In Vivo and In Vitro Characterization of the Recently Emergent PRRSV 1-4-4 L1C Variant (L1C.5) in Comparison with Other PRRSV-2 Lineage 1 Isolates

The recently emerged PRRSV 1-4-4 L1C variant (L1C.5) was in vivo and in vitro characterized in this study in comparison with three other contemporary 1-4-4 isolates (L1C.1, L1A, and L1H) and one 1-7-4 L1A isolate. Seventy-two 3-week-old PRRSV-naive pigs were divided into six groups with twelve pigs/group. Forty-eight pigs (eight/group) were for inoculation, and 24 pigs (four/group) served as contact pigs. Pigs in pen A of each room were inoculated with the corresponding virus or negative media. At two days post inoculation (DPI), contact pigs were added to pen B adjacent to pen A in each room. Pigs were necropsied at 10 and 28 DPI. Compared to other virus-inoculated groups, the L1C.5-inoculated pigs exhibited more severe anorexia and lethargy, higher mortality, a higher fraction of pigs with fever (>40 °C), higher average temperature at several DPIs, and higher viremia levels at 2 DPI. A higher percentage of the contact pigs in the L1C.5 group became viremic at two days post contact, implying the higher transmissibility of this virus strain. It was also found that some PRRSV isolates caused brain infection in inoculation pigs and/or contact pigs. The complete genome sequences and growth characteristics in ZMAC cells of five PRRSV-2 isolates were further compared. Collectively, this study confirms that the PRRSV 1-4-4 L1C variant (L1C.5) is highly virulent with potential higher transmissibility, but the genetic determinants of virulence remain to be elucidated.

The ORF5 sequences of these five PRRSV-2 isolates propagated in ZMAC cells were determined using the Sanger method, as previously described [37].Their ORF5 sequences, together with the reference sequences representing different sublineages within lineage 1 [5], were aligned using MAFFT v7.407 [38], and the maximum likelihood tree was constructed using MEGA6 [39] with the bootstrap analysis of 1000 replicates.The locations of the five PRRSV isolates in the phylogenetic tree are shown in Figure 1.

Animal Source and Approval
Seventy-two PRRSV-naive pigs at three weeks of age were purchased, weaned, and transported to the Iowa State University Laboratory Animal Resources (ISU LAR) facility.The pigs were pre-screened eight days before delivery to confirm they were negative for PRRSV, porcine circovirus type 2 (PCV2), and porcine circovirus type 3 (PCV3) by PCR on serum samples, and negative for influenza A virus (IAV) and Mycoplasma hyopneumoniae (MHP) by PCR on nasal swabs.The pigs were also confirmed negative for PRRSV

Animal Source and Approval
Seventy-two PRRSV-naive pigs at three weeks of age were purchased, weaned, and transported to the Iowa State University Laboratory Animal Resources (ISU LAR) facility.The pigs were pre-screened eight days before delivery to confirm they were negative for PRRSV, porcine circovirus type 2 (PCV2), and porcine circovirus type 3 (PCV3) by PCR on serum samples, and negative for influenza A virus (IAV) and Mycoplasma hyopneumoniae (MHP) by PCR on nasal swabs.The pigs were also confirmed negative for PRRSV antibodies using a PRRS X3 ELISA kit (IDEXX Laboratories, Westbrook, ME) on serum samples.After pigs arrived at the ISU LAR facility, serum samples were tested again before inoculation to confirm they were still negative for PRRSV by PCR and PRRS X3 ELISA antibody assay.The pigs were also confirmed IAV negative in both nasal swabs and oral fluids using IAV PCR before inoculation.The study was approved by the Iowa State University Institutional Animal Care and Use Committee (approval number IACUC-21-124) and the Institutional Biosafety Committee (approval number IBC-21-054).

Animal Study Design
The experimental design is shown in Figure 2. On the day of arrival, Excede ® (Ceftiofur crystalline free acid), at a dose of 5 mg/kg, was given to each pig intramuscularly in the post-auricular region of the neck.Each pig was microchipped to monitor body temperature.The microchips and microchip readers were purchased from Destron FearingTM.The microchip (Bio-ThermoTM) was intramuscularly implanted at the base of the left ear on the scutiform cartilage of each pig.All pigs were weighed and blocked by weight and then randomly divided into six groups with 12 pigs per group.Six rooms of the same size and condition were used in this study.Each room included two adjacent pens of equal measure (Pen A and Pen B).On the arrival day, forty-eight pigs were housed in Pen A of six rooms (8 pigs per group per room) as inoculation pigs, and the remaining 24 pigs were housed in two additional large rooms with 12 pigs per room to serve as contact pigs later on.
Viruses 2023, 15, x FOR PEER REVIEW 5 of 29 antibodies using a PRRS X3 ELISA kit (IDEXX Laboratories, Westbrook, ME) on serum samples.After pigs arrived at the ISU LAR facility, serum samples were tested again before inoculation to confirm they were still negative for PRRSV by PCR and PRRS X3 ELISA antibody assay.The pigs were also confirmed IAV negative in both nasal swabs and oral fluids using IAV PCR before inoculation.The study was approved by the Iowa State University Institutional Animal Care and Use Committee (approval number IACUC-21-124) and the Institutional Biosafety Committee (approval number IBC-21-054).

Animal Study Design
The experimental design is shown in Figure 2. On the day of arrival, Excede ® (Ceftiofur crystalline free acid), at a dose of 5 mg/kg, was given to each pig intramuscularly in the post-auricular region of the neck.Each pig was microchipped to monitor body temperature.The microchips and microchip readers were purchased from Destron Fear-ingTM.The microchip (Bio-ThermoTM) was intramuscularly implanted at the base of the left ear on the scutiform cartilage of each pig.All pigs were weighed and blocked by weight and then randomly divided into six groups with 12 pigs per group.Six rooms of the same size and condition were used in this study.Each room included two adjacent pens of equal measure (Pen A and Pen B).On the arrival day, forty-eight pigs were housed in Pen A of six rooms (8 pigs per group per room) as inoculation pigs, and the remaining 24 pigs were housed in two additional large rooms with 12 pigs per room to serve as contact pigs later on.After seven days of acclimation, the inoculation pigs in groups 1 through 5 were inoculated with the respective ZMAC isolate at the appropriate dilution via the intramuscular route (2 mL/pig) in the neck muscle area and the intranasal route (2 mL/nostril) using mucosal atomization devices (MADs) fitted to a syringe that generated mist or sprays by manual pressure, which resulted in particle sizes of 30-100 µm [40], with each pig After seven days of acclimation, the inoculation pigs in groups 1 through 5 were inoculated with the respective ZMAC isolate at the appropriate dilution via the intramuscular route (2 mL/pig) in the neck muscle area and the intranasal route (2 mL/nostril) using mucosal atomization devices (MADs) fitted to a syringe that generated mist or sprays by manual pressure, which resulted in particle sizes of 30-100 µm [40], with each pig receiving 10 6 TCID 50 of the respective virus inoculum in total.The inoculation pigs in group 6 were inoculated with a virus-negative medium following the same administration routes and the same volume.At 2 days post inoculation (DPI; corresponding to 0 day post contact (DPC)), four contact pigs were added to Pen B in each of the six rooms (Figure 2).Four inoculated pigs per group were randomly selected and necropsied at 10 DPI to check for gross and microscopic lesions.If any pigs died or were euthanized due to severe body conditions before the scheduled days, pigs were necropsied accordingly.At 28 DPI, all the remaining pigs were euthanized and necropsied.
Pigs were weighed at -1 DPI, 10 DPI, 28 DPI, and any day when pigs had to be euthanized before the scheduled necropsy days.The weight difference between two time points were divided by the number of days in the interval to calculate the average daily weight gain (ADG).Serum and oral fluid samples were collected at 0, 2, 4, 7, 10, 14, 21, and 28 DPI.Blood was collected using BD Vacutainer™ SST™ tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) via the anterior vena cava and centrifuged at 3000 rpm for 15 min.The serum was aliquoted and stored at −80 • C until testing.Cotton ropes were used to collect oral fluids from each pen as previously described [41].At necropsy, gross lung lesions were scored; fresh and formalin-fixed tissues were collected and included lung, tonsil, brain, tracheobronchial lymph nodes, thymus, heart, spleen, and kidney.

Sample Processing
Fresh lung, tonsil, and brain tissues were processed by placing 2.5 g of tissue into 22.5 mL of Dulbecco's Modified Eagle's Medium (DMEM, Sigma, Cibolo, TX, USA) in a 50 mL conical tube, followed by grinding for 30 s using a geno grinder homogenizer (Thermo Fisher Scientific, Waltham, MA, USA) to obtain a 10-20% solution.After centrifugation at 4200× g for 10 min, the tissue homogenates were harvested and stored at −80 • C until testing.

Nucleic Acid Extraction and Quantitative PRRSV Real-Time RT-PCR
Nucleic acids were extracted from 100 µL of clinical samples using a MagMAX Pathogen RNA/DNA extraction kit (Thermo Fisher Scientific, Waltham, MA, USA) and a Kingfisher Flex instrument (Thermo Fisher Scientific) following the manufacturer's instructions.Nucleic acids were eluted into 90 µL of elution buffer.A commercial PRRSV RT-qPCR (VetMAX PRRSV NA&EU One-Step RT-PCR assay, Thermo Fisher Scientific), including the TaqMan Fast 1-Step RT-PCR master mix and primers and probes mix v2, was used following the previously described protocols [42].A Ct < 37 was considered positive, and Ct ≥ 37 was considered negative for PRRSV.Standard curves generated using known concentrations of in vitro transcribed viral RNA provided in the assay kits were used to quantify the genomic copies of the virus in the samples.

PRRSV Antibody Measurement
A PRRS X3 ELISA antibody kit (IDEXX Laboratories, Westbrook, ME, USA) was used to test serum samples collected at 0, 7, 14, 21, and 28 DPI from four virus-inoculated pigs that survived after 10 DPI and all contact pigs.According to the manufacturer, samples with sample-to-positive (S/P) ratios ≥0.4 were considered positive for antibody against PRRSV.
The same set of serum samples was tested by indirect fluorescent antibody (IFA) assay [43] using the ISU-P isolate [44] as the indicator virus.
The same set of serum samples was also tested for neutralizing antibodies using the fluorescent focus neutralization (FFN) assay and the homologous virus isolate as the indicator virus.For example, serum samples from the L1C.5 (L1C variant) group were tested for neutralizing antibody against L1C.5 isolate MN/01775GA/2021.Serum samples were first inactivated at 56 • C for 30 min, then 2-fold serially diluted from 1:2 to 1:256 dilutions in 96-well plates with a volume of 100 µL per well after dilution.Subsequently, 100 µL of the respective PRRSV isolate was mixed with an equal volume of diluted sera (in the serum-virus mixture, the serum dilution was adjusted from 1:4 to 1:512).After incubation for 1 h at 37 • C with 5% CO 2 , 100 µL of the serum-virus mixture (containing 100-200 TCID 50 of virus) was transferred to 96-well plates with a MARC-145 cell monolayer.The plates were incubated at 37 • C with 5% CO 2 for 48 h, fixed with cold 80% acetone, and stained with the mixture of PRRSV N protein-specific monoclonal antibody SDOW17 and SR30-conjugated to FITC (Rural Technologies Inc., Brookings, SD, USA) at a 1:100 dilution for 60 min.The staining was examined under a fluorescent microscope.The reciprocal of the highest serum dilution resulting in >90% reduction in staining compared to the antibody-negative serum control was defined as the FFN antibody titer of the serum sample.An FFN antibody titer of ≥8 was considered positive.

Bacterial Culture on Lung and Brain Homogenates
In order to determine if pigs were infected with any bacteria, the lung and brain homogenate samples from all 72 pigs in the study were submitted for general bacterial culture at the Bacteriology Section of Iowa State University Veterinary Diagnostic Laboratory (ISU VDL).

Gross Lung Pathology, Histopathology, and Immunohistochemistry
At necropsy, lung tissue from each pig was examined in a blind fashion by a single pathologist and given a subjective score for the severity of gross lung lesions using an established scoring system [15] that estimated the percentage of lung tissues affected by pneumonia and consolidation.Formalin-fixed tissues of the lung (two sections from the caudal lobe and one from each of middle, accessory, and cranial lung lobes), tonsils, tracheobronchial lymph nodes, thymus, heart, spleen, kidney, and brain were submitted to ISU VDL for histopathological examination.Lung and brain tissues were also tested by PRRSV immunohistochemistry (IHC).For microscopic and IHC lung scores, one pathologist blind to treatments scored each of the five lung sections for all pigs and then averaged those numbers to give a unique value per pig.The microscopic sections were examined and assigned a score for the severity of interstitial pneumonia (0 to 6) as previously described [15,45].The IHC score was based on a 0 to 3 system, as previously described [46].

Multistep Growth Curve in Cell Culture
For in vitro characterization, the growth curves of five PRRSV-2 lineage 1 isolates included in this study were compared in ZMAC cell line derived from primary alveolar macrophages.Each stock virus was diluted to 1 × 10 6 TCID 50 /mL based on the titration conducted in ZMAC cells.Monolayers of ZMAC cells grown in 24-well plates were inoculated with each PRRSV-2 isolate at a multiplicity of infection (moi) of 0.1.After 1 h absorption at 37 • C in 5% CO 2 incubator, the virus inoculum was discarded, and two milliliters of fresh medium was added to each well of cells, and this time point was designated time zero with respect to infection.Seven 24-well plates were used for the entire experiments, with one plate for each time point and duplicate wells for each virus at each time point.The cell plates were incubated at 37 • C with 5% CO 2 .At 0, 6, 12, 24, 48, 72, and 96 h post infection (hpi), the plates were frozen at −80 • C.After one freeze-thaw cycle, the cell lysates were centrifuged at 1200× g for 10 min, and the supernatant was saved at −80 • C for titration.Each isolate at each hpi was titrated using both aliquots.The supernatants were 10-fold serially diluted and titrated in ZMAC cells grown in 96-well plates with triplicate wells per dilution.Virus titers were determined according to the Reed and Muench method [49] and expressed as TCID 50 /mL.

Statistical Analysis
Two-way ANOVA with interaction was applied for analyzing virus titers in a multistep growth curve.The two factors in the analysis were virus groups and hours post infection.Temperatures, anorexia and lethargy scores, virus loads in serum determined by quantitative PRRSV real-time RT-PCR.PRRSV antibody (PRRS X3 ELISA) S/P ratios, PRRSV IFA antibody titers converted into log2 format, and PRRSV FFN antibody titers converted into log2 format over time were analyzed using a linear mixed model.The ADG, PRRSV RNA levels in tissues (e.g., lung, tonsil, and brain), and gross lung lesion scores were analyzed using one-way ANOVA with Tukey-Kramer HSD.The microscopic lesion scores in lungs and IHC scores in lung and brain tissues were analyzed using a linear mixed model.No statistical analysis was carried out on oral fluid data.For all analyses, SAS was used, and a p-value < 0.05 was considered significant.

Clinical Observations (Anorexia and Lethargy, Mortality, Microchip Temperature, and ADG) in Inoculated Pigs
The mock-inoculated pigs did not show clinical signs of illness throughout the experiment.Temperature and respiratory disease scores in this negative control group were within the normal ranges.
Pigs inoculated with the 1-4-4 L1C.5 isolate or 1-7-4 L1A isolate became more lethargic and were off-feed faster than other virus-inoculated groups.Regarding the anorexia and lethargy scores, the 1-4-4 L1C.5 isolate-inoculated group was significantly different from other virus-inoculated groups at different time points, such as 1, 4, 5, 8-13, and 16-21 DPI.The overall anorexia and lethargy scores in different inoculation groups are shown in Figure 3A.Based on an overall statistical analysis of the anorexia and lethargy scores from 0 to 21 DPI, pigs inoculated with the 1-4-4 L1C.5 isolate had significantly higher scores (a mean score of 2) compared to other groups; the 1-4-4 L1C.1 inoculation group had the lowest scores among all virus-inoculated groups.
The mortality was calculated by counting the pigs that naturally died or were euthanized due to severe body conditions based on the IACUC protocol.As shown in Table 2, pigs inoculated with the 1-4-4 L1C.5 isolate had higher mortality (6 out of 8 pigs) than other virus-inoculated groups (1-2 pigs out of 8 pigs).Labels with different letters indicate significant differences; for example, a and b have a significant difference, but a and ab have no significant difference.
The mortality was calculated by counting the pigs that naturally died or were euthanized due to severe body conditions based on the IACUC protocol.As shown in Table 2, pigs inoculated with the 1-4-4 L1C.5 isolate had higher mortality (6 out of 8 pigs) than other virus-inoculated groups (1-2 pigs out of 8 pigs).Note: The pigs that naturally died or were euthanized due to the severe body conditions were counted as mortality.Note: The pigs that naturally died or were euthanized due to the severe body conditions were counted as mortality.
Similar to the viremia level, the 1-4-4 L1C.5 virus-inoculated pen had a numerically higher virus load in oral fluid than all other virus-inoculated pens, while the 1-7-4 L1A virus-inoculated pen had a numerically higher virus load than 1-4-4 L1C.1, 1-4-4 L1A, and 1-4-4 L1H virus-inoculated pens at 2 and 3 DPI (Figure 4B).The 1-4-4 L1H virus-inoculated pen had a higher virus load than all other virus-inoculated pens at 7 and 28 DPI.Notably, no statistical analysis was conducted on viral loads in oral fluid samples because there was only one oral fluid sample from each inoculation pen at each time point.Also, no oral fluid was collected from the 1-4-4 L1C.5 virus-inoculated pen at 10 DPI.At 14 DPI, no oral fluids were collected from any virus-inoculated pen.Similar to the viremia level, the 1-4-4 L1C.5 virus-inoculated pen had a numerically higher virus load in oral fluid than all other virus-inoculated pens, while the 1-7-4 L1A virus-inoculated pen had a numerically higher virus load than 1-4-4 L1C.1, 1-4-4 L1A, and 1-4-4 L1H virus-inoculated pens at 2 and 3 DPI (Figure 4B).The 1-4-4 L1H virus-inoculated pen had a higher virus load than all other virus-inoculated pens at 7 and 28 DPI.Notably, no statistical analysis was conducted on viral loads in oral fluid samples because there was only one oral fluid sample from each inoculation pen at each time point.Also, no oral fluid was collected from the 1-4-4 L1C.5 virus-inoculated pen at 10 DPI.At 14 DPI, no oral fluids were collected from any virus-inoculated pen.

PRRSV RNA Load in Tissue Samples of Inoculated Pigs
A PRRSV quantitative real-time RT-PCR was used to detect PRRSV RNA in lung, tonsil, and brain tissue samples in inoculated pigs necropsied at 10 DPI or during 9-10 DPI (Figure 7A) and at 28 DPI or during 10-28 DPI (Figure 7B).All lung and tonsil samples from each virus inoculation group were PRRSV PCR-positive at both time points.Brain tissues were not collected from the three pigs that died naturally before 10 DPI, all of which were from the 1-4-4 L1C.5, 1-4-4 L1H, and 1-7-4 L1A inoculation groups; hence, only three brain samples from each of those groups were available at 10 DPI.For the brain samples, 2/4 up to 3/3 pigs at 10 DPI and 1/4 up to 3/4 pigs at 28 DPI were PRRSV PCRpositive among all virus-inoculated groups (Figure 7A,B).Although numerical differences were observed among some groups, PRRSV RNA loads in lung homogenates were not significantly different among five virus-inoculated

PRRSV RNA Load in Tissue Samples of Inoculated Pigs
A PRRSV quantitative real-time RT-PCR was used to detect PRRSV RNA in lung, tonsil, and brain tissue samples in inoculated pigs necropsied at 10 DPI or during 9-10 DPI (Figure 7A) and at 28 DPI or during 10-28 DPI (Figure 7B).All lung and tonsil samples from each virus inoculation group were PRRSV PCR-positive at both time points.Brain tissues were not collected from the three pigs that died naturally before 10 DPI, all of which were from the 1-4-4 L1C.5, 1-4-4 L1H, and 1-7-4 L1A inoculation groups; hence, only three brain samples from each of those groups were available at 10 DPI.For the brain samples, 2/4 up to 3/3 pigs at 10 DPI and 1/4 up to 3/4 pigs at 28 DPI were PRRSV PCR-positive among all virus-inoculated groups (Figure 7A,B).groups at 10 DPI or 28 DPI.Similar results and conclusions were obtained for tonsil and brain homogenates.

PRRSV Immunohistochemistry Staining in Brain Tissues
Since brain homogenates from numerous virus-inoculated pigs were PRRSV PCRpositive, we further investigated the microscopic lesions and PRRSV IHC staining in brain tissues, with results summarized in Table 3 and Figure 8.The data suggested that pigs inoculated with PRRSV isolates, such as 1-4-4 L1C.5, 1-4-4 L1A, 1-4-4 L1H, and 1-7-4 L1A, developed encephalitis to different degrees, which was corroborated by positive IHC staining and relatively low PCR CT values.This was found not only in virus-inoculated pigs but also in the contact pigs of the 1-4-4 L1A, 1-4-4 L1H, and 1-7-4 L1A groups.Neurological signs including ataxia, incoordination, posterior paresis, and convulsion were observed in four pigs; these included two from 1-4-4 L1A group (one inoculated pig (#5) showing signs at 12 DPI and one contact pig (#38) showing signs at 15 DPC) and two from 1-4-4 L1H group (one inoculated pig (#33) showing signs at 9 DPI and one contact pig (#37) showing signs at 10 DPC).However, in the 1-4-4 L1C.1 group, regardless of inoculated pigs or contact pigs, no infection in brain tissue was observed by PRRSV IHC staining, although some pigs were PRRSV PCR-positive with relatively high CT values (26.3-30.5).Bacteria culture in brain tissue homogenates revealed no growth of bacteria in most pigs (Table 3).Although numerical differences were observed among some groups, PRRSV RNA loads in lung homogenates were not significantly different among five virus-inoculated groups at 10 DPI or 28 DPI.Similar results and conclusions were obtained for tonsil and brain homogenates.

PRRSV Immunohistochemistry Staining in Brain Tissues
Since brain homogenates from numerous virus-inoculated pigs were PRRSV PCRpositive, we further investigated the microscopic lesions and PRRSV IHC staining in brain tissues, with results summarized in Table 3 and Figure 8.The data suggested that pigs inoculated with PRRSV isolates, such as 1-4-4 L1C.5, 1-4-4 L1A, 1-4-4 L1H, and 1-7-4 L1A, developed encephalitis to different degrees, which was corroborated by positive IHC staining and relatively low PCR C T values.This was found not only in virus-inoculated pigs but also in the contact pigs of the 1-4-4 L1A, 1-4-4 L1H, and 1-7-4 L1A groups.Neurological signs including ataxia, incoordination, posterior paresis, and convulsion were observed in four pigs; these included two from 1-4-4 L1A group (one inoculated pig (#5) showing signs at 12 DPI and one contact pig (#38) showing signs at 15 DPC) and two from 1-4-4 L1H group (one inoculated pig (#33) showing signs at 9 DPI and one contact pig (#37) showing signs at 10 DPC).However, in the 1-4-4 L1C.1 group, regardless of inoculated pigs or contact pigs, no infection in brain tissue was observed by PRRSV IHC staining, although some pigs were PRRSV PCR-positive with relatively high C T values (26.3-30.5).Bacteria culture in brain tissue homogenates revealed no growth of bacteria in most pigs (Table 3).We further examined the possible correlation between PRRSV IHC scores and PCR CT values in brain tissues.In the 1-4-4 L1C.5 group, two inoculated pigs had a PRRSV IHC score of 1, and one inoculated pig had a PRRSV IHC score of 3, with the corresponding PRRSV PCR CT values of 27.9, 20.2 and 17.5 (Table 3).In the 1-4-4 L1A group, three inoculated pigs had a PRRSV IHC score of 1 with CT values ranging from 24.1 to 33.9, and one contact pig had a PRRSV IHC score of 2 with a CT of 22.3.In the 1-4-4 L1H group, two inoculated pigs had a PRRSV IHC score of 2 with CT values ranging from 21.5 to 22.9, and We further examined the possible correlation between PRRSV IHC scores and PCR C T values in brain tissues.In the 1-4-4 L1C.5 group, two inoculated pigs had a PRRSV IHC score of 1, and one inoculated pig had a PRRSV IHC score of 3, with the corresponding PRRSV PCR C T values of 27.9, 20.2 and 17.5 (Table 3).In the 1-4-4 L1A group, three inoculated pigs had a PRRSV IHC score of 1 with C T values ranging from 24.1 to 33.9, and one contact pig had a PRRSV IHC score of 2 with a C T of 22.3.In the 1-4-4 L1H group, two inoculated pigs had a PRRSV IHC score of 2 with C T values ranging from 21.5 to 22.9, and one contact pig had a PRRSV IHC score of 3 with a C T of 20.3.In the 1-7-4 L1A group, two inoculated pigs had a PRRSV IHC score of 1 with C T values ranging from 20.1 to 24.9, and one contact pig had a PRRSV IHC score of 1 with a C T of 20.8.All of the four pigs with neurological signs (pigs #5, 38, 33, and 37) showed infection in brain by IHC, with the scores ranging from 1 to 3 and PRRSV C T values from 20.3 to 24.1.Although some brain homogenates in the 1-4-4 L1C.1-inoculated and contact pigs were PCR-positive, no brain infection was detected by PRRSV IHC.The overall trend is that brain tissues with lower PCR C T values were more likely to be PRRSV IHC-positive, but there could be some exceptions.

Antibody Responses in Inoculated Pigs
PRRSV ELISA antibody results are shown in Figure 9A.All pigs in the six inoculation groups were seronegative at 0 DPI.All pigs in the mock-inoculated group were negative throughout the study.At 7 DPI, three out of four pigs were seropositive in the 1-4-4 L1C.5 and 1-4-4 L1H inoculation groups, two out of four pigs were seropositive in the 1-4-4 L1A and 1-7-4 L1A inoculation groups, and one out of four pig was seropositive in 1-4-4 L1C.1 inoculation group.There was no significant difference in ELISA S/P ratios between the virus-inoculated groups.At 14, 21, and 28 DPI, all pigs in each virus inoculation group were seropositive.At 14 DPI, the 1-4-4 L1C.1 group had significantly higher ELISA S/P ratios than all other virus inoculation groups except the 1-4-4 L1C.5 inoculation group.Similarly, the 1-4-4 L1C.1 inoculation group had substantially higher ELISA S/P ratios than the 1-4-4 L1H inoculation group at 21 and 28 DPI, and had a significantly higher ELISA S/P ratio than the 1-7-4 L1A inoculation group at 21 DPI.
Viruses 2023, 15, x FOR PEER REVIEW 17 of 29 one contact pig had a PRRSV IHC score of 1 with a CT of 20.8.All of the four pigs with neurological signs (pigs #5, 38, 33, and 37) showed infection in brain by IHC, with the scores ranging from 1 to 3 and PRRSV CT values from 20.3 to 24.1.Although some brain homogenates in the 1-4-4 L1C.1-inoculated and contact pigs were PCR-positive, no brain infection was detected by PRRSV IHC.The overall trend is that brain tissues with lower PCR CT values were more likely to be PRRSV IHC-positive,but there could be some exceptions.

Antibody Responses in Inoculated Pigs
PRRSV ELISA antibody results are shown in Figure 9A.All pigs in the six inoculation groups were seronegative at 0 DPI.All pigs in the mock-inoculated group were negative throughout the study.At 7 DPI, three out of four pigs were seropositive in the 1-4-4 L1C.5 and 1-4-4 L1H inoculation groups, two out of four pigs were seropositive in the 1-4-4 L1A and 1-7-4 L1A inoculation groups, and one out of four pig was seropositive in 1-4-4 L1C.1 inoculation group.There was no significant difference in ELISA S/P ratios between the virus-inoculated groups.At 14, 21, and 28 DPI, all pigs in each virus inoculation group were seropositive.At 14 DPI, the 1-4-4 L1C.1 group had significantly higher ELISA S/P ratios than all other virus inoculation groups except the 1-4-4 L1C.5 inoculation group.Similarly, the 1-4-4 L1C.1 inoculation group had substantially higher ELISA S/P ratios than the 1-4-4 L1H inoculation group at 21 and 28 DPI, and had a significantly higher ELISA S/P ratio than the 1-7-4 L1A inoculation group at 21 DPI.PRRSV IFA antibody responses were determined in serum samples of inoculated pigs using the PRRSV-2 ISU-P isolate as the indicator virus, and the results are summarized in Figure 9B.At 0 DPI, serum samples from all inoculation groups were IFA antibody-negative.At 7 DPI, all pigs in 1-4-4 L1C.1 and 1-4-4 L1A inoculation groups, and three out of four pigs in the 1-4-4 L1C.5, 1-4-4 L1H, and 1-7-4 L1A inoculation groups were IFA antibody-positive.At 14, 21, and 28 DPI, all pigs in all five virus inoculation groups were IFA antibody-positive.Regarding PRRSV IFA antibody titers, no significant differ- PRRSV IFA antibody responses were determined in serum samples of inoculated pigs using the PRRSV-2 ISU-P isolate as the indicator virus, and the results are summarized in Figure 9B.At 0 DPI, serum samples from all inoculation groups were IFA antibodynegative.At 7 DPI, all pigs in 1-4-4 L1C.1 and 1-4-4 L1A inoculation groups, and three out of four pigs in the 1-4-4 L1C.5, 1-4-4 L1H, and 1-7-4 L1A inoculation groups were IFA antibody-positive.At 14, 21, and 28 DPI, all pigs in all five virus inoculation groups were IFA antibody-positive.Regarding PRRSV IFA antibody titers, no significant difference was found between virus inoculation groups during 7-28 DPI.All pigs in the mock-inoculated group remained IFA antibody-negative over time and were significantly different from the virus-inoculated groups (p < 0.05) at 7, 14, 21, and 28 DPI.
PRRSV homologous FFN antibody testing results for the serum samples of inoculated pigs are shown in Figure 9C.From 0 to 14 DPI, the mock-inoculation group and all five virus inoculation groups were FFN antibody-negative.At 21 DPI, low FFN antibody titers in the range of 1 to 3.5 log2 were detected; the l-4-4 L1C.5 inoculation group had significantly higher FFN antibody titer than 1-4-4 L1H and 1-7-4 L1A groups but not significantly different from the 1-4-4 L1C.1 and 1-4-4 L1A inoculation groups.At 28 DPI, slightly higher FFN antibody titers in the 2-4.5 log2 range were detected; all virus inoculation groups, except the 1-4-4 L1H group, had similar FFN antibody titers.
The nucleotide length of 5 UTR, each ORF, 3 UTR, and the amino acid length of the proteins encoded by the respective ORF are summarized in Table 4. Compared to the VR-2332 isolate, the length differences were mainly found in the 5 UTR, 3 UTR, and nsp2 regions of other five PRRSV-2 lineage 1 isolates.In addition, some nucleotide point mutations in ORF5a resulted in early translation stop codons and shorter ORF5a proteins on some virus isolates (Table 4).The lengths of nucleotides in nsp1, nsp3-nsp12, ORF2a, ORF2b, ORF3, ORF4, ORF5, ORF6, ORF7, and their corresponding encoded proteins are the same among the VR-2332 isolate and the PRRSV-2 lineage 1 isolates evaluated in this study (Table 4).

Growth Characteristics of Virus Isolates in ZMAC Cells
The growth characteristics of five PRRSV-2 lineage 1 isolates evaluated in this study were compared in ZMAC cells, and the growth curve is shown in Figure 13.At 0 hpi, the baseline mean titers of the viruses ranged from 2.4 to 3 log10 (TCID50/mL) but with no significant titer differences.At 6 hpi, the mean virus titers ranged from 2.4 to 3 log10 (TCID50/mL), with no significant differences among the virus isolates.At 12 hpi, the mean virus titers ranged from 5.4 to 6.4 log10 (TCID50/mL); the mean titer of 1-4-4 L1C.5 isolate was significantly higher than the 1-4-4 L1C.1, 1-4-4 L1A and 1-7-4 L1A isolates, but no significant difference was observed between the 1-4-4 L1C.5 and 1-4-4 L1H isolates.At 24 hpi, the mean virus titers ranged from 5.4 to 6.5 log10 (TCID50/mL); the mean titer in the
Virulence is a comparative or quantitative term that defines the severity or intensity of the disease caused by the pathogen in the infected individuals [51,52].In the case of PRRSV, the terms "virulent" and "highly virulent" have been used loosely to describe different PRRSV isolates because there are no unequivocal quantitative criteria to define virulence phenotypes of PRRSV isolates.Ruedas-Torres et al., 2021 [53] have proposed some criteria to define PRRSV virulence, but those have not been widely accepted for use.They believe that correlates of PRRSV virulence could include the following: (1) viral genetic determinants of virulence; (2) clinical signs, temperature, and lesions; (3) wider tissue distribution and higher viral load; (4) higher and expanded tissue tropism; and (5) immunological aspects [53].Generally speaking, "virulent" or "highly virulent" PRRSV isolates may cause increased mortality and morbidity; severe clinical signs, such as elevated fever, severe respiratory scores, anorexia and lethargy, higher viremia and high viral load in different tissues, severe interstitial pneumonia associated with the involvement of secondary bacterial infections; and altered innate and adaptive immune responses [53].Some parameters seem to correlate with the PRRSV virulence phenotype.For example, high fever, anorexia, and lethargy are generally observed in pigs infected with virulent PRRSV strains [20,[53][54][55][56][57][58][59].Viremia levels have been reported to be higher for infections with virulent PRRSV-1 isolates or PRRSV-2 isolates [53,59,60]; however, the PRRSV-1 isolate SU1-bel induced lower and shorter viremia in comparison with the reference Lelystad isolate, although SU1-bel isolate caused a greater clinical score and gross lung lesions [61].However, regarding the immunological parameters, there have been controversial reports between cytokine production and the virulence of PRRSV isolates [53].Nonetheless, how to consistently define the "virulence" criteria for PRRSV isolates remains to be determined.
Another interesting aspect is that this study provides evidence that some PRRSV isolates may replicate better in the central nervous system than other isolates, allowing the virus to induce neurologic clinical signs and brain infection.We found that all of the L1 PRRSV-2 isolates used in this study, except the 1-4-4 L1C.1 isolate, were able to induce brain infection, which was confirmed by lesions of viral encephalitis and PRRSV immunohistochemistry.This is an area that warrants further studies.
In the current study, a higher number of contact pigs in the 1-4-4 L1C.5 group became viremic at 2 days post contact, implying that the L1C.5 isolate may have higher transmissibility than the other PRRSV-2 lineage 1 isolates evaluated in this study.Higher viremia levels and higher viral load in oral fluid samples were observed at the initial DPI in both the virus-inoculated and contact pens of the 1-4-4 L1C.5 group, supporting this conclusion.However, the transmissibility of the 1-4-4 L1C.5 virus needs to be confirmed with a study involving more pigs because our study used only four contact pigs per group.
This study showed that the routinely used IDEXX PRRSV ELISA antibody assay can detect antibodies against the emergent 1-4-4 L1C.5 isolate.For pigs being exported from the USA to some countries, the PRRSV IFA antibody assay is still used.After the emergence of 1-4-4 L1C.5 (L1C variant) in October 2020, we received inquiries whether the routinely conducted PRRSV IFA antibody assay can detect antibodies against the 1-4-4 L1C.5 virus strain.The data in this study demonstrated that IFA antibody assay based on the ISU-P isolate can readily detect antibodies against the 1-4-4 L1C.5 isolate.All five lineage 1 isolates evaluated in this study only induced a low level of homologous neutralizing antibodies during 21-28 DPI.
It is well accepted that the virulence of PRRSV isolates correlates with some genetic determinants of virulence present in the PRRSV genome.However, the genetic determinants of virulence of PRRSV appear to involve multiple genes and have not been fully understood.Whole genome sequence analyses have revealed high genetic variations in the nsp2 region.In a recent study, based on the systematic analysis of insertion and deletion patterns of nsp2 in PRRSV-2 sequences, five large patterns and 25 subdivided groups have been suggested [28].Compared to the VR-2332 isolate that belongs to the nsp2 pattern 1.0 [28], the 1-4-4 L1C.5, 1-7-4 L1A, and 1-4-4 L1A fall close to the nsp2 pattern 3.0 with the continuous deletion of 100 amino acids.The 1-4-4 L1C.1 isolate used in this study falls close to the nsp2 pattern 2.1, and the 1-4-4 L1H isolate falls close to the nsp2 pattern 2.4.1.The deletion patterns in nsp2 observed in the PRRSV isolates used in our study were different from the HP-PRRSV strain associated with the 2006 epidemic of atypical PRRSV outbreak in China, which had 1 amino acid and 29 amino acid deletions in the nsp2 region and belonged to the nsp2 pattern 4.0 [20,28].Moreover, the HP-PRRSV strain associated with the 2006 PRRSV outbreak in China belongs to lineage 8 [22], but the PRRSV isolates evaluated in this experimental study belong to lineage 1 (L1A, L1C.5, L1C.1, and L1H).The nsp2 protein is the most variable non-structural protein among different PRRSV strains [62].Besides the protease activity of the papain-like protease 2 (PLP2) domain at N-terminal [63,64], nsp2 may play a role in PRRSV pathogenesis and modulate host immune responses by its deubiquitinating activities to inhibit ubiquitin-dependent antiviral pathways [65] and regulate the ubiquitin-dependent innate immunity of type I IFN activation [66].However, the exact role of nsp2 in contributing to the virulence differences of various PRRSV isolates remains to be determined.In addition to nsp2, considerable variations were observed in other genomic regions and proteins among the PRRSV-2 isolates evaluated in this study (1-4-4 L1C.5, 1-4-4 L1C.1, 1-4-4 L1A, 1-4-4 L1H, and 1-7-4 L1A); the genetic determinants responsible for virulence differences of these PRRSV-2 isolates are still unclear.PRRSV strains with distinct glycosylation patterns may alter the immunogenicity of the virus and induce different neutralizing antibody production [67].A recent study revealed the different N-glycosylation patterns associated with the emergence of new genetic variants of PRRSV-2 in the USA [68].The PRRSV-2 isolates evaluated in our current study also have different N-glycosylation patterns, but their correlation with virus phenotypic functions remains to be determined.
When five PRRSV-2 lineage 1 isolates evaluated in this study were compared for their growth in ZMAC cells, the L1C.5 isolate replicated to higher titers at 12, 24 and 48 hpi compared to some other isolates.However, more studies are needed to determine whether growth characteristics of PRRSV isolates in ZMAC cells can reliably predict the virulence of PRRSV isolates.

Conclusions
This study provides experimental data in weaned pigs regarding the clinical impact, virulence, transmissibility, and antibody detection of the newly emergent L1C.5 virus (1-4-4 L1C variant), along with comparisons with other PRRSV-2 lineage 1 isolates.The findings confirm that L1C.5 virus is highly virulent in weaned pigs.The higher number of contact pigs becoming viremic at 2 days post contact implies that the L1C.5 virus may have higher transmissibility than other PRRSV isolates, although it needs to be confirmed in a study involving more pigs.Brain infection by PRRSV is also an area needing further investigation.The whole genome sequence comparisons of five PRRSV-2 lineage 1 isolates revealed variations in numerous genomic regions at both the nucleotide and amino acid levels, but the genetic determinants for virulence differences of these virus isolates remain to be elucidated.The L1C.5 isolate replicated to higher titers in ZMAC cells at some time

Figure 2 .
Figure 2. Experimental design of the pig study.On the (left), information of groups and the corresponding virus inoculum is provided.On the (right), a schematic diagram describes the pen in each room and the corresponding inoculation pigs and contact pigs.

Figure 2 .
Figure 2. Experimental design of the pig study.On the (left), information of groups and the corresponding virus inoculum is provided.On the (right), a schematic diagram describes the pen in each room and the corresponding inoculation pigs and contact pigs.
mean score of 2) compared to other groups; the 1-4-4 L1C.1 inoculation group had the lowest scores among all virus-inoculated groups.

Figure 3 .
Figure 3. Clinical observations in inoculated pigs.(A) The overall average anorexia and lethargy scores in different groups from 0 to 21 DPI.For each group, the available pigs were averaged for their anorexia and lethargy scores for each day.Therefore, there are 22 spots for each group corresponding to each day from 0 to 21 DPI.(B) Percentage of pigs showing fever (>40 °C) in each group at each time point.(C) Microchip body-temperature changes of inoculated pigs over time.The mean microchip temperature in degree Celsius is shown on the Y-axis.(D) Mean average daily weight gain (ADG) of inoculated pigs between −1 DPI and 10 DPI, with significance denoted by letters.Pigs that died or were euthanized due to severe body conditions before 10 DPI are shown by * in (D).Labels with different letters indicate significant differences; for example, a and b have a significant difference, but a and ab have no significant difference.

Figure 3 .
Figure 3. Clinical observations in inoculated pigs.(A) The overall average anorexia and lethargy scores in different groups from 0 to 21 DPI.For each group, the available pigs were averaged for their anorexia and lethargy scores for each day.Therefore, there are 22 spots for each group corresponding to each day from 0 to 21 DPI.(B) Percentage of pigs showing fever (>40 • C) in each group at each time point.(C) Microchip body-temperature changes of inoculated pigs over time.The mean microchip temperature in degree Celsius is shown on the Y-axis.(D) Mean average daily weight gain (ADG) of inoculated pigs between −1 DPI and 10 DPI, with significance denoted by letters.Pigs that died or were euthanized due to severe body conditions before 10 DPI are shown by * in (D).Labels with different letters indicate significant differences; for example, a and b have a significant difference, but a and ab have no significant difference.

Figure 4 .
Figure 4. PRRSV RNA load in different specimens of inoculated pigs.(A) PRRSV RNA detected in serum samples of inoculated pigs by quantitative real-time RT-PCR.The average PRRSV RNA levels in the unit of log10(genomic copies/mL) of each group in serum samples at each time point are shown.The number of pigs confirmed positive by PRRSV PCR and statistical analysis are shown in the bottom.Pigs that died or euthanized between 8 and 10 DPI were included for counting at 10 DPI.Similarly, dead pigs between 11 and 14 DPI were counted at 14 DPI, dead pigs between 15 and 21 DPI were counted at 21 DPI, and dead pigs between 21 and 28 DPI were counted at 28 DPI.Labels with different letters indicate significant differences; for example, a and b have a significant difference, but a and ab have no significant difference.(B) PRRSV RNA log10(genomic copies/mL) in penbased oral fluids of inoculated pigs.Oral fluids could not be collected at some time points due to the inactivity of pigs.

Figure 4 .
Figure 4. PRRSV RNA load in different specimens of inoculated pigs.(A) PRRSV RNA detected in serum samples of inoculated pigs by quantitative real-time RT-PCR.The average PRRSV RNA levels in the unit of log 10 (genomic copies/mL) of each group in serum samples at each time point are shown.The number of pigs confirmed positive by PRRSV PCR and statistical analysis are shown in the bottom.Pigs that died or euthanized between 8 and 10 DPI were included for counting at 10 DPI.Similarly, dead pigs between 11 and 14 DPI were counted at 14 DPI, dead pigs between 15 and 21 DPI were counted at 21 DPI, and dead pigs between 21 and 28 DPI were counted at 28 DPI.Labels with different letters indicate significant differences; for example, a and b have a significant difference, but a and ab have no significant difference.(B) PRRSV RNA log 10 (genomic copies/mL) in pen-based oral fluids of inoculated pigs.Oral fluids could not be collected at some time points due to the inactivity of pigs.

Figure 5 .
Figure 5. Gross lesions, microscopic lesions, and PRRSV immunohistochemistry (IHC) scores in lung tissues of inoculated pigs at 10 DPI.Percentage of macroscopic lung lesions due to PRRSV in inoculated pigs (A) and percentage of macroscopic lung lesions due to bacteria in inoculated pigs (B) are shown.(C) Microscopic lung lesion scores (in the range of 0-6) in inoculated pigs at 10 DPI.(D) PRRSV IHC staining scores (in the range of 0-3) in lung tissues of inoculated pigs at 10 DPI.A cluster graph was used to present the data with standard error of the mean.The statistical analysis was conducted between groups, with significance denoted by letters on the individual plot.Labels with different letters indicate significant differences; for example, a and b have a significant difference, but a and ab have no significant difference.

Figure 5 .
Figure 5. Gross lesions, microscopic lesions, and PRRSV immunohistochemistry (IHC) scores in lung tissues of inoculated pigs at 10 DPI.Percentage of macroscopic lung lesions due to PRRSV in inoculated pigs (A) and percentage of macroscopic lung lesions due to bacteria in inoculated pigs (B) are shown.(C) Microscopic lung lesion scores (in the range of 0-6) in inoculated pigs at 10 DPI.(D) PRRSV IHC staining scores (in the range of 0-3) in lung tissues of inoculated pigs at 10 DPI.A cluster graph was used to present the data with standard error of the mean.The statistical analysis was conducted between groups, with significance denoted by letters on the individual plot.Labels with different letters indicate significant differences; for example, a and b have a significant difference, but a and ab have no significant difference.

Figure 6 .
Figure 6.Representative images showing gross lesions, microscopic lesions, and PRRSV immunohistochemistry (IHC) staining in lung tissues at 10 days post inoculation (DPI).The inoculation groups are shown at the top.Gross lung pathology, microscopic lung lesions, and PRRSV IHC staining in lung tissues are exemplified in (A-F), (G-L), and (M-R), respectively.

Figure 6 .
Figure 6.Representative images showing gross lesions, microscopic lesions, and PRRSV immunohistochemistry (IHC) staining in lung tissues at 10 days post inoculation (DPI).The inoculation groups are shown at the top.Gross lung pathology, microscopic lung lesions, and PRRSV IHC staining in lung tissues are exemplified in (A-F), (G-L), and (M-R), respectively.

Figure 7 .
Figure 7. PRRSV RNA load in different specimens of inoculated pigs.(A) PRRSV RNA log10(genomic copies/mL) in lung, tonsil, and brain tissues of inoculated pigs necropsied at 10 DPI or during 9-10 DPI.(B) PRRSV RNA log10(genomic copies/mL) in lung, tonsil, and brain tissues of inoculated pigs necropsied at 28 DPI or during 10-28 DPI.Number of pigs confirmed positive by PRRSV PCR is indicated on top of each histogram.

Figure 7 .
Figure 7. PRRSV RNA load in different specimens of inoculated pigs.(A) PRRSV RNA log 10 (genomic copies/mL) in lung, tonsil, and brain tissues of inoculated pigs necropsied at 10 DPI or during 9-10 DPI.(B) PRRSV RNA log 10 (genomic copies/mL) in lung, tonsil, and brain tissues of inoculated pigs necropsied at 28 DPI or during 10-28 DPI.Number of pigs confirmed positive by PRRSV PCR is indicated on top of each histogram.
gliosis, vasculitis/perivasculitis, and neuronal necrosis were each scored in the range of 0-3, and the average score of the four categories of lesions was calculated.Pigs with PRRSV IHC-positive staining in brain are highlighted in green.Pigs with relatively low PCR CT values in brain but negative IHC staining in brain are highlighted in orange.For those pigs marked with ×, brain samples were not collected.

Figure 8 .
Figure 8. Representative images showing microscopic lesions and PRRSV immunohistochemistry (IHC) staining in brain tissues at 10 days post inoculation (DPI).The inoculation groups are shown at the top.Histopathological changes and PRRSV IHC staining in brain tissues are exemplified in (A-F) and (G-L), respectively.

Figure 8 .
Figure 8. Representative images showing microscopic lesions and PRRSV immunohistochemistry (IHC) staining in brain tissues at 10 days post inoculation (DPI).The inoculation groups are shown at the top.Histopathological changes and PRRSV IHC staining in brain tissues are exemplified in (A-F) and (G-L), respectively.

Figure 9 .
Figure 9. PRRSV antibody responses in serum samples of inoculated pigs over time.(A) PRRSV ELISA antibody detected by HerdChek ® PRRS X3 ELISA (IDEXX).The average PRRSV ELISA antibody serum to positive (S/P) ratio of each group in serum samples at each time point is shown on the Y-axis.(B) PRRSV IFA antibody detected in serum samples of inoculated pigs.The average PRRSV IFA antibody titer of each group in serum samples at each time point is shown on the Y-axis.(C) Homologous PRRSV fluorescent focus neutralization (FFN) antibody titer (log2 format) is shown on the Y-axis.The statistical analysis was conducted between groups at each time point, with significance denoted by letters.Labels with different letters indicate significant differences; for example, a and b have a significant difference, but a and ab have no significant difference.

Figure 9 .
Figure 9. PRRSV antibody responses in serum samples of inoculated pigs over time.(A) PRRSV ELISA antibody detected by HerdChek ® PRRS X3 ELISA (IDEXX).The average PRRSV ELISA antibody serum to positive (S/P) ratio of each group in serum samples at each time point is shown on the Y-axis.(B) PRRSV IFA antibody detected in serum samples of inoculated pigs.The average PRRSV IFA antibody titer of each group in serum samples at each time point is shown on the Yaxis.(C) Homologous PRRSV fluorescent focus neutralization (FFN) antibody titer (log 2 format) is shown on the Y-axis.The statistical analysis was conducted between groups at each time point, with significance denoted by letters.Labels with different letters indicate significant differences; for example, a and b have a significant difference, but a and ab have no significant difference.

Figure 10 .
Figure 10.Microchip body-temperature and PCR data in serum and oral fluid samples collected from contact pigs.(A) The mean microchip body temperature in degree Celsius is shown on the Yaxis for contact pigs over time.(B) PRRSV RNA detected in serum samples of contact pigs by quantitative real-time RT-PCR.The number of pigs confirmed positive by PRRSV PCR and statistical analysis are shown in the bottom.The statistical analysis was conducted between groups at each time point, with significance denoted by letters.Labels with different letters indicate significant differences; for example, a and b have a significant difference, but a and ab have no significant difference.(C) PRRSV RNA log10(genomic copies/mL) in pen-based oral fluids of contact pigs.Oral fluids could not be collected at some time points from some groups due to the inactivity of pigs.

Figure 10 .
Figure 10.Microchip body-temperature and PCR data in serum and oral fluid samples collected from contact pigs.(A) The mean microchip body temperature in degree Celsius is shown on the Y-axis for contact pigs over time.(B) PRRSV RNA detected in serum samples of contact pigs by quantitative real-time RT-PCR.The number of pigs confirmed positive by PRRSV PCR and statistical analysis are shown in the bottom.The statistical analysis was conducted between groups at each time point, with significance denoted by letters.Labels with different letters indicate significant differences; for example, a and b have a significant difference, but a and ab have no significant difference.(C) PRRSV RNA log 10 (genomic copies/mL) in pen-based oral fluids of contact pigs.Oral fluids could not be collected at some time points from some groups due to the inactivity of pigs.

Figure 11 .
Figure 11.PRRSV antibody responses in contact pigs.(A) PRRSV ELISA antibody detected in serum samples of contact pigs.(B) PRRSV IFA antibody detected in serum samples of contact pigs.The statistical analysis was conducted between groups at each time point, with significance denoted by letters.Labels with different letters indicate significant differences; for example, a and b have a significant difference, but a and ab have no significant difference.

Figure 11 .
Figure 11.PRRSV antibody responses in contact pigs.(A) PRRSV ELISA antibody detected in serum samples of contact pigs.(B) PRRSV IFA antibody detected in serum samples of contact pigs.The statistical analysis was conducted between groups at each time point, with significance denoted by letters.Labels with different letters indicate significant differences; for example, a and b have a significant difference, but a and ab have no significant difference.

Figure 12 .
Figure 12.Partial nsp2 protein sequences of five PRRSV-2 isolates included in this study in comparison with the PRRSV-2 prototype isolate VR-2332.The positions evident in the figure represent positions of the nsp2 amino acid sequence in reference to that of VR-2332.The deletion of the amino acids in each PRRSV isolate is highlighted in blue.

Figure 12 .
Figure 12.Partial nsp2 protein sequences of five PRRSV-2 isolates included in this study in comparison with the PRRSV-2 prototype isolate VR-2332.The positions evident in the figure represent positions of the nsp2 amino acid sequence in reference to that of VR-2332.The deletion of the amino acids in each PRRSV isolate is highlighted in blue.

Figure 13 .
Figure 13.Multistep growth curve analysis of five PRRSV-2 isolates in ZMAC cells.(Top) The virus titers Log10(TCID50/mL) are shown on the Y-axis with the standard error of the mean presented.(Bottom)The statistical analysis was conducted on the mean Log10(TCID50/mL) between groups at each hour post infection (hpi).Labels with different letters indicate significant differences; for example, a and b have a significant difference, but a and ab have no significant difference.

Figure 13 .
Figure 13.Multistep growth curve analysis of five PRRSV-2 isolates in ZMAC cells.(Top) The virus titers Log 10 (TCID 50 /mL) are shown on the Y-axis with the standard error of the mean presented.(Bottom) The statistical analysis was conducted on the mean Log 10 (TCID 50 /mL) between groups at each hour post infection (hpi).Labels with different letters indicate significant differences; for example, a and b have a significant difference, but a and ab have no significant difference.

Table 1 .
Information of PRRSV isolates used in this study.

Table 2 .
Proportion of mortality observed in pigs in different virus inoculated groups.

Table 2 .
Proportion of mortality observed in pigs in different virus inoculated groups.

Table 3 .
Summary of pig brain infection after experimental inoculation with different PRRSV isolates.

Table 3 .
Summary of pig brain infection after experimental inoculation with different PRRSV isolates.
* Meningitis, gliosis, vasculitis/perivasculitis, and neuronal necrosis were each scored in the range of 0-3, and the average score of the four categories of lesions was calculated.Pigs with PRRSV IHC-positive staining in brain are highlighted in green.Pigs with relatively low PCR C T values in brain but negative IHC staining in brain are highlighted in orange.For those pigs marked with ×, brain samples were not collected.

Table 4 .
Summary of genomic sequence comparisons of the five PRRSV isolates evaluated in this study together with VR-2332 isolate.