Evolution Characterization and Pathogenicity of a Porcine Reproductive and Respiratory Syndrome Virus Isolate from a Pig Farm in Shandong Province, China

In recent years, porcine reproductive and respiratory syndrome virus (PRRSV) strains have been experiencing extensive recombination in Chinese swine farms. This recombination usually happens in NADC30/34 strains and highly pathogenic (HP) PRRSV strains. This study identified a new PRRSV isolate that shared 99% and 99.1% nucleotide identity with CH-1a and CH-1R at the genomic level, respectively. After purification by viral plaque assay, this isolate was named PRRSV CSR1801. The isolate did not experience any recombination with other PRRSV strains common in swine herd epidemics in China, which means it still maintains the stable features of the classical PRRSV strain and did not easily recombine with other PRRSV strains. Further analysis of the pathogenicity of the PRRSV isolate CSR1801 was performed in piglets. The results indicated that none of the inoculated piglets showed the typical clinical manifestations of PRRS, which presented with runny noses, rough back hair, rectal temperatures always below 40.5 °C, and no deaths. Additionally, no obvious histopathological lesions such as severe interstitial pneumonia could be observed in the lungs of the piglets. Hence, the PRRSV isolate CSR1801 should be classified as a classical-like PRRSV strain. This classical PRRSV strain showed genetic stability and maintained low pathogenicity. This study may provide new clues for further understanding the genetic evolution and pathogenicity of PRRSV and may also be an important reference for the prevention and control of PRRS in swine farms.


Introduction
Porcine reproductive and respiratory syndrome (PRRS) has become one of the most economically significant pig diseases all over the world since it was first reported in 1986 and 1990 in North America and Europe, respectively [1]. PRRS virus (PRRSV), horse arteritis virus, simian hemorrhagic fever virus, and lactate dehydrogenase-elevating virus are all members of the Arteriviridae family. The PRRSV genome comprises a positive-sense single-stranded RNA of approximately 15 kb in length and encodes 10 open reading frames (ORFs) [2]. The ORF1 (ORF1a and ORF1b), which encodes the RNA replicase polyprotein in the PRRSV genome, accounts for approximately 80% of the entire genome. The enzyme can be cleaved into 13 non-structural proteins with different biological functions (Nsp1α, Nsp1β, Nsp2-Nsp12), of which Nsp2 shows the greatest variation [3].

Sample Collection and Virus Isolation
PRRSV-positive lung samples were collected from a PRRS suspected pig farm uninoculated with PRRSV vaccine from Shandong Province, China, in 2018. These samples were homogenized with Dulbecco's modified Eagle medium (DMEM, Life Technologies Corp., Grand Island, NY, USA). The treated samples were inoculated after passing through 0.22 µm filters to MARC-145 cells. The inoculated cells were maintained in DMEM supplemented with 2% fetal bovine serum (FBS) and 100 U/mL of penicillin plus 100 µg/mL streptomycin at 37 • C in a 5% CO 2 atmosphere. When approximately 80% of the cells exhibited a cytopathic effect (CPE) in the fourth generation, the supernatant of the virus culture was collected after two freeze-thaw repeats and stored at −80 • C for further use.

Plaque Assay
For purification of the isolated virus, a plaque assay was performed using MARC-145 cells as previously described [14]. Briefly, the stock virus was 10-fold serially diluted and overlaid on a~90% confluent monolayer of MARC-145 cells cultured in 6-well cell culture plates (0.5 mL/well). After a 1.5 h adsorption period at 37 • C, the plates were washed with sterile PBS and overlaid with melted agarose (2 mL/well). Two days of infection later, 2 mL/well of 0.01% neutral red staining solution (Solarbio, Beijing, China) was added for staining at 37 • C for 3 h. Then, a clear and uniform plaque was picked out and inoculated with new MARC-145 cells in 96-well cell culture plates. When approximately 80% of the Viruses 2022, 14, 1194 3 of 14 cells displayed visible CPEs, the cells were frozen at −80 • C and thawed twice. After centrifugation at 10,000× g for 5 min, the supernatant was subjected to two additional rounds of plaque purification.

Immunofluorescence Assay (IFA)
MARC-145 cells were infected with PRRSV strain CSR1801 at a multiplicity of infection (MOI) of 0.1 when the cells reached approximately 90% confluence. At 24 h post-infection (hpi), the cells were fixed with 4% paraformaldehyde and then permeabilized with 0.3% Triton X 100. After three washes with PBS, they were blocked with 5% fetal bovine serum albumin at 37 • C for 1 h. The cells were incubated overnight at 4 • C with the monoclonal antibody (mAb) against PRRSV N protein at a dilution of 1:5000. Then, the cells were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (Proteintech, Wuhan, China) at 37 • C for 1 h. After a final washing, the cells were observed using a fluorescent microscope (Leica, SPE, Buffalo Grove, IL, USA).

Phylogenetic and Recombination Analysis
The evolutionary relationship of the CSR1801 isolate with other represented Chinese PRRSV isolates was analyzed using MegAlign in the Lasergene software (DNASTAR). Phylogenetic trees were constructed using the MEGA software (version X) using the neighbor-joining method, and the reliability of the tree was assessed by bootstrap analysis with 1000 replications. The nucleotide and deduced amino acid (AA) sequences were aligned using the MegAlign program of the DNASTAR software (DNASTAR) to determine the sequence homology. A phylogenetic tree was constructed using the MEGA5 software with the neighbor-joining method; bootstrap values were calculated for 1000 replicates for alignment with multiple sequences of representative PRRSV sequences available in GenBank.
Furthermore, the software MegAlign was used to compare the full-length gene sequences of VR2332, CH-1R, JXA1, NADC30, and HLHDZD32-1901 (NADC34-like) strains. The above file was saved and homologous recombination analysis was performed using Simplot software (version 3.5.1). The CSR1801 strain was selected as the recombined parent sequence and Do BootScan was used to carry out a homologous recombination analysis.  1 The position is determined based on the representative strain ATCC VR-2332 (U87392).

Animal Experiments
The experimental protocol for the pig studies was approved by the Shandong Agricultural University Animal Care and Use Committee (Approval Number: # SDAUA-2019-022). The animal experiments were conducted in strict accordance with the Chinese Regulations on Laboratory Animals and the Guidelines for the Care of Laboratory Animals (the Ministry of Science and Technology of the People's Republic of China).
A virus challenge experiment in piglets was performed to evaluate the pathogenicity of the PRRSV CSR1801 strain. A total of 10 28-day-old PRRSV-and antibody-double-negative piglets, as assessed by RT-PCR and enzyme-linked immunosorbent assay (ELISA; IDEXX PRRS X3, Westbrook, ME, USA), were randomly divided into two groups. The piglets in group 1 (n = 5) were all nasally inoculated with 2 mL/pig of the PRRSV CSR1801 strain at a dose of 1 × 10 5 TCID 50 . The piglets in group 2 (n = 5) were inoculated with the cell culture medium as the negative control. The piglets were monitored daily for clinical signs of PRRS, including fever (assessed by rectal temperature), respiratory symptoms (scored as described previously [16]), and abnormal behavior; the mental status, appetite status, and fecal status of the animals were also monitored to detect clinical signs of PRRS. Serum samples were collected from the piglets at 0, 3, 5, 7, 10, 14, and 21 days post-inoculation (dpi) and used in assays to measure the cytokine level and viral load. At 21 dpi, piglets were euthanized to collect lungs, lymph node, and spleen. The collected sample tissues were fixed in 4% formalin buffer and used for histopathological examination via hematoxylin and eosin (H&E) staining.

Real-Time Quantitative PCR (RT-qPCR)
The viral loads of PRRSV in the sera collected at serial timepoints (0, 3, 5, 7, 10, 14, and 21 dpi) were determined by conducting absolute RT-qPCR as reported previously [17]. First, a standard curve was established according to the results of six 10-fold gradients of recombinant plasmid amplified with the primers: forward, 5 -ACCAGGCGTTTCGCATCT-3 ; and reverse, 5 -ACTCTCTGCACTCACGGAAGG-3 . The extracted total RNA was then subjected to reverse transcription and RT-PCR, and the virus RNA quantity of the sample was calculated from a linear extrapolation of the Ct value plotted against the standard curve.

Histopathological Examination
Histopathological examinations of the collected lungs were performed using the H&E method. Briefly, the dried tissue sections were passed through the following alcohol gradient: xylene I (15 min), xylene II (15 min), alcohol xylene (1:1) (5 min), 100% alcohol (3 min), 90% alcohol (3 min), 80% alcohol (3 min), 70% alcohol (3 min), and deionized water (4 min). The sections were then dyed with hematoxylin for 5 min and rinsed for 1 s. The sections were subsequently covered with 1% hydrochloric acid alcohol for 2-3 s and flushed for 16 min. Next, the slices were placed in 85% alcohol for 2 s, 1% eosin for 3 s, dipped in 95% alcohol, 95% alcohol for 3 min, 100% alcohol for 2 min, and xylene for 10-30 min. Finally, the slices were sealed with neutral resin and the pathological changes in the lungs were observed under a light microscope (Optika, Ponteranica, Italy).

Data Analysis
The data are expressed as the mean ± standard deviation (X ± SD), Duncan's multisample test was used to analyze the differences between the groups, and SPSS 19.0 was used for statistical analysis. p < 0.05 was considered statistically significant.

Virus Isolation and Identification
MARC-145 cells were inoculated with the grinding fluid of PRRSV-positive lung samples, and typical PRRSV-induced CPE appeared at 48 h after inoculation from the fourth-generation virus ( Figure 1A). One of the viral plaques was picked out from the virus stock by a virus plaque assay as shown in Figure 1B and was subsequently amplified in MARC-145 cells and named PRRSV strain CSR1801. Then, the presence of PRRSV was confirmed by IFA staining with a PRRSV-specific mAb against the virus N protein. As shown in Figure 1C, after 24 h inoculation of MARC-145 cells with the CSR1801 strain, PRRSV-specific fluorescence was observed in the field of vision; by contrast, MARC-145 cells in mock-infection could not react with the mAb against N protein.

Analysis of Full-Length Genomic Sequence
The full-length genome of the PRRSV CSR1801 strain was determined by segmental gene sequencing and splicing, which is 15,219 bp excluding the poly (A) tail (GenBank accession number: OM743305). To investigate the genetic relationships between the PRRSV CSR1801 strain and other PRRSV strains, the whole genome sequence of the CSR1801 strain and the representative PRRSV reference strains or vaccine strain registered in GenBank were used for phylogenetic tree analysis. Based on the phylogenetic tree result of full-length genome sequence analysis, the PRRSV isolate CSR1801 was classified into the CLASSIC-like PRRSV group (Figure 2A), which shared 99% and 99.1% nucleotide identity with CH-1a and CH-1R at the genomic level, respectively. Meanwhile, RDP4 and SimPlot software were used to analyze the recombination within the whole genome sequences between the CSR1801 strain and other representative PRRSV strains. The results showed that there was no gene fragment recombination among the strains of CSR1801, VR2332, CH-1R, JXA1, NADC30, and HLHDZD32-1901 (NADC34-like), as shown in Figure 2B. Moreover, to further characterize the CSR1801 strain, the Nsp2 amino acid sequences were aligned with the PRRSV strains, including HP-PRRSV, classical PRRSV, NADC30, and NADC34. Nsp2 possesses highly variable features and is recognized as a molecular marker in HP-PRRSV-like and NADC30/34-like strains, which are currently epidemic in swine farms. Sequence alignment suggested that Nsp2 amino acids of the CSR1801 strain did not contain recombination or deletion positions ( Figure 2C). The analysis of the full-length genome sequence and Nsp2 amino acid sequences suggested that classical PRRSV strains are still epidemic but have weak recombination characteristics in some swine farms. NADC30, and NADC34. Nsp2 possesses highly variable features and is recognized as a molecular marker in HP-PRRSV-like and NADC30/34-like strains, which are currently epidemic in swine farms. Sequence alignment suggested that Nsp2 amino acids of the CSR1801 strain did not contain recombination or deletion positions ( Figure 2C). The analysis of the full-length genome sequence and Nsp2 amino acid sequences suggested that classical PRRSV strains are still epidemic but have weak recombination characteristics in some swine farms. Finally, we analyzed the amino acid mutation of the CSR1801 strain compared with CH-1R and CH-1a strains, which showed that the ORF1a (2503 aa  Finally, we analyzed the amino acid mutation of the CSR1801 strain compared with CH-1R and CH-1a strains, which showed that the ORF1a (2503 aa

Viral Load in Sera
The viral titers of PRRSV in the sera collected from piglets at regular intervals were detected by RT-qPCR. The results showed that the PRRSV CSR1801 isolate infection induced a low level of viremia. The viral titer peaked at 3 dpi, remained constant at about 4.2 log copies per microliter until 14 dpi, and declined rapidly to near zero after 14 dpi ( Figure 3A), indicating the end of viremia.

Clinical Symptoms and Histopathological Lesion
In general, the piglets inoculated with the PRRSV CSR1801 isolate showed mild clinical signs. In the animal challenge experiment, the rectal temperature of piglets inoculated with PRRSV CSR1801 strain increased to 39.7 • C at two dpi and it always fluctuated around 39.5 • C the rest of the time. The rectal temperature of the mock-inoculated pigs was relatively stable and almost always about 39.0-39.2 • C ( Figure 3B). Mild clinical signs were observed in the challenged pigs. Their respiratory disease scores are summarized in Figure 3C. All five piglets showed mild dyspnea when stressed or at rest from three dpi to 10 dpi. Other symptoms included a runny nose and rough back hair in infected piglets from eight dpi to 16 dpi, but all were in good mental status and showed a good appetite. There were no deaths. By contrast, the mock-inoculated piglets behaved normally without any clinical signs throughout the experiment.
Lymphocytic interstitial pneumonia characterized by a diffuse interstitial proliferation could be observed in the lungs in the PRRSV CSR1801 strain-inoculated piglets (Figure 4), which was consistent with the results of a previous study [18], while the mock-inoculated piglets showed normal lung structure.

Viral Load in Sera
The viral titers of PRRSV in the sera collected from piglets at regular intervals were detected by RT-qPCR. The results showed that the PRRSV CSR1801 isolate infection induced a low level of viremia. The viral titer peaked at 3 dpi, remained constant at about 4.2 log copies per microliter until 14 dpi, and declined rapidly to near zero after 14 dpi ( Figure 3A), indicating the end of viremia.

Clinical Symptoms and Histopathological Lesion
In general, the piglets inoculated with the PRRSV CSR1801 isolate showed mild clinical signs. In the animal challenge experiment, the rectal temperature of piglets inoculated with PRRSV CSR1801 strain increased to 39.7 °C at two dpi and it always fluctuated around 39.5 °C the rest of the time. The rectal temperature of the mock-inoculated pigs  Figure 3B). Mild clinical signs were observed in the challenged pigs. Their respiratory disease scores are summarized in Figure 3C. All five piglets showed mild dyspnea when stressed or at rest from three dpi to 10 dpi. Other symptoms included a runny nose and rough back hair in infected piglets from eight dpi to 16 dpi, but all were in good mental status and showed a good appetite. There were no deaths. By contrast, the mock-inoculated piglets behaved normally without any clinical signs throughout the experiment. Lymphocytic interstitial pneumonia characterized by a diffuse interstitial proliferation could be observed in the lungs in the PRRSV CSR1801 strain-inoculated piglets (Figure 4), which was consistent with the results of a previous study [18], while the mockinoculated piglets showed normal lung structure. Figure 4. Histopathological results. The piglets in group 1 (n = 5) were all nasally inoculated with 2 mL/pig of the PRRSV CSR1801 strain at a dose of 1 × 10 5 TCID50. The piglets in group 2 (n = 5) were inoculated with the cell culture medium as the mock-inoculated control. Histopathological examination of lung tissue from the PRRSV CSR1801-and of the mock-inoculated piglets at 21 dpi was performed using routine hematoxylin and eosin staining method, respectively. The results showed a mild lymphocytic interstitial pneumonia in lungs from the PRRSV CSR1801-inoculated piglets. Magnification, 200×.

Concentrations of the Corresponding Cytokines in Sera
In the animal experiment, the concentrations of the corresponding swine cytokines, including IFN-α, IFN-γ, IL-10, and TGF-β1 in pig sera were determined by ELISA. Ten days after the challenge, the concentration of IL-10 did not change significantly. At 10 dpi and 14 dpi, the concentration of IL-10 in the CSR1801-inoculated piglets was significantly higher than that of the mock-inoculated piglets and then decreased rapidly ( Figure 5A). The characteristic of TGF-β1 change was similar to that of IL-10, and its concentration in the virus-inoculated piglets was significantly higher than that of the control group at 14 dpi ( Figure 5B). On the other hand, as cytokines play a role in cellular immunity, the levels of IFN-α and IFN-γ from the CSR1801-inoculated piglets was elevated at seven dpi and peaked at 10 dpi, then decreased to normal levels thereafter ( Figure 5C,D), which demon- Figure 4. Histopathological results. The piglets in group 1 (n = 5) were all nasally inoculated with 2 mL/pig of the PRRSV CSR1801 strain at a dose of 1 × 10 5 TCID 50 . The piglets in group 2 (n = 5) were inoculated with the cell culture medium as the mock-inoculated control. Histopathological examination of lung tissue from the PRRSV CSR1801-and of the mock-inoculated piglets at 21 dpi was performed using routine hematoxylin and eosin staining method, respectively. The results showed a mild lymphocytic interstitial pneumonia in lungs from the PRRSV CSR1801-inoculated piglets. Magnification, 200×.

Concentrations of the Corresponding Cytokines in Sera
In the animal experiment, the concentrations of the corresponding swine cytokines, including IFN-α, IFN-γ, IL-10, and TGF-β1 in pig sera were determined by ELISA. Ten days after the challenge, the concentration of IL-10 did not change significantly. At 10 dpi and 14 dpi, the concentration of IL-10 in the CSR1801-inoculated piglets was significantly higher than that of the mock-inoculated piglets and then decreased rapidly ( Figure 5A). The characteristic of TGF-β1 change was similar to that of IL-10, and its concentration in the virus-inoculated piglets was significantly higher than that of the control group at 14 dpi ( Figure 5B). On the other hand, as cytokines play a role in cellular immunity, the levels of IFN-α and IFN-γ from the CSR1801-inoculated piglets was elevated at seven dpi and peaked at 10 dpi, then decreased to normal levels thereafter ( Figure 5C,D), which demonstrates the similar change characteristics.

Discussion
The PRRSV epidemic strains in China have experienced recombination since 2015 due to the introduction of the PRRSV NADC30 strain from North America that has undergone genetic exchange with the HP-PRRSVs in China [19][20][21], producing a series of PRRSV NADC30-like strains that have affected most of the swine farms in China for ~5 years [10,11,22,23]. In 2017, another PRRSV strain, which originated from NADC34 from North America, called NADC34-like, was first detected in a swine farm in Liaoning Province and then in Fujian Province [24,25], indicating that NADC34-like PRRSV might have the potential ability to develop an endemic strain in swine farms in China. The ratio of NADC34-like positives increased to 11.5% and 28.6% in 433 PRRSV-positive clinical samples from eight provinces in 2020 and 2021 [26]. A common characteristic of both NADC30 and NADC34 is strong recombination ability, and the recombination happens among NADC34-like, NADC30-like, or HP-PRRSV strains from local swine farms in China [26]. No recombination between NADC30/NADC34 and classical PRRSV has been reported in China. Maybe the weak recombination ability of classical PRRSV strains leads to its low positive percentage in the positive swine farms.
In our study, the PRRSV CSR1801 strain still shows the classical PRRSV features, and no recombination or deletion was found in the amino acids (AAs) of Nsp2, the most variable genes in the whole genome of PRRSV and NADC30/NADC34/HP-PRRSV [11,[26][27][28]. However, as an RNA virus, the CSR1801 strain also experienced an evolution process. These mutations happened discontinuously and occasionally from ORF1 to ORF7, but whether these mutations are related to pathogenicity warrants further investigation.
The pathogenicity of the PRRSV CSR1801 strain for the piglets was mild and mainly manifested in clinical signs, rectal temperature, viral load, and lung lesions, which is consistent with the previous report [18]. Whether due to its genetic evolution or its pathogenicity, the PRRSV isolate CSR1801 showed the characteristics of a classical strain and

Discussion
The PRRSV epidemic strains in China have experienced recombination since 2015 due to the introduction of the PRRSV NADC30 strain from North America that has undergone genetic exchange with the HP-PRRSVs in China [19][20][21], producing a series of PRRSV NADC30-like strains that have affected most of the swine farms in China for 5 years [10,11,22,23]. In 2017, another PRRSV strain, which originated from NADC34 from North America, called NADC34-like, was first detected in a swine farm in Liaoning Province and then in Fujian Province [24,25], indicating that NADC34-like PRRSV might have the potential ability to develop an endemic strain in swine farms in China. The ratio of NADC34-like positives increased to 11.5% and 28.6% in 433 PRRSV-positive clinical samples from eight provinces in 2020 and 2021 [26]. A common characteristic of both NADC30 and NADC34 is strong recombination ability, and the recombination happens among NADC34-like, NADC30-like, or HP-PRRSV strains from local swine farms in China [26]. No recombination between NADC30/NADC34 and classical PRRSV has been reported in China. Maybe the weak recombination ability of classical PRRSV strains leads to its low positive percentage in the positive swine farms.
In our study, the PRRSV CSR1801 strain still shows the classical PRRSV features, and no recombination or deletion was found in the amino acids (AAs) of Nsp2, the most variable genes in the whole genome of PRRSV and NADC30/NADC34/HP-PRRSV [11,[26][27][28]. However, as an RNA virus, the CSR1801 strain also experienced an evolution process. These mutations happened discontinuously and occasionally from ORF1 to ORF7, but whether these mutations are related to pathogenicity warrants further investigation.
The pathogenicity of the PRRSV CSR1801 strain for the piglets was mild and mainly manifested in clinical signs, rectal temperature, viral load, and lung lesions, which is consistent with the previous report [18]. Whether due to its genetic evolution or its pathogenicity, the PRRSV isolate CSR1801 showed the characteristics of a classical strain and should be classified as a genetically stable classical strain of PRRSV.
In China, PRRSV MLVs are used extensively in swine farms, and the MLV strains include VR-2332, R98, CH-1R, PC, JXA1-P80, HuN4-F112, GDr180, and TJM-F92, which can offer effective protection in homologous PRRSV strain challenges [29][30][31][32][33]. In recent years, the inactivated CH-1a vaccine has been widely used after one year of African swine fever introduction, although its efficiency is controversial. No study has reported that anyone PRRSV vaccine could completely protect against the main epidemic PRRSV strains of NADC30-like or NADC34-like in the field. According to the efficiency reports of commercial MLVs used in swine farms against different NADC30-like strains, these PRRSV MLVs showed limited or no cross-protection efficacy against some NADC30-like strains [21,23,[34][35][36]. In particular, PRRSVs pools in swine farms increase viral recombination frequency and make the clinical prevalence and control of PRRS more complicated. Since the NADC30 invasion in 2015, variant strains that recombine with PRRSV MLVs originating from HP-PRRSV are often found in PRRSV-positive swine farms [11,37,38]. In these studies, although PRRSV NADC30 or NADC34 strains easily recombine and produce many NADC30-like or NADC34-like strains with local HP-PRRSV (-like) strains, the recombination of both NADC30 or NADC34 and the local classical PRRSV strain was not found.
In summary, this study looked at a new classical PRRSV strain from a swine farm in Shandong province, China, which maintained stable features and did not recombine with other PRRSV strains (NADC30-like, NADC34-like, or HP-like strains) as is common in swine farms. This study may provide clues for further understanding the genetic evolution and pathogenicity of PRRSV and may also have important reference value for the efficient prevention and control of PRRS in swine farms.