L Grau-Roma

P M H Heegaard

C K Hjulsager

M Sibila

C S Kristensen

A Allepuz

M Piñeiro

L E Larsen

J Segalés

L Fraile

Pig major acute-phase protein and haptoglobin serum concentrations correlate with PCV2 viremia and the clinical course of postweaning multisystemic wasting

Keywords: Haptoglobin, pig-major acute phase protein (pig-MAP), acute phase proteins (APPs), acute phase reaction (APR), porcine circovirus type 2 (PCV2), postweaning multisystemic

Pig major acute-phase protein and haptoglobin serum concentrations correlate with PCV2 viremia and the clinical course of postweaning multisystemic wasting syndrome

A c c e p t e d M a n u s c r i p t 2

Abstract

The aim of the present longitudinal study was to assess the evolution of two acute phase proteins (APPs), pig-major acute phase protein (pig-MAP) and haptoglobin (HPT), in serum from pigs that developed postweaning multisystemic wasting syndrome (PMWS) in comparison to healthy and wasted non-PMWS affected pigs. In addition, evidence of infection with other pathogens and its relation with variations in APPs concentrations was also assessed. Fourteen independent batches of 100 to 154 pigs were monitored from birth to PMWS outbreak occurrence in 11 PMWS affected farms. Pigs displaying PMWS-like signs and age-matched healthy controls were euthanized during the clinical outbreak. PMWS was diagnosed according to internationally accepted criteria and pigs were classified as: i) PMWS cases, ii) wasted non-PMWS cases and iii) healthy pigs. At the moment of PMWS occurrence, pig-MAP and HPT concentration in PMWS affected pigs were higher than in healthy ones (p<0.0001). No differences in APPs serum concentrations between subclinically PCV2 infected pigs and healthy non-PCV2 infected pigs (based on quantitative PCR on serum results) were detected. Results showed a significant correlation between PCV2 loads and both pig-MAP (R=0.487 to 0.602, p<0.0001) and HPT (R=0.326 to 0.550, p<0.05 to 0.0001) concentrations in serum of PMWS affected pigs, indicating that the acute phase response in PMWS affected pigs occurred concomitantly to PCV2 viremia. No other pathogen, apart from PCV2, was consistently related with variations in APPs concentrations. A ROC analysis, made to determine the capacity of discrimination of both APPs between PMWS affected and non-affected pigs, showed higher sensitivity and specificity values using pig-MAP compared to HPT. These results suggest that pig-MAP might be a better indicator of PMWS status than HPT. Moreover, the fact that APR occurred some days A c c e p t e d M a n u s c r i p t

Introduction

The acute phase response (APR) is an innate, non-specific immune response which occurs after many different stimuli such as infections, tissue damage, neoplastic growth or immunological disorders [START_REF] Baumann | The acute phase response[END_REF]Gruys et al., 2005a). This complex reaction is mediated by pro-inflammatory cytokines and involves both local and systemic reactions, including fever, increase in muscle protein catabolism, alterations in sleep and appetite patterns, and changes in the concentration of a group of plasma proteins which are called acute phase proteins (APPs) (Gruys et al., 2005a).

These proteins are mainly synthesised in the liver and are classified according to the direction of change (positive APPs if their concentration increases, negative if it decreases), and according to the extent to which their concentrations change (minor, intermediate or major) during the APR [START_REF] Petersen | Application of acute phase protein measurements in veterinary clinical chemistry[END_REF]Gruys et al., 2005a).

APPs have been described as useful for assessing health in humans and animals [START_REF] Petersen | Application of acute phase protein measurements in veterinary clinical chemistry[END_REF]Gruys et al., 2005b). In pigs, APPs concentrations have been demonstrated to be increased after experimental infections with several bacteria, viruses or parasites, as for instance Streptococcus suis (Sorensen et al., 2006), porcine reproductive and respiratory syndrome virus (PRRSV) [START_REF] Asai | Elevated serum haptoglobin in pigs infected with porcine reproductive and respiratory syndrome virus[END_REF] and A c c e p t e d M a n u s c r i p t 4 Toxoplasma gondii [START_REF] Jungersen | Pathogenicity of selected Toxoplasma gondii isolates in young pigs[END_REF]. Moreover, increases in APPs concentrations have also been detected in pigs affected by tail-and ear-biting or after stress induced by transport conditions or changes in the pattern of food administration [START_REF] Parra | Porcine acute phase protein concentrations in different diseases in field conditions[END_REF][START_REF] Piñeiro | Characterisation of the pig acute phase protein response to road transport[END_REF][START_REF] Salamano | Changes in haptoglobin, C-reactive protein and pig-MAP during a housing period following long distance transport in swine[END_REF]. Specifically, haptoglobin (HPT) and pig-major acute phase protein (pig-MAP) have been suggested as the most robust APPs as indicators of infection in pigs (Sorensen et al., 2006).

Porcine circovirus type 2 (PCV2) is considered the essential infectious agent for postweaning multisystemic wasting syndrome (PMWS) development, but it is also known that other triggering factors are necessary for the full expression of the clinical disease [START_REF] Harding | The clinical expression and emergence of porcine circovirus 2[END_REF][START_REF] Ghebremariam | Postweaning multisystemic wasting syndrome (PMWS) in pigs with particular emphasis on the causative agent, the mode of transmission, the diagnostic tools and the control measures. A review[END_REF][START_REF] Segalés | Porcine circovirus diseases[END_REF]. The internationally accepted criteria for diagnosis of PMWS include the presence of compatible clinical signs (mainly wasting and respiratory distress), moderate to severe lymphocyte depletion with granulomatous inflammation in lymphoid tissues, and detection of moderate to high amounts of PCV2 within these lesions [START_REF] Sorden | Update on porcine circovirus and postweaning multisystemic wasting syndrome[END_REF][START_REF] Segalés | Porcine circovirus diseases[END_REF]. Although it is known that APPs serum concentrations are increased in PMWS affected pigs [START_REF] Segalés | Haptoglobin and pigmajor acute protein are increased in pigs with postweaning multisystemic wasting syndrome (PMWS)[END_REF][START_REF] Parra | Porcine acute phase protein concentrations in different diseases in field conditions[END_REF], few field data are available in the literature. Moreover, the longitudinal evolution of APPs in animals that finally develop the disease has not been assessed.

The aim of this study was to assess the evolution of two APPs, pig-MAP and HPT, in serum throughout the productive life of pigs that developed PMWS in comparison to healthy pigs and pigs that developed wasting without fulfilling PMWS diagnosis.

Moreover, the infection dynamics of PCV2 and several other pathogens was monitored by quantitative PCR, nested PCR and/or serological techniques to determine potential

Materials and methods

Study design

Two longitudinal case-control studies in PMWS affected farms, one in Spain and one in Denmark, were performed as previously described [START_REF] Grau-Roma | Infection, excretion and seroconversion dynamics of porcine circovirus type 2 (PCV2) in pigs from postweaning multisystemic wasting syndrome (PMWS) affected farms in Spain and Denmark[END_REF]. Briefly, both studies were carried out using similar designs, and were performed on 13 independent farm batches (6 in Spain and 7 in Denmark) of 100 to 154 animals per batch, coming from 10 different farms (3 from Spain and 7 from Denmark). Studied piglets were ear-tagged at 1 week of age and monitored until the occurrence of the PMWS outbreak. Nasal and rectal swabs as well as blood samples were serially collected from those piglets at established weeks of age (1,3,7 and 11 in Spain,and 1,4,6 and 9 in Denmark) and at the time of PMWS outbreak occurrence (necropsy).

When the PMWS compatible clinical picture [START_REF] Segalés | Update on postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome diagnostics[END_REF] appeared at the studied farms, pigs displaying PMWS-like signs and healthy age-matched pigs were selected, euthanized and necropsied. At necropsy, sections of lymphoid tissues were collected and fixed by immersion in neutral-buffered 10% formalin to assess the pathological status of both clinically healthy and diseased animals.

All treatments, housing, husbandry and slaughtering conditions followed the European Union Guidelines and Good Clinical Practices.

Histopathology

A c c e p t e d M a n u s c r i p t 6 Formalin fixed tissues were dehydrated and embedded in paraffin blocks. Two consecutive 4 µm thick sections containing collected lymphoid tissues from each pig were cut from each block. One section was processed for haematoxylin and eosin stain, while the other was used for PCV2 nucleic acid detection by in situ hybridization (ISH) [START_REF] Rosell | Pathological, immunohistochemical, and in-situ hybridization studies of natural cases of postweaning multisystemic wasting syndrome (PMWS) in pigs[END_REF] for Spanish cases, and for immunohistochemistry (IHC) for PCV2 antigen detection [START_REF] Jensen | Distinction between porcine circovirus type 2 enteritis and porcine proliferative enteropathy caused by Lawsonia intracellularis[END_REF] for Danish cases. Pathological evaluation and PMWS diagnosis was carried out using a previously described scoring system evaluating the PCV2 amount and the intensity of lymphoid depletion and granulomatous infiltration present in lymphoid tissues [START_REF] Grau-Roma | Infection, excretion and seroconversion dynamics of porcine circovirus type 2 (PCV2) in pigs from postweaning multisystemic wasting syndrome (PMWS) affected farms in Spain and Denmark[END_REF]. Three different categories of pigs were established: i) PMWS cases: corresponding to pigs showing clinical wasting and with moderate to severe lymphoid lesions and moderate to high amount of PCV2 antigen/nucleic acid; ii) wasted non-PMWS cases: corresponding to pigs showing clinical wasting but without or with slight PMWS characteristic histopathological lesions and no or low amount of PCV2 antigen/nucleic acid within lymphoid tissues; iii) Healthy pigs: corresponding to pigs showing good clinical condition, without or with slight PMWS characteristic histopathological lesions and no or low amount of PCV2 antigen/nucleic acid within lymphoid tissues.

Acute phase proteins determination in serum

Two APPs, HPT and Pig-MAP, were measured in serum of studied pigs. Both APPs were determined in all longitudinally collected sera from Spanish necropsied pigs (107 pigs coming from 3 different farms). For the Danish pigs, serum samples collected at 1, 6 or 9 weeks of age and at necropsy from only those pigs with available serum at the moment of necropsy were analyzed.(53 pigs coming from 7 different batches). This kit was based on an ELISA previously developed and validated (Piñeiro et al., 2009b). HPT concentration was determined by a sandwich ELISA as previously described (Sorensen et al., 2006). Pig-MAP and HPT results were expressed as mg per millilitre of serum (mg/ml). PCV2 DNA was quantified using two previously described qPCR techniques on Spanish [START_REF] Olvera | Comparison of porcine circovirus type 2 load in serum quantified by a real time PCR in postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome naturally affected pigs[END_REF] and Danish [START_REF] Hjulsager | Inter-laboratory and inter-assay comparison on two real-time PCR techniques for quantification of PCV2 nucleic acid extracted from field samples[END_REF] samples, respectively. The performance of both qPCRs was compared in a previous study based on field samples [START_REF] Hjulsager | Inter-laboratory and inter-assay comparison on two real-time PCR techniques for quantification of PCV2 nucleic acid extracted from field samples[END_REF]. This latter work showed a highly significant linear association between results of the two qPCR assays. However, the method performed by the Danish laboratory had higher sensitivity and yielded systematically higher PCV2 load values than the one used in the Spanish laboratory. All PCV2 qPCR results were expressed as log 10 (number of PCV2 copies per ml of sera). to detect antibodies against European and American PRRSV strains (Sorensen et al., 1998), ELISAs to detect antibodies against PPV [START_REF] Madsen | Detection of antibodies against porcine parvovirus nonstructural protein NS1 may distinguish between vaccinated and infected pigs[END_REF] and Lawsonia intracellularis [START_REF] Boesen | Evaluation of a novel enzyme-linked immunosorbent assay for serological diagnosis of porcine proliferative enteropathy[END_REF], and haemagglutination inhibition (HI) test to detect antibodies against H1N1 and H3N2 subtypes of SIV, which was essentially carried out according to the OIE manual (OiE manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2008). A seroconversion against studied pathogens was considered to be present when an increase in the antibody titres in two consecutive samples or when a positive result preceded by a previous negative result was detected.

Quantitative PCV2 PCR

Mycoplasma hyopneumoniae nested PCR

It is known that the appearance of antibodies to M. hyopneumoniae can be delayed as much as 8 weeks after infection [START_REF] Sitjar | Relationships among seroconversion to Mycoplasma hyopneumoniae, lung lesions, and production parameters in pigs[END_REF]. Thus, in order to avoid underestimating its potential prevalence in studied animals, a nested PCR (nPCR) assay to detect M. hyopneumoniae was performed in the Spanish nasal swabs collected at the A c c e p t e d M a n u s c r i p t 9 moment of necropsy [START_REF] Calsamiglia | Application of a nested polymerase chain reaction assay to detect Mycoplasma hyopneumoniae from nasal swabs[END_REF]. For 1 out of 107 Spanish pigs the nasal swab sample was lacking.

Statistical analyses

For statistical evaluation, the SAS 9.1.3 software was used (SAS institute Inc., Cary, North Carolina, USA). A repeated measurement analysis of variance according to the general linear model procedure (PROC MIXED) and LSMEANS follow-up test was used to evaluate APPs mean concentrations differences between: i) PMWS cases, wasted non-PMWS and healthy pigs at each sampling time and within them at different samplings points; ii) healthy PCV2 subclinically infected pigs (PCV2 qPCR positive) and healthy non-infected pigs (PCV2 qPCR negative) at different sampling points; iii) seroconverted and non-seroconverted pigs against studied pathogens at each sampling; and iv) M. hyopneumoniae infected (nPCR positive) and non-infected (nPCR negative) at necropsy. Constant correlation was assumed between repeated measurements. Posthoc multiple comparisons were addressed using the Tukey test. PCV2 qPCR data were log10 transformed prior to graphic representation, and negative results were coded as 0.

A Pearson's chi-square test was used to compare the proportions of seroconverted animals to each studied pathogen at the moment of necropsy between the three categories of pigs studied; same comparison was also established using M. hyopneumoniae nPCR results. The level of significance for all analyses (α) was set to p<0.05.

To determine the capacity of discrimination of both Pig-MAP and HPT between PMWS affected and healthy pigs, a Receiver Operating Characteristic (ROC) analysis from samples collected at necropsy was carried out using Statsdirect (version 2.6.6, A c c e p t e d M a n u s c r i p t 10 CamCode, Ashwell, UK). Moreover, ROC analysis was also performed to determine the diagnostic performance of both APPs on clinically affected animals. For this later analysis, only wasted pigs were considered, considering PMWS as "diseased" and wasted non-PMWS as "non-diseased" animals. Taking into account that APPs concentration was determined by the same laboratorial tests in both studies, all samples collected at necropsy coming from Spain and Denmark were analysed together.

Results

Studied animals, histopathology and acute phase proteins

The number of pigs from which APPs concentrations were analysed and their pathological classification (PMWS, healthy and wasted non-PMWS) is detailed in Table 1. Mean±SD of weeks of age at necropsy for PMWS, healthy and wasted non-PMWS pigs were 14.0±2.1, 14.7±2.5, 15.2±2.7, respectively in Spain, and 11.2±1.5, 10.0±3.5, 10.9±1.9, respectively, in Denmark.

The longitudinal evolution of pig-MAP serum concentrations is displayed in figure 1.

Pig-MAP concentration in PMWS affected pigs was higher than in non-PMWS affected ones (healthy and wasted non-PMWS) either in Spain (p<0.0001) and Denmark (p<0.001) at the moment of necropsy. Although no statistically significant differences were found between healthy and wasted non-PMWS pigs, the mean pig-MAP serum concentration at the moment of necropsy in wasted non-PMWS was higher than in healthy ones (Spain: p=0.0715; Denmark: p=0.1472). Moreover, the Pig-MAP concentration in PMWS pigs at the sampling prior to necropsy in Spain was higher than its concentration in non-PMWS affected pigs (healthy and wasted non-PMWS) (p<0.01). Accordingly, in the Spanish pigs, the increase of pig-MAP concentration in A c c e p t e d M a n u s c r i p t 11 PMWS affected pigs was already significant at the sampling prior to necropsy, compared to basal pig-MAP concentrations detected at weeks 1 to 7. In Denmark, a significant increase in Pig-MAP concentration was only detected at necropsy in PMWS pigs. No statistical significant changes in pig-MAP concentration were found among all samplings in healthy and in wasted non-PMWS pigs from both countries.

The longitudinal evolution of HPT serum concentrations is displayed in figure 2. HPT concentration at the moment of necropsy was higher in PMWS than in healthy pigs either in Spain (p<0.0001) and Denmark (p<0.01). On the other hand, wasted non-PMWS could not be distinguished from the other two groups in Denmark, but had significantly lower HPT concentration than PMWS (p<0.001) in Spain. Regarding the longitudinal evolution of HPT concentration within each group, HPT increased progressively in both PMWS and wasted non-PMWS pigs in Spain from 11 weeks of age (p<0.01) to the necropsy moment (p<0.0001). In Denmark, only PMWS affected pigs gave significant increase in HPT concentrations with respect to the basal concentrations detected at the 1 st week of age (p<0.01). No statistically significant variation was found among the HPT concentrations of healthy pigs neither in Spain nor Denmark.

Pig-MAP concentrations obtained at the moment of necropsy were statistically correlated with the global average scoring (histopathology plus PCV2 detection in lymphoid tissues) in Spain (R=0.622; p<0.0001) and also in Denmark (R=0.524; p<0.0001). On the other hand, significant correlation between HPT concentration at the moment of necropsy and the global average scoring was detected in Spain (R=0.443; p<0.0001), but only a tendency was observed in Denmark (R=0.254; p=0.066).

PCV2 qPCR

PCV2 loads in sera are schematically displayed together with APPs results in figures 1 and 2. PCV2 loads increased progressively from the 1 st week of age until necropsy in Denmark and during the last two samplings in Spain. Maximum PCV2 loads were observed at the moment of disease outbreak (necropsy) in both countries. Significant correlations were observed between pig-MAP serum concentrations and PCV2 loads in serum (Spain: R=0.278, p<0.0001; Denmark: R=0.47, p<0.001).

Considering the 3 groups of pigs separately (figure 3), PMWS affected pigs corresponded to the group with higher correlation between PCV2 load and pig-MAP (Spain: R=0.487, p<0.0001; Denmark: R=0.602, p<0.0001), followed by the correlations observed in wasted non-PMWS pigs (Spain: R=0.218, p<0.01; Denmark: R=0.437, p<0.01). On the contrary, no significant correlation was found between pig-MAP concentrations and PCV2 loads when only healthy pigs were considered (Spain: R=-0.152, p=0.07; Denmark: R=0.2, p=0.194) (figure 3).

Significant correlations were also found between HPT serum concentrations and PCV2 loads in serum (Spain: R=0.443, p<0.0001; Denmark: R=0.326, p<0.001). Considering the 3 groups of pigs separately, the group of pigs with PMWS had again the highest correlation (Spain: R=0.550, p<0.0001; Denmark: R=0.326, p<0.05), followed by the correlations observed in wasted non-PMWS pigs (Spain: R=0.437, p<0.0001; Denmark: R=0.295, p=0.057). The lowest correlation between HPT concentration and PCV2 load Taking into account that more than half of healthy Spanish pigs gave negative results at all samplings by PCV2 qPCR, mean APPs concentrations in healthy PCV2 infected and non-infected pigs were also compared. No statistical differences were seen between both groups of pigs neither in pig-MAP nor HPT concentrations. Such comparison was not done in any other group of pigs due to the low number of negative PCV2 qPCR results.

Serology and PCR against different swine pathogens

The number of pigs that had seroconverted against studied pathogens and PCV2 qPCR results at the moment of necropsy in both countries, and M. hyopneumoniae nPCR in Spain, are displayed in Table 2.

No serological evidence of infection to ADV was detected. Evidence of M. hyopneumoniae, PPV or PRRSV infections in Spain and SIV infection in Denmark were found in less than 10% of analysed pigs. Prevalence of SIV and Salmonella spp. in Spain and PPV, PRRSVe, PRRSVu and L. intracellularis in Denmark was detected in 25 to 55% of the studied pigs.

No differences in the proportions of infected and/or seroconverted animals to studied pathogens between PMWS, wasted non-PMWS and healthy pigs were observed.

Moreover, no significant differences in APPs concentrations between seroconverted and non-seroconverted pigs to studied pathogens were detected. However, the 5 pigs that

ROC analysis

Optimal cut-off values for both studied APPs maximizing sensitivity (Sn) and specificity (Sp) to discriminate diseased and non-diseased pigs are displayed in Table 3.

Sp and Sn values were higher when comparing PMWS versus healthy pigs than comparing PMWS versus wasted non-PMWS animals. Moreover, Sn and Sp values obtained using pig-MAP concentrations were always higher than when HPT was used.

Further exploration of the data showed that, when comparing PMWS and healthy pigs, Sp values were slightly improved when the presence of PCV2 (detected by qPCR) was considered together with the APPs concentration. Thus, a potential pre-mortem diagnosis of PMWS could be established only when APP concentrations were above optimal threshold and qPCR gave a positive result. Following this condition, pigMAP and HPT maintained their Sn at 85.7% (73.8-93.6) and 67.9% (54.0-79.7), respectively, and Sp increased to 93.5% (82.1-98.6) in both cases.

Discussion

The present study represents the first longitudinal determination of pig-MAP and HPT concentrations in pigs that subsequently developed PMWS. Both APPs increased in parallel to the increase of PCV2 load in sera and reached the maximum values at the moment of disease manifestation. Pig-MAP and HPT concentrations were higher in PMWS affected pigs than in healthy ones, which is in agreement with previous reports A c c e p t e d M a n u s c r i p t 15 [START_REF] Segalés | Haptoglobin and pigmajor acute protein are increased in pigs with postweaning multisystemic wasting syndrome (PMWS)[END_REF][START_REF] Parra | Porcine acute phase protein concentrations in different diseases in field conditions[END_REF]. This situation is probably a reflection of the systemic inflammatory status suffered by PMWS affected pigs [START_REF] Rosell | Pathological, immunohistochemical, and in-situ hybridization studies of natural cases of postweaning multisystemic wasting syndrome (PMWS) in pigs[END_REF].

It is known that APPs serum concentrations might vary with the age (Piñeiro et al., 2009a). However, in the present study, samples from all pigs were collected at previously established weeks of age to accomplish a longitudinal study (Grau-Roma et al., 2009). Moreover, at the moment of necropsy, pigs displaying PMWS-like signs and healthy pigs were euthanized in a age-matched way. Therefore, the differences observed between the three studied groups of pigs should not be influenced by a potential ageeffect.

The higher correlation between PCV2 load and both APPs detected in PMWS affected pigs, together with the absence or weak correlation detected in healthy pigs, indicates that the APR in PMWS affected pigs occurs concurrently with PCV2 infection, further supporting the idea that PCV2 is the essential infectious agent for PMWS development [START_REF] Harding | The clinical expression and emergence of porcine circovirus 2[END_REF][START_REF] Ghebremariam | Postweaning multisystemic wasting syndrome (PMWS) in pigs with particular emphasis on the causative agent, the mode of transmission, the diagnostic tools and the control measures. A review[END_REF][START_REF] Segalés | Porcine circovirus diseases[END_REF]. Based on qPCR PCV2 results, an important percentage of healthy and wasted non-PMWS pigs were also infected by PCV2. However, significant increase in APPs concentrations was only clearly observed in PMWS affected animals. It has been reported that infections with pathogens like Streptococcus suis produce an increase of APPs in both clinically and subclinically infected pigs (Sorensen et al., 2006). However, present data showed that, in accordance with previous results [START_REF] Segalés | Haptoglobin and pigmajor acute protein are increased in pigs with postweaning multisystemic wasting syndrome (PMWS)[END_REF], no differences between subclinically PCV2 infected pigs (healthy PCV2 qPCR positive pigs) and healthy non-PCV2 infected pigs (healthy PCV2 qPCR negative pigs) were detected. These findings suggest that sole infection with PCV2 does not imply a systemic inflammatory status A c c e p t e d M a n u s c r i p t 16 causing a detectable APR; only when the disease develops, HPT and pig-MAP concentrations increase significantly. Taking into account that PMWS affected pigs have higher serum viral loads than non-affected pigs [START_REF] Brunborg | Quantitation of porcine circovirus type 2 isolated from serum/plasma and tissue samples of healthy pigs and pigs with postweaning multisystemic wasting syndrome using a TaqMan-based realtime PCR[END_REF][START_REF] Olvera | Comparison of porcine circovirus type 2 load in serum quantified by a real time PCR in postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome naturally affected pigs[END_REF][START_REF] Grau-Roma | Infection, excretion and seroconversion dynamics of porcine circovirus type 2 (PCV2) in pigs from postweaning multisystemic wasting syndrome (PMWS) affected farms in Spain and Denmark[END_REF], a certain level of serum PCV2 load might be associated with the increase of HPT and pig-MAP serum concentrations. Therefore, it is likely that when the animal limits the PCV2 replication to certain levels and/or the virus is not able to overcome the defences of the host, the APR becomes much less evident or even non-detectable using HPT and pig-MAP determinations in sera.

It is difficult to collectively interpret the results of the pigs suffering wasting but without a PMWS diagnosis (wasted non-PMWS animals). This group of animals might include pigs suffering from wasting due to PMWS-unrelated causes, such as other infectious or non-infectious diseases and/or management conditions [START_REF] Harding | Recognizing and diagnosing porcine multisystemic wasting syndrome (PMWS)[END_REF], but might also include PMWS convalescent pigs or pigs that were able to overcome or limit the disease expression [START_REF] Segalés | Update on postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome diagnostics[END_REF][START_REF] Krakowka | Features of porcine circovirus-2 disease: correlations between lesions, amount and distribution of virus, and clinical outcome[END_REF]. The obtained pig-MAP concentration profile in wasted non-PMWS pigs was similar to the one obtained in healthy pigs. However, an increase in HPT concentration was detected at the two last samplings in Spain. Moreover, in Denmark, HPT concentrations at the moment of necropsy in wasted non-PMWS animals could not be distinguished from those in PMWS affected pigs. These results, together with the wider APPs concentration variation observed in wasted non-PMWS pigs compared to healthy ones, suggest that at least a proportion of wasted non-PMWS animals mounted an APR around the PMWS outbreak. On the other hand, this group of pigs had lower PCV2 serum loads than PMWS-affected ones and higher than healthy pigs during PMWS outbreak [START_REF] Grau-Roma | Infection, excretion and seroconversion dynamics of porcine circovirus type 2 (PCV2) in pigs from postweaning multisystemic wasting syndrome (PMWS) affected farms in Spain and Denmark[END_REF]. Moreover, a correlation between PCV2 viral load and APPs concentration The fact that an increase of both pig-MAP and HPT proteins was seen in Spanish pigs prior to the PMWS outbreak suggests that the detectable APR occurred some days before the start of clinical signs. Therefore, those APPs could provide valuable prognostic information if proper timing of sampling is assured [START_REF] Murata | Current research on acute phase proteins in veterinary diagnosis: an overview[END_REF].

These increased values before the PMWS outbreak were not detected in the Danish animals, probably due to the low number of samples analyzed at week 9 in each group.

Despite it is known that APPs are generic markers of inflammation and therefore are not specific of any disease [START_REF] Petersen | Application of acute phase protein measurements in veterinary clinical chemistry[END_REF]Gruys et al., 2005a), the previously described potential use of APP as markers of health (Gruys et al., 2005b;[START_REF] Parra | Porcine acute phase protein concentrations in different diseases in field conditions[END_REF] prompted us to evaluate their diagnostic performance for PMWS. Both HPT and pig-MAP serum concentrations were significantly correlated. Nevertheless, pig-MAP gave a better diagnostic value of PMWS, since this protein displayed higher Sp and Sn values than HPT in the ROC analysis. Moreover, pig-MAP was able to differentiate PMWS from non-PMWS affected pigs (comprising wasted non-PMWS and healthy pigs) even at the sampling prior to PMWS development (at least in Spain). The previously described individual higher variation in HPT concentrations (Piñeiro et al., 2009a) could partially explain the lower diagnostic value of HPT. In any case, as previously indicated, the APPs values of single reactants are not sensitive enough to detect a special subject in a population livestock (Gruys et al., 2005a). It has been suggested that the acute phase signal situation obtained for an individual animal can be A c c e p t e d M a n u s c r i p t 19 PMWS and not on infection with PCV2 alone, as previously suggested [START_REF] Segalés | Haptoglobin and pigmajor acute protein are increased in pigs with postweaning multisystemic wasting syndrome (PMWS)[END_REF].

  APPs concentrations and the moment of infection for each pathogen.

  concentration was determined by a sandwich enzyme-linked immunosorbent assay (ELISA), using a commercial kit based on two anti-Pig-MAP specific monoclonal antibodies (PigCHAMP Pro Europa S.A.), according to the manufacturers' instructions.

  PCV2 real time quantitative PCR (qPCR) was performed on all longitudinally collected serum samples. DNA was extracted from 200 µl of serum from Spanish (Nucleospin® Blood, Macherey-Nagel, GmbH & Co KG, Düren, Germany) and Danish samples (QIAamp DNA Mini Kit, Qiagen® GmbH, Germany), according to the manufacturers' instructions. DNA was eluted in 100 µl and 200 µl of elution buffer in Spain and Denmark, respectively.

  performed on all longitudinally collected serum samples. In Spain, ELISAs were used to detect antibodies against porcine reproductive and respiratory syndrome virus (PRRSV) (HerdCheck PRRS virus antibody test, IDEXX, Inc., USA), Aujeszky's disease virus (HerdCheck anti-PRV gpI, IDEXX, Inc., USA), porcine parvovirus (PPV) (Ingezim PPV, Ingenasa, Spain), swine influenza virus (SIV) (Civtest Suis Influenza, Laboratorios Hipra, Spain), Mycoplasma hyopneumoniae (Civtest Suis Mycoplasma Hyopneumoniae, Laboratorios Hipra, Spain), and Salmonella spp. (Salmonella covalent mix-ELISA, Svanovir TM , Svanova, Sweden). In Denmark, assays used were previously described immunoperoxidase monolayer assays (IPMAs)

  -MAP and HPT concentrations were significantly correlated (Spain: R=0.462, p<0.0001; Denmark: R=0.576, p<0.0001).

  healthy pigs, being significant in Spain (R=0.186, p<0.05) but not in Denmark (R=0.273, p=0.73).

  results for M. hyopneumoniae in Spain had higher pig-MAP mean serum concentrations (mean±SD=4.47±4.49) than the 101 pigs that gave negative nPCR (2.35±2.13) (p<0.05).

  Taken together, these findings suggest that the APR detected in the groups of wasted non-PMWS affected pigs might also be associated with PCV2 infection.

Figure 1 .Figure 2 .

 12 Figure 1. Pig-MAP concentrations (mg/ml) (lines) and PCV2 loads (copies of PCV2/ml of sera)

  Figure 2R1

Table 1 .

 1 Number of pigs within each pathological category from which haptoglobin and pig-MAP concentrations were determined in each country.

		PMWS	Healthy	Wasted non-PMWS	Total
	Spain	35	29	43	107
	Denmark	21	17	15	53
	Total	56	46	58	160

Acknowledgements

This work was funded by the Projects No. 513928 from the Sixth Framework Programme of the European Commission, GEN2003-20658-C05-02 (Spanish Government) and Consolider Ingenio 2010 -PORCIVIR (Spanish Government). We are grateful to all the production team from Vallcompanys S.A. for their great collaboration during the study, and to M. Pérez, M. Mora, E. Huerta, H. Nielsen, H. Tran and I. Larsen for their excellent technical assistance. PhD studies of Mr. Grau-Roma are funded by a pre-doctoral FPU grant of Ministerio de Educación y Ciencia of Spain. Matilde Piñeiro was funded by Programa Torres Quevedo (Ministerio de Educación y Ciencia y Fondo Social Europeo) and Linea 4B del ADE (Junta de Castilla y León).

APPs determination in the search for unhealthy subjects among a population of apparently normal animals (Gruys et al., 2005a).

PMWS is considered a multifactorial disease in which the occurrence of PCV2 infection is necessary but not sufficient [START_REF] Harding | The clinical expression and emergence of porcine circovirus 2[END_REF][START_REF] Ghebremariam | Postweaning multisystemic wasting syndrome (PMWS) in pigs with particular emphasis on the causative agent, the mode of transmission, the diagnostic tools and the control measures. A review[END_REF][START_REF] Segalés | Porcine circovirus diseases[END_REF]. Present results showed that, among all studied pathogens, PCV2 was the only one systematically present in PMWS affected farms and pigs. This fact suggests that none of the other studied pathogens are necessary for the PMWS development under field conditions. Moreover, no significant differences in APP serum concentrations between those pigs showing evidences of infection for pathogens different from PCV2 at the moment of clinical PMWS outbreak were detected, with the only exception of M. hyopneumoniae in Spain. Specifically, the 5 pigs that gave positive PCR results for M. hyopneumoniae had higher pig-MAP concentration than the 101 pigs with negative results. In fact, the two pigs with higher pig-MAP concentration (10 and 9 mg/ml) corresponded to 2 out of the 5 M. hyopneumoniae nPCR positive, suggesting that PCV2 co-infection with other pathogens could increase the APR, aggravating the severity of the disease [START_REF] Ellis | Porcine circovirus-2 and concurrent infections in the field[END_REF]. The potential influence of other not studied pathogens on the observed clinical signs and, therefore, on the APR of the PMWS affected animals, can not be ruled out.

In conclusion, the present results indicate that the acute phase response in PMWS affected pigs occurred concomitantly to PCV2 viremia. In addition, present work supports the hypothesis that the increase of APPs levels depends on the development of


Lihong Liu

Jaruwan Kampa

Sándor Belák

Claudia Baule
email: claudia.baule@bvf.slu.se

Virus recovery and full-length sequence analysis of atypical bovine pestivirus Th/04_KhonKaen 1

Abstract

Phylogenetic analysis of recently identified bovine "atypical" pestiviruses, performed based on different gene regions, has revealed unclear relationships with other established species, therefore, their phylogenetic position could not been determined so far. In this study, the atypical pestivirus Th/04_KhonKaen was recovered from serum of a naturally infected calf and the complete genome sequence was determined and analysed, as means to define its position. The viral genome is 12 337 nucleotides (nt) long, and comprises a 5´ UTR of 383 nt, a 3´ UTR of 254 nt and an open reading frame of 11700 nt, without duplication of viral sequences or insertions of cellular sequences. The phylogenetic analyses of the full-length sequence, performed by Neighbor-joining, maximum likelihood, and the Bayesian approach, unanimously placed Th/04_KhonKaen in a single lineage, distinct from the established pestivirus species, and close to bovine viral diarrhea virus types 1 and 2. Furthermore, Th/04_KhonKaen and two previously reported atypical pestiviruses D32/00_'HoBi' and CH-KaHo/cont formed a well-supported monophyletic clade in trees based on the complete N pro and E2 gene regions. The finding provides conclusive classification of the Th/04_KhonKaen virus and confirms the standing of the bovine "atypical" pestiviruses as a novel pestivirus species. Keywords: BVDV, genotyping, pestivirus, phylogeny, Th/04_KhonKaen

A c c e p t e d M a n u s c r i p t

Introduction

The pestivirus genome is a single-stranded, positive-sense RNA molecule, which comprises two untranslated regions (UTRs) at the 5' and 3' ends, and one open reading frame (ORF) encoding a large polyprotein. This polyprotein is co-and post-translationally processed into 11-12 polypeptides in the following order: N-terminal autoprotease (N pro ), capsid protein (C), envelope proteins (E rns , E1, and E2), p7, and non-structural (NS) proteins , NS4A, NS4B, NS5A, and NS5B)] (reviewed by [START_REF] Rümenapf | Molecular Biology of Pestiviruses[END_REF]. Recombination or point mutations in the viral genome have been shown to correlate with induction of cytopatogenic effects (CPE) on cell culture following infection with BVDV (Thiel et al., 2006). Accordingly, two biotypes of BVDV can be distinguished: the non-cytopathogenic (ncp), that replicates without cytopathic effect, and cytopathogenic (cp), that leads to death of infected cells [START_REF] Thiel | Pestiviruses[END_REF].

The genus Pestivirus of the family Flaviviridae includes four recognized species: Bovine viral diarrhea virus 1 (BVDV-1), Bovine viral diarrhea virus 2 (BVDV-2), Border disease virus (BDV), Classical swine fever virus (CSFV), and a fifth tentative species Pestivirus of giraffe (Seventh Report of the International Committee on Taxonomy of Viruses; http://www.virustaxonomyonline.com). A recently identified new pestivirus, D32/00_'HoBi', was isolated from a batch of fetal calf serum originating from Brazil and was proposed as a sixth pestivirus species [START_REF] Schirrmeier | Genetic and antigenic characterization of an atypical pestivirus isolate, a putative member of a novel pestivirus species[END_REF]. Additional atypical pestiviruses of bovine-origin have also been described, including: Brz buf 9, originally isolated from a buffalo in Brazil; CH-KaHo/cont, a cell culture contaminant possibly A c c e p t e d M a n u s c r i p t -4 -originating from a batch of fetal calf serum produced in South America [START_REF] Stalder | Genetic heterogeneity of pestiviruses of ruminants in Switzerland[END_REF]; and Th/04_KhonKaen from a naturally infected calf in Thailand [START_REF] Ståhl | Natural infection of cattle with an atypical 'HoBi'-like pestivirus-implications for BVD control and for the safety of biological products[END_REF]. All these pestiviruses were shown to be closely related to D32/00_'HoBi', based on trees generated from the 5´UTR and N pro regions, however their final phylogenetic classification could not be determined.

The Th/04_KhonKaen virus was first detected by ELISA tests and preliminarily characterized by virus neutralization and partial sequence analysis of the 5' UTR [START_REF] Ståhl | Natural infection of cattle with an atypical 'HoBi'-like pestivirus-implications for BVD control and for the safety of biological products[END_REF]. Further genetic characterization of this pestivirus required a virus isolate, but the fact that the serum had been inactivated hampered virus isolation by conventional methods. In this study, a viable Th/04_KhonKaen virus was recovered by transfection of bovine turbinate cells with RNA extracted from an inactivated serum sample, and the complete sequence was determined. Comparative sequence analyses show that the Th/04_KhonKaen virus, together with atypical pestiviruses D32/00_'HoBi' and CH-KaHo/cont represent a new species within the pestivirus genus.

Materials and Methods

Virus recovery from an inactivated serum sample

The serum sample was obtained from a calf in a herd in Thailand. According to the legislation in Sweden the sample had to be heat-inactivated at 56C for 90 min, therefore it was no longer suitable for virus isolation by conventional methods. To recover virus, RNA A c c e p t e d M a n u s c r i p t -5 -was extracted from this sample using TRIzol Reagent (Invitrogen, Carlsbad, USA), and purified with phenol-chloroform. The RNA was electroporated into BVDV-free bovine turbinate (BT) cells in a cell suspension containing 1  10 6 cells, using a Bio-Rad Gene Pulser Xcell electroporator. The cells were grown in Eagle's minimum essential medium (EMEM) containing 10% horse serum, 1% L-glutamine, and 100 U/ml of penicillin and 100 µg/ml of streptomycin, at 37C in a CO 2 incubator. Confluent cells were split and grown for four days. The cells were then frozen and thawed and the lysates were clarified by centrifugation. The supernatants were used to infect fresh sub-confluent BT cells, as performed routinely for virus propagation. After incubation at 37C for four days, total RNA extracted from lysates of the cells was used to confirm presence of viral RNA with a pan-pestivirus RT-PCR targeting the 5´ UTR.

Immunoperoxidase test

The immunoperoxidase (IPX) test was performed to demonstrate presence of BVDV specific antigens in cells infected with the recovered virus. Briefly, serial dilutions of the supernatant from virus-infected BT cells were used to infect BT cells grown in a 96-well plate. Following incubation at 37C for 4 days, the cells were washed once with 0.01 M PBS, pH 7.6, fixed with fixation fluid and dried. After re-dehydration of the cells, an antiserum directed against BVDV-1 was added to each well and the plates were incubated at room temperature for 15 min. The cells were washed three times and 50 µl of diluted rabbit anti-bovine horseradish peroxidase conjugate was added, followed by incubation A c c e p t e d M a n u s c r i p t -6 -and washing as before. For detection, 50 µl of substrate solution (5 ml of 0.05 M sodium acetate, 6 µl of 3% H 2 O 2 , and 300 µl of chromogen solution) was used.

2.3. Amplification, sequencing and analysis of the full-length genome

RNA extraction

When presence of virus was confirmed by RT-PCR, infected cells were washed twice with cold PBS without Mg 2+ and Ca 2+ , and lysed with a lysis buffer containing 100 mM Tris-HCl, pH 8.0, 500 mM LiCl, 5 mM DTT, 10 mM EDTA, pH 8.0 and 5% Lithium Duodecyl Sulphate. Total RNA was purified from the cell lysates by phenol/chloroform and precipitated in an equal volume of isopropanol. After centrifugation, the RNA pellet was washed once with 70% of ice-cold ethanol. The dried pellet was re-suspended in 30 µl RNase-free water.

Synthesis of double-stranded cDNA

SuperScript II (Invitrogen, Carlsbad, USA) was used to synthesize the first strand cDNA using 2 µM of a gene-specific primer (PAS1) in a 20-µl volume, following conditions recommended by the manufacturer. The second strand was prepared in 150-µl reaction volume, containing the 1 st strand reaction, E. coli DNA ligase (10 U), E. coli DNA polymerase (40 U), E. coli RNAse H (2 U) in 1  second strand buffer. The reaction was performed at 16°C for 2 hours. The cDNA was purified with phenol/chloroform and A c c e p t e d M a n u s c r i p t -7 -precipitated in 95% ethanol at 20°C overnight. The cDNA pellet was washed with 75% ethanol, and resuspended in 20 µl of distilled water.

PCR amplification

The primers were designed based on conserved sequences of established pestiviruses and the sequence of the D32/00_HoBi virus. The list of primers for PCR amplification and DNA sequencing is presented in Table 1. The viral genome sequence, excluding the 5´and 3´ ends, was amplified in two overlapping fragments by PCR using primers TF1-TR1, and TF2-TR2. PCR was performed in a 25-µl volume, containing 0.5 µM of forward and reverse primers, 0.5 mM dNTP mix, 1.25 U of PfuUltra High-Fidelity or 2.5 U EXL DNA polymerase (Stratagene, La Jolla, CA, WSA), 1 µl of cDNA, and 1  reaction buffer with MgCl 2 . After initial denaturation at 92C for 2 min, a three-step cycling of 30 cycles was performed with denaturation at 92C for 30 sec, annealing at 55C for 30 sec, and elongation at 72C for 7 min. Following amplification, the PCR products were separated on 0.8% agarose gels, and the designed band was excised from the gel and purified with the Wizard SV Gel and PCR Clean-Up System (Promega Co., Madison, WI, USA).

Determination of the 5´and 3´ends by RNA ligase-mediated RACE

To determine the 5´and 3´ends, a GeneRacer Kit (Invitrogen, Carlsbad, USA) was used, following manufacturer's instruction. In brief, the GeneRacer RNA oligo, and RNA oligo A c c e p t e d M a n u s c r i p t -8 -2 (Table 1) were ligated to the 5´end and 3´end of the viral genome, respectively. Ten units of T4 RNA ligase were used in the reactions and ligation was performed at 37C for 1 h. The reactions were then treated with proteinase K, followed by phenol/chloroform purification, and precipitation of the RNA in 95% ethanol in the presence of 0.1M sodium acetate. The RNA was pelleted by centrifugation, washed with 70% ethanol and resuspended in 30 µl of RNase-free water. The GeneRacer 5´primer from the kit and genespecific primer T5U-R1 were used to amplify the target 5´end using a one-step RT-PCR kit (Qiagen, Hilden, Germany), with the following cycling profile: 50°C for 30 min, 95°C for 15 min, 35 cycles of 95°C for 15 sec, 54°C for 20 sec, and 72°C for 1 min, and a final extension at 72°C for 7 min. To amplify the 3´end, the gene-specific primer T3U-F1 and M13 forward primer (complementary to the 5´end of the RNA oligo) were used. The same reaction was performed as for amplification of the 5´end.

The purified PCR products were cloned into pCR4-TOPO (Invitrogen, Carlsbad, USA).

Four clones containing inserts of the 5´ and 3´ends of the genome were used for DNA sequencing with M13 forward and reverse primers (Invitrogen, Carlsbad, USA).

Phylogenetic analysis

For phylogenetic analysis, a total of 24 full-length genome sequences of pestiviruses were retrieved from GenBank, including 9 of CSFV, 3 of BDV, 7 of BVDV-1 and 4 of BVDV-2, and Pestivirus of giraffe. The GenBank accession numbers are included in Table 2.

Multiple sequence alignment was performed with software BioEdit version 7.0.5.2 [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF], using CLUSTAL W [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting[END_REF]. To avoid bias due to different methods, phylogenetic analysis of complete genome sequences was performed with three methods: Neighbor-joining, maximum likelihood and the Bayesian approach. All gaps created by insertions were deleted by the software MEGA prior to the analyses. MEGA 4 [START_REF] Tamura | MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0[END_REF] was used to reconstruct the Neighbor-joining tree under the nucleotide substitution model of Tamura 3-parameter, and tree reliability was evaluated by bootstrapping 1000 replicates. PHYML (v2.4.4) was used for phylogeny inference according to maximum likelihood criterion. The analysis settings were: Base frequency estimates (ML); Proportion of invariable sites (estimated); Nucleotide substitution model (estimated). Following tree reconstruction, 1000 non-parametric bootstrap analyses were performed to test the robustness of the hypothesis. Bayesian inference analysis was performed with the software MrBayes 3.1 [START_REF] Huelsenbeck | MRBAYES: Bayesian inference of phylogenetic trees[END_REF][START_REF] Ronquist | MrBayes 3: Bayesian phylogenetic inference under mixed models[END_REF], as previously described (Xia et al., 2007). In brief, the model settings were a Dirichlet prior for both substitution rates (Nst=6) and state frequencies (# states=4).

Rate variation across sites was modeled using a -distribution with a proportion of sites invariable (rates=invgamma). The MCMC search was run with four chains for 2 000 000 generations, sampling the Markov chain every 1000 generations. The first 25% trees were discarded as "burn-in". Each analysis was performed three times and a representative consensus tree is shown in this paper.

In order to demonstrate the relationships among these recently described atypical pestiviruses, sequences of the complete N pro and E2 coding regions from 64 pestiviruses, corresponding to the GenBank accession numbers listed in Table 2, were analysed with the Bayesian approach. The same parameters applied for the full-length phylogeny analysis were used. The RNA extracted from lysates of infected cells yielded a specific PCR product on amplification with a pan-pestivirus RT-PCR targeting 5´ UTR. The recovered virus was confirmed by the IPX test that showed presence of pestivirus-specific proteins upon propagation following cell transfection. The Th/04_KhonKaen virus reacted with the BVDV-1 antiserum, which also recognizes BVDV-2 and D32/00_'HoBi'. Cytopathic effect (CPE) was not observed on isolation and propagation of the virus, supporting the recovery of a non-cytopathogenic pestivirus from the serum sample.

Results

Virus recovery

Sequence analysis of the viral genome

The full-length sequence was assembled from partial sequences obtained from the two RT-PCR fragments and the 5´and 3´ends of the genome. The complete genome contains 12 337 nucleotides (nt), comprising 383 nt at the 5´ UTR, 254 nt at the 3´ UTR and an ORF of 11 700 nt. The ORF involves the 11 gene regions that correspond to predicted products of polyprotein processing: the pestivirus-specific N-terminal protease N pro (504 nt), capsid (303 nt), E rns (681 nt), E1 (585 nt), E2 (1119 nt), p7 (210 nt), NS2-3 (3408 nt), NS4A (192 nt), NS4B (1041 nt), NS5A (1491 nt), and NS5B (2166 nt). Neither duplication of viral sequences nor insertions of cellular sequences were found in the genome. Comparative sequence analysis showed that this virus shares 67.3-68.3% identity with other pestivirus species, with the highest identity of 68.3 to BVDV-1 and -67.9% to BVDV-2, respectively (Table 3). The polyprotein (3899 aa) was used as a query protein for searching in the Protein Data Bank (PDB). The protein BLAST revealed several conserved domains, including the N pro endopeptidase C53 that is unique to pestiviruses; the RNase T2 in the Erns region; the pestivirus NS3 polyprotein peptidase S31 and DEXH-box helicases located in NS3; and RNA-dependent RNA polymerase within NS5B. Such analysis provided additional support for the classification of Th/04_KhonKaen as a pestivirus.

Phylogenetic analysis of Th/04_KhonKaen sequence

Three methods, Neighbor-joining, maximum likelihood, and the Bayesian approach were exploited for phylogenetic analysis. By applying the later method, we have previously shown that a total evidence approach could be used for confident classification of BVDV into subgroups (Xia et al., 2007). All three methods produced a single, reliable phylogenetic hypothesis that was supported by significantly high bootstrap values and posterior probability (Fig. 1). The established species BVDV-1, BVDV-2, BDV, CSFV separated into four monophyletic clades, and the tentative pestivirus of Giraffe formed a single branch. Th/04_KhonKaen also formed a single branch positioned distinctly from the four established pestivirus species and from pestivirus of Giraffe, and closely related to BVDV-1 and BVDV-2. The position of Th/04_KhonKaen in the consensus trees was supported by bootstrap values of 99% (Neighbor-joining), 93% (ML), and posterior probability of 1.00 (Bayesian approach).

A c c e p t e d M a n u s c r i p t

-13 -To demonstrate the relationship between the Th/04_KhonKaen virus and other atypical pestiviruses D32/00_'HoBi' and CH-KaHo/cont, single N pro and E2 gene analyses were performed. As shown in Fig. 2 (a andb), all three atypical pestiviruses formed a monophyletic clade strongly supported by bootstrap value and posterior probability. The position of this clade in the tree of E2 gene was same as that of Th/04_KhonKaen virus in the tree based on full-length sequence analysis. As in the tree of Figure 1, this clade is genetically distant from the established bovine pestivirus species BVDV-1 and BVDV-2, as well as distinct from BDV, CSFV and pestivirus of Giraffe. The four recognized species BVDV-1, BVDV-2, BDV and CSFV also formed monophyletic clades.

Discussion

Phylogenetic analysis is useful to classify novel viruses, and to reveal their evolutionary history. Particularly for pestiviruses, whole-genome phylogeny has been used to classify two novel species, Giraffe-1 and Reindeer-1 [START_REF] Avalos-Ramirez | Evidence for the presence of two novel pestivirus species[END_REF], although the latter has been re-classified as BDV [START_REF] Becher | Genetic and antigenic characterization of novel pestivirus genotypes: implications for classification[END_REF].

In this study the complete genome sequence of the recently identified pestivirus Th/04_KhonKaen was determined and analysed, aiming to establish the phylogenetic positioning of this virus and to determine its relationship with known pestivirus species.

Following successful recovery of the virus from an inactivated serum sample, the genome organization of Th/04_KhonKaen was shown to be reminiscent of that of an ncp pestivirus, consistent with the recovery of a virus that did not induce CPE. The full-length A c c e p t e d M a n u s c r i p t -14 -nucleotide identity of the Th/04_KhonKaen with the recognized BVDV species is around 68%, which is close to the range of 69.5% that separates BVDV-2 from BVDV-1 (Table 3), motivating its discrimination as a distinct pestivirus species of bovine origin.

A single phylogenetic tree topology was produced by all three methods, highly supported by strong bootstrap and posterior probability values, on analysis of the full-length sequence. This analysis unambiguously positioned the Th/04_KhonKaen virus as a unique species in the consensus tree, distinct from the four established pestivirus species and pestivirus of Giraffe, and closely related to the bovine species BVDV-1 and BVDV-2.

Remarkably, analysis of sequences of the complete N pro and E2 coding region showed that the three atypical pestiviruses formed a well-supported single clade, demonstrating that these atypical bovine pestiviruses are genetically distinct from the current well established pestivirus species. This relationship has been unequivocally confirmed by the analysis of a sequence dataset combining the 5´UTR, and the complete N pro and E2 gene regions of 56 pestiviruses, published in [START_REF] Liu | Phylogeny, classification and evolutionary insights into pestiviruses[END_REF].

Recovery of the Th/04_KhonKaen virus enables further studies currently in progress to define cross-reactivity patterns with polyclonal and monoclonal antibodies, as well as the characterization of in vitro and in vivo biological properties of this group of viruses.

In conclusion, the atypical pestivirus Th/04_KhonKaen was isolated from an inactivated serum sample. Sequence analysis supported positioning of the Th/04_KhonKaen virus as a new species within the pestivirus genus. Comparisons of sequences of the N pro and E2

  2.3.5. DNA sequencingAn ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) was used for DNA sequencing. Sequencing reactions were performed in a total volume of 10 l, containing 1.5 µl of ABI 5  sequencing buffer, 0.5 µM primer, 1.0 µl of BigDye, and 1.0 µl of the purified PCR product. The thermo profile A c c e p t e d M a n u s c r i p t -9 -was 25 cycles of denaturation at 96°C for 15 sec, annealing at 50°C for 10 sec, and extension at 60°C for 4 min. To remove contaminating salts and unincorporated dye terminators from the sequencing reactions, the Montage SEQ96 sequencing reaction cleanup kit (Millipore Co., Billerica, MA, USA) was used, following the manufacturer's instructions. Capillary electrophoresis was carried out in an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA, USA). Sequences were analyzed with multiple programs of the Lasergene package (DNASTAR, Inc., Madison, WI).

  ; Number of substitution rate categories (4); Gamma distribution parameter

Fig. 1 .

 1 Fig.1. Consensus phylogenetic tree based on analysis of the full-length sequence of the Th/04_KhonKaen virus and those of pestivirus species available in the GenBank. The Neighbor-joining tree was reconstructed under the nucleotide substitution model of Tamura 3-parameter using MEGA 4. PHYML (v2.4.4) was used for phylogeny inference according to maximum likelihood (ML) criterion, and MrBayes 3.1 for Bayesian inferring. The robustness of the tree was evaluated by bootstrapping 1000 replicates by Neighbor-joining and maximum likelihood, and by posterior probability from Bayesian analysis. The supporting value for major clades is shown in the order of NJ/ML/PP. "1.00" indicates 100%/100%/1.00. A representative, un-rooted consensus tree is shown in

Figure

  Figure

Fig. 2 .

 2 Fig. 2. Phylogenetic trees based on analysis of sequences of the complete N pro (a., left) and E2 (b., right) gene regions. The relationship of atypical pestiviruses Th/04_KhonKaen, D32/00_'HoBi' and CH-KaHo/cont with representatives of established pestivirus species retrieved from GenBank is shown. The un-rooted consensus trees are inferred by Bayesian approach. Numbers indicate posterior probability values for major clades. The bar indicates changes per site.

Figure

  Figure

Table 1 .

 1 Nucleotide sequence of primers used for PCR amplification and sequencing of the Th/04_KhonKaen virus.

	Primer name	Nucleotide sequence a	Position
	TF1	GCTAGCCATGCCCTTAGTAGGA	98-119
	TR1	CCCTAGTTCGGCCCATTGAGTC	5382-5403
	TF2	GGGCCGAACTAGGGTGGTGT	5390-5409
	TR2	GTGCGTTGAGTGTAGTGTTGTC	12247-12268
	TF3	ATGGAGTTGTTAAACTTTGAAC	384-405
	TR3	GCAGCTTCCTACCCAGATGG	868-878
	TF5	GACCTCAGTTGTAAGCCTGAG	2457-2477
	TR5	CCCCCTAGCTCCTTGTTCAGTT	3554-3575
	TF4	ACCCCACCAAAAACAAACCAGA	1090-1111
	TR4	TGGGCCTATCTCATTGGTCTCA	2495-2516
	TF6	TCTTGGGTGGGAGGTATGTGCT	3505-3526
	TR6	TTAGGCCCCTTGCTTCTTTCTC	4648-4669
	TF7	CAAGGCCCCAGAGCAAAGACG	4431-4451
	TF8	CGAGCAGCGGCAGAATCAGTAT	5913-5934
	TR8	GTCCGGCCAGTCCAACC	7120-7136
	TF9	CAAATGCCATAGAATCAGGAGTA	6496-6518
	TF10	TGATCACCCGGAACCCATACAG	6923-6944
	TF11	AGGCAGGCTGGGGAGTGAAAC	7619-7639
	TF12	AGGCAAAAGAATTAGCGGAAAGA	8317-8339
	TR9	TGCCCAGGCTGAGTGCYTCYA	9649-9669
	TR10	TCCTTCCCGTTCCAGTTGTTCT	10283-10304
	TR11	GCGGCTTGTTACCTGAGTGTC	10965-10985
	TF13	CTCTAAAATGAAGAAAGGGTGYG	10895-10917
	TR12	CTGTGGTTTGCCAGATTCATAC	11327-11348
	TR13	GGGGATATACATTTTGCCAGTC	11885-11906
	TF14	AGGCACACCAGCAAGAGACTAC	11790-11811
	PAS1	CTGTGTGCRTTRARTGTAGTG	12252-12273
	T3U-F1	TTATTACTTAGGCTGATGAGAAC	12214-12236
	T5U-R1	CAGGGAGCTCACTGCCACCAC	139-159
	GeneRacer RNA oligo CGACUGGAGCACGAGGACACUGACA
		UGGACUGAAGGAGUAGAAA
	GeneRacer 5´primer	CGACTGGAGCACGAGGACACTGA
	RNA oligo 2	CGCAAAUGGGCGGUAGGCGUGCUGG
		CCGUCGUUUUAC

a Bold bases indicate mismatches with the sequence of the Th/04_KhonKaen virus.

Table

The full-length sequence of the Th/04_KhonKaen virus has been deposited in GenBank under accession number FJ040215.

Acknowledgements

The authors thank Dr. Karl Ståhl, The National Veterinary Institute, and Prof. Stefan Alenius, Department of Clinical Sciences, Swedish University of Agricultural Sciences for fruitful discussions. We are grateful to Dr. Hongyan Xia, Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, for valuable comments on the manuscript.

A c c e p t e d M a n u s c r i p t -15 -gene regions with those of the atypical pestiviruses D32/00_'HoBi', Brz buf 9, and CH-KaHo/cont confirmed a high sequence similarity among these viruses, demonstrating that they belong to a same, new group of pestiviruses of bovine origin. Xia, H., Liu, L., Wahlberg, N., Baule, C., Belák, S., 2007

a Including unclassified pestiviruses b Same as genomic sequence c Sequence is not included in the analysis


Maribel Casas
email: maribel.casas@cresa.uab.es

Sonia Pina

Nilsa De Deus

Bibiana Peralta

Marga Martín

Joaquim Segalés

Pigs orally inoculated with swine hepatitis E virus are able to infect contact sentinels

Keywords: hepatitis E virus (HEV), swine, experimental infection, oral inoculation, contact sentinels

A c c e p t e d M a n u s c r i p t 2

Abstract

The purpose of the present study was to explore the most likely natural route of infection of swine hepatitis E virus (HEV) by oral inoculation of pigs and to investigate the potential infection by direct contact exposure. A preliminary experiment was performed to assess the infectiousness of the bile used as source of virus. Once confirmed, sixteen pigs were inoculated via oral drop with an HEV positive bile suspension containing 2x10 5 genome equivalents per pig. Nine animals were kept as contact sentinels and twelve more pigs were used as negative controls. A number of pigs from the three groups were euthanized at 16, 32 and 64 days post-inoculation. From the HEV inoculated group, three pigs shed virus in faeces, two had virus RNA in bile at necropsy and two seroconverted. In the contact group, two animals showed presence of HEV RNA in bile. This study demonstrates that pigs orally inoculated with a single HEV dose got infection, although few animals had evidence of infection. Moreover, the virus was successfully transmitted to direct contact exposed pigs.

A c c e p t e d M a n u s c r i p t

Introduction

Hepatitis E virus (HEV) infection in humans is one of the most important causes of acute clinical hepatitis in developing countries. Transmission of the virus occurs primarily by the faecal-oral route through contaminated drinking water in areas with poor sanitary conditions. Although the case fatal rate is generally low (<1%), it can reach up to 25% in pregnant women [START_REF] Kumar | Hepatitis E in pregnancy[END_REF].

The existence of sporadic HEV infection cases in individuals of industrialized countries with no history to travel to endemic areas has suggested the possibility of an animal reservoir [START_REF] Pina | HEV identified in serum from humans with acute hepatitis and in sewage of animal origin in Spain[END_REF][START_REF] Pavio | Hepatitis E: a curious zoonosis[END_REF]. After the first description of swine HEV in USA [START_REF] Meng | A novel virus in swine is closely related to the human hepatitis E virus[END_REF], sequence analyses have shown high nucleotide identity between swine and human isolates from the same geographic area [START_REF] Huang | Detection by reverse transcription-PCR and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United States[END_REF]. The facts that human clinical hepatitis E has been associated with consumption of undercooked meat products [START_REF] Tei | Zoonotic transmission of hepatitis E virus from deer to human beings[END_REF][START_REF] Li | Hepatitis E virus transmission from wild boar meat[END_REF] and swine HEV strains can infect nonhuman primates (Meng et al., 1998b) provide more evidence that HEV should be considered an agent with zoonotic potential.

The lack of a practical animal model has hindered a better knowledge of human HEV infection mechanisms. Different experimental infections have been done in swine, most of them using the intravenous (IV) inoculation as the route of exposure. In an experiment performed in 1998 (Meng et al., 1998a), pigs got infection after IV inoculation of a swine strain of HEV and were able to infect non-inoculated contact animals. In the same study, pigs could not become infected by some human HEV strains (Mex-14 and Sar-55).

However, in a subsequent study from the same research group, another human strain (US-2) was infectious for pigs (Meng et al., 1998b). Following these studies, a comparison of pathogenesis of pigs infected with swine and human strains of HEV was performed [START_REF] Halbur | Comparative pathogenesis of infection of pigs with hepatitis E viruses recovered from a pig and a human[END_REF]. When pigs are inoculated IV, liver seems to be the primary site of virus replication [START_REF] Lee | Localisation of swine hepatitis E virus in experimentally infected pigs[END_REF]. Then, the virus can be accumulated into the gallbladder, A c c e p t e d M a n u s c r i p t 4 probably as a consequence of HEV liver replication in large amounts. The virus seems to be excreted into the bile duct and finally shed in faeces. [START_REF] Williams | Evidence of extrahepatic sites of replication of the hepatitis E virus in a swine model[END_REF] showed the existence of extrahepatic sites of HEV replication, including the small intestines, colon, and hepatic and mesenteric lymph nodes, as the most important tissues.

As previously said, almost all experimental studies focused on swine HEV have used the IV inoculation as the route of infection. Taking into account that the most likely natural route of transmission among pigs is the faecal-oral one [START_REF] Meng | Swine hepatitis E virus[END_REF][START_REF] De Deus | Hepatitis E virus infection dynamics and organic distribution in naturally infected pigs in a farrow-to-finish farm[END_REF], the pathogenic events that occur under natural conditions are not really known. Few experimental infections have used oral inoculation as the route of exposure.

The first one was reported in 1990 by [START_REF] Balayan | Brief Report: Experimental Hepatitis E Infection in Domestic Pigs[END_REF], in which two young piglets were inoculated with a 1:10 faecal suspension containing a human HEV strain of genotype 1. Even though they attributed the infection to the virus inoculated from the human stool, further studies demonstrated that those pigs were already infected with a swine HEV strain of genotype 3 [START_REF] Lu | Complete sequence of a Kyrgyzstan swine hepatitis E virus (HEV) isolated from a piglet thought to be experimentally infected with human HEV[END_REF]. In 2002, Kasorndorkbua et al. tested the faecal-oral route infecting two pigs, one inoculated with a stomach tube and the other using an oral drop system. None of them got infection although the inoculum contained a virus titer of 10 6 genome equivalents-GEs/ml. In a subsequent study from the same research group using the oral route of inoculation [START_REF] Kasorndorkbua | Routes of transmission of swine hepatitis E virus in pigs[END_REF], three pigs were inoculated by an oral gavage with 15 g of fresh swine faeces for 3 consecutive days. Only one animal shed virus in faeces (from 21 to 35 days post-inoculation (dpi)) and seroconverted by 56 dpi. No direct contact exposure was evaluated in this work. Besides, the oral route of transmission has also been investigated using manure slurry as inoculum, although no evidence of infection was obtained [START_REF] Kasorndorkbua | Infectious swine hepatitis E virus is present in pig manure storage facilities on United States farms, but evidence of water contamination is lacking[END_REF].

Taking into account the poorly documented infectiousness of HEV exposed orally to pigs, the objectives of the present study were to explore the most likely natural route of infection A c c e p t e d M a n u s c r i p t 5 by oral inoculation of a larger group of pigs and to investigate the possibility of infection by direct contact to sentinel animals.

Materials and methods

Virus inoculum

The swine HEV inoculum used in this study was prepared from bile of a naturally infected pig. The bile was tested positive for HEV by a semi-nested RT-PCR as described previously [START_REF] Martín | Association of hepatitis E virus (HEV) and postweaning multisystemic wasting syndrome (PMWS) with lesions of hepatitis in pigs[END_REF] and had a titer of 10 6 GEs/ml. The phylogenetic analysis revealed that the HEV strain used belonged to genotype 3 (Peralta et al., 2008a). A second bile, negative by semi-nested RT-PCR, was used as negative control. Biles were kept at -80ºC until used.

Experimental design

Preliminary infectivity study

In order to test the infectivity of the inocula, four 2-week-old piglets with very low values of IgG antibody to HEV measured by an in-house ELISA [START_REF] Martín | Association of hepatitis E virus (HEV) and postweaning multisystemic wasting syndrome (PMWS) with lesions of hepatitis in pigs[END_REF] and free of swine HEV in faeces and blood were housed in a biosecurity level 3 (BL-3) facility. After one week of acclimatization, each piglet was inoculated IV with 1 ml of a 1:5 bile suspension (infectious dose: 10 5 GEs/ml) in physiological saline solution. Clinical signs and rectal temperature were recorded every 2 to 3 days through a 28 day-period. Serum and rectal swabs were taken twice a week to be analyzed by a semi-nested RT-PCR [START_REF] Martín | Association of hepatitis E virus (HEV) and postweaning multisystemic wasting syndrome (PMWS) with lesions of hepatitis in pigs[END_REF]. Serum samples were also analyzed by ELISA to detect anti-HEV IgG antibodies. Bile collected at the necropsy day (28 dpi) was also tested to detect the presence of HEV RNA. All samples were stored at -80ºC until analysis.

Transmission study

A c c e p t e d M a n u s c r i p t 6 Thirty-seven 2-week-old piglets with no or very low HEV IgG antibody OD ELISA values and negative by semi-nested RT-PCR to the virus in blood and faeces were included in the study. Pigs were kept together for 1-week acclimatization period in the same experimental box in a BL-3 facility until the moment of challenge (0 dpi). Then, animals were divided in two groups (group A, n=25; group B, n= 12), taking into account the ELISA OD values of each animal; therefore, no statistical significant difference in mean OD between groups were observed. Sixteen animals of group A were inoculated orally using a syringe with 2 ml of a 1:10 suspension (infectious dose: 10 5 GEs/ml) of a RT-PCR positive bile in physiologic saline serum. The remaining nine pigs were kept as sentinels (contact controls) and were inoculated with 2 ml of 1:10 suspension of a RT-PCR negative bile in physiologic saline serum. Pigs from group B (negative controls) received orally the same inoculum as the sentinels.

Clinical signs and rectal temperature were monitored every 2 to 3 days until the end of the experiment at 64 dpi. Five or 6 pigs from the inoculated group, 3 from the contact group (sentinels) and 4 from the control group were randomly euthanized at 16, 32 and 64 dpi.

Serum and faecal samples were collected twice a week to be analyzed by semi-nested RT-PCR; bile obtained at necropsy was also tested for the mentioned technique. Serum samples were studied for anti IgG HEV antibodies by ELISA. Faecal samples were collected directly from the rectum and diluted 1:10 in PBS. After vigorous vortexing, faecal samples were centrifuged at 16.000 × g for 30 min and supernatants were taken to perform RT-PCR. All samples were stored at -80ºC until further use. Liver samples obtained at necropsy were also taken and fixed by immersion in 10% neutral-buffered formalin for histopathological studies.

Both experiments were performed in accordance with the guidelines of the Good Experimental Practices (GEP), under the supervision of the Ethical Welfare Committee on

Detection of anti-HEV antibodies

Serum and bile used as inoculum samples were tested for specific anti-HEV IgG antibodies by an in-house enzyme-linked immunosorbent assay (ELISA) previously described [START_REF] Martín | Association of hepatitis E virus (HEV) and postweaning multisystemic wasting syndrome (PMWS) with lesions of hepatitis in pigs[END_REF]. This assay was performed using a purified ORF2 truncated protein as antigen, which was obtained from a local strain of swine HEV genotype 3 that was expressed into recombinant baculovirus (Peralta et al., 2008b). To minimize the background on the ELISA results, each serum was analyzed both in coated and un-coated wells. The working solution for the anti-IgG conjugate was 1:40.000 (Serotec). The reaction was stopped with H 2 SO 4 2M and the optical density (OD) of each plate was read at 450 nm. Samples that had an OD value (antigen-coated well OD value less uncoated well OD value) equal or greater than the cut-off value (0.30) were considered positive [START_REF] Martín | Association of hepatitis E virus (HEV) and postweaning multisystemic wasting syndrome (PMWS) with lesions of hepatitis in pigs[END_REF]. All positive ELISA results were further tested by a Dot-blot test as previously described [START_REF] Casas | Retrospective serological study on hepatitis E infection in pigs from 1985 to 1997 in Spain[END_REF].

Detection of HEV RNA

Total RNA from serum, bile, rectal swabs and faecal suspension was extracted with Nucleospin® RNA virus kit (Macherey-Nagel), reverse-transcribed, and subjected to a semi-nested RT-PCR as previously described [START_REF] Martín | Association of hepatitis E virus (HEV) and postweaning multisystemic wasting syndrome (PMWS) with lesions of hepatitis in pigs[END_REF]. Sequencing analysis was performed on all resulting positive samples to verify that the HEV strain found in pigs was the same as the inoculated one. The resulting sequences were also compared with the one used as positive control in the RT-PCR to check that no cross contamination occurred. Serial dilutions of serum, faeces and bile were used to determine the titer of viral genomes by semi-nested RT-PCR. Viral concentrations were expressed as GE per A c c e p t e d M a n u s c r i p t 8 microliters of sample (serum, faecal suspension or bile) as described elsewhere (Meng et al., 1998a).

Histopathological studies

Fixed liver samples were dehydrated, embedded in paraffin and stained with hematoxylin and eosin. Microscopic analysis was performed with special emphasis for the presence or absence of hepatitis, the main lesion found in HEV infected pigs [START_REF] Meng | A novel virus in swine is closely related to the human hepatitis E virus[END_REF][START_REF] De Deus | Detection of hepatitis E virus in liver, mesenteric lymph node, serum, bile and faeces of naturally infected pigs affected by different pathological conditions[END_REF]. Hepatic lesions were scored following a previously described staging system (from I to IV) proposed by [START_REF] Rosell | Hepatitis and staging of hepatic damage in pigs naturally infected with porcine circovirus type 2[END_REF] to evaluate liver inflammation associated to porcine circovirus type 2 infection.

Results

Preliminary infectivity study

All four pigs inoculated IV with the swine HEV inoculum remained clinically healthy during the whole experimental period. Rectal temperatures remained within the normality rank throughout the study with an average (avg) of 39.7ºC (standard deviation (SD): 0.22).

RT-PCR results of HEV RNA detection in faeces and bile are shown in Table 1. Three out of 4 animals became infected. HEV was firstly detected in faeces on 3 dpi in animal A and on 7 dpi in animal B. Faecal shedding of pig A lasted for 1 week and only 4 days for pig B.

Viraemia was not detected in any studied animal. HEV RNA was not detected in faeces of pig C, although virus genome was detected in bile at necropsy (28 dpi). The sequencing of all RT-PCR products demonstrated a 100% nucleotide identity with respect to the inoculated virus. Pig D did not show any evidence of HEV infection by RT-PCR.

Transmission study

Control animals remained healthy during the whole experimental period. Pigs from the inoculated and contact group did not show clinical signs attributable to HEV during the experiment; however five animals, three from the inoculated group and two from the contact group, showed pyrexia and different clinical signs (prostration, difficult breathing and shivers), dying during the first 3 weeks of experiment. Rectal temperatures remained within normality in all the remaining animals: inoculated group, avg: 39.48ºC / SD: 0.27; sentinel group, avg: 39.52ºC / SD: 0.35; control group: avg: 39.54ºC / SD: 0.4. Bacteriological culture of different swabs (from cerebrospinal fluid and pericardium, thoracic and abdominal cavities) obtained at necropsy, revealed the presence of colonies compatibles with common pathogens of swine farms such as Haemophilus parasuis, Streptoccocus suis and Escherichia coli (data not shown).

No significant gross lesions were observed in any of the pigs from all three experimental groups (besides those that died because of systemic bacterial infections, which showed fibrino-purulent meningitis, polyserositis and/or polyarthritis). Slight perilobular or periportal hepatic fibrosis was histologically seen in animals 16 (inoculated) and 59 (contact), respectively. Slight or mild periportal hepatitis (stage I of [START_REF] Rosell | Hepatitis and staging of hepatic damage in pigs naturally infected with porcine circovirus type 2[END_REF]) was observed in pigs 17 and 89 (inoculated), 59 (contact) and 82 (control). No microscopic hepatic lesions were seen in the remaining pigs. Some animals had antibody titers of IgG at the beginning of the study: 8 from the HEV inoculated group; 1 from the contact group and 3 from the control group. Most of these animals lost maternal antibodies after 8 dpi.

A total of 4 out of 16 pigs presented one or more evidences of HEV infection (Table 2).

Three pigs from the HEV inoculated group shed virus in faeces, none of them had Nevertheless, these animals were able to infect contact sentinels; two contact pigs got infection, with presence of HEV in bile. None of the contact pigs had virus faecal shedding, viraemia or seroconversion at 64 dpi. Finally, control pigs remained seronegative throughout the study. None of them had HEV RNA in faeces, blood or bile during all the experimental period. Positive RT-PCR products showed a 100% nucleotide identity with respect of the inoculated virus. Faecal shedding was detected between 22 and 36 dpi (Table 2). Only one animal (pig 87) had HEV RNA detectable in faeces during different time points of the experiment. Pig 94 (euthanized at 16 dpi) had detectable virus in bile, but it was never recorded as positive in faeces. Pig 16 died at 22 dpi and was HEV RT-PCR positive both in bile and faeces at necropsy; serial dilutions of positive bile and faeces of this pig revealed that the genomic titer of HEV in bile (10 3 -10 4 GEs/ml) was higher than that in faeces (10 GE/ml). Seroconversion of inoculated pigs appeared 43 days after challenge onwards. In the contact group of pigs, virus was only detected in bile of two animals euthanized at 64 dpi.

Discussion

Porcine HEV was firstly described in 1997 [START_REF] Meng | A novel virus in swine is closely related to the human hepatitis E virus[END_REF]; since then, several experimental studies have been performed to clarify the pathogenesis of this viral infection.

Most of them used IV inoculation although oral HEV exposure would be the most natural route of infection. Few experimental infections have been performed using the latter route of exposure, clearly indicating the difficulty to cause HEV infection [START_REF] Kasorndorkbua | Use of a swine bioassay and a RT-PCR assay to assess the risk of transmission of swine hepatitis E virus in pigs[END_REF][START_REF] Kasorndorkbua | Routes of transmission of swine hepatitis E virus in pigs[END_REF][START_REF] Kasorndorkbua | Infectious swine hepatitis E virus is present in pig manure storage facilities on United States farms, but evidence of water contamination is lacking[END_REF]. The present report further investigated the oral route of administration of HEV, demonstrating its feasibility, and describes for the first time the successful transmission of HEV from orally inoculated pigs to sentinel ones. Other experiments A c c e p t e d M a n u s c r i p t 11 showed that pigs with direct contact exposure to positive controls got also infection, but those controls were inoculated IV (Meng et al., 1998a;[START_REF] Kasorndorkbua | Routes of transmission of swine hepatitis E virus in pigs[END_REF][START_REF] Bouwknegt | Estimation of hepatitis E virus transmission among pigs due to contact-exposure[END_REF].

Most of HEV experimental infections have mainly used faeces from infected pigs as inocula (Meng et al., 1998b;[START_REF] Kasorndorkbua | Use of a swine bioassay and a RT-PCR assay to assess the risk of transmission of swine hepatitis E virus in pigs[END_REF][START_REF] Kasorndorkbua | Routes of transmission of swine hepatitis E virus in pigs[END_REF][START_REF] Lee | Localisation of swine hepatitis E virus in experimentally infected pigs[END_REF]. In contrast, this study used bile from a naturally infected pig; this animal had higher amount of HEV genome copies in bile than faeces (data not shown). The used bile was previously tested by ELISA to ensure that specific IgG antibodies that may hinder HEV infection progress were not present.

The preliminary study performed demonstrated that the bile recovered from a naturally infected animal was infectious for pigs when inoculated IV. Two of the pigs (A and B) got infection because of the IV inoculation of the bile; however, a third pig only showed evidence of infection by RT-PCR at the end of the experiment, suggesting that infection probably occurred by oro-faecal transmission due to contact with the other two. The lack of virus detection in faeces and blood of this animal previous to its detection in bile may suggest that liver is the primary site of HEV replication, as described in other IV experimental infection [START_REF] Lee | Localisation of swine hepatitis E virus in experimentally infected pigs[END_REF]; therefore, bile would be one of the most important samples to find the virus, as has also been suggested under natural infection conditions [START_REF] De Deus | Detection of hepatitis E virus in liver, mesenteric lymph node, serum, bile and faeces of naturally infected pigs affected by different pathological conditions[END_REF]. It is worthy to note that a fourth animal did not show evidence of infection during the whole experimental period, which suggests an inadequate IV inoculation but also an apparent difficulty of horizontal transmission from infected pigs.

Faecal-oral route is believed to be the natural route of HEV transmission among pigs [START_REF] Meng | Swine hepatitis E virus[END_REF][START_REF] De Deus | Hepatitis E virus infection dynamics and organic distribution in naturally infected pigs in a farrow-to-finish farm[END_REF]. In fact, a recent study demonstrated the potential of HEV to cause epidemics in populations of pigs since the basic reproduction ratio (R 0 ) for contact-exposure was estimated to be 8.8 [START_REF] Bouwknegt | Estimation of hepatitis E virus transmission among pigs due to contact-exposure[END_REF]. In the present study, pigs could be infected orally and they were able to infect animals in contact.

A c c e p t e d M a n u s c r i p t 12

A previous study demonstrated that faeces containing a high titer of HEV (10 6 GEs/g) was able to infect animals when they were inoculated orally, but three inoculations of 15 g of faeces each were necessary to infect only one out of 3 animals [START_REF] Kasorndorkbua | Routes of transmission of swine hepatitis E virus in pigs[END_REF]. Although the present work has reproduced HEV infection successfully by oral inoculation, the infection rate was less efficient than when using IV inoculation, and orally inoculated animals showed faecal shedding and seroconversion later compared with IV inoculations [START_REF] Tsarev | Infectivity titration of a prototype strain of hepatitis E virus in cynomolgus monkeys[END_REF][START_REF] Kasorndorkbua | Routes of transmission of swine hepatitis E virus in pigs[END_REF][START_REF] Kasorndorkbua | Infectious swine hepatitis E virus is present in pig manure storage facilities on United States farms, but evidence of water contamination is lacking[END_REF]. Moreover, in the study cited above, pigs inoculated IV with an HEV dose of 10 6 GEs shed the same concentration of viral particles in faeces [START_REF] Kasorndorkbua | Routes of transmission of swine hepatitis E virus in pigs[END_REF]. In contrast, pigs from the present study inoculated orally with an HEV dose of 10 5 GEs excreted much lower concentrations of virus in faeces (data not shown). [START_REF] Mccaustland | Hepatitis E virus infection in chimpanzees: a retrospective analysis[END_REF] described that multiple variables may influence the outcome of HEV infection: virus titer, the ratio of infectious to defective particles, the route of infection and host factors such as immune status and age of exposure. The present results showed that a single dose of 10 5 GEs is insufficient to induce a large infection rate, since only four out of 16 inoculated pigs got measurable infection. Moreover, these four pigs could only infect two contact animals. Thus, it remains unclear whether oral route explains the high incidence of HEV infection in pig population [START_REF] Meng | Swine hepatitis E virus[END_REF][START_REF] Leblanc | Presence of hepatitis E virus in a naturally infected swine herd from nursery to slaughter[END_REF][START_REF] Satou | Transmission dynamics of hepatitis E among swine: potential impact upon human infection[END_REF][START_REF] Seminati | Distribution of hepatitis E virus infection and its prevalence in pigs on commercial farms in Spain[END_REF]. Some studies attributed this difference between experimental and natural conditions to the repeated exposure of animals to HEV that is probably needed for a successful transmission [START_REF] Kasorndorkbua | Use of a swine bioassay and a RT-PCR assay to assess the risk of transmission of swine hepatitis E virus in pigs[END_REF][START_REF] Kasorndorkbua | Routes of transmission of swine hepatitis E virus in pigs[END_REF][START_REF] Kasorndorkbua | Infectious swine hepatitis E virus is present in pig manure storage facilities on United States farms, but evidence of water contamination is lacking[END_REF]. On the other hand, pigs kept under experimental facilities imply box cleaning every day and, therefore, pigs probably do not have the same HEV infectious pressure as pigs in conventional farms where slurry is not often removed during all the growing period.

Another point that could influence the success of infection is a possible strain-dependent A c c e p t e d M a n u s c r i p t 13 infectivity, since many different subtypes have been described within genotype 3. Even though, this question has not yet been clarified [START_REF] Chobe | Full genome sequence and analysis of Indian swine hepatitis E virus isolate of genotype 4[END_REF].

How host factors influence HEV infection when pigs are inoculated orally is still not known [START_REF] Kasorndorkbua | Routes of transmission of swine hepatitis E virus in pigs[END_REF]. In the present study, all 16 inoculated pigs received the same dose, but a variety of responses was seen. Several facts might explain such response variability to infection. On one hand, conventional pigs usually have remaining humoral immunity of maternal origin. In the present study, half of these inoculated animals had residual IgG antibodies at the beginning of the study. Although this fact would apparently be detrimental to achieve infection, 3 out of the 4 pigs that got infection (No. 87, 94 and 95) had some residual HEV antibodies, while only one of them was seronegative to the virus. On the other hand, it has been described that host mucosal immunity in the alimentary tract could play a role in the susceptibility to HEV infection [START_REF] Kasorndorkbua | Use of a swine bioassay and a RT-PCR assay to assess the risk of transmission of swine hepatitis E virus in pigs[END_REF]. It is feasible that IgA may neutralize the virus on mucosal surfaces [START_REF] Tizard | Immunity at body surfaces[END_REF], which would explain the highest efficiency of IV inoculations than oral ones. Recent studies have demonstrated that IgA maternal antibodies can be transferred to piglets [START_REF] De Deus | Hepatitis E virus infection dynamics and organic distribution in naturally infected pigs in a farrow-to-finish farm[END_REF]. On the other hand, at least in dogs, this immunoglobulin can be present in bile to high amounts [START_REF] German | Measurement of IgG, IgM and IgA concentrations in canine serum, saliva, tears and bile[END_REF]; it can not be ruled out that a strong IgA positivity in bile used as inoculum might interfere the outcome of HEV infection. In the present work, IgA antibodies were not determined, so it remains unknown the role of them in the success of oral infections.

Data obtained on virus shedding and seroconversion agree with results showed in a previous oral inoculation study in which only one animal got infection [START_REF] Kasorndorkbua | Routes of transmission of swine hepatitis E virus in pigs[END_REF]. This animal started to shed virus in faeces from 21 dpi to 35 dpi and seroconverted by 56 dpi; bile was not tested in the mentioned study. In contrast, some differences are observed when comparing data from oral inoculations and IV ones. When animals are inoculated IV, virus in faeces is firstly detected at 7 dpi and lasts up to 3 A c c e p t e d M a n u s c r i p t weeks. Virus in bile is firstly detected at 3 dpi and seroconversion appears only 2 weeks after challenge [START_REF] Halbur | Comparative pathogenesis of infection of pigs with hepatitis E viruses recovered from a pig and a human[END_REF]. Oral intake of HEV needs to pass through the intestinal tract, which implies a delayed appearance of virus in faeces and bile as well as seroconversion compared to IV inoculation [START_REF] Kasorndorkbua | Routes of transmission of swine hepatitis E virus in pigs[END_REF][START_REF] Bouwknegt | Estimation of hepatitis E virus transmission among pigs due to contact-exposure[END_REF]. Moreover, the same dose given by one or the other route show different bioavailability, which means that the real amount of virus inoculated per os reaching the systemic circulation, can be rather low.

It has been described that HEV RNA is more frequently detected in faeces and for longer periods than in blood [START_REF] Wu | Spread of hepatitis E virus among different-aged pigs: two-year survey in Taiwan[END_REF][START_REF] De Deus | Detection of hepatitis E virus in liver, mesenteric lymph node, serum, bile and faeces of naturally infected pigs affected by different pathological conditions[END_REF][START_REF] Leblanc | Presence of hepatitis E virus in a naturally infected swine herd from nursery to slaughter[END_REF].

Therefore, to certain degree, it should not be surprising that viraemia was not detected in the present study. It is possible that a localized HEV replication in the intestines, low amounts of HEV particles in serum as a consequence of the low infection pressure, or an intermittent viraemia could explain the negative results of RT-PCR of studied pigs in sera.

Transmission of HEV infection to contact sentinels appeared approximately 1 month after HEV-excretion occurrence in inoculated pigs. In an experimental infection performed to quantify the transmission of HEV among pigs showed that this period was only 6 days for contact sentinels exposed to pigs previously in contact with IV infected animals [START_REF] Bouwknegt | Estimation of hepatitis E virus transmission among pigs due to contact-exposure[END_REF]. This result is in contrast with data from the present study, but the source of infection (IV versus oral) may explain a much lower infection pressure for the latter inoculation route, thus, reducing the probability of infection and prolonging the period between exposure and infection.

The histopathological liver lesions observed in only five animals were slight and could not have any relation to virus detection since only one of these pigs got infection. In fact, it has been described that mild, non-suppurative hepatitis can be considered as normal background of pig liver [START_REF] Halbur | Comparative pathogenesis of infection of pigs with hepatitis E viruses recovered from a pig and a human[END_REF]. In regards the five animals that died naturally during the second experiment, they probably were carriers of common pathogens from the In conclusion the results from this study demonstrated that pigs orally inoculated with a single HEV dose of 10 5 GEs got infection. Although few animals had evidence of infection, thus confirming the more difficult reproduction of an oral HEV infection than IV one, those animals were able to successfully transmit the virus to direct contact exposure sentinels.

  A c c e p t e d M a n u s c r i p t Human and Animal Experimentation of the Universitat Autònoma de Barcelona (CEEAH), Spain.

  Pig A seroconverted to anti-HEV IgG by 21 dpi (confirmed by ELISA and Dot-blot). No other pig seroconverted at any time of the experimental period. Bile used as inoculum did not contain specific anti-HEV IgG antibodies.

9

  None of the animals had significant macroscopic and microscopic lesions.

  two had virus RNA in bile at the day of necropsy and two of them seroconverted to IgG anti-HEV antibodies (confirmed by ELISA and Dot-blot).

  , when specific maternal immunity waned, bacteria were able to cause disease in a limited number of pigs.

Acknowledgements

The authors thank N. Galofré, M. Pérez and R. Valle for technical assistance and to all the personal of biosafety level 3 facilities of CReSA. This study was supported by the research grant AGL2004/06688 and Consolider-Ingenio 2010 (CSD2006-0007) from the Spanish government. Maribel Casas and Bibiana Peralta are funded by a pre-doctoral FI grant of the Government of Catalunya and Nilsa de Deus has a fellowship from CReSA.