Emergence and Pathogenicity of a Novel PRRSV-1 Strain GD18-2 in Southern China

TL;DR

A new strain of PRRSV-1 called GD18-2 is causing serious illness in pigs in China, even in vaccinated herds, and can be passed from mother pigs to their unborn offspring.

Contribution

The study identifies and characterizes GD18-2, a novel PRRSV-1 strain with unique genetic features and high pathogenicity in southern China.

Findings

GD18-2 has a unique genome with 81.4% to 83.9% nucleotide identity to classical PRRSV-1 strains.

The strain causes severe respiratory disease and mortality in piglets and reproductive failure in pregnant sows.

GD18-2 exhibits a deletion in Nsp2 and mutations in GP3 and GP4, distinguishing it from other PRRSV-1 strains.

Abstract

A pig disease caused by the Porcine Reproductive and Respiratory Syndrome Virus type 1 is becoming more common in China, making it harder to control outbreaks on farms. In this study, we investigated a new strain of this virus, GD18-2, found on a pig farm in Guangdong that had vaccinated its animals against a related virus type. Our goal was to understand how this new strain is different and how harmful it is to pigs. We found that GD18-2 has a unique genetic makeup that sets it apart from previously known strains. When tested in young pigs, it caused severe breathing problems, fever, and some deaths. In pregnant mother pigs, it did not make the mothers very sick, but it spread to the unborn piglets, causing many to be born dead or very weak. These results show that this new virus strain poses a serious threat to pig health and farm productivity, especially because it can spread from mother to offspring before birth. Our work highlights the need for closer monitoring and updated prevention strategies to protect pigs from this emerging virus.

The detection rate of Porcine Reproductive and Respiratory Syndrome Virus type 1 (PRRSV-1) in China has been increasing, with its growing genetic diversity and evolving pathogenicity posing significant challenges to disease control. In this study, a novel PRRSV-1 strain, designated GD18-2, was identified from a pig farm in Guangdong Province that experienced an outbreak despite vaccination with a PRRSV-2 vaccine. Whole-genome sequencing indicated that the GD18-2 strain possesses a genome length of 14,932 bp and exhibits 81.4% to 83.9% nucleotide identity with classical PRRSV-1 strains. Phylogenetic analyses based on both the complete genome and the ORF5 gene indicated that GD18-2 belongs to a distinct, new lineage. A unique amino acid deletion (positions 306–357) was identified in the non-structural protein Nsp2, along with specific mutations within the hypervariable regions of the structural proteins GP3 and GP4. Pathogenicity assessment demonstrated that GD18-2 induced fever, respiratory symptoms, and mortality in piglets. In pregnant sows, it caused reproductive failure (abortion, stillbirth, weak piglets) and was capable of vertical transmission via the placenta. This study highlights the emergence of a PRRSV-1 strain with a unique genetic background and high pathogenicity in southern China, underscoring the necessity for enhanced molecular epidemiological surveillance and updated control strategies. Recombination analysis using RDP4 revealed no significant recombination events in GD18-2.

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1. Introduction

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). The disease is primarily characterized by respiratory symptoms and increased mortality in piglets, as well as reproductive failure in sows (e.g., abortion, stillbirth, mummified fetuses), causing severe economic losses to the global swine industry [1].

PRRSV is an enveloped, positive-sense single-stranded RNA virus with an approximately 15 kb genome that encodes at least eleven open reading frames (ORFs). ORF1a and ORF1b encode polyprotein precursors for non-structural proteins, while ORF2–ORF7 encode the viral structural proteins [2,3]. Based on genomic divergence, PRRSV is classified into two major types: Type 1 (European) and Type 2 (North American), which share only about 55–70% nucleotide homology [4]. Since the initial isolation and identification of the Lelystad virus in the Netherlands in 1991 [5], PRRSV-1 has been reported in numerous countries across Europe, the Americas, and Asia [6,7].

The epidemiology of PRRSV-1 in China has become increasingly complex. Following its first detection in imported pigs in 1997, the virus has undergone accelerated local evolution. The isolation of strains such as BJEU06-1 and NMEU09-1 in 2011 marked the establishment of stable circulation of PRRSV-1 within Chinese swine populations [8,9]. Currently, the predominant PRRSV-1 subtypes circulating in China include NMEU09-1-like, Amervac-like, HKEU16-like, and BJEU06-1-like, with continuous reports of new genetic variants emerging [10,11,12].

Despite this genetic diversity, PRRSV-1 has traditionally been considered less pathogenic than PRRSV-2. However, highly pathogenic PRRSV-1 strains have emerged globally in recent years. For instance, the PR40/2014 strain isolated in Italy can cause high fever, dyspnea, and high mortality in infected pigs [13]. In China, pathogenic PRRSV-1 strains have also been reported, such as GZ11-G1, which induces fever in piglets [14], and the 181187-2 and ZD-1 strains reported in 2023, which exhibit moderate pathogenicity in piglets [15,16]. The increasing prevalence of PRRSV-1, coupled with its ongoing genetic evolution and shifts in pathogenicity, poses significant challenges to the swine industry [17,18,19,20].

Although cross-protection between PRRSV-1 and PRRSV-2 is limited due to their genetic divergence, the emergence of GD18-2 in a farm routinely vaccinated with a PRRSV-2 vaccine highlights the ongoing challenge of PRRSV-1 incursion and adaptation in China’s swine population, underscoring the need for updated vaccine strategies [21].

This study successfully isolated a PRRSV-1 strain, named GD18-2, from a clinical outbreak. Through whole-genome sequencing, phylogenetic analysis, and animal challenge experiments in both piglets and pregnant sows, we systematically investigated the molecular characteristics and pathogenicity of this strain. The aim is to provide data to support molecular epidemiological surveillance of PRRSV-1 and inform the development of control strategies.

2. Materials and Methods

2.1. Clinical Sample Collection and Virus Isolation

Specific-pathogen-free (SPF) piglets/sows were obtained from a PRRSV-negative herd. Animals were housed in individual isolation units with controlled temperature and humidity, and provided with feed and water ad libitum. Samples were collected from a Wens pig farm that practiced vaccination with the PRRSV-2 VR2332 modified live virus vaccine but experienced a disease outbreak. Twenty-eight-day-old piglets presented with sudden death, clinical signs of high fever (40.5–41.5 °C), dyspnea, and skin cyanosis. Lung tissue, serum, and whole blood samples were collected from affected piglets and stored at −80 °C. All samples were collected within 2 h post-mortem or from live animals using sterile procedures.

Following homogenization, lung tissues underwent multiple freeze–thaw cycles and were subsequently centrifuged to collect the supernatant for further analysis. Supernatants from serum and whole blood samples were obtained by direct centrifugation. Total RNA was extracted using the RNeasy Kit (Qiagen, Hilden, Germany), and PRRSV was identified via reverse transcription polymerase chain reaction (RT-PCR). Supernatants from RT-PCR-positive samples were filtered through a 0.22-μm membrane and inoculated onto primary porcine alveolar macrophages (PAMs) for virus propagation. Cells were cultured at 37 °C with 5% CO_2_. Cytopathic effects (CPE) were observed daily. The virus was purified through three consecutive passages, yielding the purified strain GD18-2.

2.2. Genomic Amplification and Sequencing

Viral RNA was extracted from the supernatant of infected PAM cultures. Twelve pairs of specific (primer sequences are listed in Table 1) covering the entire viral genome were designed based on PRRSV-1 reference sequences from GenBank. The sequences and characteristics of all twelve primer pairs are provided in Table 1. PCR was performed under the following conditions: 95 °C for 5 min; 35 cycles of 95 °C for 30 s, 55–60 °C (primer-specific) for 30 s, 72 °C for 1–3 min (amplicon-dependent); and a final extension at 72 °C for 10 min. Reverse transcription was performed using a PrimeScript RT-PCR Kit (TaKaRa Bio Inc., Dalian, China). The resulting cDNA served as the template for PCR amplification using PrimeSTAR GXL DNA Polymerase (TaKaRa Bio Inc., Dalian, China). The PCR amplicons were ligated into the pMD19-T vector and transformed into Escherichia coli DH5α competent cells. Multiple positive clones were selected and sent to Shengong Bioengineering (Shanghai) Co., Ltd. (Shanghai, China) for sequencing. The complete genomic sequence of GD18-2 was assembled using Lasergene SeqMan software (version 17, DNASTAR, Madison, WI, USA).

2.3. Indirect Immunofluorescence Assay (IFA)

The GD18-2 strain was inoculated onto PAMs and MARC-145 cells, with uninfected controls established in parallel. After 48 h of incubation, cells were fixed and permeabilized for subsequent staining. Cells were incubated overnight at 4 °C with an anti-PRRSV nucleocapsid (N) protein monoclonal antibody as the primary antibody, followed by incubation with Cy3-labeled goat anti-mouse IgG secondary antibody (Abcam, Cambridge, UK). Staining was observed, and images were captured using an inverted fluorescence microscope (DMi8, Leica, Wetzlar, Germany). GD18-2 was not adapted to MARC-145 cells; IFA was performed to assess cross-reactivity.

2.4. Sequence Analysis

Complete genome sequences of PRRSV-1 reference strains were downloaded from GenBank. Nucleotide homology between GD18-2 and reference strains was assessed using MegAlign software (DNASTAR, version 11.1.0.54, Madison, WI, USA). Phylogenetic trees based on the complete genome and the ORF5 gene were constructed using the neighbor-joining (NJ) and maximum likelihood (ML) methods in MEGA 7.0. For ML analysis, the best-fit nucleotide substitution model was determined using the Model Selection tool within MEGA. Branch support was assessed with 1000 bootstrap replicates for both NJ and ML methods. Amino acid sequences of Nsp2, GP3, and GP4 from GD18-2 were aligned with those from reference strains using ClustalX software (version 17) to analyze deletion and mutation sites.

Recombination Analysis

Recombination events in the complete genome of GD18-2 were detected using RDP4 software (version 4.1) against a broad set of PRRSV-1 reference sequences. Statistical significance was determined with a threshold of p < 0.05.

2.5. Piglet Pathogenicity Trial

The challenge dose was back-titrated to confirm the actual titer administered. Rectal temperature was measured daily at 8:00 AM. Thirty-five-day-old piglets confirmed negative for PRRSV were randomly allocated to two groups, with five piglets per group. Piglets in the challenge group received an intramuscular inoculation of 2 mL of GD18-2 viral stock (10^5^ TCID_50_/mL), while the control group received an equal volume of Dulbecco’s modified Eagle’s medium. Rectal temperature and clinical signs (scored 0–4 based on mental status, respiratory symptoms, etc.)Rectal temperature and clinical signs were recorded daily. A comprehensive clinical scoring system (0–4 per category, maximum total score of 32 per piglet) was used to quantify disease severity based on eight parameters: feed intake, mental status, skin condition, respiratory effort, coughing, nasal discharge, neurological signs, and diarrhea. Each parameter was graded as follows: 0 = normal; 1 = mild; 2 = moderate; 3 = severe; 4 = extremely severe. The total clinical score for each piglet was the sum of the scores across all eight categories, recorded daily. Serum samples were collected weekly to determine viral load and PRRSV-N antibody levels. The trial lasted 21 days, and mortality was recorded. Surviving piglets were euthanized at the end of the experiment, and lung tissues were collected for pathological examination.

2.6. Pregnant Sow Pathogenicity Trial

The challenge dose was back-titrated to confirm the actual titer administered. Pregnant sows at 90 days of gestation, tested PRRSV-negative, were randomly assigned to two groups, each comprising four sows. The challenge group was inoculated intramuscularly with 4 mL of GD18-2 virus culture (10^5^ TCID_50_), and the control group received an equal volume of DMEM. Rectal temperature was monitored daily. Blood, oral swabs, and anal swabs were collected periodically for viral load detection. Serum antibody levels were tested weekly. Weak piglets were defined as those with birth weight < 0.8 kg, inability to stand or suckle within 1 h, or signs of lethargy/dyspnea. All weak piglets in the control group were tested for PRRSV nucleic acid to rule out cross-contamination. Farrowing outcomes were recorded, including the total number of piglets born, live births, stillbirths, and weak piglets. PRRSV nucleic acid was detected in the umbilical cord blood of newborn piglets.

2.7. Histopathological Examination

Lung tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Pathological changes were examined under a light microscope (Cyvital Biological Technology Co., Ltd, Guangzhou, China).

2.8. Statistical Analysis

Data were analyzed using SPSS version 22.0 (IBM, Corp., Armonk, NY, USA). For continuous variables with normal distribution and homogeneity of variance, one-way ANOVA followed by Tukey’s HSD test was applied. For variables where the control group showed zero variance (e.g., clinical scores, viral loads), the non-parametric Mann–Whitney U test was used to compare groups. A p-value < 0.05 was considered statistically significant.

3. Results

3.1. Virus Isolation and Identification

Filtered homogenates from PRRSV-positive lung tissue were inoculated onto PAMs, and distinct cytopathic effects were subsequently observed under microscopy 48 h later, which included cell rounding, clustering, blurred cell margins, and eventual cell detachment and death. Immunofluorescence assay (IFA) results indicated the absence of fluorescent signals in the control group, while specific red fluorescence was detected in the cytoplasm of GD18-2-infected PAMs, demonstrating effective viral replication and N protein expression by the GD18-2 strain (Figure 1a). Purified virus particles were visualized by transmission electron microscopy, revealing an approximate diameter of 55 nm and an enveloped morphology characteristic of the Arteriviridae family (Figure 1b). IFA on MARC-145 cells showed weak fluorescence, consistent with the typical poor adaptation of PRRSV-1 to this cell line.

3.2. Genetic and Phylogenetic Analysis

The genomic sequencing results for the isolated strain were assembled using DNASTAR software (version 17), and phylogenetic trees were constructed based on both the GP5 gene and the complete genome. Both NJ and ML phylogenetic analyses (based on the complete genome and ORF5 gene) consistently positioned GD18-2 within a novel, distinct clade, separate from all known PRRSV-1 subtypes (1–4) circulating in China, Southeast Asia, and Europe, confirming it belongs to a new lineage (Figure 2). Recombination analysis using RDP4 revealed no evidence of recombination events in the GD18-2 genome.

3.3. Amino Acid Sequence Analysis of Nsp2, GP3, and GP4

Sequence alignment using MAFFT v7.0 identified a 52-amino-acid deletion (positions 306–357) in the Nsp2 protein of GD18-2. BLASTp (conducted via the NCBI web portal, https://blast.ncbi.nlm.nih.gov/Blast.cgi, accsessed on 1 February 2026) search against the NCBI non-redundant protein database confirmed this deletion pattern has not been reported in any previously characterized PRRSV strains (Figure 3a). In GP3, a single-residue deletion at position 243 was observed within its hypervariable region, together with amino acid changes at positions 233 and 236 (Figure 3b). Similarly, analysis of GP4 revealed a two-residue deletion (positions 62–63) and a substitution at position 49 in its hypervariable region (Figure 3c). These mutational profiles distinguish GD18-2 from previously characterized classical strains.

3.4. Results of the Piglet Pathogenicity Trial

During the trial, the mean rectal temperature of piglets in the control group remained below 40 °C. Meanwhile, piglets in the GD18-2 group exhibited sustained fever following challenge, characterized by a significant temperature rise from day 2 post-inoculation that persisted until the end of the experiment, peaking at 41.2 ± 0.3 °C on day 7 (Figure 4a). Clinical score in the challenged group tended to increase and peak on day 8, and tended to decrease until the end of the experiment, whereas no clinical disease score was detected in the control group. Clinical symptom scores (calculated as the sum of eight individual parameters, see Materials and Methods) were significantly higher in the GD18-2 group than in the control group (Figure 4b). Following the challenge, piglets inoculated with GD18-2 exhibited hallmark signs of PRRS, such as lethargy, dyspnea characterized by abdominal breathing, and cyanosis of the extremities. Viral load monitoring demonstrated that the GD18-2 strain induced a high level of viremia in piglets and was consistently shed via oral and rectal routes (Figure 4c–e). Serological testing revealed that all challenged piglets seroconverted by day 10 post-inoculation, with the specific antibody titer against the PRRSV-N protein showing a sustained upward trend (Figure 4f). The final survival rate was 4/5 (80%) in the GD18-2 group, compared with 5/5 (100%) in the control group (Figure 4g). Necropsy findings indicated localized hemorrhagic foci and areas of consolidation in the lung tissues of the GD18-2 group. Lung histopathology from the challenged group identified interstitial pneumonia lesions, notably alveolar septal thickening accompanied by mononuclear cell infiltration and prominent hyperplasia of alveolar macrophages (Figure 5).

3.5. Results of the Pregnant Sow Pathogenicity Trial

During the trial period, the challenged pregnant sows in the inoculated group did not exhibit acute clinical signs and maintained stable rectal temperature (Figure 6a). Serological monitoring results indicated that PRRSV-N antibodies were detectable in sows of the GD18-2 group from day 7 post-inoculation onward, with antibody levels showing a continuous upward trend (Figure 6b). Viral load results suggested that GD18-2 was able to establish infection in pregnant sows, accompanied by sustained viral shedding via the respiratory and digestive tracts (Figure 6c). Although no mortality occurred in either sow group, farrowing performance data indicated that the piglet survival rate was significantly reduced to 50.7% (36/71) in the GD18-2 group, compared to 100% (64/64) in controls. This was associated with a significant increase in the average numbers of stillbirths (6.5 vs. 0 per sow), weak piglets (1.5 vs. 1.3), and mummified fetuses (2.0 vs. 0) (Table 2; Figure 6d). Moreover, PRRSV nucleic acid was detected in 100% of umbilical cord blood samples from newborn piglets in the GD18-2 group, confirming efficient transplacental vertical transmission of the virus (Figure 6e). All weak piglets in the control group tested negative for PRRSV nucleic acid, confirming that there was no cross-contamination in the facility.

4. Discussion

In this study, we isolated and characterized a novel PRRSV-1 strain, GD18-2, from a farm despite routine vaccination with a PRRSV-2 vaccine in southern China. Key findings include its phylogenetic divergence into a new lineage [10,19], possession of a unique large deletion in Nsp2, and a pathogenic profile marked by significant virulence in piglets and efficient transplacental transmission in sows [13,16,17]. These characteristics distinguish GD18-2 from previously circulating PRRSV-1 strains in China and warrant closer attention.

Notably, the outbreak occurred on a farm routinely vaccinated with a PRRSV-2 MLV vaccine. This field observation suggests a lack of effective cross-protection against the emerging PRRSV-1 strain, highlighting a potential gap in control strategies that rely solely on PRRSV-2 vaccination in regions with increasing PRRSV-1 circulation. This underscores the need for type-comprehensive surveillance.

The continuous spread and evolution of PRRSV-1 in China’s swine industry have become critical variables affecting disease control efficacy. This study successfully isolated and systematically characterized a novel PRRSV-1 strain, GD18-2, from a clinical outbreak occurring despite routine vaccination with a PRRSV-2 vaccine. Its most significant finding is its phylogenetic position, forming a distinct branch independent of all known indigenous prevalent subtypes (e.g., NMEU09-1-like, Amervac-like). This genetic distinctness suggests that GD18-2 did not evolve directly from the major circulating lineages in China. Its origin remains unclear but could involve several scenarios: (i) localized evolutionary events that escaped surveillance, (ii) introduction via unique cross-regional transmission chains, (iii) recombination events potentially driven by vaccine-induced immune pressure [11,22]. This highlights the dynamic and complex nature of the PRRSV-1 gene pool in China. This further underscores the complexity and dynamism of the PRRSV-1 gene pool in China, highlighting the urgency for proactive surveillance of emerging variants.

At the molecular level, GD18-2 harbors a large deletion of 52 amino acids (positions 306–357) within its Nsp2 protein, which contrasts with the smaller, more common deletions observed in other indigenous strains. This could potentially impact the assembly of the viral replication complex or its ability to modulate host innate immune signaling, ultimately contributing to its distinct pathogenic phenotype. The above suggests a potential structural or functional perturbation that may be a key determinant of its distinct pathogenic profile, including enhanced vertical transmission. It should be noted, however, that this association does not imply causality, and the underlying mechanism remains speculative. Concurrently, the specific point mutations and short deletions within the hypervariable regions of GP3 and GP4 may interfere with glycoprotein conformation or interactions with host receptors, thereby potentially aiding the virus in evading pre-existing neutralizing antibodies [23,24]. Collectively, these variations constitute a molecular fingerprint distinguishing GD18-2 from other strains.

The animal experiments in this study revealed the non-negligible pathogenic characteristics of GD18-2. GD18-2 induced severe clinical disease and mortality in piglets in our experimental model. While its virulence appears notable, direct comparisons with other PRRSV-1 strains (e.g., 181187-2, ZD-1) are limited by the absence of side-by-side challenge controls in this study [16]. Notably, GD18-2 exhibited a pronounced capacity to cause severe reproductive failure through efficient vertical transmission. This pathogenic feature is worthy of attention, though cross-study comparisons with strains like SL-01 should be made cautiously due to variations in experimental conditions [13,15,17]. This shift in pathogenic focus highlights an emerging and concerning trend among PRRSV-1 variants. This suggests a spectrum of pathogenicity among emerging PRRSV-1 variants in China. Of greater concern is its highly efficient vertical transmission capacity demonstrated in the pregnant sow model. Although challenged, sows themselves did not exhibit severe clinical signs; the virus efficiently crossed the placental barrier, leading to fetal infection. This resulted in a dramatic reduction in piglet survival rate by approximately 50%, along with numerous stillbirths and weak piglets. The capacity for efficient vertical transmission and the induction of reproductive failure distinguish GD18-2 from strains whose primary impact is on respiratory disease in nurseries. This poses a significant and stealthier challenge to breeding herd productivity, as infections may not be accompanied by overt clinical signs in sows. This “subclinical infection-vertical transmission-reproductive failure” pattern presents greater challenges for the early detection and precise elimination of this strain within farms. Therefore, we recommend enhancing surveillance for similar variants by incorporating molecular assays targeting their distinct genetic markers and by systematically testing cases of reproductive failure.

In summary, GD18-2 is a novel PRRSV-1 variant with a novel genetic background, relatively high pathogenicity in piglets, reproductive failure in pregnant sows, and prominent vertical transmission ability. The identification of GD18-2 expands the known genetic diversity of PRRSV-1 in China [10,11,19] and raises the possibility that viral evolution may be selecting for variants with increased tropism for and pathogenicity in the reproductive tract, a shift with serious implications for swine health management. Consequently, reliance on traditional surveillance systems focused on respiratory symptoms may be insufficient to guard against such strains. Future research priorities should include: first, developing molecular diagnostic tools capable of specifically identifying strains with this unique Nsp2 deletion pattern; second, systematically evaluating under laboratory conditions whether existing commercial vaccines (especially PRRSV-1 vaccines) can provide sufficient cross-protection against GD18-2, particularly regarding the blockage of vertical transmission [21,25]; and third, conducting in-depth studies on the specific molecular mechanisms by which the large Nsp2 deletion and the GP3/GP4 mutations regulate placental tropism and pathogenicity. Collectively, our findings provide direct data to inform the design of molecular surveillance programs and the updating of immunization strategies against emerging PRRSV-1 lineages in China. These investigations will provide a crucial scientific basis for formulating targeted prevention and control strategies against such emerging highly pathogenic, vertically transmissible PRRSV-1 strains.

5. Conclusions

We report here the isolation and comprehensive characterization of a novel PRRSV-1 strain, designated GD18-2. Its genomic characteristics are significantly distinct from known strains, constituting a new genetic group. GD18-2 possesses unique amino acid deletions and mutations in the Nsp2, GP3, and GP4 proteins. Animal experiments confirmed its high pathogenicity in piglets, causing fever, respiratory symptoms, and mortality. In pregnant sows, it primarily induced reproductive failure and was capable of vertical transmission via the placenta. The discovery of this strain holds significant guiding importance for the epidemiological surveillance and comprehensive control of PRRSV-1 in China.