Isolation and pathogenicity comparison of two novel natural recombinant porcine reproductive and respiratory syndrome viruses with different recombination patterns in Southwest China

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses in the swine industry. Frequent mutations and recombinations account for PRRSV immune evasion and the emergence of novel strains. In this study, we isolated and characterized two novel PRRSV-2 strains from Southwest China exhibiting distinct recombination patterns. They were designated SCABTC-202305 and SCABTC-202309. Phylogenetic results indicated that SCABTC-202305 was classified as lineage 8, and SCABTC-202309 was classified as lineage 1.8. Amino acid mutation analysis identified unique amino acid substitutions and deletions in ORF5 and Nsp2 genes. The results of the recombination analysis revealed that SCABTC-202305 is a recombinant with JXA1 as the major parental strain and NADC30 as the minor parental strain. At the same time, SCABTC-202309 is identified as a recombinant with NADC30 as the major parental strain and JXA1 as the minor parental strain. In this study, we infected piglets with SCABTC-202305, SCABTC-202309, or mock inoculum (control) to study the pathogenicity of these isolates. Although both isolated strains were pathogenic, SCABTC-202305-infected piglets exhibited more severe clinical signs and higher mortality, viral load, and antibody response than SCABTC-202309-infected piglets. SCABTC-202305 also caused more extensive lung lesions based on histopathology. Our findings suggest that the divergent pathogenicity observed between the two novel PRRSV isolates may be attributed to variations in the genetic information encoded by specific genomic regions. Elucidating the genetic determinants governing PRRSV virulence and transmissibility will inform efforts to control this devastating swine pathogen. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most critical pathogens impacting the global swine industry. Frequent mutations and recombinations have made the control of PRRSV increasingly difficult. Following the NADC30-like PRRSV pandemic, recombination events involving PRRSV strains have further increased. We isolated two novel field PRRSV recombinant strains, SCABTC-202305 and SCABTC-202309, exhibiting different recombination patterns and compared their pathogenicity in animal experiments. The isolates caused higher viral loads, persistent fever, marked weight loss, moderate respiratory clinical signs, and severe histopathologic lung lesions in piglets. Elucidating correlations between recombinant regions and pathogenicity in these isolates can inform epidemiologic tracking of emerging strains and investigations into viral adaptive mechanisms underlying PRRSV immunity evasion. Our findings underscore the importance of continued genomic surveillance to curb this economically damaging pathogen.

PRRSV can be classified into two genotypes: PRRSV-1, represented by the prototype strain Lelystad, and PRRSV-2, represented by the prototype strain VR-2332.These two genotypes differ by approximately 60% at the nucleotide level (5).Shi et al. (1) con structed a global classification system for PRRSV based on a comprehensive analysis of complete ORF5 gene sequences.This system further delineates PRRSV-2 into four lineages: lineage 1 (NADC30-like), lineage 3 (QYYZ-like), lineage 5 (VR2332-like), and lineage 8 (JXA1-like and CH-1a-like) (1).PRRSV-2 has predominated in China since emerging in the late 1990s, undergoing continuous diversification at a relatively rapid evolutionary pace (6).In 2006, an outbreak of highly pathogenic PRRSV (HP-PRRSV) belonging to sublineage 8.7 resulted in over 2 million pig fatalities (7).Although the modified live virus vaccine (MLV) temporarily curbed HP-PRRSV outbreaks, the genetic heterogeneity and rapid mutation rate of PRRSV-2 enables viral immune evasion, impeding sustained control (6).During 2013-2014, circulation of NADC30-like PRRSV strains (lineage 1.8) was documented across several provinces in China, with these viruses gradually becoming epidemiologically predominant owing to their increased recombination and mutation frequencies (7).First identified in China in 2017, NADC34like PRRSV (lineage 1.5) has since disseminated rapidly nationwide (8).The co-circulation of multiple PRRSV genotypes in Chinese swine herds has caused substantial economic damage to the pork industry while intensifying prospects for viral recombination.
The rapid evolution and recombination of PRRSV have caused the emergence of novel, highly virulent viruses that induce clinical PRRS outbreaks (9).In recent years, researchers have reported the emergence of various novel recombinant PRRSV strains derived from NADC30-like PRRSV outbreaks exhibiting different pathogenicity in China (7,10,11).Previous studies have indicated that NADC30-like and JXA1-like PRRSV recombinant strains manifest great virulence, approximating that of JXA1-like strains, frequently eliciting abortion, stillbirth, and neonatal mortality in sow herds (12)(13)(14).
In this study, we isolated two novel recombinant PRRSV strains (SCABTC-202305 and SCABTC-202309) from suspected PRRSV-infected swine farms in Southwest China.To further identify these isolates, we determined their complete genome sequences and evaluated pathogenicity in piglets.Phylogenetic analysis classified SCABTC-202305 as lineage 8, and SCABTC-202309 as lineage 1.8.Recombination analysis revealed that two isolates in this study exhibited different recombination patterns.Furthermore, we evaluated their virulence properties in 1-week-old piglets, and SCABTC-202305-infected piglets exhibited relatively more severe clinical signs and higher mortality, viral load, antibody response, and lung lesions compared to SCABTC-202309-infected piglets.Our study suggests that differences in specific genomic regions may contribute to the pathogenicity and epidemic capacity.

Isolation and identification of PRRSV
Clinical serum samples were collected from suspected PRRSV-infected, unvaccinated piglets in two independent swine farms in Southwest China.The affected pigs exhibited high fever, respiratory distress, and mortality rates of 18.52% (75/405) and 8.49% (79/930) in Sichuan and Guizhou, respectively (Table 1).Following filtration of PRRSV-positive sera through 0.22-µm membranes, 1-mL aliquots were aseptically inoculated onto Marc-145 cells and were incubated for 1 hour (37°C, 5% CO 2 ).The inoculated cells were subsequently propagated in Dulbecco modified Eagle medium (DMEM; Thermo Fisher, Shanghai, China) supplemented with 3% Gibco fetal bovine serum (FBS; Thermo Fisher, Shanghai, China) for 3 days and were monitored for cytopathic effects (CPE).Upon CPE manifestation, the infected cells underwent two freeze-thaw cycles at −80°C, centrifugation at 5,000 g for 5 minutes, and collection of viral supernatants.The viral supernatants were utilized to infect Marc-145 cells and were serially passaged five times.PRRSV was detected by real-time quantitative PCR (RT-qPCR) in the supernatant from each viral passage.PRRSV RNA in testis was quantified by established qRT-PCR (15).cDNA was reverse transcribed from 1-µg total RNA using the PrimeScript RT Reagent Kit (Takara, Beijing, China).Primers were 5′-GCAAGTACATTCTGGCCCCT-3′ and 5′-CAATGTGC CGTTGACCGTAG-3′.Cycling conditions were 95°C for 30 seconds, then 40 cycles of 95°C for 5 seconds and 60°C for 20 seconds.PRRSV RNA was normalized to a housekeeper gene for relative quantification.
Indirect immunofluorescence assay (IFA) was conducted using a PRRSV N proteinspecific monoclonal antibody (GeneTex, California, USA) and fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Proteintech, Wuhan, China) to detect PRRSV.In detail, Marc-145 cells were infected with PRRSV and were incubated for 48 hours.The cells were fixed in 4% paraformaldehyde for 10 minutes and were permeabilized with 0.2% Triton X-100 for 5 minutes.After blocking with 5% bovine serum albumin (BSA) at 37°C for 1 hour, the cells were incubated with PRRSV N protein-specific monoclonal antibody (1:500 dilution) overnight at 4°C.Then, the cells were incubated with FITC-con jugated secondary antibody (1:1,000 dilution) at 37°C for 1 hour.Nuclei were counter stained with 4′,6-diamidino-2-phenylindole.Cells presenting green fluorescence in the cytoplasm under fluorescence microscopy were counted as positive.
The isolates underwent three rounds of plaque purification followed by six passages in MARC-145 cells and complete genomic sequencing.Virus titers were determined by the Reed-Muench method.Viral samples were sent to Chengdu Lilai Biotechnology Co., Ltd, for transmission electron microscopy photography.

Whole genome sequencing and genetic analysis
Total RNA was extracted from PRRSV-infected cell suspensions using RNAiso Plus (Takara, Kyoto, Japan) according to the manufacturer's instructions.Subsequently, the RNA samples were submitted to Shanghai Tanpu Biotechnology Co., Ltd, for whole genome sequencing (WGS).
To investigate the evolutionary relationship of SCABTC-202305 and SCABTC-202309, sequence alignments and phylogenetic analyses were conducted using 41 represen tative PRRSV strains from GenBank (Table S1).Nucleotide sequences were aligned employing the ClustalX tool in MEGA version 7.0 (NIH, Maryland, USA) (16).Maximum likelihood phylogenetic trees were constructed using 1,000 bootstrap replicates and were compared to the complete gene sequences of PRRSV reference strains in GenBank (17).

Amino acid mutation analysis
To characterize amino acid mutations in SCABTC-202305 and SCABTC-202309, we employed the ClustalW module in DNAStar software version 7.0 (DNASTAR Inc., Wisconsin, USA) (18) to compare the amino acid sequences of the PRRSV ORF5 and Nsp2 genes to determine sequence similarity.

Animal challenge experiment
To evaluate the pathogenicity of SCABTC-202305 and SCABTC-202309, 15 1-week-old PRRSV, pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV)-seronegative piglets were purchased from a farm in Chengdu.The piglets were randomly divided into three groups and were housed in separate rooms with appropriate care and feeding throughout the experiment.The challenge experiment commenced following an acclimatization period.Group 1 (n = 5) received 2 mL (2 × 10 5 TCID 50 /mL) of SCABTC-202305 intranasally.Group 2 (n = 5) received 2 mL (2 × 10 5 TCID 50 /mL) of SCABTC-202307 intranasally.The control group (n = 5) was sham-inoculated with DMEM medium intranasally.Rectal temperatures, body weights, survival rates, and clinical manifestations were documented daily for each group.Clinical scores were evaluated utilizing a 0-20 scale (28) based on the percentage of lung area affected.Serum, nasal, throat, and anal swabs were collected at 0, 3, 5, 7, and 14 days post-inoculation (dpi).Viral loads in serum, nasal, oropharyngeal, and rectal swabs were detected using RT-qPCR to assess viremia and patterns of viral excretion.Specific antibodies against the N protein of PRRSV in serum were detected using the IDEXX HerdChek PRRS X3 enzyme-linked immunosorbent assay (ELISA) kit (IDEXX, Shanghai, China).After 14 dpi, all surviving piglets were euthanized by an overdose of sodium pentobarbital (Sinopharm, Beijing, China) dosed by intravenous route, and a complete necropsy was performed.The animal studies were approved by the Sichuan Agricultural University Laboratory Animal Management Committee (Approval Number SYXK2019-187).The studies were conducted in accordance with the local legislation and institutional requirements.Written informed consent was obtained from the owners for the participation of their animals in this study.

Serological and viremia test
Viral loads in serum, nasal, oropharyngeal, and rectal swabs were quantified by RT-qPCR to evaluate viremia and viral shedding patterns.Serum antibodies specific to PRRSV nucleocapsid protein were detected using the IDEXX HerdChek PRRS X3 ELISA kit (IDEXX, Shanghai, China).Upon necropsy, heart, liver, spleen, lung, kidney, inguinal lymph node, and testicular tissue samples were collected from piglets.The viral load in each organ was then measured by qRT-PCR.

Gross lung pathological and histopathology examination
Gross lung pathology was performed postmortem on pigs that died during the trial, or necropsy was conducted on surviving pigs at 14 dpi.The scoring system applied has been previously described (29).Each piglet received a score from 0 to 100, with each lung lobe assigned a number reflecting its approximate percentage of total lung volume.The right cranial lobe, right middle lobe, cephalic portion of the left cranial lobe, and caudal portion of the left cranial lobe each received 10 points, while the accessory lobes received 5 points.The right and left caudal lobes each received 27.5 points, totaling 100 points for the entire lung.

Immunohistochemical (IHC) analyses
IHC staining was performed on the lung sections.Tissue sections were treated with 3% H 2 O 2 solution (pH 7.6) for 10 minutes and then with 5% BSA (Thermo Fisher, Shanghai, China) for 30 minutes.The sections were then incubated overnight at 4°C with a rabbit monoclonal antibody against PRRSV nucleocapsid protein (1:500; Chixun, Guangzhou, China) as the primary antibody.The sections were washed three times with phosphatebuffered saline (PBS) and were incubated at 37°C for 30 min with biotin-conjugated affinity-purified goat anti-rabbit IgG (Proteintech, Wuhan, China) as the secondary antibody.After further incubation with streptavidin-biotin complexes (Boster, Wuhan, China) at 37°C for 30 minutes, the specific binding to the sections was visualized using diaminobenzidine (Boster, Wuhan, China).The percentage of positive cells was calculated for each section using the ImageJ software version 1.5.1 (NIH, Maryland, USA).

Statistical analysis
Experimental data were analyzed for significance using GraphPad Prism version 8.0.2 (Graphpad Software, California, USA) and are expressed as mean ± standard deviation.P < 0.05 was considered significant, and P < 0.01 or P < 0.001 was considered highly significant.

Virus isolation and identification
Serum samples positive for PRRSV were inoculated into Marc-145 cells under favorable growth conditions for virus isolation and identification.PRRSV was successfully isolated from the serum samples.The isolates showed increased CPE with increasing incubation.At 24 hours post-infection (hpi), minimal CPE was observed by aggregation of a few cells.At 48 hpi, the cells had aggregated, shrunk, and rounded significantly.At 72 hpi, numerous cells had clumped or detached (Fig. 1a).Typical CPE persisted after five passages, with viral nucleic acid levels in the culture medium gradually increasing with each passage.The IFA showed that both SCABTC-202305 and SCABTC202309 groups exhibited significant green fluorescence in contrast to the mock group, which showed no fluorescence (Fig. 1b).Viral particles of isolated strains can be visualized by projection electron microscopy (Fig. 1c).Virus titers are shown in Figure 1d and e.In this study, two PRRSV isolates, designated SCABTC-202305 and SCABTC-202309, were isolated and identified.
We searched the similar strains of the isolates in this experiment using the NCBI database (Table 3).The results showed that SCABTC-202305 was highly similar to the strain SC/DJY (98.43%) detected in Chengdu, Sichuan Province, suggesting that it might be a novel mutant of the endemic strain SC/DJY.On the other hand, SCABTC-202309 was identified as a new recombinant strain reported in Zunyi, Guizhou Province.The most similar strain, SDYG1606, exhibited only 90.94% similarity.

Amino acid mutation analysis
Previous studies have shown that among the different proteins of PRRSV, GP5 and NSP2 have the lowest amino acid similarity, suggesting that the GP5 and NSP2 proteins are most susceptible to mutation (30).Therefore, they are usually used as targets for epidemiological analysis (31).
The major envelope protein GP5, encoded by ORF5, is one of the most variable PRRSV proteins (15).Results with other representative strains show a wide range of amino acid substitutions in the signal peptide, hypervariable regions 1 and 2 (HVR1 and HVR2), and potential neutralizing epitopes (PNE) of ORF5.Several amino acid substitutions, including S 16   3a).
Nsp2 is one of the most variable proteins of PRRSV and contains different patterns of deletions and insertions compared to the prototype strain VR2332 (32).In this study, isolates SCABTC-202305 and SCABTC-202309 exhibited a discontinuous deletion pattern of 131 amino acids (111 + 1 + 19) characteristic of NADC30-like strains compared to the prototype PRRSV-2 strain VR2332.Interestingly, SCABTC-202305 contained an additional five amino acid deletion in Nsp2 from positions 462-466, while SCABTC-202309 had an additional three amino acid deletion in Nsp2 from positions 499-501 (Fig. 3b).

Recombination analysis
To identify possible recombination events in the isolated strains, we performed recombination analyses using SimPlot and RDP software.The results revealed that both SCABTC-202305 and SCABTC-202309 strains isolated in this study underwent genetic recombination.SCABTC-202305 was a recombinant of JXA1 and NADC30, with one recombination event identified (Fig. 4a).The major parental strain was JXA1, and the minor parental strain was NADC30.Recombination event occurred at nucleic acids 1110-4089 nt (Region B).Phylogenetic analyses were conducted on the whole genome and the recombination regions.The phylogenetic tree showed that the major region of SCABTC-202305 belongs to lineage 8, while the recombination region belongs to lineage 1.8 (Fig. 4c).SCABTC-202309 was a recombinant of NADC30 and JXA1, with three recombination events identified.The major parental strain was NADC30, and the minor parental strain was JXA1 (Fig. 4b).Recombination event 1 occurred at nucleic acids 1-561 nt.Recombination event 2 occurred at nucleic acids 1321-2041 nt.Recombination event 3 occurred at nucleic acids 7161-8201 nt.Phylogenetic analyses were performed on the whole genome and the recombination regions.The genome-wide phylogenetic tree showed that SCABTC-202309 belongs to lineage 1.8, while all recombination regions belong to lineage 8 (Fig. 4d).

Clinical signs
The pathogenicity of the SCABTC-202305 and SCABTC-202309 strains was compared in 1-week-old piglets.All PRRSV-infected piglets exhibited severe respiratory impairment (Fig. 5a).In particular, clinical signs were more pronounced in piglets infected with SCABTC-202305 than in those infected with SCABTC-202309.Control piglets showed no significant clinical signs.Piglets infected with SCABTC-202305 showed a febrile response from 1 dpi, with rectal temperatures remaining above 40°C from 3 to 9 dpi and peaking at 42.9°C at 7 dpi (Fig. 5c).Clinical signs such as coughing, sneezing, and anorexia began at 3 dpi, and severe respiratory symptoms, including dyspnea, tachypnea, and shivering, started at 7 dpi.One piglet died at 11 dpi, and one at 13 dpi (Fig. 5b).Piglets infected with SCABTC-202309 had a febrile response only from 3 dpi, with temperatures above 40°C from 5 to 9 dpi and peaking at 42.4°C at 7 dpi (Fig. 5c).Coughing, sneezing, and anorexia appeared at 3 dpi, and two piglets showed dyspnea and tachypnea at 7 dpi.One piglet died at 11 dpi (Fig. 5b).Furthermore, the average daily weight gain of piglets   The red area represents the major parental regions, and the blue area represents the minor parental regions.

Viremia and viral excretion
Serum, nasal swabs, pharyngeal swabs, and anal swabs were collected from pigs at 1, 3, 5, 7, 9, 11, 13, and 15 dpi to evaluate viremia and viral shedding.Both PRRSV isolates SCABTC-202305 and SCABTC-202309 were detected in serum starting at 1 dpi, with peak viral titers observed at 9 dpi.Following the peak, serum viral titers decreased slightly.Specifically, SCABTC-202305 inoculation resulted in significantly higher serum viral RNA levels at 3, 5, and 9 dpi, but lower levels at 11 dpi compared to SCABTC-202309.No viremia was detected in the control groups at any point in time (Fig. 6a).Both PRRSV strains were detected in nasal, pharyngeal, and anal swabs starting at 3 dpi, with peak viral shedding occurring at 7-9 dpi.Respiratory viral shedding was significantly higher in SCABTC-202305-infected pigs compared to SCABTC-202309 at 7 and 11 dpi.No significant difference in gastrointestinal shedding was observed between the two PRRSV strains (Fig. 6b through d).

Tissue tropism analysis
The viral load of different tissues in piglets after the PRRSV attack was determined by qRT-PCR (Fig. 7a).The viral RNA was not detected in any of the samples from the mock group.The tissues with the highest viral load were the lungs and lymph nodes, followed by the heart, spleen, and testes.The liver, kidney, and intestines had the lowest viral load.The SCABTC-202305 group had a significantly higher viral load in the testes, hilar lymph nodes, and submandibular lymph nodes than the SCABTC-202309 group.The SCABTC-202309 group had a significantly lower viral load in the liver and kidney than the SCABTC-202305 group.There was no significant difference in the viral load of the spleen, intestine, and inguinal lymph nodes between the two groups.

Humoral immune response
After PRRSV infection, pig sera were collected at 1, 3, 5, 7, 9, 11, 13, and 15 dpi to detect PRRSV IgG antibodies.Analysis of PRRSV antibody levels showed seroconversion in all piglets of both infected groups at 7 dpi, with antibody titers continuing to increase until plateauing at 15 dpi.Antibody titers were significantly higher in pigs infected with SCABTC-202305 compared to those infected with SCABTC-202309 at 7, 9, and 11 dpi.At 1, 3, 5, 13, and 15 dpi, no significant difference in antibody titers was observed between the two infected groups.The control group remained negative for PRRSV-spe cific antibodies throughout the experiment (Fig. 7b).

Clinical anatomical, histopathology, and immunohistochemical analysis
At necropsy, PRRSV infection mainly caused lung consolidation and interstitial pneumo nia in piglets (33).Lung lesions in piglets infected with SCABTC-202305 were severe, with diffuse interstitial pneumonia, lung swelling, hard, rubbery texture, and diffuse bleeding at the edge of the lung.The lungs of piglets infected with SCABTC-202309 showed mild interstitial pneumonia and pulmonary edema (Fig. 8a and b).Microscopic histopathological examination also showed that piglets infected with PRRSV developed marked thickening of the alveolar septum, necrosis of alveolar epithelial cells, and an increase in inflammatory cells, suggesting the development of interstitial pneumonia (Fig. 8c and d).The results showed that the gross and microscopic lung lesions of piglets infected with SCABTC-202305 were more significant than those of piglets infected with SCABTC-202309, while no obvious lesions were observed in the lungs of in the control group.The distribution of PRRSV in the lungs was studied using IHC staining.No positive signals were detected in the control group, whereas PRRSV-positive signals were observed in the infected group.These signals appeared brown and were distributed in alveolar cells and macrophages, etc. (Fig. 8e and f).The mean optical density of the positive signals was significantly higher in SCABTC-202305-infected piglets compared to SCABTC-202309 (P < 0.05).

DISCUSSION
Since 2006, the emergence of lineage 8 (HP-PRRSV strains) has caused significant harm to the Chinese pig industry.After 2010, the MLV has been widely used to control the disease (6,34).However, the high mutation and recombination rates of PRRSV have led to its recurrence, presenting challenges in containing PRRSV (14,35).After 2013, lineage 1.8 (NADC30-like strains) emerged and spread rapidly and widely.Currently, the NADC30-like strain is prevalent in multiple provinces in China, causing an abortion storm and may have become the main epidemic strain of PRRSV (30).Our findings are consistent with the epidemiological trend.In this study, two novel PRRSV isolates, SCABTC-202305 and SCABTC-202309, were isolated from different regions of Southwest China.Phylogenetic analysis based on the complete genome sequences revealed that SCABTC-202305 belongs to lineage 8, and SCABTC-202309 belongs to lineage 1.8.
To determine the prevalence of the isolated strains across various geographical regions, we searched the NCBI database for strains that are similar to the isolates in this study.SCABTC-202305 appears to be a novel variant of SC/DJY strain, previously reported in Chengdu, Sichuan Province, in 2020 (31).This strain remains predominantly confined to Sichuan Province, and no cases have been reported in other regions.It is worth noting that Sichuan Province serves as the primary center for pig breeding in Southwest China (36).This situation potentially contributes to the rapid cross-regional transmission of the strain through frequent mutations and recombina tions (37).Therefore, it is vital to continuously monitor the prevalence of SCABTC-202305 over an extended period and develop an associated vaccine.In contrast, SCABTC-202309 represents a novel recombinant strain, exhibiting only 90.94% similarity to its closest strain, SDYG1606 (38).This significant differentiation is attributed to a distinct and intricate recombination pattern involving three recombination events.The emergence of this novel recombination pattern raises concerns regarding the epidemiology of PRRSV and necessitates ongoing monitoring of potential risks.only have a modest effect on the virulence of the strain.addition, the Nsp8 and Nsp9 recombination could potentially have affected viral replication (52).
Notably, SCABTC-202305 exhibits greater virulence and replication proficiency compared to SCABTC-202309, chiefly attributed to its predominant parental derivation from the highly pathogenic strain JXA1.It is worth noting that recent evidence has revealed that alterations in the genetic information of specific genes in the genome may directly affect the pathogenicity of novel PRRSV strains (53).PRRSV recombination can sometimes generate strains with enhanced virulence, depending on the genomic regions exchanged and parental viruses involved (6).Studies have shown that the recombination of NADC30-like PRRSV with other strains can increase pathogenicity, as evidenced by recombinant strains such as GZgy17 and SD-YL1712 (40,54).The regions encoding the major structural proteins GP5, M, and N in SCABTC-202305 originate from the JXA1 strain.In contrast, the structural proteins in SCABTC-202309 were derived from the NADC30 strain, which may result in greater decay of infectivity and virulence.Furthermore, the region derived from the JXA1 strain in SCABTC-202305 encodes minor structural proteins GP2, GP3, and GP4 implicated in immunomodulation through receptor interactions (35).Alterations in these proteins may enable SCABTC-202305 to suppress or circumvent host immunity more potently than SCABTC-202309.Critically, nonstructural proteins such as Nsp8, Nsp9, and Nsp10 involved in genomic replica tion and transcription originate from the JXA1 parent in SCABTC-202305, preserving the maximal replication proficiency of JXA1, unlike the origins of these proteins in SCABTC-202309.
In summary, two novel PRRSV strains with distinct recombination patterns, designa ted SCABTC-202305 and SCABTC-202309, were isolated from piglet sera collected in Southwest China.Phylogenetic analyses demonstrated that SCABTC-202305 clustered within lineage 8, and SCABTC-202309 clustered within lineage 1.8.Additionally, multiple novel amino acid and nucleotide deletions were identified in the ORF5 and Nsp2 genes.Animal studies in piglets showed both isolates were highly pathogenic, with SCABTC-202305 exhibiting greater virulence and replication capacity compared to SCABTC-202309.The results of recombination analysis showed that the virulence genes of PRRSV isolates were changed due to recombination, which affected the pathogenicity of the strains.This finding provides a reference for the surveillance and control of the PRRSV epidemic in Southwest China.

FIG 2
FIG 2Phylogenetic tree based on the PRRSV complete gene sequence.The phylogenetic tree was constructed by the maximum likelihood method in MEGA7.0.ITOL version 6.9 (Biobyte, Heidelberg, Germany) was used to modify the genetic evolutionary tree, using different colors to distinguish different lineages and reference strains with GenBank sequence numbers.The circles (•) indicate the isolated strain in this study.

FIG 3 FIG 4
FIG 3 Amino acid sequence alignment the ORF5 and Nsp2 HVR.(a) Multiple alignments of GP5 amino acid sequences of SCABTC-202305 and SCABTC-202309 and 18 PRRSV reference strains.(b) Multiple alignments of Nsp2 HVR amino acid sequences of SCABTC-202305 and SCABTC-202309 and 18 PRRSV reference strains.Deleted amino acids are indicated in red boxes, with different colors distinguishing the various PRRSV lineages.

FIG 8
FIG 8 (a) Gross lesions in piglet lungs.Piglets inoculated with SCABTC-202305 developed severe interstitial pneumonia.Piglets inoculated with SCABTC-202309 exhibited moderate interstitial pneumonitis with consolidation.Piglets in the mock group showed no gross lung lesions.(b) Scores of lung gross examination of the inoculated piglets.The lung gross lesions were scored based on the percentage of lung area affected.(c) Microscopic lesions of the lung of piglets.Piglets inoculated with SCABTC-202305 exhibited thickened alveolar septa, alveolar epithelial cell degeneration, and inflammation (magnification of 400×).Piglets inoculated with SCABTC-202309 exhibited thickened alveolar septa (magnification of 400×).Piglets in the mock group had no evident lung pathology (magnification of 400×).(d) Scores of lung microscopic lesions examination of the inoculated piglets.The microscopic lung lesions were scored based on the extent and severity of interstitial pneumonia.(e) Detection of PRRSV in lung tissues of piglets by IHC (magnification of 400×).(f) The mean density of lung IHC staining sections of inoculated piglets.Mean density is calculated by dividing the total optical density of the positive signals in each field of view by the area of the effective target distribution.

TABLE 2
Pairwise genomic identity of the whole genome between the identified strains and representative strains of PRRSV

TABLE 3
Information on the related recombinant strains of the isolates