences, where RBS-1 is completely removed), and a further deletion mutant where the deletion was extended with an additional 303 nt towards the 3’ end of the gene. Still all of these variants carried the sequence characteristics of the TGEV identified during this study, when only looking at the non-deleted parts of the gene. All of these deletions retained the functionality of the remaining portion of the S protein, as the number of nucleotides missing did not alter the reading frame of the gene. Serology Serological tests were done by the scientists at the Large Animal Clinic of the Faculty of Veterinary Medicine located in Üllő. The results are summarized in Table 2, where the ELISA positivity of pigs from the original TGEV cases is shown both for TGEV and for PRCoV. Table 2. The ELISA results from blood samples collected from 15 primiparous and the 15 multiparous sows in a TGEV-PRCoV differentiating test. TGEV ELISA Primiparous No. % Older sows No. % Positive 0 0 Positive 5 33,33 Suspect 6 40 Suspect 8 53,33 Negative 9 60 Negative 2 13,33 PRCoV ELISA Positive 11 73,33 Positive 15 100 Suspect 0 0 Suspect 0 0 Negative 4 26,67 Negative 0 0 16 DISCUSSION The first appearance of transmissible gastroenteritis (Doyle and Hatchings, 1946) when pig herds without any immunity encountered the virus was devastating. This epizootic form of the virus was typical in newly affected herds, with 100% mortality rate of newborn animals. In older animals the clinical signs were restricted to a mild diarrhea and decreased production. As the epizootic proceeded worldwide and more and more herds seroconverted, the mortality rate dropped to 10-50% in newborns, depending on maternal antibody levels. Still, even with its sporadic epizootic and characteristic enzootic form TGE was one of the major viral diseases of the swine industry until the appearance and gradual spread of the mutant PRCoV strains. The origin of PRCoV is not known, it was suspected that some attenuated vaccine started an individual spread worldwide. But the sequence differences and differences in the site and size of deletions among the identified strains indicated otherwise (Figure 6). It seems more likely that changes in the environment, mostly the pig itself, led to the simultaneous appearance of the mutated viruses (Tuboly, 1995). Although the protection of the PRCoV induced antibodies is only limited against TGEV, as an essential receptor binding site of the PRCoV-S protein is usually missing from these viruses, the constant presence of the new virus and the PRCoV antibodies resulted in a decrease of the incidence of clinical TGEV in the PRCoV infected herds. By the mid 1990s only sporadic cases of TGE were reported. Today TGEV is usually considered as a disease of the past, something that nature itself got rid of. However, there has been occasional reports of TGEV seropositivity (Elvander et al., 2000, Brendtsson et al., 2006), indicating that the virus was still present, but at levels below the threshold of clinical manifestation. 17 1 100 200 300 374 1 aa 400 1447 aa TGEV-S 219 1 aa 92 and 94 1-1 aa 21 224 aa 15 207 aa 16 226 aa 62 227 aa 289 242 222 218 1 aa 245 374 2 aa TGEV with lower pathogenicity PRCV in Europe PRCV in North America Figure 6. Summary of the mutated TGEV/PRCoV variants detected worldwide, based on GenBank data (Tuboly, unpublished). The upper line indicates the globular amino terminal half of the TGEV-S protein in a linearized form. The boxed areas show deletion mutants of TGEV. Numbers above the lines show the position in the number of amino acids (aa) from the initial part of the gene, those bellow the lines indicate the size of the deletion also expressed as number of aa. Based on the results of this study, the herd where clinical signs and histopathology raised the suspicion of a TGEV infection, was indeed proven to be TGEV positive. This is based on both serological and polymerase chain reaction tests. When the survey was extended to other herds, the presence of TGEV could be confirmed with PCR in four pig farms. From the re-emergence one would expect that a new, perhaps more virulent TGEV strain is emerging, one that is capable of breaking through the immunity induced by PRCoV. However, the sequencing results indicated that the viruses were very similar to already known TGE viruses, namely to those of the Miller M6 strain (Zhang et al., 2007). In order to decide if this truly is a new genetic variant of the virus with higher virulence, other regions of the genome must be amplified and sequenced. 18 The PRCoV sequences detected in this study can be separated into two different groups one with a 672 nt deletion and in the other group the deletion was altogether 975 nt long. The 672 nt deletion (compared to the TGEV-S gene) was identical to what was observed for the PRCoV strains widespread in Europe, where a 224 aa coding region was deleted starting at the position of amino acid number 21 of the S gene (Figure 6). It is however peculiar that after several years of establishing the genetic characteristics of the PRCoV strains in Europe the same deletion mutant is still present in pig herds, considering the genetic instability of coronaviruses discussed in the introduction. The presence of the PRCoV genomes with an even longer deletion within the S gene was surprising as such large deletions starting at the same site as the previous one and extending 303 nt further into the 3’ direction has not been reported previously. It raises the question if such deletion mutants can form infectious particles, or if they can only survive when packaged into virions produced by the co-existing longer genomes. The possibility that this deletion mutant remains infective is likely as the altogether 975 nt deletion by itself should not thwart virus infection. This is so because the RBS for aminopeptidase-N is encoded from nucleotide 1215 of the gene, which is further downstream on the sequence as regards to the end of the deletion. Without structural studies of such an S protein variant it is of course difficult to tell how the lack of such a long protein stretch may change the conformation of the second receptor binding site. Our results cannot explain the re-emergence of the virus in clinical conditions, but from this limited survey it seems that primiparous sows did not completely seroconvert to TGEV, therefore their piglets probably were not fully protected. PRCoV seronegativity was detected in a limited number of primiparous sows, indicating that cross-protection might also be suboptimal. These results indicate that the answer to why TGE is re-emerging lies not within the genetics of the virus but most likely in the immune response of the pigs. It is known that porcine circoviruses (PCV) are present worldwide and they are strongly immunosuppressive (Ramamoorthy and Meng, 2009). Vaccine or infection induced immunity is generally limited in heavily infected pig herds, even if clinical signs of the circovirus infection are not apparent. Although we have no direct proof of this assumption, namely that the presence of PCV is responsible for the TGEV problem, serological results show that primiparous pigs may remain seronegative both for TGEV or PRCoV. Similar results had been reported from China (Chen, 2010, personal communication) the world's largest pork producer, where TGEV again is becoming an important economic threat. 19 Specific prevention of the disease is not possible as a TGEV vaccine is currently unavailable in Hungary. We have no data on the safety and efficacy of coronavirus vaccines developed for other species (e.g. cats) used in sows. Back-feeding of intestinal contents of succumbed or euthanized piglets to pregnant gilts could be a way of exposing gilts to the virus and boosting maternal antibody levels, however, this technique cannot be recommended as it may be the source of further infections. As a final remark it should be noted that no disease can be fully forgotten and considered a disease of the past. The environment and its inhabitants in which we keep pigs is subject to constant change. These changes are as unpredictable as the lottery and might within a short period of time provide an entirely new playing field, where diseases considered something of the past might re-emerge and again cause problems. 20 ACKNOWLEDGEMENTS The study was financed in part by the EU SSA-NMSACC-PCVD 518432 grant. The author is in great debt to his supervisors providing unequaled assistance and guidance in his work to prepare this thesis. Without their support it would not have been possible to either perform or finish the work needed. The author thanks Dr. Attila Cságola for her help with RNA extraction and PCR. The skilled technical help from Irénke Herbák, laboratory technician, is appreciated. The colleagues at the Large Animal Clinic were very helpful in providing data about the clinical, pathological and serological results. It must be stated that it has been an altogether pleasant experience working together with this fantastic group of people. Finally I am as always, greatly appreciative of my partner Mia Karlsson for providing full support when ever needed, making sure that I always land on my feet. 21 SUMMARY Transmissible gastroenteritis (TGE) of pigs is an enteric disease caused by a porcine coronavirus. It can affect animals of any age but is most commonly affecting younger animals. TGEV belongs to the Coronaviridae family, which constitutes enveloped viruses with an unusually large single stranded RNA genome (up to 28-30 kilobases) of positive orientation. Although the virus can infect pigs at any age, the clinical signs are less pronounced in adults. Piglets however, especially during the first week of life develop devastating intestinal infections. Hallmark clinical signs are mainly vomiting and diarrhea. Mortality rate in piglets without maternal immunity can reach 100%. TGE was among one of the most important swine diseases until the mid 1980’s. Then a deletion mutant, namely the porcine respiratory coronavirus (PRCoV) with no or very limited pathogenicity emerged and spread around the world. The widespread PRCoV infection led to the gradual disappearance of TGE, due to the cross-protective immunity induced by the new virus. Recently more and more cases of piglet diarrhea with an unclear aetiology have been reported. This study is a summary of such a case, where TGEV genome was detected by polymerase chain reaction (PCR) in a swine herd. In this case it was pigs at the age of weaning that were showing signs of diarrhea. Other viral, bacterial or parasitic infections were excluded by appropriate laboratory investigations, but a parallel presence of PRCoV in the affected animals was detected. The re-emergence of TGEV was confirmed by sequencing the PCR generated amplicons (targeting the spike, S gene). Apart from the deletion at the site of the S gene the genome sequences were identical in PRCoV and TGEV cases. The reason for the reemergence of the pathogenic coronavirus is not fully understood. But based on the widespread presence of porcine circoviruses, which are well known for their immunosuppressive nature, it was speculated and later confirmed by serological tests that there is a decreased cross protection between PRCoV and TGEV. This decrease in cross protection could be in the background of the TGEV induced clinical problems. 22 ÖSSZEFOGLALÁS A transmissible gastroenteritis (TGE) egy sertés coronavírus által okozott emésztőszervi megbetegedés, amely valamennyi korcsoportot érintheti, leggyakrabban azonban a fiatal állatokban okoz jelentős klinikai tüneteket. A kórokozó a Coronaviridae családba tartozik, egy szokatlanul nagy, egyszálú, pozitív irányultságú RNS genomot (28-30 kilobázis) tartalmazó burkos vírus. Bár a vírus minden korcsoportot megbetegíthet, felnőtt állatokban a klinikai tünetek kevéssé kifejezettek, elsősorban egy hetes kor alatti malacokban fejlődik ki a súlyos emésztőszervi fertőzés. A tünetek döntően hányás és hasmenés, maternális immunitás nélkül a mortalitás elérheti a 100%-ot is. A TGE az 1980-as évek közepéig az egyik legfontosabb vírusos eredetű sertésbetegség volt világszerte, amíg egy nem, vagy kevéssé patogén deléciós mutáns, név szerint a sertések légzőszervi coronavírusa (porcine respiratory coronavirus, PRCoV) tűnt fel és terjedt el a korábban TGE-vel fertőzött állományokban. A PRCoV elterjedése a TGE fokozatos eltűnéséhez vezetett, mivel az új vírus keresztvédettséget képes indukálni. Újabban mind gyakrabban jelentkeznek tisztázatlan etiológiájú hasmenéses megbetegedések sertésekben. Ez a tanulmány egy olyan eset összefoglalása, amelyben TGE vírus genomot mutattunk ki polimeráz láncreakció (polymerase chain reaction, PCR) segítségével egy sertéstelepen, választás körüli malacok hasmenéses eseteiből. Más vírusos, baktériumos és parazitás fertőzést nem sikerült laboratóriumi módszerekkel detektálni, de párhuzamosan a PRCoV jelenléte kimutatható volt az érintett állatokban. A TGE vírus megjelenését igazolta a PCR termék (a spike, S gén) szekvencia elemzése. Az S génen lévő deléciós helyek alapján a PRCoV és a TGE vírus megkülönböztethető. A patogén coronavírus ismételt felbukkanásának oka még nem teljesen tisztázott, de a sertés circovírus elterjedésének és immunszuppresszív természetének köszönhetően felmerült, majd később szerológiai teszttel igazolódott, hogy a TGE és PRCoV közötti keresztvédettség csökkent. 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