Axel Mauroy

Alexandra Scipioni

Elisabeth Mathijs

Claude Saegerman

Jan Mast

Janice C Bridger

Dominique Ziant

Christine Thys

Etienne Thiry
email: etienne.thiry@ulg.ac.be

EPIDEMIOLOGICAL STUDY OF BOVINE NOROVIRUS INFECTION BY RT-PCR AND A VLP-BASED ANTIBODY ELISA

Keywords: norovirus, epidemiology, seroprevalence, PCR, bovine

Noroviruses, belonging to the family Caliciviridae, have been identified in human beings and in several animal species including cattle. The distribution of bovine norovirus infections was investigated by both RT-PCR to detect norovirus genomes and a virus-like particles-based ELISA to detect genotype 2 bovine norovirus antibodies. During a one-year systematic study, a virus prevalence of 7.5% (CI 95% : [3.7 ; 13.4%]) (10 out of 133 samples) was found in stool samples from diarrhoeic calves screened by RT-PCR. Nucleotide sequencing performed on the polymerase region classified all the norovirus amplicons in the bovine norovirus genotype 2. Rather surprisingly, some rotavirus sequences were also detected. On the basis of the polymerase region, genotype 1 bovine norovirus was not identified. Other enteropathogens were found in all samples. By ELISA, a genotype 2 seroprevalence of 93.2% (CI 95% : [90.4 ; 95.3%]) was found from calves and adult cattle. Antibody levels against genotype 2 bovine noroviruses rose in the first 6 months of life and were maintained in adults.

Together the results of virus prevalence and seroprevalence studies suggest that bovine norovirus infection occurs early in life and that re-infection with serologically related bovine noroviruses strains could occur in adult cattle as reported for rotaviruses. The antibody rise against genotype 2 bovine noroviruses in the adult cattle also suggests a short lived and/or strain specific immunity as already shown in human noroviruses. Genotype 2 bovine noroviruses are endemic in the region investigated.

Introduction

Noroviruses (NoV) belong to the family Caliciviridae and have been identified in the human being and in several animal species including the bovine (Scipioni et al., 2008b;[START_REF] Green | Caliciviridae : The Noroviruses[END_REF].

Noroviruses are small, round, non-enveloped viruses, with a 25-40 nm diameter and have a positive-sense, single-stranded RNA genome with three open reading frames (ORF). The non structural proteins, including the viral RNA dependent RNA polymerase (RdRp), the single capsid protein (VP1) and the minor structural protein are encoded by ORF1, 2 and 3 respectively [START_REF] Green | Caliciviridae : The Noroviruses[END_REF]. Ninety dimers of VP1 form the capsid which presents an icosahedral symmetry under electron microscopy (EM). Capsid protein VP1 has a molecular weight of around 55-60 kDa and is organised into two domains: a shell (S) domain (Nterminal part of VP1) and a protruding (P) domain (C-terminal part of VP1) [START_REF] Prasad | X-ray crystallographic structure of the Norwalk virus capsid[END_REF]. The P domain supports receptor recognition and immunogenic functions [START_REF] Lochridge | Epitopes in the P2 domain of norovirus VP1 recognized by monoclonal antibodies that block cell interactions[END_REF]. Noroviruses are difficult to grow in cell culture but abundant quantities of viruslike particles (VLP) can be obtained by expressing VP1 in the baculovirus protein expression system. These VLP are morphologically and antigenically similar to the native strain [START_REF] Jiang | Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein[END_REF].

Bovine norovirus (BoNoV) prototype strains identified so far are Newbury2 strain (NB2), previously known as Newbury Agent 2, and the Jena virus (JV). The NB2 and JV were identified by EM in the faeces of diarrhoeic calves in 1978 in Great Britain [START_REF] Woode | Isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves[END_REF] and in 1980 in Germany [START_REF] Gunther | Studies on diarrhoea in young calves. 7. Serrated virus (Jena-Agent 117/80) -a new diarrhoea pathogen in calves[END_REF]. They were later genetically identified as belonging to the genus Norovirus [START_REF] Liu | Molecular characterization of a bovine enteric calicivirus: relationship to the Norwalk-like viruses[END_REF][START_REF] Oliver | Molecular characterization of bovine enteric caliciviruses: a distinct third genogroup of noroviruses (Norwalk-like viruses) unlikely to be of risk to humans[END_REF] where all BoNoV strains fall into the genogroup III [START_REF] Ando | Genetic classification of Norwalk-like viruses[END_REF]. Two genotypes are described in the BoNoV genogroup: viruses genetically related to JV were assigned to the genotype 1 and viruses genetically related to NB2 to the genotype 2 [START_REF] Ando | Genetic classification of Norwalk-like viruses[END_REF].

A c c e p t e d M a n u s c r i p t 4 Their relative environmental stability, their main faecal-oral transmission route, their low infectious dose and their large strain diversity increase the risk of cattle infection with these viruses [START_REF] Green | Caliciviridae : The Noroviruses[END_REF]. In gnotobiotic calves, BoNoV induce non-hemorrhagic enteritis, mild diarrhoea, transient anorexia and malabsorption [START_REF] Woode | Isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves[END_REF]. But the real impact of these viruses in the field is currently poorly evaluated. The two genotypes of BoNoV were recently shown to be endemic in Great Britain and Germany (Oliver et al., 2007b). They were also isolated in all continents and thus seem to be widespread (Scipioni et al., 2008b).

In this work, an original combined virological and serological approach was used to characterise the epidemiology of BoNoV infection in cattle. In a first virological approach, the molecular prevalence was investigated in diarrhoeic calf samples during a one year study.

In a second serological approach, a VLP-based ELISA was developed and the cattle seroprevalence against BoNoV was estimated in the same region. Together the results suggest an association of BoNoV infections with the age of the animals. Four different primer pairs were used to amplify segments of the polymerase gene and the beginning of the capsid region: CBECu [START_REF] Smiley | Reverse transcription-PCR assays for detection of bovine enteric caliciviruses (BEC) and analysis of the genetic relationships among BEC and human caliciviruses[END_REF], JV12/13 [START_REF] Vinje | International collaborative study to compare reverse transcriptase PCR assays for detection and genotyping of noroviruses[END_REF], P289/290 [START_REF] Jiang | Design and evaluation of a primer pair that detects both Norwalk-and Sapporo-like caliciviruses by RT-PCR[END_REF] and BEC [START_REF] Ike | Identification of bovine enteric Caliciviruses (BEC) from cattle in Baden-Wurttemberg[END_REF]. These primers were selected in order to perform the broadest detection of NoV including a possible zoonotic transmission. Out of the BoNoV specific primers (CBECu and BEC), CBECu pair was seen to detect not only genotypes 2 but also genotypes 1 BoNoV with an apparent sensitivity of about 83% relative to the p289/290 pair in a previous study [START_REF] Smiley | Reverse transcription-PCR assays for detection of bovine enteric caliciviruses (BEC) and analysis of the genetic relationships among BEC and human caliciviruses[END_REF]. RT-PCR program consisted in a reverse transcription step of 45 min at 45°C followed by the PCR reaction. It consisted in an initial denaturation step of 2 min at 94°C followed by 40 PCR cycles (30 sec denaturation step at 94°C; 1 min annealing step at 48°C for CBECu, JV12/13 and BEC primers, 51°C for p289/290 primers; 2 min extension step at 68°C) and a final extension step of 10 min at 68°C. BLAST tool (available at http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). Phylogenetic inference was performed with the MEGA version 4 software package [START_REF] Tamura | MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0[END_REF].

Phylogenetic tree was constructed by neighbor-joining analysis where evolutionary distances were computed using the Maximum Composite Likelihood method. The confidence values of the internal nodes were calculated by performing 1,000 replicates bootstrap values.

Construction of a recombinant baculovirus for the BoNoV capsid protein

A long fragment product (RdRp region to the poly-A tail) from the genome of a genotype 2 BoNoV strain previously isolated in the laboratory (named B309) was generated by RT-PCR and cloned into pTopoXL (Invitrogen, Carlsbad, CA, USA). The insert was sequenced by primer walking. The sequence coding for the ORF2 was isolated and mutations in this sequence were evaluated with the vector NTI 6 software (InforMax, North Bethesda, MD, USA). Specific forward and reverse PCR primers to amplify the ORF2 were selected using the Vector NTI program (InforMax, North Bethesda, MD, USA). These primers include the (5,000 U/ml)-streptomycin (5 mg/ml) (Invitrogen, Carlsbad, CA, USA) and subcultured when they reached confluence. Transfection of Sf9 cells was conducted in 6-wells according to the manufacturer protocol to generate a recombinant baculovirus for the BoNoV capsid protein.

Expression and purification of the capsid protein of a bovine norovirus

A recombinant baculovirus stock was generated and monolayers of Sf9 insect cells were infected at a multiplicity of infection of 10. Five days post-infection, cells and supernatants were harvested and submitted to three freezing/thawing cycles. It was followed by low speed centrifugation to remove cell debris and by high speed centrifugation through 5% sucrose cushion in a Beckman SW28 rotor at 100,000 x g for 2 h. The pellet was suspended in phosphate buffered saline (PBS) and centrifuged through a 35% to 65% sucrose gradient at 100,000 x g for 18 h in a Beckman SW28 rotor. The gradient was fractionated and each fraction was dialysed separately against PBS in 25 kDa porous Spectra/Por membrane (VWR, Leuven, Belgium). Protein concentration in each gradient fraction was evaluated by BCA (Pierce, Rockford, IL, USA). Fractions were then subjected to verification for the presence of either capsid protein as VLP.

Validation of the production of the bovine norovirus-like particles

An horse serum and the 1,000-fold diluted calf serum. After three washes with PBST 0.05%, the membrane was incubated for 1h in PBS containing 20% horse serum and a horseradish peroxidase (HRP) conjugated, 10,000-fold diluted, anti-bovine immunoglobulins (Ig) G rabbit IgG. After three washes with PBST 0.05%, the chemiluminescent peroxidase substrate (Sigma-Aldrich, Salt Lake city, UT, USA) was added on the membrane and it was revealed in dark room on Kodak BioMax Light film (Sigma-Aldrich, Salt Lake City, UT, USA).

Serum sampling

Blood samples were taken from 439 calves and adult cattle from different premises in Belgium. Serum samples originated from the same area that stool samples but from different premises. The sera were grouped according to the age of the animals. Seven age classes were determined: 1 week to 1 month old (n=20), 1 to 6 months old (n=29), 6 months to 1 year old (n=76), 1 to 3 years old (n=105), 3 to 6 years old (n=123), 6 to 9 nine years old (n=59) and 9 years old and older (n=27). In the addition to the 439 sera, a collection of 30 serum samples of colostrum deprived newborn calves from the same region was used as a negative serum control. A commercial bovine anti-rabbit IgG antibody (Bethyl laboratories, Montgomery, TX, USA), purified by affinity chromatography, was also used to test the specificity of the ELISA. The serum of the NB2 experimentally infected calf P131 was used as a positive control [START_REF] Bridger | Characterization of a calici-like virus (Newbury agent) found in association with astrovirus in bovine diarrhea[END_REF].

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

Molecular prevalence of bovine norovirus infection

A molecular prevalence of 7.5% (CI 95% : [3.7 ; 13.4%]) for BoNoV infection was found in the stool samples of diarrhoeic calves. Twelve potentially positive amplicons were generated by use of the different primer pairs: 9 with the CBECu pair, 1 with both the CBECu and the BEC pairs, 1 with both the CBECu and the JV12/13 pairs and 1 only with the BEC pair. No amplicon was generated with the p289/290 pair. By BLAST and phylogenetic analysis, 10 amplified polymerase sequences were genetically related to NoV genogroup III genotype 2 strains (Fig. 1). The two other ones, amplified with the CBECu pair at a lower molecular size than expected (about 418 bp), matched with about 90% identity with bovine rotavirus polymerase (VP1) gene sequence. These two samples were also confirmed rotavirus-positive by ELISA based on the rotavirus VP6 detection.

BoNoV were found equally in diagnostic and necropsy samples. Another etiology (rotavirus, coronavirus, Cryptosporidium parvum or Escherichia coli) was found in all the 10 BoNoV positive samples by different tests performed in a diagnostic laboratory. Remarkably, the sample BV119 was positive by ELISA for bovine rotavirus but no rotavirus nucleotidic sequence was amplified with the CBECu pair in this sample.

Virus-like particles of the bovine norovirus isolate B309

The B309 BoNoV isolate was previously related to the genotype 2 BoNoV in its RdRp region. By BLAST after the sequencing of its C-terminal part, the B309 isolate shared 89% of nucleotide and 98% of amino acid identity with the NB2 in the entire capsid gene. The B309_EcoRI/B309_HindIII amplified ORF2 sequence was compared to the original sequence and two mutations were found. The first mutation was a transition in the 5 th codon and modified the composition in amino acid (Asp 5 → Asn 5 ). The second mutation was located in to 40 nm range diameter with typical calicivirus cup-shaped depressions were observed under EM examination of some positive fractions of the gradient (Fig. 3).

Western blot analysis

By western blot analysis, fractions containing B309 VLP reacted at about 57 kDa with a serum resulting from the immunization of a calf with NB2 strain. A weak positive band was also obtained in the lane containing Sf9 cells infected by a wild type baculovirus (Fig. 4A).

Western blot performed under non denaturing conditions revealed a band at about 120 kDa with VLP but not against mock infected or Sf9 cell components recovered after wild type baculovirus infection (Fig. 4B).

Seroprevalence against BoNoV genotype 2 in the Belgian cattle

Only fractions positive to EM examination (visualization of VLP) were used in the ELISA format. Analysis of sera from colostrum deprived newborn calves allowed the definition of a cut-off value of 0.3 for the ELISA. Optical density values obtained for the bovine anti-rabbit IgG serum (mean: 0.1) were similar to those of colostrum deprived newborn calves. By statistical analysis, a high seroprevalence of 93.2% (CI 95% : [90.4 ; 95.3%]) was found in Belgian cattle sera. Significant statistical differences were found between the mean OD value of different age groups of animals: between the colostrum deprived calves and the 1 week to 1 month old calf groups, the 1 to 6 months old and 6 months to 1 year old groups, and the 6 months to 1 year old group and the 1 to 3 years old group. In the boxplot graph, median value of the "1 week to 1 month old" class rose dramatically compared to the median value of the A c c e p t e d M a n u s c r i p t 12 newborn class. This value was maintained during the "1 month to 6 months old" period and was followed by a new rise in the "6 months to 1 year old" age class. A drop in the values of medians was shown between this last class and the "1 to 3 year old" class. Medians were maintained thereafter in the adult period (Fig. 5). Remarkably, during this period, some OD values were below the cut-off (dots below the line at 0.3 OD value in fig. 5) and were similar to the values recorded during the colostral period.

A c c e p t e d M a n u s c r i p t 13

Discussion

Although HuNoV are known to be widely distributed and already recognized as a threat to public health, the impact of BoNoV is still poorly studied. In Europe, very few countries have performed epidemiological studies. The one year prospective study reported in the present paper endorsed that genotype 2 BoNoV are endemic in European cattle. A BoNoV prevalence of 7.5% was found in stool samples of diarrhoeic calves. In the serological approach, a strong seroprevalence (93.2%) against genotype 2 BoNoV was determined with a VLP-based antibody ELISA in cattle of the same region. Together, the results of prevalence and seroprevalence allowed the description of the evolution of the antibody levels against genotype 2 BoNoV infection during the life of the cattle.

The molecular prevalence of BoNoV in samples coming from the Belgian diagnostic laboratory was in accordance with studies in Great Britain [START_REF] Milnes | Retrospective study of noroviruses in samples of diarrhoea from cattle, using the Veterinary Laboratories Agency's Farmfile database[END_REF] and Korea where Park et al. [START_REF] Park | Molecular epidemiology of bovine noroviruses in South Korea[END_REF] report also a majority of genotype 2 infection. The genotype of the strains detected in the British study was not clarified. The prevalence determined by RT-PCR analysis in this study is also similar to the results obtained by an antigen capture ELISA in Germany and developed using a genotype 1 JV strain [START_REF] Deng | Studies of epidemiology and seroprevalence of bovine noroviruses in Germany[END_REF]. As genotypes 1 and 2 BoNoV are antigenically distinct [START_REF] Oliver | Genotype 1 and genotype 2 bovine noroviruses are antigenically distinct but share a cross-reactive epitope with human noroviruses[END_REF], the true BoNoV prevalence could be higher or a preferential geographic distribution could exist. In contrast, [START_REF] Mattison | Human noroviruses in swine and cattle[END_REF] detected only 1.6% positive in stool samples from a random study in Canadian cattle but [START_REF] Smiley | Reverse transcription-PCR assays for detection of bovine enteric caliciviruses (BEC) and analysis of the genetic relationships among BEC and human caliciviruses[END_REF] detected up to 72% of samples positive in two farms in USA. In a longitudinal study in The Netherlands, prevalences of 31.6% and 4.2% were found in farm stool samples and individual samples of dairy cattle respectively [START_REF] Van Der Poel | Epidemiology of Norwalk-like virus infections in cattle in The Netherlands[END_REF]. Thus, prevalence results by RT-PCR seem to depend on the continent, the country, the sampling strategy and especially on the test conditions (e.g.

A c c e p t e d

M a n u s c r i p t 14 annealing temperature, primers, use of internal control as mentioned in Scipioni et al., 2008a).

Also the type of study, cross-sectional versus longitudinal as a part of the Dutch study, could explain some differences. BoNoV infection seems to be strongly related to the age with a higher rate in the first year of the animal life. Positive results were obtained along all the sampling period (data not shown) but further research is needed to investigate a potential seasonality for the infection unlike as described for HuNoV [START_REF] Mounts | Cold weather seasonality of gastroenteritis associated with Norwalk-like viruses[END_REF]. In our results, we detected more isolates with the CBECu pair than with all the other pairs. Also, the CBECu primers allowed the detection of two bovine rotavirus infections with however a small difference in the molecular weight for the amplicon (418 bp versus 532 bp). To our knowledge, such observation has never been reported and it could account for some differences between RT-PCR prevalence results depending on the primers used. Nucleotide similarities were found between the CBECu primers and the prototype bovine rotavirus sequence to which they matched by BLAST. On the 23 nt of the CBECu-F sequence, 14 nt including the last five nt matched exactly with the sequence of the bovine rotavirus polymerase (VP1, genbank accession number: J04346). Nucleotide similarity for CBECu-R was smaller. The CBECu-F primer was designed to match the widely conserved YGDD motif in RNA-dependent RNA polymerases of RNA viruses [START_REF] Bruenn | Relationships among the positive strand and double-strand RNA viruses as viewed through their RNA-dependent RNA polymerases[END_REF] and this could explain the detection of bovine rotavirus. Some strains were detected with primers designed to detect HuNoV (JV pair) but at a low rate, as previously reported [START_REF] Van Der Poel | Epidemiology of Norwalk-like virus infections in cattle in The Netherlands[END_REF]. No

HuNoV sequences were identified despite the use of two primer sets designed for their detection, suggesting at least a very low prevalence of these human viruses in cattle. Despite the variety of the primers used in the present study, the lack of detection of the genotype 1

BoNoV could be explained by suboptimal primer use for the RT-PCR (Oliver et al., 2007b).

The age of the cattle population studied was also suggested as an explanation for the lower detection of genogroup III genotype 1 NoV (Oliver et al., 2007b). As RT-PCR primers used strains, the possibility that some positive samples contained chimaeric bovine noroviruses could be another explanation for the low apparent prevalence of GIII.1 noroviruses.

Interestingly, few amino acid mutations were detected between the native B309 strain sequence (year of isolation: 2003) and the NB2 strain sequence (year of isolation: 1976) (Scipioni et al., submitted). Nearly all mutations were located in the P domain with surprisingly relatively few mutations in the P2 sub-domain, the most variable region of VP1 known to support antigenic determinants and drift-induced antigenic variation in NoV strains [START_REF] Allen | Analysis of Amino Acid Variation in the P2 Domain of the GII-4 Norovirus VP1 Protein Reveals Putative Variant-Specific Epitopes[END_REF][START_REF] Lochridge | A single-amino-acid substitution in the P2 domain of VP1 of murine norovirus is sufficient for escape from antibody neutralization[END_REF].

The two mutations detected in the VLP constructed in the baculovirus system compared to the native B309 strain, did not affect the antigenicity by their effect and their localisation in the amino acid sequence. Oliver et al. (2007a) reported that the P131 calf serum did not react in western blotting with BoNoV genotype 2 VLP in denaturing reaction but react at a higher molecular weight than expected under non denaturing conditions. In our conditions this serum reacts against a fraction containing VLP but also with infected Sf9 cells, in denaturing conditions. To test the specificity of this reaction against infected Sf9 cells, western blots were carried out with foetal calf serum and serum from a colostrum deprived newborn calf used in this study. With these two sera, a positive band at the same molecular weight was observed with baculovirus infected Sf9 cells under denaturing conditions whereas no reaction was detected in non denaturing conditions (Mauroy and Thiry, unpublished results). These data confirm the non specific binding of bovine immunoglobulins on denatured protein extracts from baculovirus infected Sf9 cells. In non denaturing conditions, the serum only reacts against VLP. Thus, B309 VLP are representative of the genotype 2 BoNoV.

A high seroprevalence (93.2%) against genotype 2 BoNoV was found by ELISA. Genotypes 1 and 2 in the BoNoV genogroup, although antigenically distinct, were shown to cross-react A c c e p t e d M a n u s c r i p t 16 in ELISA to the heterologous VLP but at low serum dilution (log 10 1.7) [START_REF] Oliver | Genotype 1 and genotype 2 bovine noroviruses are antigenically distinct but share a cross-reactive epitope with human noroviruses[END_REF].

However the true genotype 2 seroprevalence should still remain high, even corrected for a lower sensitivity and specificity of the test. The OD values obtained with the serum raised against rabbit IgG in bovine confirmed the selected cut-off. Almost all colostrum deprived calves did not have antibodies against BoNoV as previously described [START_REF] Deng | Studies of epidemiology and seroprevalence of bovine noroviruses in Germany[END_REF]. It suggests that BoNoV infection is mainly localised in the intestine, according to the observations with HuNoV [START_REF] Mattner | Risk groups for clinical complications of norovirus infections: an outbreak investigation[END_REF], and that vertical transmission of the virus is rare. The rise in antibody levels in one or two weeks-old calves suggests an acquisition of antibodies through colostrum absorption or from early infection. Information on prevalence brought by the RT-PCR analysis suggests that the risk of infection during the first six months of life is high. The antibody levels determined by ELISA rise again in the post-colostral period (6 months to one year-old). These results could be explained by a large exposure during this period and the development of natural immunity.

The finding that cattle aged 3 to 9 years still had antibodies suggests either a long lasting immunity against BoNoV or frequent re-infections. Several characteristics of NoV (environmental resistance, fecal-oral transmission) and farming practices in developed countries could increase the risk of re-infection in cattle, whereas such re-infection was not observed during a prospective RT-PCR study in The Netherlands [START_REF] Van Der Poel | Epidemiology of Norwalk-like virus infections in cattle in The Netherlands[END_REF] and suggest that antibody levels decrease in some adult cattle reinforcing the hypothesis of a short lived immunity.

The zoonotic risk associated with animal NoV is widely discussed but is hypothetical.

However, several recent data showed that HuNoV replication occurred experimentally in gnotobiotic animals including calves [START_REF] Souza | Pathogenesis and immune responses in gnotobiotic calves after infection with the genogroup II.4-HS66 strain of human norovirus[END_REF]. Furthermore, [START_REF] Mattison | Human noroviruses in swine and cattle[END_REF] have identified HuNoV sequences in swine and cattle. Shellfish like oysters are known as a main route of food-associated HuNoV outbreaks. As they bind specifically HuNoV to their digestive tissues (Le [START_REF] Guyader | Norwalk virus-specific binding to oyster digestive tissues[END_REF] and as BoNoV sequences were also detected in these molluscs [START_REF] Costantini | Human and animal enteric caliciviruses in oysters from different coastal regions of the United States[END_REF], new recombinant strains could appear through mixed infection. Such event was already reported with human strains [START_REF] Symes | Norovirus mixed infection in an oyster-associated outbreak: an opportunity for recombination[END_REF]. Mixed infections could occur in countries with high population densities of human and production animals and with high prevalence of BoNoV in premises. However, this study did not detect any contaminations of cattle with HuNoV.

Experimental infection of calves with BoNoV has shown their low pathogenicity [START_REF] Bridger | Characterization of a calici-like virus (Newbury agent) found in association with astrovirus in bovine diarrhea[END_REF] but their impact in association with classic enteric pathogens in superinfections and in economic losses (weight gain, health status, veterinary treatment cost) has not been studied.

In this study, BoNoV were found both in diagnostic samples and necropsy samples and Results for some fractions are reported here (lanes 1 to 9). Proteins of about 57 kDa, the expected molecular weight for the B309 capsid protein, were shown. Supernatant strain [START_REF] Bridger | Characterization of a calici-like virus (Newbury agent) found in association with astrovirus in bovine diarrhea[END_REF]), mo: month, wk: week, yr: year.

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

  stool samples and genetic analysis Stool samples from calves aged 1 week to 6 months were systematically received from a Belgian diagnostic laboratory each month through the year 2007. Samples came from different places out of five provinces of Belgium (Hainaut, Namur, Liege, Luxembourg, Walloon Brabant). Their origin was either from diarrhoeic samples (n=74) submitted for etiologic diagnosis of gastroenteritis or from necropsy samples (n=59) where gastroenteritis lesions were found. This stool bank (n=133) was screened by one step RT-PCR for the presence of BoNoV genome sequences with Quick Access kit (Promega, Madison, WI, USA).

  Positive amplicons were purified from agarose gel and cloned into pGEMt Easy (Promega, Madison, WI, USA). Sequencing reactions were carried out on three clones of each amplicon with the BigDye terminator kit version 3.1 (AppliedBiosystem, Foster City, CA, USA) and resolved with an ABI 3730 automatic capillary sequencer (AppliedBiosystem, Foster City, ). Nucleotidic sequences were analysed with the BioEdit Sequence Editor version 7.0 software. Nucleotidic similarity with the NCBI genetic database was assessed using the

  site upstream and downstream of the start and stop codon of the ORF2 respectively. The PCR product was cloned into the EcoRI/HindIII digested pFastBac Dual plasmid of the bac-to-bac system (Invitrogen, Carlsbad, CA, USA). A recombinant plasmid was used to transform MAX Efficiency DH10bac competent cells (Invitrogen, Carlsbad, CA, USA) where transposition between the recombinant pFastBac Dual and a baculovirus shuttle vector (bacmid) occurred. Recombinant bacmid purification was performed from positive clones with the Plasmid Purification Mega kit (Qiagen, Valencia, CA, USA). The transposition was controlled by PCR with M13 primer pair. Monolayers of Spodoptera frugiperda (Sf9) insect cells were grown in Sf-900 II medium Carlsbad, CA, USA) supplemented with a 10% association of penicillin

  aliquot of each fraction was analysed by Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) with the NuPAGE kit (Invitrogen, Carlsbad, CA, USA) to select fractions containing proteins at a molecular weight of about 55-60 kDa. These fractions were analysed under 80 kV EM examination (Philips EM208 transmission electron microscope). Prior to the examination, samples were submitted to ultracentrifugation on grids with increased hydrophilicity by Alcian blue pre-treatment and subjected to negative staining by phosphotungstic acid or uranyl acetate.

  calf experimentally infected with NB2[START_REF] Bridger | Characterization of a calici-like virus (Newbury agent) found in association with astrovirus in bovine diarrhea[END_REF] was used in western blot to detect the B309 BoNoV capsid protein. Briefly, a positive fraction, non infected Sf9 cells and wild type baculovirus infected Sf9 cells were blotted to a nitrocellulose membrane after electrophoresis under denaturing or non denaturing conditions. The membrane was blocked overnight at room temperature with PBS complemented with 0.05% tween 20 (PBST 0.05%) and 20% horse serum. The second day, blocking solution was removed and the membrane was incubated for 1h at room temperature in PBS containing 20%

  codon and was a silent mutation. A recombinant bacmid for the B309 ORF2 was generated and 72 hours after recombinant baculovirus infection, proteins of about 57 kDa could be detected by SDS-PAGE analysis in several fractions obtained by the gradient applied on harvested cells and supernatants (Fig.2). Bioinformatic analysis with the Vector NTI software predicted a similar molecular weight for the B309 capsid protein. Particles from 30

  only target the viral polymerase region and thus miss potential recombinant

  during a cross-sectional RT-PCR study performed by ourselves on stool from up to 6 months old cattle (n=47) housed in two different premises (Mauroy and Thiry, unpublished results). However in a retrospective study in Great Britain, Milnes et al. (2007) have amplified norovirus sequences by RT-PCR in adult stool samples. The detection of high levels of antibodies in the adult also suggests their presence in the colostrum. It could explain the antibody rise in neonates after colostrum intake. Remarkably, in contrast to the 1 week to 1 year old group, some OD values of adult cattle were below the cut-off of the test. These data A c c e p t e d M a n u s c r i p t

  always in association with other pathogens. The role of BoNoV as pathogens in the bovine species should be considered, taking into account their endemicity in the investigated situation and their presence in diarrhoeic calves More specific primers, primers targeting both the ORF1-end and the ORF2 regions, a broader sampling strategy, in particular regarding the age of the animals, and an ELISA including VLP of the genotype 1 could refine epidemiological data for BoNoV infection. An Ig discriminative ELISA format (IgA, IgM, IgG1 and 2) could define the status of the animals during their first year of life.

Figure 2 .

 2 Figure 2. Supernatants of the baculovirus expression of the capsid protein of the B309 isolate

  infected by a wild type baculovirus (lane WT) was used as negative control. MwM : molecular weight marker.

Figure 3 .

 3 Figure 3. Electron micrographs of Virus-like particles (VLP) of the bovine norovirus B309

Figure 4 .

 4 Figure 4. Western blotting of the expressed capsid protein of B309, mock infected Sf9 cells

Figure 5 .

 5 Figure 5. Seroprevalence against bovine noroviruses in the Belgian cattle. The OD means of

  Figure 1

  Figure 3

Figure 5

 5 Figure 5

2.6. Evaluation of the seroprevalence against BoNoV by ELISA Positive

  gradient fractions by both SDS-PAGE analysis and EM examination were selected for the ELISA. Ninety-six well Microlon plates (Greiner Bio-one, Piscataway, NJ, USA) were

	coated with 100 µl of a VLP solution in PBS (2 ng/µl) and incubated 1 h at 37°C. They were
	washed once with PBS and then blocked with PBS complemented with 0.1% tween20 (PBST
	0.1%) and with a 4% casein hydrolysate (PBST/HC). Plates were washed once with PBS and
	then incubated with the 100-fold diluted bovine serum samples in triplicate at 37°C. After 1 h
	and three washes with PBST 0.1%, 10,000-fold diluted, HRP conjugated rabbit IgG

anti-bovine IgG were applied to each well. After incubation at 37°C for 1 h, wells were washed three times with PBST 0.1% and 50 µl of the substrate of HRP, tetramethylbenzidine (Sigma-Aldrich, Salt Lake city, UT, USA), were added. The reaction was stopped after 5 min by addition to each well of 50 µl of H 2 SO 4 2M. Optical densities (OD) at 450 nm were measured with a Thermo Labsystems Multiskan microplate reader and analysed with Ascent Software for Multiskan version 2.6 (Thermo Labsystems, Waltham, MA, USA). All sera were analysed on the same day. The cut-off value for the indirect ELISA test was obtained from the serum samples of the 30 colostrum deprived calves (mean + 1.96 standard deviation) and the apparent prevalence was determined on the 439 bovine serum samples with a 95% confidence interval assuming a binomial exact distribution. All statistical analyses were carried out with the STATA/SE software (Stata Corp., College Station, TX, USA). The OD means in the different age groups of animals (unequal variance and non normal distribution) were compared with the Wilcoxon rank-sum test

[START_REF] Dagnelie | Statistique théorique et appliquée. Tome 2. Inférence statistique à une et à deux dimensions[END_REF]

. The limit of statistical significance of the conducted test was defined as P≤0.05.

Acknowledgements

This study was funded by the Federal Public Service of Health, Food Chain Safety and Environment (RF6185), the Belgian policy "Science for a Sustainable Development" (SD/AF/01) and by the Région Wallonne (415701). The authors would like to thank C. Généreux (Parasitology, Faculty of Veterinary Medicine, ULg) for providing neonate sera, J.

Piret (Histology, electron microscopy unit, Faculty of Veterinary Medicine) for some electron microscopic analysis, the Association Régionale de Santé et d'Identification Animales (ARSIA, Dr Marc Saulmont), veterinarians and farmers for accessing bovine stool and serum samples.

During this period, the immunological status of cattle against BoNoV varies both by colostrum intake and by natural infection.

Conflict of interest statement

The authors ensure that there are no conflict of interest with any person or organisation.


M L Carrozza

M Mazzei
email: mmazzei@vet.unipi.it

D Lacerenza

L Del Chiaro

M Giammarioli

C Marini

D Rutili

S Rosati

F Tolari

Del Chiaro

Seroconversion against SU5 derived synthetic peptides in sheep experimentally infected with different

Keywords: Small Ruminant Lentiviruses, envelope glycoprotein, SU5 peptides, type-specific seroconversion

Synthetic peptides were generated, corresponding to SU5 domain of envelope glycoprotein of Italian SRLV isolates It-561 and It-Pi1, belonging respectively to MVV-and CAEV-like genotypes. The peptides, encompassing an N-terminal variable and a C-terminal conserved antibody-binding site, were used in an ELISA assay to analyse the sera of two groups of sheep experimentally infected with these isolates. The kinetics and specificity of the humoral response to the homologous and heterologous antigen and the affinity maturation of the sera were evaluated. Seroconversion occurred between week 3 and 8. The response to SU5 antigen was mostly type-specific. The few broadly reacting sera may reflect the production of antibodies Manuscript 2 directed to the SU5 constant antibody-binding site. All sera underwent with time avidity maturation, resulting in the appearance of high affinity antibodies. This study suggests constant monitoring of the circulating viral variants to develop a panel of diagnostic peptides representative of local genotypes.

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

Introduction

Maedi Visna Virus (MVV) and Caprine Arthritis Encephalitis Virus (CAEV) are non oncogenic lentiviruses of the Retroviridae family, presently referred to as Small Ruminant Lentiviruses (SRLVs) [START_REF] Zanoni | Phylogenetic analysis of small ruminant lentiviruses[END_REF]. A recent phylogenetic classification of these viruses defines two main groups, A and B, including respectively MVV-like and CAEV-like genotypes [START_REF] Shah | Phylogenetic analysis and reclassification of caprine and ovine lentiviruses based on 104 new isolates: evidence for regular sheep-to-goat transmission and worldwide propagation through livestock trade[END_REF].

SRLV infections occur almost worldwide and cause relevant economic losses. Therapy and effective vaccine are not yet available [START_REF] Gonzalez | Mucosal immunization of sheep with a Maedi-Visna virus (MVV) env DNA vaccine protects against early MVV productive infection[END_REF]Petursson et al., 2005;Torsteindottir et al., 2007;[START_REF] Reina | Mucosal immunization against ovine lentivirus using PEI-DNA complexes and modified vaccinia Ankara encoding the gag and/or env genes[END_REF][START_REF] Reina | Mucosal immunization against ovine lentivirus using PEI-DNA complexes and modified vaccinia Ankara encoding the gag and/or env genes[END_REF], therefore eradication and prevention of the infection largely depend on early, efficient and correct identification of infected animals [START_REF] De Andrés | Diagnostic tests for small ruminant lentiviruses[END_REF]. This is routinely done by serological analysis, the ELISA assay being the most sensitive and suitable technique both for large scale screening and individual examination. Several ELISA protocols have been developed so far, mostly based on antigens from a single viral strain. The specificity of these assays is generally high, but the sensitivity shows extensive variability [START_REF] De Andrés | Diagnostic tests for small ruminant lentiviruses[END_REF] due to antigenic heterogeneity of demonstrated that gag-encoded capsid (CA) and matrix (MA) proteins carry type-specific epitopes and most sheep and goat sera reacted to these antigens in a type-specific manner, irrespective of the species of origin [START_REF] Grego | Genetic heterogeneity of Small Ruminant Lentiviruses involves immunodominant epitope of capsid antigen and affects sensitivity of single-strain-based immunoassay[END_REF][START_REF] Grego | Genetic and antigenic characterization of the matrix protein of two genetically distinct ovine lentiviruses[END_REF]. Furthermore, homologous CA-MA fusion protein was able to detect seroconversion at an earlier stage compared to the heterologous protein [START_REF] Lacerenza | Antibody response in sheep experimentally infected with different small ruminant lentivirus genotypes[END_REF]. The present study extends the analysis of the humoral response induced by It-561 and It-Pi1 to SU5, one of the major epitopes of the envelope (Env) protein [START_REF] Bertoni | B-cell epitopes of the envelope glycoprotein of caprine arthritis-encephalitis virus and antibody response in infected goats[END_REF][START_REF] Valas | Variability and immunogenicity of caprine arthritis-encephalitis virus surface glycoprotein[END_REF]. Two SU5 synthetic peptides were generated, corresponding to It-561 and It-Pi1 sequences. The peptides were used as antigens to assay sera from two groups of sheep experimentally infected with these viruses, in order to investigate the kinetics and specificity of the humoral response to the homologous and heterologous antigen.

Materials and Methods

Viral strains and experimental infection

SRLV strains It-561 (genotype A) and It-Pi1 (genotype B) [START_REF] Grego | Genetic heterogeneity of Small Ruminant Lentiviruses involves immunodominant epitope of capsid antigen and affects sensitivity of single-strain-based immunoassay[END_REF] were used in the present study. Infection of sheep with these strains has been described by [START_REF] Lacerenza | Antibody response in sheep experimentally infected with different small ruminant lentivirus genotypes[END_REF]. Detection of viral sequences in blood of experimental sheep was done by LTR PCR [START_REF] Extramiana | Evaluation of a LTR-PCR technique for the detection of Maedi-Visna proviral DNA in blood, milk and tissue samples of naturally infected sheep[END_REF] and Pol PCR [START_REF] Grego | Genetic heterogeneity of Small Ruminant Lentiviruses involves immunodominant epitope of capsid antigen and affects sensitivity of single-strain-based immunoassay[END_REF].

Cloning, sequencing and sequence analysis of env region coding for SU5

An env gene fragment encompassing the 75bp "SU5 total" domain [START_REF] Mordasini | Analysis of the antibody response to an immunodominant epitope of the Envelope glycoprotein of a lentivirus and its diagnostic potential[END_REF] coding sequence (nt 7800-7874) was amplified from DNA of foetal ovine lung fibroblasts infected respectively with It-561 and It-Pi1 viral stocks. Primer sequences [START_REF] Bertoni | B-cell epitopes of the envelope glycoprotein of caprine arthritis-encephalitis virus and antibody response in infected goats[END_REF] were (1990; Genbank accession number M33677). Amplified fragments were cloned and sequenced.

Genetic distances were calculated with MEGA 3 [START_REF] Kumar | MEGA2: molecular evolutionary genetics analysis software[END_REF] and used to construct a neighbor-joining tree with the Tamura-Nei two-parameter distance option [START_REF] Tamura | Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees[END_REF].

Peptide ELISA

Peptides corresponding to the 25aa "SU5 total" domain of both viruses, were resuspended in 0.1M carbonate buffer pH 9.6 at 5 g/mL. ELISA plates were coated overnight at 37°C (50L/well), incubated for 1 hour at 37°C with sera diluted 1/20 and for 1 hour at room temperature with a 1:8000 dilution of peroxidase-labelled anti-sheep/goat IgG monoclonal antibody (Sigma). Optical density was measured at 405 nm wavelength after 40 min incubation with ABTS (Sigma).

Cut-off

A panel of sera collected before experimental infection as well as sequential sera from two mock-infected sheep, were repeatedly assayed: no significant interplate variation of the absorbance was observed. The cut-off was calculated as mean absorbance of negative sera + 3x Standard Deviation. Samples with absorbance higher than 0.3 were considered positive with a 99% confidence level. A serum collected from a sheep belonging to a long term seronegative flock was loaded on each plate.

Antibody avidity measurements

The avidity index values of SU5-specific antibodies were measured by testing the stability of the antigen-antibody complexes following a wash in 8 M urea [START_REF] Mordasini | Analysis of the antibody response to an immunodominant epitope of the Envelope glycoprotein of a lentivirus and its diagnostic potential[END_REF]. Antibodies with avidity indexes <30% were considered to be of low affinity; those with values between 30% and 50% of intermediate avidity and those with values >50% of high avidity.

Kinetics of antibody avidity maturation

The avidity maturation of sera was assayed at week 8 p.i., corresponding to the first antibody peak, at week 35 and at week 78. The results obtained for group A sheep are depicted in fig. 3 a. Sheep 2, 3 and 4 underwent a marked increase of antibody avidity over the period analyzed.

All avidity indexes of sheep 1 were low; therefore additional time points were analyzed.

Maturation to high avidity was detected at week 56, followed by a gradual decrease to low avidity by the end of the experiment (data not shown. Group B sheep underwent a marked avidity maturation, reaching the highest values at week 35 p.i (fig. 3b).

Discussion

Most ovine isolates collected in Italy belong to the CAEV-like group [START_REF] Grego | Genetic heterogeneity of Small Ruminant Lentiviruses involves immunodominant epitope of capsid antigen and affects sensitivity of single-strain-based immunoassay[END_REF] and coinfection of goats with both MVV-and CAEV-like strains in an Italian flock has recently been reported [START_REF] Pisoni | Demonstration of coinfection with and recombination by caprine Arthritis-Encephalitis Virus and Maedi-Visna Virus in naturally infected sheep[END_REF]. In the last years serological assays based on Gag antigens derived from Italian isolates belonging to both phylogenetic groups were developed and applied in field conditions: the response obtained was largely type-specific, indicating the opportunity to use antigens representative of both genotypes to improve the sensitivity of the diagnostic assays [START_REF] Grego | Genetic heterogeneity of Small Ruminant Lentiviruses involves immunodominant epitope of capsid antigen and affects sensitivity of single-strain-based immunoassay[END_REF]2005). Furthermore, when sequential sera collected from sheep experimentally infected with either It-561 or with It-Pi1, were analyzed by ELISA with both homologous and heterologous CA-MA fusion proteins, the homologous antigen detected infected animals up to months before the heterologous antigen [START_REF] Lacerenza | Antibody response in sheep experimentally infected with different small ruminant lentivirus genotypes[END_REF]. In this It-Pi1 SU5 peptides. Five epitopes have been identified in the SU subunit of Env [START_REF] Bertoni | B-cell epitopes of the envelope glycoprotein of caprine arthritis-encephalitis virus and antibody response in infected goats[END_REF][START_REF] Valas | Variability and immunogenicity of caprine arthritis-encephalitis virus surface glycoprotein[END_REF]. SU5 in particular, a 25 aminoacid sequence located at the C terminus of SU, is immunodominant and partly type-specific: it carries at least three antibody-binding sites, one in the N-terminal conserved region, one within the C-terminal most variable domain, and the third encompassing the junction between the variable and conserved regions [START_REF] Mordasini | Analysis of the antibody response to an immunodominant epitope of the Envelope glycoprotein of a lentivirus and its diagnostic potential[END_REF]. In this study, two env gene fragments encoding respectively It-561 and Alternatively, this sheep may present some failure in the mechanisms that underly the ability to maintain the production of high affinity antibodies. A failure of T helper function may be responsible for the loss of high affinity antibodies as well as the associated decay of the antibody titer, that have been reported in individuals who later developed AIDS [START_REF] Chargelegue | A 7 year analysis of anti-gag (p17 and p24) antibodies in HIV-1 seropositive patients with haemophilia: immunoglobulin titre and avidity are early predictors of clinical course[END_REF][START_REF] Chargelegue | The affinity of IgG antibodies to gag p24 and p17 in HIV-1 infected patients correlates with disease progression[END_REF][START_REF] Thomas | Differential maturation of avidity of IgG antibodies to gp41, p24 and p17 following infection with HIV-1[END_REF]. SRLV infection, however, does not involve the lymphocyte compartment, and sheep 1 antibody titer does not decrease, therefore a different mechanism may be responsible. A comparison of the results obtained in this study and in [START_REF] Lacerenza | Antibody response in sheep experimentally infected with different small ruminant lentivirus genotypes[END_REF] shows that both CA-MA fusion antigens and SU5 peptides detect infection at early stages, from week three onwards. Furthermore, while It-561 drives typespecific antibody response towards both Gag and Env antigens, It-Pi1 seems to drive production of type-specific antibody only against SU5. In fact, all group B sheep reacted against the heterologous Gag subunits as well [START_REF] Lacerenza | Antibody response in sheep experimentally infected with different small ruminant lentivirus genotypes[END_REF]. Since the humoral response to SU5 is mainly directed towards the variable antibody-binding site, a constant monitoring of the circulating viral variants will be necessary to develop a panel of SU5 peptides representative of most local genotypes.

  analysis of two Italian SRLV isolates, MVV-like It-561 and CAEV-like It-Pi1,

  ). The nucleotide positions refer to the CAEV sequence published bySaltarelli et al.

  A c c e p t e d M a n u s c r i p t Results Cloning and sequencing of SU5-encoding env region The env sequences encompassing respectively the SU5 domain of It-561 and It-Pi1 strains (Genbank accession numbers: It-561: EU702487: It-Pi1: EU709743) were aligned with MVV and CAEV prototypic env sequences of worldwide origin. It-561 clusters with representatives of group A genotypes and It-Pi1 with B genotypes (fig.1 a), in agreement with the results of a phylogenetic analysis of gag and pol sequences (Grego et al., 2002; 2005). The SU5 aminoacid sequences of It-561 and It-Pi1 share homology in the N-terminal region, which is well conserved among MVV and CAEV strains, and diverge in the C-terminal, more variable portion (fig.1 b). Peptide ELISA Peptide ELISA assays were performed on a panel of sera obtained over a period of up to 92 weeks p.i. from three groups of experimentally infected sheep: group A, sheep 1 to 5, infected with It-561; group B, sheep 6 to 9, infected with It-Pi1 (fig.2 a, b) and group C, four mockinfected sheep. Group C sheep remained seronegative throughout the study (data not shown). Group A sheep 1 to 4 seroconverted to the homologous antigen between week 4 and 8 and remained positive throughout the study. Sheep 1 to 3 were completely negative to the heterologous It-Pi1 peptide, while sheep 4 raised a weak humoral response to It-Pi1, from week 77 onwards. Sheep 5 did not seroconvert and was the only one negative to both LTR and Pol PCR assays performed throughout the study to confirm the presence of virus in the infected animals (data not shown). Group B sheep seroconverted to the homologous antigen between week 3 and 8; all sheep remained positive until the end of the experiment, with high absorbance values. Only sheep 9 responded to the heterologous antigen from week 8 onwards. TheA c c e p t e d M a n u s c r i p t 6 absorbance, which was about two-to threefold lower than for the homologous antigen, reached a peak at week 22 and gradually decreased thereafter, to values just above the cut-off.

  panel of sequential sera was analyzed with an Env ELISA based on It-561 and

  It-Pi1 SU5, were sequenced. Alignment of these fragments with sequences of MVV and CAEV prototypic strains of worldwide origin showed that It-561 clusters with the MVV strains and It-Pi1 is phylogenetically related to classical CAEV-like prototypes. The SU5 N-terminal aminoacid sequences of the two strains share extensive homology; the C-terminal region is more divergent, confirming the results of[START_REF] Mordasini | Analysis of the antibody response to an immunodominant epitope of the Envelope glycoprotein of a lentivirus and its diagnostic potential[END_REF]. Seroconversion kinetics of group A and B sheep were similar, with antibodies being mostly type-specific. The few broadly reacting sera may reflect the production of antibodies directed to the constant antibody-binding site. The antibody levels varied between animals and for the same animal during the time course of the experiment. In group A, the first antibody peak was followed by a slight decrease of reactivity and then by a rise to three to fourfold higher values. One sheep of this group did not seroconvert. This result was most likely caused by a technical failure during the experimental infection, since the sheep was repeatedly negative to both LTR and pol PCR assays which were performed on blood samples of the experimental animals to confirm the presence of viral genomes. Group B sheep developed immediately an antibody response well over the cut-off. All sera underwent with time avidity maturation, resulting in the appearance of high affinity antibodies, although with different kinetics. It-Pi1 sera, which developed higher antibody levels from the beginning, underwent high avidity maturation at an earlier time point than It-561 sera.

  a peculiar pattern of avidity maturation, with indexes decreasing from week 56 onwards, to return to low values by the end of the experiment, in spite of a persisting high antibody level. The reason for sheep 1 behaviour is unclear; the genetic variability of SU5 could possibly explain the disappearance of the high affinity antibody-producing clone(s).

  Fig.1:

Fig. 2 :

 2 Fig. 2:

Fig. 3 :

 3 Fig.3:

  Figure 1

  Figure 2

Figure 3

 3 Figure 3

Acknowledgements

This work was supported by the Italian MIUR.

Conflict of interest statement


Sílvia C Barros

Benedita Cruz

Tiago M Luís

Fernanda Ramos

Teresa Fagulha

Margarida Duarte

Margarida Henriques

Miguel Fevereiro
email: miguel.fevereiro@lniv.min-agricultura.pt

A DIVA system based on the detection of antibodies to non-structural protein 3 (NS3) of Bluetongue virus

Keywords: Bluetongue virus, Non-structural proteins, NS3, ELISA, DIVA

Vaccination programs for the control of bluetongue (BT) in ruminants have limitations due to difficulties in differentiating between vaccinated and virus infected animals (DIVA). To overcome this problem a DIVA test that looks at a differential immune response to bluetongue virus (BTV) non-structural protein 3 (NS3) was developed. The NS3 encoding gene of strain BTV4/22045/PT04 was inserted into expression vector pET-28a and expressed in Escherichia coli strain JM109. Recombinant NS3 protein was used as an antigen in an indirect ELISA (NS3-ELISA) to measure the serologic response to NS3 protein in cattle and sheep. Following a cattle vaccination/challenge experiment with a bivalent inactivated BTV 2-4 vaccine, NS3 antibodies were detected at approximately 15 days post-infection in control unvaccinated animal, while vaccinated animals did not develop detectable NS3 antibodies and, with exception of one, remained negative even after virus challenge. The inactivated vaccine induced antibodies against the major core structural protein (VP7) of BTV as well as neutralizing antibodies in all animals.

To evaluate the applicability of NS3-ELISA in field scenario, a total of 562 serum samples collected from uninfected, BTV infected and vaccinated animals were tested for NS3 antibodies. Taken together, the results confirm that NS3 antibodies were induced to the greatest levels in animals infected with BTV in comparison to the levels induced in animals immunized with inactivated BTV vaccines, implying that antibody response to NS3 allows the differentiation between infected and vaccinated animals.

A c c e p t e d M a n u s c r i p t Introduction Bluetongue is a vector-borne viral disease of ruminants, including sheep, goats and cattle, caused by bluetongue virus (BTV). Clinical signs are seen mainly in sheep but recently clinical cases have been also observed in cattle [START_REF] Toussaint | Bluetongue in northern Europe[END_REF]. BTV is a member of the Orbivirus genus within the family Reoviridae, possessing 10 doublestranded RNA segments enclosed by three consecutive capsid layers of multiple proteins. In addition to the seven structural (VP1 to VP7), there are three non-structural (NS1, NS2 and NS3) proteins. The smallest RNA segment (S10) of BTV encodes two closely related proteins, the NS3 (25.5 kDa) and NS3A (24 kDa), which are read from alternative in frame methionine initiation codons [START_REF] Roy | Structure of bluetongue virus genome and its encoded proteins[END_REF]. The NS3A expression is more variable and generally occurs at a much lower level than that of NS3 (Wu et al., 1992). The NS3 protein is essential for both virus assembly and release from infected cells [START_REF] Bansal | Membrane organization of bluetongue virus non-structural glycoprotein NS3[END_REF][START_REF] Hyatt | Release of bluetongue virus-like particles from insect cells is mediated by BTV non-structural protein NS3/NS3A[END_REF][START_REF] Wirblich | Non-structural protein 3 of bluetongue virus assists virus release by recruiting ESCRT-I protein Tsg101[END_REF].

Since 1998, seven BTV serotypes (BTV-1, 2, 4, 6, 8, 9 and 16) have invaded Europe (Maan et al., 2007;[START_REF] Toussaint | Bluetongue in northern Europe[END_REF]Ref OIE: 7458/October/2008). In Portugal the first outbreak of BTV-4 occurred in 2004 [START_REF] Barros | Molecular epidemiology of bluetongue virus in Portugal during 2004-2006 outbreak[END_REF] in the South of the country. In 2007, a new incursion of BTV, this time of serotype 1, was reported (Ref OIE: 6248/September/2007) and since then the virus has spread across the country.

Vaccination is the most effective mean to protect animals against disease and to reduce the spread of virus after infection. Unfortunately, vaccination interferes with the serological detection of infected animals. Inactivated vaccines against serotypes 1, 2, 4, and 8 of BTV were recently developed and have been used in the European Union, replacing the conventional live attenuated vaccines. In Portugal, a bivalent inactivated vaccine against serotypes 2 and 4 was firstly used in 2005 but was subsequently superseded by monovalent vaccines for BTV-4 and BTV-1. These vaccines offer The non-structural NS3 protein is produced in large quantities in infected cells, but is not packaged in the virion. Since inactivated vaccines for BTV are primarily made with whole virions, a differential antibody response should be observed between naturally infected and vaccinated animals. Therefore, NS3 antibody response is related in vivo to the virus activity.

For both trade and surveillance purposes, it is important not only to differentiate naturally infected and vaccinated ruminants, but also to identify vaccinated animals that become infected with BTV.

The main goal of the present study was to establish a diagnostic test for differentiation between vaccinated and BTV infected animals. We describe the development of an indirect ELISA with a DIVA function that uses a recombinant non-structural NS3 protein as an antigen for the detection of BTV-NS3 antibodies in cattle and sheep. This NS3-ELISA was evaluated with sera collected from naive, vaccinated and infected animals and the performance of the method was compared with a commercial competitive ELISA (cELISA), serum neutralization test (SN) and with RT-PCR to determine BTV infection status.

Materials and Methods

Expression and purification of recombinant NS3 protein

The DNA fragment encoding NS3, from BTV4/22045/PT04 (GenBank accession number EF434180), contained in a pCR2.1 vector (Invitrogen) was amplified by using the primers: NheI-NS3-F (5'-GCTAGCATGCTATCCGGGCTGAT-3') and EcoRI-NS3-R (5'-GAATTCTCGGTTAATGGTAATTCG-3') inserting NheI and EcoRI A c c e p t e d M a n u s c r i p t 5 cleavage sites. PCR was undertaken for 40 cycles (30 s at 94 ºC; 30 s at 55 ºC, 1 min 20 s at 72 ºC) with a final extension at 72 ºC for 5 min. Amplified fragments were digested with NheI and EcoRI and cloned in the expression vector pET-28a(+) (Novagen) inframe with amino and carboxy terminals 6xHis-tag epitopes. The recombinant plasmid pET28a/NS3 was then transformed into E. coli JM109. Cells were grown at 37 ºC in LB medium containing 25 g/ml of kanamycin, and protein expression induced with 1 mM IPTG when the cell density (OD 600 ) reached 0.6. The cultivation was continued for 4 h and cells were harvested by centrifugation at 5000 x g for 10 min at 4 ºC.

Recombinant NS3 was solubilized from inclusion bodies as previously described [START_REF] Lu | Development and validation of a 3ABC indirect ELISA for differentiation of foot-and-mouth disease virus infected from vaccinated animals[END_REF], with minor modifications. The concentrated bacterial pellets were washed twice in 0.25 x culture volume buffer (20 mM Tris-HCl, pH 7.5) and ressuspended in inclusion body (IB) lysis buffer (20 mM Tris-HCl, pH 7.5; 10 mM EDTA; 1% Triton X-100) supplemented with lysozyme to a final concentration of 100 g/ml. The mixture was incubated at room temperature for 15 min, sonicated on ice until the solution was no longer viscous, and the inclusion bodies were recovered by centrifugation. After two additional washes in lysis buffer, the inclusion bodies were solubilized by the addition of IB solubilization buffer (50 mM CAPS, pH 11; 0.3% N-lauroylsarcosine; 1 mM DTT). The supernatant containing the solubilized protein was collected for subsequent binding to NI-NTA His binding resin (Qiagen), according to manufacture's instructions.

The amount of total proteins was determined by Pierce BCA kit.

Indirect ELISA procedure for detection of NS3 antibodies (NS3-ELISA)

The optimal concentration of antigen and dilution of the serum were determined by performing a checkerboard titration of the NS3 antigen and positive and negative bovine and ovine control sera. Based on this initial titration, 150 ng/ 100µl of antigen and 1:10 A c c e p t e d M a n u s c r i p t 6 serum dilution were used in all further NS3-ELISA. Microtiter plates were coated with NS3 antigen diluted in carbonate-bicarbonate buffer, pH 9.6 and incubated overnight at 4 ºC. Following 4 washes with PBS/0.05% Tween-20, serum samples diluted 1:10 in dilution buffer (PBS containing 5% skimmed milk and 3% E.coli lysate), were added at 100 l/well and incubated for 30 min at room temperature. Plates were washed and donkey anti-sheep/goat-IgG-HRP (1:20000) or sheep anti-bovine-IgG-HRP conjugate (1:1500) both from Serotec, was added, 100 l/well, in the same dilution buffer and incubated for more 30 min at room temperature. After four wash cycles, colour was developed with 100 l TMB reagent (Sigma) and the reaction was allowed to proceed for 15 min before being stopped by adding 0.5 M H 2 SO 4 . The optical density (OD) was read at 450 nm using a microplate reader (Tecan). The samples and positive control OD values (OD sample and OD pos ) were corrected by subtracting the OD value of the negative control (OD neg ): Percentage Positivity (PP) = [(OD sample -OD neg ) / (OD pos -OD neg )] x 100%. Samples with a PP ≥40% were considered positive, between 35-40% were considered inconclusive and those <35% were negative. The test was considered valid if OD pos / OD neg >5.

Source of sera

A group of 7 bovines (numbered, N1 to N7) maintained in vector-free premises were immunised with a bivalent inactivated vaccine (BTVPUR AlSap 2-4, Merial). Two subcutaneous immunizations were performed with 3 weeks apart. Sixty days after the first immunization the 7 vaccinated and one control animal (N8) were challenged intravenously with strain BTV4/22045/PT04 (6.5 ± 1.1 x 10 -5 PFU/5ml). Blood samples collected before and after challenge were tested by NS3-ELISA.

Commercial ELISA test

Bluetongue virus antibody test kit (VMRD, Inc., Pullman, WA, USA), a competitive ELISA (cELISA) was used as a standard for comparison. The test is based on the detection of antibodies specific to the highly conserved VP7 protein of BTV.

Serum neutralization test (SN)

Samples were tested for the presence of neutralizing antibodies. Serial dilutions (1:10) of heat-inactivated sera were incubated for 1h at 37 ºC with an equal volume (50 μl) of BTV (100 TCID50), in a 96-well microplate. Approximately 10 4 BHK 21 cells in 100 µl of medium were added per well and after incubation for 5 days at 37 ºC with 5% CO 2 , the microplate was read using an inverted microscope. Two replicates of each serum were tested with BTV-1 (BTV1/PT/29058/07), BTV-2 (BTV2/26629/PT05) or BTV-4 (BTV4/22045/PT/05). Neutralizing antibody titers were calculated according to Spearman-Kärber's formula and expressed as the last dilution of serum that neutralizes CPE in 100%. Serum samples with a virus neutralisation titer of ≥1.3 log10 were considered positive.

Real time RT-PCR (rRT-PCR)

The Pan BTV (NS1 based) rRT-PCR was routinely used as reference test to determine the true status of BTV infection in animals, as previously described (Barros et al., probe-5'FAM-TTTGAGTCAGAAACAAACCCAATCAGCCGT-3'TAMRA).

Results

Generation of recombinant NS3 protein for development of an indirect ELISA (NS3-ELISA)

In order to investigate the applicability of NS3 as DIVA test, the NS3 gene of BTV4/22045/PT04 isolate was cloned in vector pET-28a, creating a His-tagged recombinant NS3 protein, confirmed in the Western blot analysis by anti-His-tag monoclonal antibody as well as by BTV positive serum (Fig. 1). The expressed protein was used as antigen for developing an indirect ELISA (NS3-ELISA).

Evaluation of NS3-ELISA with sera from vaccination trial

Serological results

Sera from seven bovines that participated in a vaccination/challenge experiment with the inactivated vaccine BTVPUR AlSap 2-4 (Merial) were tested in NS3-ELISA, c-ELISA and SN assay. The animals were given one dose of vaccine subcutaneously, and 3 weeks later a booster immunisation was performed. Sixty days after the primovaccination, all the seven immunized animals and one unvaccinated control animal (N8)

were challenged with virus strain BTV4/22045/PT04. Before vaccination all animals were serologically and virologically negative for BTV. Neutralizing antibodies were first detected 14 days (BTV-2) and 21 days (BTV-4) postvaccination and by days 21 and 28 all animals had developed BTV-2 and BTV-4

neutralizing antibodies, respectively. The mean neutralizing antibody titers peaked on day 14 after boosting (2,4 log10 for BTV-2 and 2,0 log10 for BTV-4) and raised up to a mean of 2,7 log10 after challenge with BTV-4, being detectable until day 216 when the assay ended (Fig. 3).

Following challenge, all vaccinated animals remained NS3 seronegative with the exception of animal N7 that developed a weak and short lasting (~15 days) antibody response to NS3 (Fig. 2).

Before challenge the unvaccinated control animal (N8) was serological and virological negative for BTV. After inoculation with BTV4/22045/PT04 the NS3 antibody levels started to rise between 8-15 day post-infection (pi) reaching a maximum at approximately 43 pi and lasted over 141 days (Fig. 2). By using the cELISA, antibodies against VP7 were detected approximately 8 days pi, a week earlier than NS3 antibodies.

Neutralizing antibodies to BTV-4 were also detected after inoculation, peaking around 60 days pi (Fig. 3).

The exact duration of NS3 antibodies in the control animal (N8) was not determined.

Luckily an extra blood sample from this animal was received in the laboratory at day 330 pi and tested NS3-ELISA negative but VP7 cELISA positive.

A c c e p t e d M a n u s c r i p t 10

rRT-PCR results

Blood samples were analysed for the presence of viral RNA by rRT-PCR (Fig. 4). Of the seven immunised animals, two (N3 and N6) did not yield any positive rRT-PCR, three (N1, N4 and N5) were positive between 1-3 days and one (N7) was positive up to 7 days post-challenge (pc). In this animal a low level replication of the challenging virus may have occurred since a low titer and short lasting NS3 antibodies were detected (Fig. 2). On the other hand, the detection of viral RNA during the first three days pc in some of the other animals may reflect circulation of the inoculums rather than viral replication, since NS3 antibodies were not detected.

In control animal (N8), BTV-4 nucleic acid was first detected at 24h pi and was continuously detected until the end of the trial (156 days pi). As inferred by the Ct values of rRT-PCR, viral load steadily increased from day 12 up to day 20 pi, and then began to decrease (Fig. 4). The rRT-PCR was able to give a positive signal up to 15 days before antibodies could be detected by NS3-ELISA while the delay time for cELISA was 8 days. As referred above, an extra blood sample from animal N8 was obtained at day 330 pi. Interestingly enough, in this sample the challenge virus could still be detected by rRT-PCR (Ct value = 36.8) while NS3-specific antibodies were no longer detectable.

Field application study of NS3-ELISA

For further validation of the NS3-ELISA as a DIVA test, a total of 562 field serum samples originated from uninfected, vaccinated or naturally infected animals were used.

These samples were also tested by cELISA, SN assay and rRT-PCR.

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

11

The mean and SD of the OD of the sera obtained from uninfected animals were 0.11 ± 0.07 and 0.13 ± 0.05 in ovine and bovine, respectively. For vaccinated sheep and cattle, the mean and SD of OD values were respectively 0.17 ± 0.08 and 0.17 ± 0.07 (Table 1).

A good discrimination between positive and negative samples was observed; the difference between the average OD value of samples from infected (NS3 positive) and vaccinated (VP7 positive/ NS3 negative) animals was 0.84 and 0.73, respectively for sheep and cattle.

To evaluate the ability to diagnose infected animals the NS3-ELISA was compared with rRT-PCR (Table 2). The correlation between NS3-ELISA and rRT-PCR was 87% and 51% respectively for sheep and bovine positive samples, while for negative samples the correlation was respectively 98% and 100%. All discrepant samples were analysed at least three times.

Of 135 ovine BTV-1 rRT-PCR positive samples, 17 (13%) were negative by NS3-ELISA. Nine of these discrepant samples were also negative in cELISA and SN test, suggesting a very early infection prior to seroconversion. The remaining eight samples, positive for cELISA, originated from vaccinated animals with neutralizing antibodies for BTV-4 but not for BTV-1, suggesting a recent infection with BTV-1 (Table 3).

From a total of 293 rRT-PCR negatives samples, only three from sheep with clinical signs were positive by NS3-ELISA and cELISA.

Sixty-seven discrepant rRT-PCR positive and NS3-ELISA negative bovine samples were recorded (49%). These animals were separated into two groups, BTV-1 (24 animals) or BTV-4 (43 animals) rRT-PCR positive. The samples from the latter group were collected between December 2007 and February 2008, when vector activity was reduced or absent and outbreaks of BT were not registered. Two animals of this group were bled twice with one month interval and the second collection resulted rRT-PCR A c c e p t e d M a n u s c r i p t antibodies. As previously determined for bovine N8, these animals could be in a late stage of infection.

In the group of 24 BTV-1 positive cattle, originated from farms located in areas where BTV-1 outbreaks were occurring, two animals were NS3 and VP7 negative, suggesting a very early stage of infection, while the remaining samples, NS3 negative and VP7

positive, are likely to originate from recent infected animals that seroconverted to VP7

but not yet to NS3. In one case it was possible to demonstrate seroconversion to NS3 from paired samples received with one week interval.

Inter-and intra-assay variations of NS3-ELISA

To calculate the inter-assay variation (reproducibility) of NS3-ELISA, the OD values of a pool of positive sera was measured 6 times on different days. The coefficient of variation for ovine sera was 0.067 and for bovine sera was 0.028.

For intra-assay variation (variability), OD values of six replicates of the same serum were measured in six columns of the plate. The coefficient variation was 0.016 and 0.022 respectively for ovine and bovine sera.

Discussion

In the present work an indirect ELISA was developed for detection of BTV antibodies against non-structural NS3 protein, which enables the differentiation between infected and vaccinated animals (DIVA). This method is theoretically based on the assumption that inactivated BTV-vaccines mainly contain capsid proteins, so they are less likely to illicit production of antibodies against non-structural proteins.

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

13 Following a vaccination/challenge experiment, with a bivalent inactivated BTV2-4 vaccine, seven vaccinated and one unvaccinated control bovine were evaluated for the ability to develop antibodies to NS3. All vaccinated animals were negative by NS3-ELISA and, with exception of one (N7), remained negative even after challenge. In animal N7 a low level replication of the challenging virus may have occurred since viral RNA was detected up to 7th day pc. This brief viraemia could be responsible for the low titer and short lasting NS3 antibodies observed after challenge. In contrast, all vaccinated animals developed antibodies against BTV, as measured by commercial cELISA and SN test. These animals were protected since viraemia was no longer detected by rRT-PCR soon after challenging virus inoculum was cleared from blood stream (3 days pc). The efficacy of this inactivated vaccine was also demonstrated in a previous study [START_REF] Savini | Assessment of efficacy of a bivalent BTV-2 and BTV-4 inactivated vaccine by vaccination and challenge in cattle[END_REF].

The unvaccinated control animal (N8) developed NS3 and VP7 antibodies in response to challenge infection. Concerning the antibodies appearance, VP7 antibodies were detected in the serum 8 days pc, whereas NS3 antibodies appear about one week later.

The exact duration of NS3 antibodies could not be determined, but they were no longer detected on day 330 pc, while VP7 antibodies were still detectable. Surprisingly, this sample was rRT-PCR positive for challenging virus on this date. Therefore, if viral replication ever occurred in the control animal between 216-330 days pc, it was not enough to stimulate NS3 antibodies at detectable levels.

The dynamic of the appearance of NS3 antibodies were also studied in another trial involving five BTV-4 immunized sheep with inactivated vaccine BTVPUR AlSap 4 (Merial). These animals were NS3 antibody negative and VP7 antibody positive. After inoculation with BTV-1 (BTV1/PT/29058/07) the animals seroconvert to NS3 on day 10 pi (data not shown). As for the bovine trial described above, this experiment with Previous studies demonstrate that viraemia in sheep and cattle could be prolonged up to 38-54 days as determined by virus isolation [START_REF] Koumbati | Duration of bluetongue viraemia and serological responses in experimentally infected European breeds of sheep and goats[END_REF][START_REF] Bonneau | Duration of viraemia infectious to Culicoides sonorensis in bluetongue virus-infected cattle and sheep[END_REF]. However, BTV nucleic acids can be still detected by RT-PCR on a longer period, up to 180 and 222 days pi in cattle and sheep, even if it does not mean that infectious particles are present [START_REF] Katz | Diagnostic analysis of the prolonged bluetongue virus RNA presence found in the blood of naturally infected cattle and experimentally infected sheep[END_REF][START_REF] Bonneau | Duration of viraemia infectious to Culicoides sonorensis in bluetongue virus-infected cattle and sheep[END_REF]. The RT-PCR assay potentially would detect viral RNA contained in any inactivated virions or subviral particles bound to circulating erythrocytes or other blood cells (Brewer and MacLachlan, 1992). Thus, ruminants whose blood contains RT-PCR detectable BTV nucleic acid, but not infectious virus as determined by virus isolation, likely are unimportant to the epidemiology of BTV infection [START_REF] Maclachlan | Detection of bluetongue virus in the blood of inoculated calves: comparison of virus isolation, PCR assay, and in vitro feeding of Culicoides variipennis[END_REF][START_REF] Tabachnick | Culicoides variipennis and bluetongue-virus epidemiology in the United States[END_REF][START_REF] Bonneau | Duration of viraemia infectious to Culicoides sonorensis in bluetongue virus-infected cattle and sheep[END_REF].

To further evaluate the applicability of NS3-ELISA as a DIVA test, we analysed a larger number of field samples from infected, vaccinated, and from virological and serological negative animals of BTV free areas. Once again the results show that BTV infected animals develop higher levels of NS3 antibodies compared with the response elicited by inactivated vaccines, which induced little or no detectable antibodies to NS3

protein. The mean OD value of NS3-ELISA for sera from vaccinated animals was almost identical to that obtained with sera from BTV negative animals, allowing a good discrimination between positive and vaccinated animals.. Considering the bovine samples, the percentage of discrepancies (rRT-PCR positive/NS3-ELISA negative) was 49% and it can be classified in two types. As in sheep, one type of samples may be originated from animals in very early or early stages of infection. At least in one case, this assumption was confirmed with paired samples collected with a week interval in which seroconversion to NS3 was observed.

Moreover, these animals were located in the vicinity of sheep farms where outbreaks of BTV-1 were occurring.

The other type of discrepancy was observed in samples with an rRT-PCR positive result for BTV-4 in which BTV-4 neutralizing antibodies were detected but not NS3

antibodies. It has been demonstrated before that appearance of neutralizing antibodies in serum does not coincide with clearance of virus from blood and BTV may coexist with specific neutralizing antibodies in infected animal for long periods [START_REF] Richards | Comparison of virologic and serologic responses of lambs and calves infected with bluetongue virus serotype 10[END_REF]. These samples were collected between December 2007 and February 2008, when vector activity was reduced or absent and outbreaks were not registered. Two animals of this group were bled twice with one-month interval and in second collection of blood A good agreement between rRT-PCR and NS3-ELISA for negative samples was observed. From a panel of 293 rRT-PCR negative samples only three from sheep with clinical signs of BT were NS3-ELISA positive. These discordant results are difficult to explain, however, since NS3-ELISA and cELISA were concordant, it seems that limiting factors of rRT-PCR, namely pour quality of RNA, RT-PCR inhibitors, mutations that hamper primer or probe annealing or viral load below the limit of detection, can be responsible for the negative detection of viral RNA. However, these samples were several times re-analysed giving consistent results. Although maternal antibodies could be another possible explanation, the age of the animals, over one year, excludes this hypothesis. On the other hand, animals in which inactivated vaccines have been repeatedly used may develop NS3 antibodies, due to possible presence of small amounts of this protein in the vaccine. However, in the present study this hypothesis was not confirmed with the vaccines used, since many of the NS3 antibody negative field samples originated from BTV-1 or/and BTV-4 vaccinated animals that had been

  attenuated vaccines because of the absence of replicating virus.

  s c r i p t vaccinated animals with BTVPUR AlSap 4 (Merial) and/or BTV-1 Zulvac 1 (Fort Dodge), as well as samples from infected animals collected during BTV-1 and BTV-4 outbreaks in 2007 and 2008, were studied.

  BTV serotyping was carried out by rRT-PCR using specific primers for BTV-

  Figure2, none of the immunized animals developed significant antibodies levels against NS3 antigen before challenge, indicating that bivalent vaccine did not induce NS3 antibodies. In contrast, antibodies against BTV structural protein VP7 were detected by cELISA at day 21 post-vaccination in three of the seven immunized animals and 7 days later the four remaining animals also seroconverted. All these animals maintained detectable levels of VP7 antibodies until the end of the study (216 days).

  the ability of NS3-ELISA to differentiate between infected and vaccinated animals.

  Although the RT-PCR does not detect the same biological entity as the ELISA (viral nucleic acid versus antibodies), we compare both results in order to evaluate the ability of NS3-ELISA to diagnose infected animals. Between the two tests, a concordance of 87% was found for sheep positive samples. Half of the sheep discrepant samples, BTV-PCR positive but NS3-ELISA negative were also negative for cELISA, suggesting a very early infection (NS3, VP7, and SN negative/rRT-PCR positive). The remaining discrepant samples (rRT-PCR positive/NS3-ELISA negative) were cELISA positive and originated from vaccinated animals with neutralizing antibodies for BTV-4 but not for BTV-1, suggesting, an early BTV-1 infection (NS3 negative, VP7 positive, and SN negative/rRT-PCR positive). VP7 antibodies can be present due to previous BTV-4 immunization or due to a recent BTV-1 infection in which the faster antibody response to VP7 is already present but antibodies to NS3 are not yet detectable. In the vaccination trial, NS3 antibodies appeared one week later than those to VP7 in the control unvaccinated animal after challenge.

  no longer detected. These results together with the high Ct values of rRT-PCR, presence of BTV-4 neutralizing antibodies, winter time, and the absence of new BTV-4 outbreaks reported during 2008, suggest that these samples originated from animals in a late stage of infection (NS3 negative, VP7 positive, SN-BTV-4 positive and rRT-PCR-BTV-4 positive). As observed for control animal (N8), viral RNA could be detected for a longer period than NS3 antibodies.Taking into account the epidemiological data (i.e., vector activity; the season; occurrence of outbreaks; trade of animals; introduction of new serotypes), the use of NS3-ELISA may be advantageous allowing the movement of positive rRT-PCR animals in late stages of infection when viral RNA is still detected but NS3 antibodies are no longer present.

  , Iwata H., Chen S-Y, Compans R.W., Roy P. 1992. Multiple glycoproteins synthesised by the smallest RNA segment (S10) of bluetongue virus. J Virol.

a

  Uninfected animals: rRT-PCR (-), NS3-ELISA (-) and cELISA (-); b Vaccinated animals: rRT-PCR (-), NS3-ELISA (-), cELISA (+) and SN (+); c Infected animals: rRT-PCR (+), NS3-ELISA (+), cELISA (+).

Figure 1 :

 1 Figure 1: Western blot analysis of the NS3 immune response. Western blots developed with HRPconjugated anti-His-tag monoclonal antibody (lane 1), of serum from BTV-1 infected sheep (lane 2) and of serum from uninfected sheep (lane 3).

Figure 2 :

 2 Figure 1

Figure 2 AFigure 3 .

 23 Figure 2

Table 1 :

 1 OD values of NS3-ELISA of 478 field sera from uninfected, infected and vaccinated sheep and cattle.

			Sheep			Cattle
	NS3-ELISA	Uninfected a Vaccinated b Infected c Uninfected a Vaccinated b Infected c
	(OD 450nm )
	Mean (M)	0.12	0.18	1.00	0.13	0.17	0.88
	Standard deviation	0.07	0.08	0.28	0.06	0.07	0.25
	(SD)
	Total of animals	71	63	118	68	88	70

Table 1 A

 1

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

Table 2 .

 2 Comparison between the NS3-ELISA and the rRT-PCR using 562 field serum samples

					RT-PCR
			Ovine			Bovine
	NS3-ELISA	Positive	Negative	Total	Positive	Negative	Total
	Positive	118	3	121	70	0	70
	Negative	17	134	151	67	156	223
	Total	135	137	272	137	156	293

Table 2

 2

Acknowledgements

We are grateful to J. Teixeira, A. Batista, A. Almeida, R. Ferreira, F. Cordeiro and M.R.

Ferreira for technical assistance. We thank the Portuguese Veterinary Authorities involved in the BTV eradication and surveillance program.

A c c e p t e d M a n u s c r i p t 17 revaccinated every six months. Finally, a short lasting viraemia could also be an explanation as observed in animal N7 of the vaccination trial.

The purity status of the inactivated vaccines was a critical point since low levels of antibodies against non-structural proteins can be produce. The inactivated vaccines used in this study were sufficiently purified since it did not induce NS3 antibodies at levels detectable by NS3-ELISA. However, tests with different vaccines from other companies and for different BTV serotypes are needed to ascertain whether those vaccines induce antibodies against NS3 protein. In a recent inter-laboratory proficiency test carried out by the EU Community Reference Laboratory for BT (Institute for Animal Health, Pirbright, UK), the NS3-ELISA did not detect NS3 antibodies in four samples from cattle vaccinated with the 4 available BTV-8 inactivated vaccines (CZ Veterinaria, Fort Dodge, Intervet and Merial). These preliminary results suggest that NS3-ELISA is not serotype dependent.

Non-structural proteins have also been used in DIVA tests, for African horse sickness virus [START_REF] Laviada | The use of African horse sickness virus NS3 protein, expressed in bacteria, as a marker to differentiate infected from vaccinated horses[END_REF], foot-and-mouth disease virus [START_REF] Clavijo | Developments in diagnostic techniques for differentiation infection from vaccination in foot-and-mouth disease[END_REF][START_REF] Lu | Development and validation of a 3ABC indirect ELISA for differentiation of foot-and-mouth disease virus infected from vaccinated animals[END_REF] and equine influenza virus infections [START_REF] Birch-Machin | Expression of the non-structural protein NS1 of equine influenza A virus: detection of anti-NS1 antibody in post infection equine sera[END_REF]. Also a competitive ELISA based on the use of a monoclonal antibody directed against the NS1 protein of BTV allowing the discrimination between infected and vaccinated animals has been described [START_REF] Anderson | A competitive ELISA for the detection of anti-tubule antibodies using monoclonal antibody against bluetongue virus non-structural protein NS1[END_REF].

The diagnostic test described in this study demonstrates that NS3 protein of BTV can be used as a diagnostic reagent in DIVA tests. Although NS3-ELISA is less sensitive than rRT-PCR detecting earlier infections, it's a method comparatively easy and cheap for a routine diagnosis. Because of the imposed restriction to animal movement between BTV-infected and non-infected areas, the NS3-ELISA can be used as a reliable tool for massively serological survey to detect incursions of BTV into the animal population, A c c e p t e d M a n u s c r i p t