Detection Of Porcine Reproductive And Respiratory Syndrome Virus Research Articles

Simultaneous Detection of Porcine Respiratory Coronavirus, Porcine Reproductive and Respiratory Syndrome Virus, Swine Influenza Virus, and Pseudorabies Virus via Quadruplex One-Step RT-qPCR.

Porcine respiratory coronavirus (PRCoV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), and pseudorabies virus (PRV) are significant viruses causing respiratory diseases in pigs. Sick pigs exhibit similar clinical symptoms such as fever, cough, runny nose, and dyspnea, making it very difficult to accurately differentially diagnose these diseases on site. In this study, a quadruplex one-step reverse-transcription real-time quantitative PCR (RT-qPCR) for the detection of PRCoV, PRRSV, SIV, and PRV was established. The assay showed strong specificity, high sensitivity, and good repeatability. It could detect only PRCoV, PRRSV, SIV, and PRV, without cross-reactions with TGEV, PEDV, PRoV, ASFV, FMDV, PCV2, PDCoV, and CSFV. The limits of detection (LODs) for PRCoV, PRRSV, SIV, and PRV were 129.594, 133.205, 139.791, and 136.600 copies/reaction, respectively. The intra-assay and inter-assay coefficients of variation (CVs) ranged from 0.29% to 1.89%. The established quadruplex RT-qPCR was used to test 4909 clinical specimens, which were collected in Guangxi Province, China, from July 2022 to September 2023. PRCoV, PRRSV, SIV, and PRV showed positivity rates of 1.36%, 10.17%, 4.87%, and 0.84%, respectively. In addition, the previously reported RT-qPCR was also used to test these specimens, and the agreement between these methods was higher than 99.43%. The established quadruplex RT-qPCR can accurately detect these four porcine respiratory viruses simultaneously, providing an accurate and reliable detection technique for clinical diagnosis.

A smartphone-based portable biosensor for colorimetric detection of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) causes serious impacts on livestock farms worldwide, especially in the big pig-raising country of China. Development of fast and accurate PRRSV assay is of great significance in early diagnosis of PRRSV infection, which helps control large-scale disease transmission. Herein, we develop a portable and universal colorimetric biosensor for rapid detection of PRRSV with a smartphone-based platform. In the presence of target sequence, a sandwich structure is formed to anchor polydopamine-silver nanoparticles with pH indicators on cotton swabs. The amplified visual information can be captured and analyzed for accurate quantification within the range from 10 pM to 1 μM. Excellent analytical results are also validated challenging real tissue samples from PRRSV infected pigs. Meanwhile, the biosensor can be extended for nervous necrosis virus (NNV) detection from Plectropomus leopardus, demonstrating its great universal utility.

A cross-sectional assessment of PRRSV nucleic acid detection by RT-qPCR in serum, ear-vein blood swabs, nasal swabs, and oral swabs from weaning-age pigs under field conditions.

The porcine reproductive and respiratory syndrome virus (PRRSV) continues to challenge swine production in the US and most parts of the world. Effective PRRSV surveillance in swine herds can be challenging, especially because the virus can persist and sustain a very low prevalence. Although weaning-age pigs are a strategic subpopulation in the surveillance of PRRSV in breeding herds, very few sample types have been validated and characterized for surveillance of this subpopulation. The objectives of this study, therefore, were to compare PRRSV RNA detection rates in serum, oral swabs (OS), nasal swabs (NS), ear-vein blood swabs (ES), and family oral fluids (FOF) obtained from weaning-age pigs and to assess the effect of litter-level pooling on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection of PRRSV RNA. Three eligible PRRSV-positive herds in the Midwestern USA were selected for this study. 666 pigs across 55 litters were sampled for serum, NS, ES, OS, and FOF. RT-qPCR tests were done on these samples individually and on the litter-level pools of the swabs. Litter-level pools of each swab sample type were made by combining equal volumes of each swab taken from the pigs within a litter. Ninety-six piglets distributed across 22 litters were positive by PRRSV RT-qPCR on serum, 80 piglets distributed across 15 litters were positive on ES, 80 piglets distributed across 17 litters were positive on OS, and 72 piglets distributed across 14 litters were positive on NS. Cohen's kappa analyses showed near-perfect agreement between all paired ES, OS, NS, and serum comparisons (). The serum RT-qPCR cycle threshold values (Ct) strongly predicted PRRSV detection in swab samples. There was a ≥ 95% probability of PRRSV detection in ES-, OS-, and NS pools when the proportion of positive swab samples was ≥ 23%, ≥ 27%, and ≥ 26%, respectively. ES, NS, and OS can be used as surveillance samples for detecting PRRSV RNA by RT-qPCR in weaning-age pigs. The minimum number of piglets to be sampled by serum, ES, OS, and NS to be 95% confident of detecting ≥ 1 infected piglet when PRRSV prevalence is ≥ 10% is 30, 36, 36, and 40, respectively.

Critical evaluation of strategies to achieve direct real-time PCR detection of swine pathogens in oral fluids.

Based on publications reporting improvements in real-time PCR (rtPCR) performance, we compared protocols based on heat treatment or dilution followed by direct rtPCR to standard extraction and amplification methods for the detection of porcine reproductive and respiratory syndrome virus (PRRSV), influenza A virus (IAV), porcine epidemic diarrhea virus (PEDV), or Mycoplasma hyopneumoniae (MHP) in swine oral fluids (OFs). In part A, we subjected aliquots of positive OF samples to 1 of 4 protocols: protocol 1: heat (95°C × 30 min) followed by direct rtPCR; protocol 2: heat and cool (25°C × 20 min) followed by direct rtPCR; protocol 3: heat, cool, extraction, and rtPCR; protocol 4 (control): extraction and then rtPCR. In part B, positive OF samples were split into 3, diluted (D1 = 1:2 with Tris-borate-EDTA (TBE); D2 = 1:2 with negative OF; D3 = not diluted), and then tested by rtPCR using the best-performing protocol from part A (protocol 4). In part A, with occasional exceptions, heat treatment resulted in marked reduction in the detection of target and internal sample control (ISC) nucleic acids. In part B, sample dilution with TBE or OF produced no improvement in the detection of targets and ISCs. Thus, standard extraction and amplification methods provided superior detection of PRRSV, IAV, PEDV, and MHP nucleic acids in OFs.

Effect of extrinsic factors on the detection of PRRSV and a porcine-specific internal sample control in serum, oral fluid, and fecal specimens tested by RT-rtPCR.

We characterized the effect of 1) temperature × time, 2) freeze-thaw cycles, and 3) high porcine reproductive and respiratory syndrome virus (PRRSV) RNA concentrations on the detection of PRRSV and a porcine-specific internal sample control (ISC) in serum, oral fluid, and fecal samples using a commercial PRRSV RT-rtPCR assay (Idexx). In study 1, the effect of temperature × time on PRRSV and ISC detection was shown to be specimen dependent. In serum stored at 4, 10, or 20°C, PRRSV detection was consistent for up to 168 h, but storage at 30°C reduced detectable PRRSV RNA. ISC RNA was stable in serum held at 4 and 10°C, but not at 20 and 30°C. In contrast, PRRSV and ISC RNAs in oral fluid and fecal samples continuously decreased at all temperature × time treatments. Based on these data, serum samples should be stored at ≤ 20°C to optimize PRRSV RNA detection. Oral fluid and fecal samples should be frozen in a non-self-defrosting freezer until tested. In study 2, freeze-thaw cycles had little impact on PRRSV and ISC detection, but more so in oral fluids than serum or fecal samples. Thus, freeze-thaw cycles in oral fluids should be minimized before RT-rtPCR testing. In study 3, the ISC was not affected by high concentrations of PRRSV RNA in serum, oral fluid, or fecal samples. It should not be assumed that data from our PRRSV study are applicable to other pathogens; additional pathogen-specific studies are required.

Research Progress on the detection methods of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes clinical syndromes typified as reproductive disorders in sows and respiratory diseases in piglets. PRRSV remains one of the most prevalent pathogens affecting the pig industry, because of its complex infection profile and highly heterogeneous genetic and recombination characteristics. Therefore, a rapid and effective PRRSV detection method is important for the prevention and control of PRRS. With extensive in-depth research on PRRSV detection methods, many detection methods have been improved and promoted. Laboratory methods include techniques based on virus isolation (VI), enzyme-linked immunosorbent assays (ELISA), indirect immunofluorescence assays (IFA), immunoperoxidase monolayer assays (IPMA), polymerase chain reaction (PCR), quantitative real-time PCR (qPCR), digital PCR (dPCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), clustered regularly interspaced short palindromic repeats (CRISPR), metagenomic next-generation sequencing (mNGS), and other methods. This study reviews the latest research on improving the main PRRSV detection methods and discusses their advantages and disadvantages.

Rapid and Visual Detection of Type 2 Porcine Reproductive and Respiratory Syndrome Virus by Real-Time Fluorescence-Based Reverse Transcription Recombinase-Aided Amplification.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases that has brought significant economic losses to the swine industry worldwide. Rapid and accurate PRRS virus (PRRSV) detection is one of the key factors for PRRS prevention and control. This study developed a real-time fluorescence-based reverse transcription recombinase-aided amplification (RF-RT-RAA) method for type 2 PRRSV (PRRSV-2) detection. The RF-RT-RAA assay could be performed at 42 °C for 20 min with the optimal primers and a probe. RF-RT-RAA results could be monitored using real-time fluorescence read-out or visually observed with the naked eye using a portable blue light transilluminator. The method had a strong specificity; no cross-reaction was identified with the detected common swine viruses. Moreover, the technique yielded high sensitivity with the lowest detection limit of 101 copies/μL and exhibited good repeatability and reproductively with the coefficients of variation (CV) less than 10%. Eighty-seven clinical samples were tested using RF-RT-RAA and a commercial PRRSV-2 RT-qPCR detection kit. The coincidence rate was 100% between RF-RT-RAA (real-time fluorescence read-out) and RT-qPCR, and 97.7% between RF-RT-RAA (visually observed) and RT-qPCR. The RF-RT-RAA assay provides a new method for rapid and visual detection of PRRSV-2.

Effect of pooling family oral fluids on the probability of PRRSV RNA detection by RT-rtPCR

Family oral fluids (FOFs) are an aggregate sample type shown to be a cost-efficient and convenient option for determining the porcine reproductive and respiratory syndrome virus (PRRSV) status of weaning age pigs. This study investigates the effect of pooling PRRSV-positive FOF samples with PRRSV-negative FOF samples at different levels (1/3, 1/5, 1/10, 1/20) on the probability of PRRSV RNA detection by reverse-transcription real-time polymerase chain reaction (RT-rtPCR). Mathematical models were built to assess how much the probability of RT-rtPCR PRRSV detection changed with increasing proportion of PRRSV-positive samples present within pools and how partially sampling a farrowing room influenced the probability of RT-rtPCR detection of PRRSV RNA in pooled samples at different prevalence scenarios. A general example of a guideline for FOF-based sampling under different prevalence scenarios to detect PRRSV RNA by RT-rtPCR with at least 95 % certainty is presented. At the sample level, the probability of detecting PRRSV RNA by RT-rtPCR decreased from 100 % to 87 %, 68 %, and 26 % when diluting up to 1/20 for PRRSV positive FOF having an initial Cycle threshold (Ct) below 34, between 34 and 36, or above 36, respectively. When PRRSV prevalence is near-zero (1 or 2 litters positive out of 56), the most cost-efficient farrowing room sampling strategy to detect PRRSV RNA with at least 95 % certainty was pooling FOF samples up to 1/10; at higher prevalence (≥ 3 of 56 litters positive), the most cost-efficient strategy was submitting samples in pools of 20. Subsampling a farrowing room for FOF pools was also demonstrated to be a valuable cost-saving strategy. Overall, based on the conditions of this study, pooling FOFs up to 1/20 is a valid option in situations of cost constraint and regardless of pooling level chosen, capturing as many litters as possible improves the probability of PRRSV detection.

A Multiplex Crystal Digital PCR for Detection of African Swine Fever Virus, Classical Swine Fever Virus, and Porcine Reproductive and Respiratory Syndrome Virus.

African swine fever (ASF), classical swine fever (CSF), and porcine reproductive and respiratory syndrome (PRRS) are highly infectious diseases of domestic pigs and wild boars. The co-infections of ASF virus (ASFV), CSF virus (CSFV), and PRRS virus (PRRSV) have been reported in different pig farms. Early differential detection and diagnosis of ASFV, CSFV, and PRRSV in the clinical samples is very important for the effective prevention and control of these diseases. A multiplex crystal digital PCR (dPCR) was developed for differential detection of ASFV, CSFV, and PRRSV in this study, targeting p72, 5' untranslated region (UTR), and ORF7 genes, respectively. The different reaction conditions were optimized, and the specificity, sensitivity, and repeatability of the assay were evaluated. The results showed that the multiplex crystal dPCR was able to accurately and differentially detect ASFV, CSFV, and PRRSV with a limit of detection of 4.69 × 10−1 copies/μl, respectively, and could not detect other porcine viruses, i.e., foot-and-mouth disease virus (FMDV), Senecavirus A (SVA), atypical porcine pestivirus (APPV), pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), and porcine parvovirus (PPV). The assay showed excellent repeatability and reproducibility, with coefficients of variation (CV) of the intra- and inter-assay from 0.09 to 1.40%, and from 0.64 to 2.26%, respectively. The 289 clinical samples from different pig herds in Guangxi province, China, were tested by the multiplex crystal dPCR and a reference multiplex real-time quantitative RT-PCR (qRT-PCR) established previously in our laboratory. The positive rates of ASFV, CSFV, and PRRSV were 30.10, 13.49, and 22.49% by the multiplex crystal dPCR, and 24.57, 8.65, and 18.34% by the multiplex qRT-PCR, with coincidence rates of 94.66, 95.16, and 95.84%, respectively. The results indicated that the established multiplex crystal dPCR was a specific, sensitive, and accurate method for the detection and quantification of ASFV, CSFV, and PRRSV. This is the first report on the multiplex dPCR for detecting ASFV, CSFV, and PRRSV.

Detection of Multiple Lineages of PRRSV in Breeding and Growing Swine Farms.

The detection and co-circulation of multiple variants of porcine reproductive and respiratory syndrome virus (PRRSV) have been observed and reported in swine. However, the potential long-term impact of multiple prevailing PRRSV variants on pig-performance is not yet fully understood. The primary objective of this study was to describe the genetic variation of PRRSV in processing fluid (PF), oral fluid (OF), and tonsil scraping (TS) specimens from five swine farms with different production types and PRRS status over a period of time (~1 year). Furthermore, the association between PRRSV prevalence and production parameters was investigated. Results showed that PRRSV was detected by RT-qPCR in 21–25% of all types of specimens. In breeding farms, PRRSV detection in PF and/or TS samples was correlated with stillborn and mummified fetuses, and pre-weaning mortality throughout the study period. Although ORF5 sequences were obtained in <16% of all sample types, simultaneous detection of PRRSV variants including field and vaccine strains within a single sampling event was identified in both breeding and growing pig farms. Phylogenetic analyses based on the ORF5 sequence classified the detected field PRRSV into L1A and L1H, two sub-lineages of lineage 1 (L1). Our study demonstrated the presence of multiple PRRSV lineages, sub-lineages, and variants in swine herds and its potential association with swine reproductive performance under field conditions.

Comparison of virus detection, productivity, and economic performance between lots of growing pigs vaccinated with two doses or one dose of PRRS MLV vaccine, under field conditions

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a significant economic impact on swine production. It has been demonstrated that PRRS modified-live virus (MLV) vaccination of pigs, with one full dose, significantly reduces clinical consequences of wild-type PRRSV infection compared to non-vaccinates. However, there is limited information about the effect that two doses of PRRSV MLV vaccine have on the performance of growing pigs, compared to vaccination with a single dose. This study was conducted with the objectives to compare (a) the wild-type PRRSV detection in oral fluids over time, (b) key closeout productivity indicators, and (c) economic performance between lots of growing pigs vaccinated with two doses of Ingelvac PRRS® MLV vaccine and lots vaccinated with a single dose of the same vaccine. This randomized field trial included 15 lots of growing pigs from PRRSV positive-unstable sow farms and 66 lots from PRRSV positive-stable sow farms, according to the American association of swine veterinarians’ terminology. All pig lots received the first vaccination either around processing or weaning age. Lots allocated in the two doses group received the second vaccination three to four weeks after the first vaccination. The pig lots were monitored for PRRSV detection over time. Six oral fluids samples were collected in three weeks intervals and were tested for wild-type PRRSV-2 RNA by RT-qPCR and open reading frame 5 (ORF)− 5 sequencing. Regression models were used to compare wild-type PRRSV detection dynamics on oral fluids samples and to compare key closeout performance indicators between one dose group and two doses group. Additionally, a benefit-cost ratio analysis compared economic performance between one dose group and two doses group. The proportion of wild-type PRRSV detection on oral fluids samples and the log counts of viral RNA per ml of oral fluids from the two doses group was lower than the one dose group on lots originated from PRRSV positive-stable sow farms, with a risk ratio of 1.24 and a rate ratio of 1.17, respectively. The two doses group had a significantly lower mortality rate than the one dose group, with a rate ratio of 1.21. The effect size increased on lots originated from PRRSV positive-unstable sow farms, and on lots with higher frequency and diversity of wild-type PRRSV detection during the growth phase. No differences in growth performance were detected between two doses group and one dose group. The second MLV vaccination dose had a benefit-cost ratio of 1.83. For lots originated from PRRSV positive-unstable farms, the benefit-cost ratio was 4.45, and for lots originated from PRRSV positive-stable farms, the benefit-cost ratio was 0.45. Under study conditions, vaccinating growing pig lots with two doses of PRRS MLV vaccine was a useful strategy to immunize growing pigs against PRRSV, lowering the wild-type PRRSV detection, lowering mortality rate, and increasing profitability, compared to lots of growing pigs that received a single dose of the same vaccine.

Molecular characterization of porcine reproductive and respiratory syndrome virus (PRRSv) identified from slaughtered pigs in northern Uganda

BackgroundA cross sectional study was conducted to detect and characterize species of porcine reproductive and respiratory syndrome virus (PRRSv) identified from slaughtered pigs in Lira district, northern Uganda. The study was conducted from March to September 2019 in three selected slaughter slabs. Pigs brought for slaughter were randomly sampled. At necropsy, lungs were extracted from the thoracic cavity and examined for pneumonic lesions. Seventy-three (73) pigs with gross lung lesions were sampled, from which one hundred and one (101) tissue samples were taken. A real-time reverse transcriptase PCR (RT-qPCR) was used to characterize PRRSv species.ResultsA total of 20 samples tested positive for PRRSv. The respective prevalence of PRRSv type 1 and type 2 were 24.65% (n = 18) and 2.73% (n = 2) respectively. Of the pigs sampled (n = 73), only two pigs, 2.73% (n = 2) tested positive to both species. The likelihood of PRRSv detection decreased with pig age, but increased with gross pneumonic pathology.ConclusionsThis study demonstrated dual circulation of both species in northern Uganda. The association between PRRSv and lung pathology suggests that it may be an important cause of lung disease in pigs in Uganda and hence loss of production. This calls for further investigations on potential economic impacts of PRRSv on pig productivity. These findings contribute to discussions about the need of surveillance and possible vaccination strategies against PRRSv in Uganda.

Point-of-Care and Label-Free Detection of Porcine Reproductive and Respiratory Syndrome and Swine Influenza Viruses Using a Microfluidic Device with Photonic Integrated Circuits.

Swine viral diseases challenge the sector’s sustainability by affecting productivity and the health and welfare of the animals. The lack of antiviral drugs and/or effective vaccines renders early and reliable diagnosis the basis of viral disease management, underlining the importance of point-of-care (POC) diagnostics. A novel POC diagnostic device utilizing photonic integrated circuits (PICs), microfluidics, and information and communication technologies for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza A (SIV) was validated using spiked and clinical oral fluid samples. Metrics including sensitivity, specificity, accuracy, precision, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) were calculated to assess the performance of the device. For PRRSV, the device achieved a sensitivity of 83.5%, specificity of 77.8%, and DOR values of 17.66, whereas the values for SIV were 81.8%, 82.2%, and 20.81, respectively. The POC device and PICs can be used for the detection of PRRSV and SIV in the field, paving the way for the introduction of novel technologies in the field of animal POC diagnostics to further optimize livestock biosecurity.

A NADC30-like PRRSV causes serious intestinal infections and tropism in piglets

Porcine reproductive and respiratory syndrome virus (PRRSV) causes huge economic loss to China's swine industry. Currently, a novel type 2 PRRSV, called the NADC30-like strain, is epidemic in numerous provinces of China. In this study, a NADC30-Like PRRSV strain was isolated in primary alveolar macrophage (PAM) cells from fecal samples collected from a local pig farm, which suffered severe diarrhea. A pathogenicity comparison study was conducted in 6–week‐old piglets by inoculating highly pathogenic HP-PRRSV and NADC30-Like PRRSV isolates. RT-qPCR revealed detection of NADC30-Like PRRSV but not the HP-PRRSV in the intestine. PRRSV infection-related lesions were observed in the intestine were further confirmed by histopathological and immunohistochemical examination (IHC). In addition, severe virus infections were also detected by RT-qPCR. Based on clinical observation and pathogenicity experiments, we confirmed that NADC30-Like PRRSV gained more tissue tropism, especially in the small intestine. This may be the one reason explaining why NADC-Like 30 PRRSV become a major epidemic strain in China since the first outbreak in 2013.

Fluorescent immunosensor based on fluorescence resonance energy transfer between CdSe/ZnS quantum dots and Au nanorods for PRRSV detection

An ultrasensitive and selective sandwich-type fluorescent immunosensor based on fluorescence resonance energy transfer (FRET) between CdSe/ZnS quantum dots (QDs) and Au nanorods (AuNRs) was developed for the rapid detection of porcine reproductive and respiratory syndrome virus (PRRSV). Modified CdSe/ZnS QDs and AuNRs were bioconjugated with primary antibodies M and GP5, respectively. When the target antigen was present, a well-known sandwich-type form CdSe/ZnS QDs-M antibody-antigen-GP5 antibody-AuNRs was produced, and thus reducing the distance between the QDs and NRs. As a result, FRET occurred and the fluorescence intensity of CdSe/ZnS QDs decreased, and this decrease was used to determine the antigen concentration. Ultrasensitive detection of PRRSV at low concentrations in swine serum was achieved with a limit of detection of 0.55 TCID50/mL and a linear detection range of 101–3.5 ​× ​104 TCID50/mL. Therefore, the fluorescent immunosensor is selective and highly sensitive; specifically, it can detect PRRSV at low concentrations in swine serum over a large concentration range. This proposed detection method can facilitate the development of high-performance fluorescent immunosensors for the detection of PRRSV and other antigens.

First Detection of NADC34-like PRRSV as a Main Epidemic Strain on a Large Farm in China.

The newly emerged sublineage 1.5 (NADC34-like) porcine reproductive and respiratory syndrome virus (PRRSV) has posed a direct threat to the Chinese pig industry since 2018. However, the prevalence and impact of NADC34-like PRRSV on Chinese pig farms is unclear. In the present study, we continuously monitored pathogens—including PRRSV, African swine fever virus (ASFV), classical swine fever virus (CSFV), pseudorabies virus (PRV), and porcine circovirus 2 (PCV2)—on a fattening pig farm with strict biosecurity practices located in Heilongjiang Province, China, from 2020 to 2021. The results showed that multiple types of PRRSV coexisted on a single pig farm. NADC30-like and NADC34-like PRRSVs were the predominant strains on this pig farm. Importantly, NADC34-like PRRSV—detected during the period of peak mortality—was one of the predominant strains on this pig farm. Sequence alignment suggested that these strains shared the same 100 aa deletion in the NSP2 protein as IA/2014/NADC34 isolated from the United States (U.S.) in 2014. Phylogenetic analysis based on open reading frame 5 (ORF5) showed that the genetic diversity of NADC34-like PRRSV on this farm was relatively singular, but it had a relatively high rate of evolution. Restriction fragment length polymorphism (RFLP) pattern analysis showed that almost all ORF5 RFLPs were 1-7-4, with one 1-4-4. In addition, two complete genomes of NADC34-like PRRSVs were sequenced. Recombination analysis and sequence alignment demonstrated that both viruses, with 98.9% nucleotide similarity, were non-recombinant viruses. This study reports the prevalence and characteristics of NADC34-like PRRSVs on a large-scale breeding farm in northern China for the first time. These results will help to reveal the impact of NADC34-like PRRSVs on Chinese pig farms, and provide a reference for the detection and further prevention and control of NADC34-like PRRSVs.

A longitudinal study on PRRSV detection in swine herds with different demographics and PRRSV management strategies.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been one of the major health-related concerns in the swine production industry. Through its rapid transmission and mutation, the simultaneous circulation of multiple PRRSV strains can be a challenge in PRRSV diagnostic, control and surveillance. The objective of this longitudinal study was to describe the temporal detection of PRRSV in swine farms with different production types and PRRS management strategies. Tonsil scraping (n=344) samples were collected from three breeding and two growing herds for approximately one year. In addition, processing fluids (n=216) were obtained from piglet processing batches within the three breeding farms while pen-based oral fluids (n=125) were collected in the two growing pig farms. Viral RNA extraction and reverse-transcription quantitative PCR (RT-qPCR) were conducted for all samples. The sample positivity threshold was set at quantification cycle (Cq) of ≤ 37. Statistical analyses were performed using generalized linear modelling and post hoc pairwise comparisons with Bonferroni adjustments using R statistical software. The results suggested a higher probability of detection in processing fluids compared to tonsil scraping specimens [odds ratio (OR)=3.86; p=.096] in breeding farms whereas oral fluids were outperformed by tonsil scrapings (OR=0.26; p<.01) in growing pig farms. The results described herein may lead to an improvement in PRRSV diagnostic and surveillance by selecting proper specimens.

Highly Sensitive CRISPR/Cas12a-Based Fluorescence Detection of Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease of swine that is caused by PRRS virus (PRRSV). In this study, we established a fluorescence assay for highly sensitive detection of PRRSV through integration of the reverse transcription-recombinase polymerase amplification (RT-RPA)-coupled Cas12a system with an optical property of single stranded DNA-fluorescently quenched (ssDNA-FQ) reporter. This technique can achieve isothermal and visual detection of PRRSV in 25 min. In particular, the assay reaction can be completed in a single tube. The limit of sensitivity for PRRSV detection was single copy without cross-reactivity of other porcine viruses. Correlation between 11 PRRSV clinical samples measured by the quantitative reverse transcription polymerase chain reaction (RT-qPCR) and CRISPR/Cas12a assay was determined; the result showed that our results were highly accurate. To sum up, this study developed a visual, sensitive, and specific method of nucleic acid detection based on a CRISPR-Cas12a technique for the on-site detection of PRRSV.

Investigating the relationship of porcine reproductive and respiratory syndrome virus RNA detection between adult/sow farm and wean-to-market age categories.

Porcine reproductive and respiratory syndrome (PRRS) is a disease caused by the PRRS virus (PRRSV) that has spread globally in the last 30 years and causes huge economic losses every year. This research aims to 1) investigate the relationship between the PRRSV detection in two age categories (wean-to-market and adult/sow farm), and 2) examine the extent to which the wean-to-market PRRSV positive rate forecasts the adult/sow farm PRRSV positive rate. The data we used are the PRRSV RNA detection results between 2007 and 2019 integrated by the US Swine Disease Reporting System project that represent 95% of all porcine submissions tested in the US National Animal Health Network. We first use statistical tools to investigate to what extent the increase in PRRSV positive submissions in the wean-to-market is related to the PRRSV increase in adult/sow farms. The statistical analysis confirms that an increase in the PRRSV positive rate of wean-to-market precedes the increase in the adult/sow farms to a large extent. Then we create the dynamic exponentially weighted moving average control charts to identify out-of-control points (i.e., signals) in the PRRSV rates for both wean-to-market and adult/sow farms. This control-chart-based analysis finds that 78% of PRRSV signals in the wean-to-market are followed by a PRRSV rate signal in the adult/sow farms within eight weeks. We expect that our findings will help the producers and veterinarians to justify and reinforce the implementation of bio-security and bio-contaminant practices to curb disease spread across farms.

Finding PRRSV in sow herds: Family oral fluids vs. serum samples from due-to-wean pigs

The aim of this study was to compare the detection of porcine reproductive and respiratory syndrome virus (PRRSV) in due-to-wean litters in commercial swine breeding herds using family oral fluids (FOF) vs. individual piglet serum samples. FOF and piglet serum samples were collected in 199 due-to-wean litters on six farms containing 2177 piglets. All samples were individually tested for PRRSV RNA by RT-rtPCR. A litter was considered PRRSV-positive when PRRSV RNA was detected in ≥ 1 piglet serum sample or the FOF sample. Mixed effect logistic regression with farm as a random effect was used 1) to evaluate the probability of obtaining a PRRSV RNA positive FOF as a function of the proportion of viremic piglets in a litter and 2) the effect of litter size and parity on the probability that a litter would test PRRSV RNA positive in FOF. A Bayesian prevalence estimation under misclassification (BayesPEM) analysis was used to calculate the PRRSV prevalence and 95 % credible interval given the condition that all samples (FOF and serum) tested negative. In total, 34 of 199 litters (17.1 %) contained ≥ 1 viremic piglet(s), and 28 of 199 litters (14.1 %) were FOF positive. When all piglet serum samples within a litter tested negative, 1 of 165 FOF (0.6 %) tested PRRSV RNA positive. The probability of a PCR-positive FOF sample from litters with 10 %, 20 %, 30 %, 40 %, and 50 % within-litter PRRSV prevalence was 3.5 %, 35.1 %, 88.8 %, 99.2 %, and >99.9 %, respectively. The odds of a PCR-positive FOF in a first parity litter were 3.36 times (95 % CI: 2.10–5.38) that of a parity ≥ 2 litter. The odds of a positive FOF result in a litter with ≤ 11 piglets were 9.90 times (95 % CI: 4.62–21.22) that of a litter with > 11 piglets. FOF was shown to be an efficacious sample type for PRRSV detection in farrowing rooms. A risk-based approach for litter selection combined with FOF collection can be used to improve on-farm PRRSV detection with a limited sample size, compared to sampling multiple individual pigs. Finally, the BayesPEM analysis showed that PRRSV may still be present in breeding herds when all samples (serum and FOF) test PRRSV RNA negative, i.e., negative surveillance results should be interpreted with caution.