Emerging European-like PRRSV in the U.S. Implications for diagnostic and control strategies

Title: Emerging European-like PRRSV in the U.S.: Implications for diagnostic and control strategies - NPB# 04-186 Investigator: Eric A Nelson Institution: South Dakota State University Co-Investigator: Raymond R.R Rowland (Kansas State University) and Jane Christopher-Hennings Date Received: March 1, 2006 I Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) exists as two major genotypes, designated as Type (European-like) and Type (North American-like) Type isolates have only recently appeared in the U.S Therefore, the purpose of this study was to evaluate the genetic, pathogenic and immunological properties of representative U.S Type isolates and develop panels of well-characterized serum samples to serve as reference or control materials for diagnostic laboratories and other researchers Forty week-old pigs were divided into five groups (n=8) and inoculated intra-nasally with one of four different U.S Type PRRSV isolates (SD01-07, SD01-08, SD02-11 or SD03-15) or left as mock-infected controls Isolates were chosen based on their location in different branches of the current U.S Type phylogenetic tree, origin from different geographical regions and presentation of a range of clinical signs in the field Full-length sequence analysis of the four challenge isolates, as well as additional Type isolates, demonstrated that these viruses are a divergent and rapidly evolving group The presence of recombination between viruses suggests a new source of genetic diversity and that Type viruses may be more widely distributed than previously thought These results have important implications in the detection of PRRS viruses in the field Challenged pigs were monitored daily for clinical signs and blood samples were collected twice weekly for the first two weeks and weekly thereafter Three pigs from each group were euthanized at 14 dpi to evaluate acute lesions and the remaining pigs were retained for 12 weeks to monitor antibody responses, viral loads and persistence Clinical signs and pathology were variable between groups but generally remained mild Viremia was monitored using semi-quantitative real-time PCR and viral loads varied substantially within and between groups Challenged animals seroconverted, as detected by the IDEXX ELISA, by approximately 14 dpi Neutralizing antibody responses against the homologous challenge isolates reached detectable levels as early as 21 dpi and in some cases, were particularly robust, reaching neutralization titers as high as 1:512 However, sera did not neutralize the North American (Type 2) PRRSV prototype isolate (VR-2332) and demonstrated intermediate levels of neutralization against other European-like isolates and the European prototypic strain, Lelystad virus This study represents the first detailed analysis of the relative virulence, persistence potential and pathogenesis of diverse U.S Type PRRSV isolates It also provides a resource of well-characterized serum samples to serve as reference and control materials for other researchers and diagnostic laboratories II Introduction: Porcine reproductive and respiratory syndrome (PRRS) viruses can be divided into Type or Type isolates Historically, Type isolates, such as the Lelystad virus, were restricted to the European continent; whereas Type isolates, such as VR-2332, were found in North America Comparisons at the gene level show only 55-70% nucleotide identity between Type and Type isolates The first reported European-like PRRSV in North America appeared in Ontario, Canada in May 1999 (Dewey et al 2000) The source was traced to a single group of pigs imported from the Netherlands and the affected pigs were isolated with no further documented spread of the virus Soon after this time, European-like PRRSV isolates were reported by diagnostic laboratories at Minnesota (Rossow et al 2000; Mahlum et al 2000), South Dakota (Ropp et al 2002) and Missouri (Schommer et al 2002) However, unlike the Canadian outbreak, these viruses could not be traced to a specific European country of origin European-like (Type 1) PRRSV isolates have now been identified throughout the U.S and have complicated diagnostic strategies for the detection of PRRSV due to their substantial genetic and antigenic differences from North American (Type 2) PRRSV Many diagnostic laboratories have modified their PCR assays to also allow the accurate detection of European-like PRRSV Monoclonal antibody-based reagents are available for the detection or confirmation of both PRRSV genotypes by immunohistochemistry (IHC) and virus isolation, although care is required in the selection of appropriate reagents The commercially available IDEXX HerdChek ® PRRS ELISA will accurately detect an antibody response to both North American and European-like PRRSV, but does not differentiate between them However, results obtained using the common follow-up indirect fluorescent antibody (IFA) assay for PRRSV serology can be influenced by antigenic differences between virus genotypes Virus neutralization results are also highly dependant on the virus isolate or genotype used in the neutralization assay Therefore, it is essential for diagnostic laboratories to have access to well-characterized European-like PRRS virus isolates and control serums to ensure accurate serodiagnosis of European-like PRRSV These reference materials, developed during the current project, are also important for ongoing research studies and the development of improved diagnostic assays Our previous studies suggested that European-like (Type 1) isolates in the U.S were a heterogeneous group, and that these isolates were distinct from Type isolates currently circulating on the European continent (Ropp et al 2004; Fang et al 2004) These viruses have also shown substantial differences in clinical presentation in the field; ranging from completely asymptomatic to quite virulent Elimination strategies must take into account the unique genetic, antigenic and biological properties of U.S Type PRRSV The overall goal of this proposed work was to gain the first insights into the diagnostic, antigenic and molecular properties of European-like Type PRRSV now circulating in the U.S These properties will help explain their successful emergence and evolution in the field, as well as lead to the design of strategies to control their spread The results of this initial study will lead to tools that will be useful to monitor the spread of Type isolates, rapidly and accurately detect their presence by diagnostic laboratories and the development of new information for vaccine and control strategies to prevent their spread III Objectives: Objective Genomic epidemiological analysis of U.S Type isolates: Full-length genome sequencing combined with phylogenetic analysis The first step to address questions related to the spread of Type PRRSV is to establish a database containing the genome sequences of known U.S Type isolates recovered from the field The relatedness and evolution of Type viruses was studied by performing phylogenetic analysis The standard approach for comparing PRRSV isolates is to perform nucleotide sequencing on open reading frame (ORF) 5, which accounts for less than 5% of the viral genome Other regions of the genome, such as non-structural protein (nsp) 2, may be more useful for comparing isolates Therefore, genome sequencing is used to identify additional regions of the genome which are important in diagnosis and pathogenesis Finally, there is a need to understand recombination as a mechanism for how PRRS viruses, including vaccines, acquire new genetic sequences Genome sequencing of multiple isolates will provide insight into how often recombination occurs in the field Other benefits to the producer include; 1) the ability to track the source of Type outbreaks, 2) identification of unique antigenic and genetic sequences, which can be used to develop future, improved diagnostic assays, and 3) incorporation of sequence information into existing databases for bioinformatics analysis and vaccine design Objective Development of a proficiency panel containing serum samples for the accurate detection of U.S Type PRRSV isolates The IDEXX HerdChek® PRRSV ELISA is reported to detect antibodies in response to infection by Type and Type PRRSV However, many follow-up assays are somewhat strain-specific and have not been validated with respect to Type field isolates, primarily because we lack known serum standards Some diagnostic laboratories have already incorporated PCR assays designed to detect European Type isolates but proficiency panels containing known U.S Type isolates are not readily available A key component of this project is to develop panels of sequential serum standards obtained from pigs at different stages after infection with antigenically distinct U.S Type PRRSV isolates These samples will be distributed and made available to diagnostic laboratories to assess sensitivity and specificity of PCR and serological assays This objective will provide producers with more confidence in reported diagnostic results, reduced risk of transmission between farms, and the validation of new diagnostic assays will be accelerated Objective Evaluate the virus neutralizing properties of sera from pigs experimentally infected with representative U.S Type isolates Current efforts by several investigators are directed at developing a typing system for PRRSV These systems can be based on the cross-neutralization properties of mono-specific sera from experimentally infected pigs In collaboration with Dr Fernando Osorio and others, the goal of this objective is to incorporate U.S Type isolates into this classification system A large body of preliminary work performed in our laboratories demonstrates that the European-like PRRSV isolates in the U.S represent a heterogeneous group (Fang et al 2004; Ropp et al 2004) Nucleotide heterogeneity would be expected to lead to antigenic variation of the proteins, including potential changes in the virus neutralization determinants in glycoprotein (GP) and other proteins Extensive evaluation of neutralizing antibody titers against homologous and heterologous PRRSV isolates has shown substantial differences in cross-neutralization titers which may correlate with cross-protection between isolates The approach is to prepare neutralizing antisera against four different U.S Type isolates and compare cross-neutralizing activities within the Type group and between Type and North American Type isolates Benefits to the producer include; 1) an analysis of the cross-neutralizing capacity among Type viruses (inc Lelystad virus), and between Type and Type isolates; and 2) the eventual development of a practical classification scheme for PRRSV involving crossneutralization as an indicator of potential cross-protection to aid in the design of future vaccines and controlled exposure strategies Objective Evaluate pathogenic properties of U.S Type isolates The pathogenic potential of European-like PRRSV remains unknown Observation of infected herds suggests that Type isolates may exhibit a broad range of pathogenicity Therefore, the final objective is to perform the first controlled pathogenic studies with these viruses Benefits include gaining a better understanding of factors such as extended persistence or differences in antibody responses and viral loads that may complicate diagnostics Linking virulence with sequence data is critical to new vaccine design Also, the identification of possible “naturally attenuated” Type isolates may provide a starting point for more traditional modified-live vaccines IV Materials & Methods: Genomic sequencing and analysis Nucleotide sequence information for Type viruses was obtained directly from sera or after isolation on porcine pulmonary alveolar macrophages (PAMs) RNA was extracted from the pellet using a QIAamp viral RNA kit (Qiagen) and cDNA prepared by reverse transcription using Superscript II (GibcoBRL) and random hexamer oligonucleotides according to manufacturer’s recommendations The forward and reverse oligonucleotides for the PCR amplification of ORF1 were designed based on the sequence of Lelystad virus (GenBank accession number M96262) See Table for a complete listing of PCR primers PCR was performed using pfu polymerase (Stratagene) in a reaction volume of 100 ul containing Mg-free 1x PCR buffer, mM MgCl2, 200 uM of each dNTP, 1uM of each primer and 2.5 units of polymerase The PCR consisted of cycle of 95 C for minute, 30 cycles at 94 C for 30 seconds, 55-600 C for 30 seconds, 680 C for per kb, then followed by 25 cycles at 940 C for 20 seconds, 55-600 C for 30 seconds, 680 C for per kb increasing by 10 seconds per cycle The final extension was at 70 0C for 20 Sequencing reactions were prepared using a Big Dye Dideoxy cycle sequencing kit (Perkin-Elmer) and sequencing performed at the Iowa State University DNA Sequencing Facility PCR products were sequenced in both directions at least twice Sequences were assembled and analyzed using Gene Tool and Pep Tool software (BioTools) Multiple sequence alignments were performed using Vector NT software (InforMax,), which is based on the Clustal W algorithm Maximum-parsimony analysis on the Clustal W sequences was performed using PAUP 4.0 (Swofford, 2000) All characters were equally weighted and heuristic search with TBR, stepwise addition, and MULTIPARS options were used in the search for most-parsimonious trees A strict consensus tree was generated to evaluate the differences among equal most parsimonious trees, if more than a single most parsimonious tree was recovered Bootstrap analyses with 50% majority-rule consensus from 1000 replicates (PAUP) were conducted to evaluate the support of individual clades Neighbor-joining distance analysis was performed on the resulting distance matrix using MEGA with Lelystad virus as the outgroup The bootstrap option was carried out with NJBOOT from 5000 replicates to assess the robustness of interior branches of the tree Table shows a complete listing of virus isolates evaluated with state of origin, date of isolation, and associated GenBank accession numbers Table PCR primers used for amplification and sequencing Forward Primers Primer Sequence Primer 1F ATGATGTGTAGGGTATTCCC 6229F 190F ATGTCTGGGATACTTGATCGG 6501F 204F GATCTCCACCCTTTAACCATG 6581F 392F GTTCCTATCGGCATTCCTCAGG 6787F 451F GGCTCTCAGCCGTATTCCCT 6820F 490F GTAATCACAACTTCCTCCAACG 6900F 684F TGTTGACTAACTCGCCTCTGCC 6975F 693F GTGTTGACTAACTCGCCTCTGC 7101F 881F CGAGTTAGAACGTCAGGTCG 7245F 951F TGGGAGCTCACTGAGTCCCC 7567F 1321F GAGCTCATGAGCCATAAGTGGTACGGCGC 7601F 1339F GCTGGAAAGAGAGCAAGAAAA 7680F 1351F GGTTTGGAGCACATAAGTGG 8101F 1462F AATCACCACCTACTCTCCACCG 8351 F 1501F GGTCTTGTTGGTGGCACGTT 8601F 1861F ACAGACTACCCTCAGATTGT 9101F 2037F TCATCTATGCTGTTGAGAGG 9601F 2051F CTATGCCGTTGAGAGGATGT 9618F 2601F ATACTCCGTCATGTTGAGCA 10101F 2910F AAGATACTCTGGTGGCTGACGC 10601F 3040F CGGTTCACTTGCTGATGTCC 10941F 3151F CTTCACAGTTCCAGGCCGGT 11101F 3303F GTCTGAAGCAACTGGTGG 11171 F 3322 F CGGCTGGGTTAGTGCTTGAC 11601F 3651F GGATGCCTTCCCTTACTGGG 11610F 3770F TTCTTCTTGTGACGACGATTCG 11967F 3803F CCAGTCGGTTCTTCTTGTGACG 12037F 3875F TCTCTTATGTGTCATCCTTGGC 12101F 4251F ACGGTGGTTGCTGTCCCATA 12427F 4279F CTCGAGGGGGCACGTTGCATCTGGCA 12601F 4374F AGCCTACGCCTGAAGTTGTTCG 12629F 4801F AGGTAGGGATTTTTATTTTA 13101F 4981F TGCTTTGGGTGCACTCATTC 13150F 5096 F CACCTTATGACATCCACCAG 13523F 5134F TACACCTTATGACATCCACC 13601F 5351F CGTCGTCACTGCTGCTCATG 13913F 5477F AGTGGCGATTATGCCTGG 14010F 5617F GGTGCTTTCAGAACTCGAAAG 14101F 5704F ACTCGGTTCTGGTCTTGTGACG 14416F 5901F CTTCTCTGGCGCATGATGGG 14601F 6100F GATTAGACTCCTCACGGCATCC 14851F 6137F GCCGTTGAAATCGGGGCTTTTGCTGGCAGG Reverse Primers Primer Sequence Primer 390R TCTTGTCCTTAGGCTTGTAA 7565R 820R CGACCGTTGGGAGAGGAGTC 7700R Sequence CTCGAGGGGGGCCTCTCCACCGTCCA GTATCTGCTTCAAACATGAA ATTGAAGCAGCGTATGCCAAGG CTCGAGTCTCTGACTGGTGCCCTCGC CCTTAACTGGCATCCCACTCC AGAGTCGAGAGGATGAAGAAACACTGTGTGT TCGGCTTCCACAACATCAATGG CAACAGGGCTTTGATCCAAA AGCCTGACAACTGCCTTGAAGC CTCGAGGCAAAGCTTTCTGTGGAGC TCCGGACTTGACACAAAACC GAGCTCGGAGCAGTGTTTAAACTGC ATCTACTCAAGGATTTGTCC TGTTCCAAGCCCAAAACCAG AACTTGCAGGATGTGAAGAG GAGTATTATGCGTCTGCTGC TATTGCAGGCAATGAAGTTG GAGCTCGGGAAGAAGTCGAGAGTGT CCACTATTTATAGATTTGGC CTGGAGGGGAGCTGTATGCC GAATTCGGTACGAGCTCTCCGCTCC AGTAAGCTCGCCCGGCAGGG CCATACCTGCAACCTGAGAC ACAACTGGGATTGTGTTCGA CTCGAGGGTCGCTATTTCACCTGGTA CTCTCCATTCACTCTCC ACATGCGAGTTTCCCACC TTCAGGTCAAGCGGCCTGGA CTCTGTGGCTTCATCTGTTACC TGAGGAGCGTGACCATGATG ATGACAGGTGTGAGGAGC AAAGTCGTCCCAATGCCGTG AATGTCGTGAAGCCGTCG CACTCTTGCTTCTGGTGG GAATGGGACCAATTGGCTGT ACCAACTTCATTGTGGACGACC CAACCCTTGACGAGAACTTCG CAGCATCACATACACACCTA GAATTCTCTGGTAACCGAGCATACGC GGGAATGGCCAGCCAGTCAA TCCAGGTTGAGTTTATGCTG Sequence GGATCCGCTTCCCATTGGACTCTTCC GGATCCTAGCAGTTTAAACACTGCTC 1338R 1680R 2171R 2240R 3028R 3400R 3680R 4101R 4278R 4302R 4352R 4511R 5160R 5616R 5727 R 5937R 6010R 6228R 6740R 7100R 7420R GCCGTACCACTTGTGACTGCCAA TACTTGGCGTTAGGACAGGC AGGAAAACACTCGTCCAAAGAA GTGAACACAGGACAACAGCAGC TAAGGCTTGTGCGGAATGACGC GGTCCCACATGCGGGAAAGCCATTCCTGCG AAGAACGACAGAGCAAGAGAGC ACAAGGATACAAGAGAGACACG GCCCAGTAACCTGCCAAGAATGG GGATCCTAAAAAGTGCCAGATGCAACGTGCC AACAACTTCAGGCGTAGGCTGG TCAAGTAACCACAGCGGACTGC TACTGGTGGATGTCATAAGG GGATCCCTCAAGAAGAGACCCAAGCTG CTGACCTTTCCAGACATTTTGC AAGACACACCAGAAGTTGACGG GCAAAGGAAAACACGGCTCGGACCAAAACT TTCCAGTTCAGGTTTGGCAGCAA GGATCCCCCTCGGCAAAGTATCGCAAGA TTGGGAGGCGGTTTGCAGACCCTCATAATCC CGCTGTTTCAGTTACAACTAAGC 7886R 7984R 8150R 8450R 8840R 9617R 10940R 11206R 11230R 11250 R 11463R 11609R 12071R 12152R 13900 R 14243 R 14433R 14463R 14993R 15062R TTGCGGACTTGGTGTCTTGAGG TTGACTTACCATCAGACACAGG GAATCTGCGTACAAGGCGTAA TCTTCATGAATGCCTGGGTGACACCACTGA GGTCCTCGAATTTAAGCTGTTCCTCGAGGAA GGATCCTAACTCATAATTGGATCTGAGTTT GGATCCTAATTCTAGATCAGCACAAATGGC TCCCTGAAGTCTAATTTGAGTTTC TTAATTAACGTCCGACTAATGGTCTGGAA TTCCAGACCATTAGTCGGACG ACCAGACGCTGAGATTGAATGG GGATCCTAATTCAAGTTGGAAATAGGCTGT GGATCCTCAATTCAGGCCTAAAGTTG GCGGCCGCCCGCTTGACCTGAATGTTCC CCCGGTCGTCCACAATG GGTGGGTAGAATGAAAATGCAA TATGCTCGGTTACCAGACGC GCGGCCGCTCTAGAACCAGACGCTGAGATTGAATGG GGATCCATTGAATAGGTGACTCAGAGGC TTTTTTTTTTTTTAATTTCGGTCACATGGTTCCTC Table Type PRRSV isolates used for phylogenetic analysis Asterisks identify those isolates for which full genomes were prepared from this study or are available from GenBank Isolate Isolation date Accession No *1 Location within U.S Hawaii 01-07 07-26-01 *2 Iowa-Farm 99-01 09-22-99 Iowa-Farm 02-10 05-14-02 AY383632 (Nsp2); AY395079 (ORF2-7) Sequenced at SDSU AY366525 (EuroPRRS complete genome obtained from GenBank) AY383633 (Nsp2); AY385081 (ORF2-7) *4 Iowa-Farm 04-41 02-19-04 Complete sequence obtained from this study Minnesota 03-01 09-29-03 Missouri 03-08 01-03-03 *7 Nebraska-Farm 02-11 12-18-02 Nebraska-Farm 04-06 01-21-04 *9 North CarolinaFarm 01-08 07-27-01 *10 North CarolinaFarm 03-12 03-07-03 11 North CarolinaFarm-2 North CarolinaFarm 04-48 01-09-04 03-15 01-16-03 North CarolinaFarm North CarolinaFarm 03-16 01-09-03 03-17 01-16-03 AY749375 (Nsp2); AY749376 (ORF3); AY749377 (ORF5) AY749383 (Nsp2); AY749384 (ORF3);AY749385 (ORF5) AY383634 (Nsp2); AY749399 (ORF3); AY395078 (ORF5) Complete sequence obtained from this study AY749391 (Nsp2); AY749392 (ORF3); AY749393 (ORF5) AY375474 (ORF1); AY395080 (ORF2-7) Part of this genome obtained from GenBank, remainder from this study AY383635 (Nsp2); AY749401 (ORF3); AY395074 (ORF5) Complete genome obtained from sequencing performed in this study AY749409 (Nsp2); AY749410 (ORF3); AY749411 (ORF5) AY383636 (Nsp2); AY749403 (ORF3); AY395076 (ORF5) Complete genome obtained from sequencing performed in this study AY383637 (Nsp2); AY749405 (ORF3); AY395077 (ORF5) AY383638 (Nsp2); AY749407 (ORF3); AY395075 (ORF5) 04-40 01-22-04 04-42 02-24-04 04-43 03-18-04 18 North CarolinaFarm North CarolinaFarm North CarolinaFarm Ohio 03-05 04-09-03 19 Tennessee 03-10 10-09-03 20 Wisconsin 04-09 01-24-04 *12 13 14 15 *16 *17 Complete genome obtained from sequencing performed in this study Complete genome obtained from sequencing performed in this study AY749379 (Nsp2); AY749380 (ORF3); AY749381 (ORF5) AY749387 (Nsp2); AY749388 (ORF3);AY749389 (ORF5) AY749395 (Nsp2); AY749396 (ORF3);AY749397 (ORF5) Animals / challenge groups Forty week-old, PRRSV naïve pigs were obtained from a certified PRRSV-negative herd and randomly divided among groups housed separately in isolation facilities in the SDSU Animal Resource Building All animal care and handling procedures were approved and in compliance with Institutional Animal Care and Use Committee guidelines After a 4-day acclimation period, pigs from each group (n=8) were inoculated intranasally (1 ml/naris) with each U.S Type isolate at 1X104 TCID50 The four isolates were chosen based on their genetic heterogeneity, geographic diversity, year of isolation and clinical presentation in the herd Selected isolates and their phylogenetic relationships are identified in bold in Figure Isolate 01-07 was obtained from a 7-10 week old nursery pig in swine herd in Hawaii showing no morbidity or mortality in this age group Isolate 01-08 was from a group of week old pigs in North Carolina showing no significant clinical signs Isolate 02-11 was obtained from a nursery in Nebraska showing 6-30% nursery mortality while 25% of this age group were visibly ill or unthrifty throughout the nursery and grower phases Isolate 03-15, from North Carolina, was associated with a herd experiencing a preweaning mortality of 80-90% for weeks time The subsequent performance in the nursery was poor and continued through the finisher stage In the adult sow population, there was only a mild abortion storm compared to previous U.S PRRSV outbreaks The negative control group consisted of pigs mock-challenged with MARC-145 host-cell culture supernatant Virus isolates were propagated on MARC-145 cells and used at the lowest passage possible Virus inoculums were also plaque purified to ensure homogeneity and screened by PCR for adventitious agents, such as PCV-2, prior to challenge Throughout the study, the clinical condition of animals was assessed, as well as virus load, antibody responses, virus neutralization activity and gross and microscopic pathology Three pigs from each group were euthanized at 14 dpi to evaluate acute lesions and the remaining pigs were retained for 12 weeks to monitor later term antibody responses, viral loads and persistence, prior to being euthanized at 85 dpi for necropsy evaluation Sampling and storage Pigs were observed daily for clinical signs and serum samples were obtained from all pigs twice weekly for the first two weeks and weekly thereafter The study was terminated at 85 dpi and samples were cataloged by number, dispersed into appropriate aliquots for analysis and distribution and stored at -80 C Histopathology At days 14 and 85 dpi, samples of ventral and dorsal lung, liver, kidney, heart, tonsil, spleen, lymph node, ileum, brain, and nasal turbinate were collected from pigs following euthanasia and fixed in 10% phosphate buffered formalin These tissues were then processed and sectioned by standard methods, and stained with hematoxylin and eosin for examination Virus isolation / Serology For the isolation of PRRSV, serum and tissue samples were evaluated in porcine pulmonary alveolar macrophages and MARC-145 cells as previously described (Benfield et al., 1992) Briefly, serum samples were centrifuged and diluted ten-fold in MEM + 2% horse serum; then added to confluent monolayers of MARC-145 cells in 24 well plates Plates were incubated for 48 hours at 37 C in a humidified incubator Cells were then washed, fixed in 80% acetone and stained with PRRSV-specific, SDOW-17 FITC-labeled monoclonal antibody Virus titration results were reported as TCID50/ml or fluorescent focus units (FFU) /ml Serological responses were measured using the IDEXX HerdChek ® PRRS 2XR ELISA according to the manufacturer’s instructions and results were reported as a sample to positive ratio (S/P) with an S/P value of 0.4 or greater considered to be positive Group B 011 03-15 (NC2003) 03-16 (NC-2003) 03-17 (NC-2003) 025 100 03-01 (MN2003) 03-08 (MO-2003) Group A 006 03-05 (OH-2003) 100 04-09 (NE -2004) 001 53 02-10 (IA-2002) 04-06 (NE2004 03-12 (NC-2003) 006 008 82 U.S Type Isolates 021 100 001 005 87 01-08 (NC-2001) 04-48 (NC-2004) 013 96 036 003 99-01 (IA-1999) 029 03-10 (TN-2003) 055 02-11 (NE-2002) 001 047 01-07 (HI-01) 005 B1 016 Lelystad Virus 88 010 84 002 005 046 100 GB1 Lelystad-like viruses vivvviruses 5A Porcilis 026 ES1 040 024 100 ES5 ES6 052 PL15 006 035 046 PL6 D2 ES2 Figure Phylogenetic placement of Type PRRSV isolates used for European animal 007 Type PyrSvac challenge Location and year of isolation are in parentheses and isolates selected 1for 045 PRRSV 111-92 challenge are shown in bold The figure shows the neighbor-joining tree from p-distance PL9 estimation of nucleotide substitutions per site for ORF5 sequence Remaining European Type Isolates 002 015 North American Type Isolates Virus neutralization Isolate-specific virus neutralization titers were determined using a fluorescent focus neutralization (FFN) assay as previously described (Wu et al., 2001) The four challenge isolates from this study, as well as selected North American reference isolates were evaluated against all serum samples Results were reported as the greatest serum dilution resulting in a >90% reduction in viral infectivity or FFU Briefly, each test serum was heat inactivated for 30 minutes at 56 C Serum was diluted in 2-fold dilution series in MEM + 2% horse serum and an equal volume of selected virus suspensions containing 10 FFU/ml were added to 96-well tissue culture plates The mixture was incubated one hour at 37 C then transferred to a confluent monolayer of MARC-145 cells in a 96 well format with further incubation at 37 C, in a humidified atmosphere of 5% CO2 After 24 hours, the cells were fixed with 80% acetone and stained with SDOW-17 FITC-labeled PRRSV monoclonal antibody for hour at 37 C Neutralization of the virus was determined by using a 90% reduction of fluorescent foci as the endpoint Real-time, quantitative PCR For the detection of viral RNA and determination of viral load, serum and tissue samples were examined using a real-time, semi-quantitative PCR; Tetracore, Inc VetAlert™ PRRSV assay (Wasilk et al 2004) To determine viral RNA quantity, a standard curve was generated from an in-vitro transcript dilution V Results: Objective Genomic epidemiological analysis of U.S Type isolates: Fulllength genome sequencing combined with phylogenetic analysis ORF5 codes for a 201 amino acid major glycoprotein, GP5, which is considered the primary viral protein involved in the recognition of the PRRSV receptor on macrophages and is a target of neutralizing antibody Since there is no well-established serotyping system for PRRSV, ORF5 gene sequence is frequently used to identify and classify PRRSV isolates Previous analysis of ORF5 sequence indicated a close association of Type viruses, with a group of Lelystad-like viruses (Figure 1) Phylogenetic trees using ORF5 sequences of 20 U.S Type isolates were constructed using Lelystad virus (LV) as an outgroup Phylogenetic trees were constructed using the neighbor-joining and maximum-parsimony methods Neighbor-joining placed a majority of the U.S Type ORF5 sequences into one of two subgroups (see Figure 2), which for the purpose of convenience, we designated as Group A and Group B viruses Parsimony produced the same tree including the placement of viruses into the same groups (data not shown) Group A and Group B formed monomorphic clades strongly supported by bootstrap values of 100% and 97%, respectively The placement of viruses in either group did not correlate with geographic location or year of isolation Based on bootstrap values, isolates 99-01 (EuroPRRS), 02-11, 03-10 and 04-40 could not be placed in either group, but remained closely associated with the other mainland U.S isolates to form a single monomorphic group supported by a bootstrap value of 96% The 01-07 Hawaiian island isolate was well-separated from the mainland isolates and along with LV occupied a basal position A similar topology was obtained when trees were constructed using ORF3 sequence (data not shown) 10 10 03 16 03 17 04 41 (Grp A) Group B 03 15 04 09 9 10 03 05 03 08 03 01 03 10 6 02 10 04 42 10 04 06 9 04 40 01 08 03-12 6 10 Group A 02 11 99 01 01 07 Lelystad 01 Figure Phylogenetic analysis using nsp2 Viruses could be placed into similar groups according to ORF5 The one important exception is isolate 04-41, which was located in ORF5 Group A The significance of this virus in genetic recombination is discussed in more detail One interesting difference between the nsp2 and ORF5 trees was the relocation of isolate 40-41; from Group A in the ORF5 tree to Group B in the nsp2 tree A recombination event between a Group A and Group B virus could resolve this discrepancy To look at recombination, we sequenced all ORFs in a subset of eight representative U.S Type viruses We were able to include the genome sequence of virus 99-01 or EuroPRRS (Ropp et al 2004) A search for recombination was initiated using SimPlot, a computer program originally developed to identify chimeric HIV viruses SimPlot is designed to identify chimeras that result from recombination between known clades or groups of viruses Genomic sequences from isolates 01-08 (Group A), 03-15 (Group B), 04-41 (putative A/B chimera) and LV (non-A/B) were 13 included in the analysis The results in Figure showed that the 04-41 query sequence is a chimera composed of 03-15 and 01-08–like viral sequences The 03-15-like sequence in 04-41 was located in a region between nucleotides 400 and 4,200 in ORF1a through the end of the genome The 3’ break point for recombination was just downstream from the nsp2/3 junction To confirm the presence of a chimeric sequence, we constructed neighbor-joining trees of the nine U.S Type sequences that covered nucleotides 3,601-4,000 and 4,801-5,200, which flanked the break point for recombination identified in SimPlot LV was incorporated as an outgroup The tree upstream of nt 4,000 showed that the Group B, 03-15, virus as closely associated with 04-41 with a bootstrap value of 100% In contrast, the downstream 4,801-5,200 tree showed a close association between 04-41 and the Group A virus, 41-08, with a bootstrap value of 99% These results support the possibility that U.S Type viruses are undergoing genetic recombination (Figure 5) 14 nsp1 nsp2 nsp3 nsp4 nsp5 nsp6 nsp7 nsp8 ORF2 ORF3 ORF5 ORF1b ORF1a nsp9 nsp10 nsp11 nsp12 ORF4 ORF6 ORF7 03-15 01-08 LV 98 03-12 03-12 01-08 92 04-42 04-40 100 01-08 99 04-42 04-41 02-11 04-41 03-15 99-01 01-07 02-11 04-40 99-01 03-15 01-07 LV LV nt 3,601 – 4,400 nt 4,801 – 5,200 0.01 0.005 Figure Analysis for the presence of recombination in isolate 04-41 The SimPlot analysis (colored graph) shows that the 04-41 query sequence is composed of sequences from Group A viruses (green) and Group B viruses (purple) The phylograms show how the position of 04-41 changes at the break point of recombination Algorithms, such as SimPlot, are based on a search for recombination between known groups of viruses To search for recombination involving the incorporation of genetic information from an unknown source requires a different approach We conducted a search for the presence of phylogenetic incongruencies as a means to detect the presence of recombination This approach is based on the assumption that the topology of a phylogenetic tree should not change regardless of the genome region chosen for analysis Neighbor-joining trees were constructed for every 400 nt along the genome sequences of the U.S Type isolates (Table 2) with the incorporation of LV as the outgroup This resulted in the construction of 37 phylogenetic trees Each isolate was assigned a number relative to its position to LV in the cladogram For 15 example, the isolates closest and furthest from LV were assigned the numerical values and respectively A graph of the results showing the changes in tree topology (incongruencies) are shown in Figure These data suggest that recombination is a rather frequent event and appears more frequently in the 3’ end where the structural genes are located nsp1 nsp2 nsp3 nsp4 nsp5 nsp6 nsp7 nsp8 ORF2 ORF3 ORF5 ORF1b ORF1a nsp9 nsp10 nsp11 nsp12ORF4 ORF6 ORF7 Position of isolate in cladogram relative to LV 1 2000 4000 6000 8000 10,000 12,000 14,000 Figure Phylogenetic incongruencies in NA Type genomes Horizontal bars show areas where the phylogenetic tree has undergone a significant re-organization, perhaps as a result of recombination Objective Development of a proficiency panel containing serum samples for the accurate detection of U.S Type PRRSV isolates Objective Evaluate the virus neutralizing properties of sera from pigs experimentally infected with representative U.S Type isolates Objective Evaluate pathogenic properties of U.S Type isolates Studies associated with Objectives 2, and were conducted simultaneously using the same group of 40 pigs Results are described below Clinical signs Pigs in challenge group 03-15 showed minor clinical signs, including occasional coughing and slightly elevated temperatures during the first two weeks of infection Animals in all other groups appeared normal throughout the course of the study 16 Histopathology Tissue samples from infected pigs euthanized at 14 dpi were characterized by mild pulmonary and nasal epithelial changes, but with minimal lymphoid involvement All infected groups had mild subacute multifocal interstitial pneumonia, with thickening of interstitial septa focused around small airways There were common peribronchiolar inflammatory aggregates and occasional foci of alveolar macrophage proliferation in areas with interstitial inflammation Lymphoid changes were confined to mild diffuse medullary lymphoid hyperplasia in lymph nodes and gutassociated lymphoid tissue within the ileum Lesions in nasal mucosa ranged from minimal subacute inflammatory infiltrates in the submucosa to moderate diffuse suppurative rhinitis with submucosal lymphoid aggregates In general, day 14 lesions were comparable in severity among animals inoculated with 03-15, 01-07, and 02-11 viral strains, while the 01-08 inoculated group was less severe Mock inoculated control animals sacrificed at day 14 dpi showed no lesions in tissues examined Samples collected at day 85 dpi showed substantial differences among treatment groups Animals inoculated with viral strain 02-11 had inconsistent mild subacute multifocal interstitial pneumonia with peribronchiolar lymphoid aggregates, as well as advanced lymphoid changes such as medullary reticulosis and subacute diffuse lymphadenitis Nasal epithelial changes in this group also persisted to this later time point, showing a similar range and severity as seen at day 14 dpi Strain 03-15 inoculated animals had a similar spectrum of pulmonary lesions at day 85 dpi, but lacked consistent changes in lymphoid tissues and nasal epithelium Within the group inoculated with viral strain 01-08, only one animal had detectable interstitial pneumonia and rhinitis, and there were no other significant lesions noted in this group Finally, animals inoculated with viral strain 01-07 had no observed lesions at day 85 dpi, with the exception of minimal peribronchiolar lymphoid aggregates noted in the lungs of three animals As before, mock inoculated control animals were free of lesions IDEXX ELISA serology All challenged animals, except for three, sero-converted (S/P ≥0.4) by day The remaining three pigs had elevated S/P values (≥0.2) on day and seroconverted by day 16 All control pigs remained seronegative (Figure 7) 17 Figure Mean IDEXX ELISA S/P values for each animal group (N=8/group through day 13 and N=5/group for days 16-85) Virus isolation PRRSV isolation and titration results on MARC-145 cells are shown in Figure Virus isolation from serum was sporadic from days through 21, peaking at day No significant differences were noted between groups and control animals remained negative throughout the study Figure Mean virus titration values for each animal group using MARC-145 cells (N=8/group through day 13 and N=5/group for days 16-85) 18 Real-time PCR Virus load in copies/ml for serum samples is shown in Figure Peak viral loads occurred from to dpi with isolates 01-07, 02-11 and 03-15 The peak was slightly later for group 01-08 at 13 dpi Results show that virus was detected as early as dpi with both the 01-08 and 03-15 isolates and by day with 01-07 and 02-11 isolates The highest mean viral loads were associated with isolate 03-15, at 3.2X10 copies per ml, although copies/ml varied greatly among individual animals Peak mean viral loads for other groups were 1.4X10 for the 01-08 group, 1.7X10 for the 01-07 group and 5.3X106 for the 02-11 group, although no significant differences were noted between groups Peak viremia correlated with elevated temperatures presented in febrile piglets during those times within each group Viral RNA was detectable in serum up until almost two months post inoculation with isolates 01-07 and 03-15 while RNA was detectable up to 42 days with the other isolates tested All mock challenged animals remained negative by PCR throughout the duration of the study Quantitative PCR results for selected tissues are shown in Table Both lung and pooled lymphoid tissues from most pigs euthanized at 14 dpi were PCR positive with viral loads ranging from 4.5X105 to 1X108 copies/gram of tissue In pigs euthanized at 85 dpi, only one pig (from group 01-08) had a detectable level of PRRSV RNA in the lung Lymphoid tissues from all challenged pigs euthanized at 85 dpi, except one pig from group 02-11, tested positive for PRRSV with viral loads ranging from 1.8 X10 to 5.2X105 copies/gram of tissue All tissues from negative control animals tested negative for PRRSV by PCR Figure PRRS virus load in serum as determined by real-time PCR (N=8/group through day 13 and N=5/group for days 16-85) 19 Table Tissue PCR results and mean viral load at time of euthanasia for each group of pigs Group 14 dpi 85 dpi Lung Lymphoid tissue Lung Lymphoid tissue 03-15 2/3a (4.1X106)b 3/3 (6.1X106) 0/5 (negative) 5/5 (5.2X105) 01-08 3/3 (6.7X106) 3/3 (4.5X105) 1/5 (6.3X102) 5/5 (2.1X105) 01-07 3/3 (1.0X108) 3/3 (5.0X105) 0/5 (negative) 5/5 (5.1X105) 02-11 3/3 (9.5X107) 2/3 (9.5X105) 0/5 (negative) 4/5 (1.8X105) Control 0/3 (negative) 0/3 (negative) 0/5 (negative) 0/5 (negative) _ a = number animals testing positive / total number of animals euthanized at time-point b = mean viral load in copies/gram of tissue Virus neutralization Fluorescent focus neutralization (FFN) results are shown in Figure Neutralizing antibody responses were detectable as early as 21 dpi in some animals and generally peaked between 34 and 77 dpi Peak neutralization titers against homologous virus isolates were typically in the range of 1:32 to 1:128, although occasional animals demonstrated titers as high as 1:512 All challenged animals developed neutralizing antibody titers of at least 1:16 against their respective homologous challenge isolate Neutralizing antibody levels were consistently higher against the homologous virus isolates, even though the amino acid sequences of the predicted GP5 neutralization epitope (Ostrowski et al 2002; Plagemann et al 2002) for all four isolates was identical (Figure 10) The prototypic Type Lelystad Virus was partially neutralized by serum from pigs infected with U.S Type isolates with serum from pigs in group 01-08 demonstrating the highest neutralization titers against the Lelystad Virus None of the pigs challenged with U.S origin Type viruses neutralized the Type prototype isolate, VR-2332 The North American reference isolate ISU-P was neutralized at very low levels by serum from some pigs infected with Type isolates 03-15 and 01-08 A unique North American isolate, HV-1, which lacks an Nglycosylation site in the ectodomain of GP5, was neutralized at moderate levels by sera from pigs challenged with all four Type isolates Mean cross-neutralization titers of selected serum samples against additional virus isolates are shown in Table 20 Figure Mean virus neutralization titers against all challenge isolates and selected reference isolates Neutralization titers are the greatest serum dilution resulting in a 90% or greater reduction in fluorescent focus units (N=8/group through day 13 and N=5/group for days 16-85) 21 Lelystad 01-07 01-08 02-11 03-15 VR2332 MRCSHKLGRFLTPHSCFWWLFLLCTG-LSWSFAD GNGDSSTYQYIYNLTICELNGTDWLSSHFGWAVE R - . .S.N N S .K HS - . NN .N G E .S I.- S N G D E .S - . NN .A G D -M.EKC AGC.SRL.S.W.IVPFCFAVLANAS.DS HL.L L .ANK.D Figure 10 Partial GP5 amino acid sequence alignment of PRRSV isolates used in this challenge study Predicted neutralizing epitope B (aa 37-45) of North American Type PRRSV isolate VR-2332 is highlighted The Type isolates used in this study show complete amino acid identity from aa 38-55 Table Neutralization data using 85 dpi sera from selected pigs challenged with 03-15 and 01-08 Type PRRSV Note limited cross-neutralization between isolates 03-15 and 01-08 FFN Isolate SD 03-15 03-15 pig #11 1:256 03-15 pig #12 1:64 01-08 pig #14