GENOME SEQUENCES Description of a New Clade within Subtype 1 of Betaarterivirus suid 1 Causing Severe Outbreaks in Spain Gerard E. Martín-Valls,a Martí Cortey,a Alberto Allepuz,a Francesc Illas,b Montse Tello,a Enric Mateua Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain a b Grup Batallé, Riudarenes, Spain Gerard E. Martín-Valls and Martí Cortey contributed equally to this article. Author order was determined by the roll of a die. ABSTRACT This report describes 28 genome sequences from a new clade within subtype 1 of Betaarterivirus suid 1, formerly known as porcine reproductive and respiratory syndrome virus 1. All share a potential recombinant pattern, with a highly pathogenic Italian strain as the putative major parental sequence and three other possible parents. C urrently, the International Committee on Taxonomy of Viruses (ICTV) designates porcine respiratory and reproductive syndrome virus 1 (PRRSV1) as Betaarterivirus suid 1, within the Variarterivirinae subfamily (1). PRRSV1 is one of the most important pathogens of pigs and causes serious economic losses (2). Within PRRSV1, at least 3 subtypes are recognized. Subtype 1 is the most widely distributed (3), while subtypes 2 and 3 were described in Western Siberia and Eastern Europe, respectively (4). Within each subtype, the genetic diversity is considerable. For PRRSV1, recombinant strains have been repeatedly reported in European countries, occasionally involving different modified live virus (MLV) vaccines (5). Within the scope of a series of longitudinal studies aiming to evaluate the diversity of PRRSV1 quasispecies and the utility of genome segments as epidemiological markers, serum samples were collected between February and November 2021 from sows and piglets on 11 breeding farms located in Catalonia (northeast Spain), owned by different companies. All farms were suffering severe PRRS outbreaks characterized by high abortion rates and mortal- ity in the sows and increased mortality in the weaners and growers (.20%). Briefly, PRRSV1 was isolated from individual serum samples after a single passage in porcine alveolar macro- phages (PAMs). The PAMs were inoculated into Eagle’s minimal essential medium (EMEM), supplemented with penicillin-streptomycin and 7.5% bovine calf serum, and maintained. If a cytopathic effect was observed, supernatant was collected, and total RNA was extracted using TRIzol. Libraries were prepared using the NEBNext RNA library prep kit, without an ini- tial amplification step, and sequenced on the Illumina MiSeq platform (paired-end 251-bp reads). Next-generation sequencing (NGS) reads were trimmed using Trimmomatic v0.36 (6) and mapped against the PRRSV1 prototype strain Lelystad (GenBank accession number Editor Simon Roux, DOE Joint Genome NC_043487), using the Burrows-Wheeler Aligner (BWA-MEM algorithm). The SAMtools view Institute (7) was used to remove reads with a mapping quality lower than Q20. LoFreq-Star v2.0 (8) Copyright © 2022 Martín-Valls et al. This is an open-access article distributed under the terms was used to call variants. Bam-readcount v1.0.1 (9) was used to determine the frequency of of the Creative Commons Attribution 4.0 each base at each position, and this frequency was used to infer a quasispecies population International license. of 100 genomes in FASTA format. Variants with a minimum base-call quality score lower than Address correspondence to Martí Cortey, marti.cortey@uab.cat. 20 and coverage less than 5 were discarded. From this quasispecies, a consensus sequence The authors declare no conflict of interest. was generated using Consensus Maker (https://www.hiv.lanl.gov/content/sequence/ Received 6 April 2022 CONSENSUS/consensus.html). All tools were run with default parameters. The whole- Accepted 9 May 2022 genome sequences obtained (Table 1) were compared with other PRRSV1 sequences Published 2 June 2022 available in GenBank, as well as in the database of the Veterinary Laboratory for the Diagnosis July 2022 Volume 11 Issue 7 10.1128/mra.00304-22 1 Announcement Microbiology Resource Announcements TABLE 1 Sample and sequencing data for the 28 PRRSV1 genomes described Sample IDa GenBank accession no. SRA accession no. No. of reads Length (bp) %GC Coverage (×) PRRSV1/Pig-wt/CAT-Esp/R1/May-2021 OM893828 SAMN27285123 680,713 14,910 52.8 538 PRRSV1/Pig-wt/CAT-Esp/R2/Feb-2021 OM893829 SAMN27285124 674,411 14,910 52.8 845 PRRSV1/Pig-wt/CAT-Esp/R3/Feb-2021 OM893830 SAMN27285125 829,785 14,910 52.7 3,317 PRRSV1/Pig-wt/CAT-Esp/R4/Jun-2021 OM893831 SAMN27285126 345,101 14,910 52.6 1,147 PRRSV1/Pig-wt/CAT-Esp/R5/Oct-2021 OM893832 SAMN27285127 1,061,085 14,910 52.7 2,691 PRRSV1/Pig-wt/CAT-Esp/R6/Mar-2021 OM893833 SAMN27285128 477,811 14,910 52.8 812 PRRSV1/Pig-wt/CAT-Esp/R7/May-2021 OM893834 SAMN27285129 920,987 14,910 52.7 669 PRRSV1/Pig-wt/CAT-Esp/R8/Jun-2021 OM893835 SAMN27285130 86,290 14,910 52.8 169 PRRSV1/Pig-wt/CAT-Esp/R9/Jul-2021 OM893836 SAMN27285131 878,809 14,910 52.7 1,975 PRRSV1/Pig-wt/CAT-Esp/R10/Apr-2021 OM893837 SAMN27285132 5,653,071 14,910 52.6 2,182 PRRSV1/Pig-wt/CAT-Esp/R11/Apr-2021 OM893838 SAMN27285133 968,821 14,910 52.8 1,328 PRRSV1/Pig-wt/CAT-Esp/R12/May-2021 OM893839 SAMN27285134 561,265 14,910 52.7 609 PRRSV1/Pig-wt/CAT-Esp/R13/Jun-2021 OM893840 SAMN27285135 419,903 14,910 52.7 1,546 PRRSV1/Pig-wt/CAT-Esp/R14/Jul-2021 OM893841 SAMN27285136 933,866 14,910 52.7 1,547 PRRSV1/Pig-wt/CAT-Esp/R15/Nov-2021 OM893842 SAMN27285137 1,292,169 14,910 52.7 2,564 PRRSV1/Pig-wt/CAT-Esp/R16/Nov-2021 OM893843 SAMN27285138 795,071 14,910 52.7 1,855 PRRSV1/Pig-wt/CAT-Esp/R17/Apr-2021 OM893844 SAMN27285139 1,187,971 14,910 52.8 4,345 PRRSV1/Pig-wt/CAT-Esp/R18/Apr-2021 OM893845 SAMN27285140 1,260,111 14,910 52.7 4,168 PRRSV1/Pig-wt/CAT-Esp/R19/May-2021 OM893846 SAMN27285141 440,400 14,910 52.6 353 PRRSV1/Pig-wt/CAT-Esp/R20/May-2021 OM893847 SAMN27285142 763,771 14,910 52.6 1,477 PRRSV1/Pig-wt/CAT-Esp/R21/Jun-2021 OM893848 SAMN27285143 301,518 14,910 52.6 263 PRRSV1/Pig-wt/CAT-Esp/R22/Jul-2021 OM893849 SAMN27285144 790,502 14,910 52.6 1,150 PRRSV1/Pig-wt/CAT-Esp/R23/Mar-2021 OM893850 SAMN27285145 1,008,440 14,910 52.7 2,128 PRRSV1/Pig-wt/CAT-Esp/R24/Mar-2021 OM893851 SAMN27285146 886,568 14,910 52.8 2,154 PRRSV1/Pig-wt/CAT-Esp/R25/Apr-2021 OM893852 SAMN27285147 440,798 14,910 52.8 406 PRRSV1/Pig-wt/CAT-Esp/R26/May-2021 OM893853 SAMN27285148 830,917 14,910 52.7 761 PRRSV1/Pig-wt/CAT-Esp/R27/Jun-2021 OM893854 SAMN27285149 352,456 14,910 52.8 993 PRRSV1/Pig-wt/CAT-Esp/R28/Jul-2021 OM893855 SAMN27285150 716,349 14,910 52.6 839 a Includes host, origin, strain, and sampling date (mo-yr). Pig-wt, XXX; CAT-Esp, Catalonia, Spain. of Infectious Diseases of the Universitat Autònoma de Barcelona. Phylogenetic analyses were performed using MEGA X. Recombination was detected using RDP5 software and GARD. Local similarity analyses conducted using RDP and GARD revealed a common pattern among all isolates (Fig. 1A). A local similarity analysis conducted using BLAST identified a highly pathogenic Italian strain (10) as the putative major parental sequence (PR40/2014; FIG 1 (A) Local similarity analyses among the 28 PRRSV1 genomes described, including the segments associated with every best BLAST hit of genomic subregions, indicated via the color code (identified using RDP and GARD); (B) maximum likelihood phylogenetic tree built up using the whole-genome sequence and the general time-reversible model. The confidence of the main internal branches, based on 100 bootstrap replicates, is indicated. Three PRRSV1 subtypes are marked in blue, the potential recombinant clade described in this report is highlighted in red, and the three putative parental strains identified are indicated in green. ORF, open reading frame; UTR, untranslated region. July 2022 Volume 11 Issue 7 10.1128/mra.00304-22 2 Announcement Microbiology Resource Announcements GenBank accession number MF346695; 80% of the genome comprised in 5 segments, with 86 to 89% nucleotide identity) and the following three putative minor parental strains: ESP-1991-Olot91 (KC862570; 11% of the genome comprised in 2 segments, with 86% nucle- otide identity), D40 (KY434184; 7% of the genome comprised in a single segment, with 86% nucleotide identity), and an unknown strain (2% of the genome comprised in a single seg- ment, with a nucleotide identity lower than 85%). A maximum likelihood (ML) phylogenetic tree placed all the genomes in a mono- phyletic branch within PRRSV1 subtype 1 (Fig. 1B). The strains isolated in these outbreaks, characterized by high virulence, potentially origi- nated from the recombination of multiple subtype 1 strains. Its apparent greater pathogenicity should be evaluated in future studies. Furthermore, the regular detection of recombinant PRRSV strains (5) illustrates the plasticity of this virus, together with the need to use whole- genome sequences as an analytical tool. Data availability. The 28 whole-genome sequences described in this report have been deposited at GenBank under the accession numbers OM893828 to OM893855; the corresponding fastq files have been deposited in the Sequence Read Archive under the BioSample accession numbers SAMN27285123 to SAMN27285150. ACKNOWLEDGMENT M. Cortey was funded by MINECO (Ramon y Cajal grant number RYC-2015-17154). REFERENCES 1. Brinton MA, Gulyaeva AA, Balasuriya UBR, Dunowska M, Faaberg KS, Goldberg Illumina sequence data. 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