Rapid genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) using MinION nanopore sequencing.

The global distribution and constant evolution are challenges for the control of porcine reproductive and respiratory syndrome virus (PRRSV), one of the most important viruses affecting swine worldwide. Effective control of PRRSV benefits from genotyping, which currently relies on Sanger sequencing. Here we developed and optimized procedures for real-time genotyping and whole genome sequencing of PRRSV directly from clinical samples based on targeted amplicon- and long amplicon tiling sequencing using the MinION Oxford Nanopore platform. Procedures were developed and tested on 154 clinical samples (including lung, serum, oral fluid and processing fluid) with RT-PCR Ct values ranging from 15 to 35. The targeted amplicon sequencing (TAS) approach was developed to obtain sequences of the complete ORF5 (main target gene for PRRSV genotyping) and partial ORF4 and ORF6 sequences of both PRRSV-1 and PRRSV-2 species. After only 5 min of sequencing, PRRSV consensus sequences with identities to reference sequences above 99% were obtained, enabling rapid identification and genotyping of clinical PRRSV samples into lineages 1, 5 and 8. The long amplicon tiling sequencing (LATS) approach targets type 2 PRRSV, the most prevalent viral species in the U.S. and China. Complete PRRSV genomes were obtained within the first hour of sequencing for samples with Ct values below 24.9. Ninety-two whole genome sequences were obtained using the LATS procedure. Fifty out of 60 sera (83.3%) and 18 out of 20 lung samples (90%) had at least 80% of genome covered at a minimum of 20X sequence depth per position. The procedures developed and optimized in this study here are valuable tools with potential for field application during PRRSV elimination programs.

Characterization of the Subclinical Infection of Porcine Deltacoronavirus in Grower Pigs under Experimental Conditions.

This study characterized the susceptibility and dynamic of porcine deltacoronavirus infection in grower pigs under experimental conditions using a combination of syndromic and laboratory assessments. Seven-week-old conventional pigs (n = 24) were randomly distributed into PDCoV- (n = 12) and mock-inoculated (n = 12) groups. Serum was collected at -7, 0, 3, 7, 10, 14, 17, 21, 28, 35, and 42 days post-inoculation (DPI) to evaluate viremia (RT-qPCR) and antibody response (S1-based ELISA). Viral shedding and potential infectivity were determined using pen-based oral fluids and feces collected every other day between DPI 0 and 42. Pigs showed no clinical signs or viremia throughout the study. Active virus shedding was detected in feces (6-22 DPI) and oral fluids (2-30 DPI), peaking at DPI 10. IgG was first detected at DPI 10, being statistically significant after DPI 14 and increasing thereafter, coinciding with the progressive resolution of the infection. Likewise, a significant increase in proinflammatory IL-12 was detected between DPI 10 and 21 in PDCoV-inoculated pigs, which could enhance innate resistance to PDCoV infection. This study demonstrated that active surveillance based on systematic sampling and laboratory testing combining molecular and serological tools is critical for the accurate detection of subclinical circulation of PDCoV in pigs after weaning.

Evaluation of the Efficacy of an S-INDEL PEDV Strain Administered to Pregnant Gilts against a Virulent Non-S-INDEL PEDV Challenge in Newborn Piglets.

A safe and efficacious live-attenuated vaccine for porcine epidemic diarrhea virus (PEDV) is not commercially available in the United States yet. Two major PEDV strains are currently circulating in US swine: highly virulent non-S-INDEL strain and milder virulent S-INDEL strain. In this study, the safety and protective efficacy of a plaque-purified S-INDEL PEDV isolate formulated as a vaccine candidate was evaluated. Ten pregnant gilts were divided into three groups and orally inoculated at 79 days of gestation and then boosted at 100 days gestation (T01: n = 4, vaccination/challenge; T02: n = 4, non-vaccination/challenge; T03: n = 2, non-vaccination/non-challenge). None of the gilts had adverse clinical signs after vaccination. Only one T01 gilt (#5026) had viral replication and detectible viral RNA in feces. The same gilt had consistent levels of PEDV-specific IgG and IgA antibodies in serum and colostrum/milk. Farrowed piglets at 3 to 5 days of age from T01 and T02 gilts were orally challenged with 103 TCID50/pig of the virulent non-S-INDEL PEDV while T03 piglets were orally inoculated with virus-negative medium. T01 litters had overall lower mortality than T02 (T01 36.4% vs. T02 74.4%). Specifically, there was 0% litter mortality from T01 gilt 5026. Overall, it appears that vaccination of pregnant gilts with S-INDEL PEDV can passively protect piglets if there is virus replication and immune response induction in the pregnant gilts.

The N-terminal Subunit of the Porcine Deltacoronavirus Spike Recombinant Protein (S1) Does Not Serologically Cross-react with Other Porcine Coronaviruses.

Porcine deltacoronavirus (PDCoV), belonging to family Coronaviridae and genus Deltacoronavirus, is a major enteric pathogen in swine. Accurate PDCoV diagnosis relying on laboratory testing and antibody detection is an important approach. This study evaluated the potential of the receptor-binding subunit of the PDCoV spike protein (S1), generated using a mammalian expression system, for specific antibody detection via indirect enzyme-linked immunosorbent assay (ELISA). Serum samples were collected at day post-inoculation (DPI) -7 to 42, from pigs (n = 83) experimentally inoculated with different porcine coronaviruses (PorCoV). The diagnostic sensitivity of the PDCoV S1-based ELISA was evaluated using serum samples (n = 72) from PDCoV-inoculated animals. The diagnostic specificity and potential cross-reactivity of the assay was evaluated on PorCoV-negative samples (n = 345) and samples collected from pigs experimentally inoculated with other PorCoVs (n = 472). The overall diagnostic performance, time of detection, and detection rate over time varied across different S/P cut-offs, estimated by Receiver Operating Characteristic (ROC) curve analysis. The higher detection rate in the PDCoV group was observed after DPI 21. An S/P cut-off of 0.25 provided 100% specificity with no serological cross-reactivity against other PorCoV. These results support the use of S1 protein-based ELISA for accurate detection of PDCoV infections, transference of maternal antibodies, or active surveillance.

Development and Clinical Applications of a 5-Plex Real-Time RT-PCR for Swine Enteric Coronaviruses.

A PEDV/PDCoV/TGEV/SADS-CoV/XIPC 5-plex real-time RT-PCR was developed and validated for the simultaneous detection and differentiation of four swine enteric coronaviruses (PEDV, PDCoV, TGEV and SADS-CoV) in one PCR reaction (XIPC serves as an exogenous internal positive control). The 5-plex PCR had excellent analytical specificity, analytical sensitivity, and repeatability based on the testing of various viral and bacterial pathogens, serial dilutions of virus isolates, and in vitro transcribed RNAs. The 5-plex PCR had comparable diagnostic performance to a commercial PEDV/TGEV/PDCoV reference PCR, based on the testing of 219 clinical samples. Subsequently, 1807 clinical samples collected from various U.S. states during 2019-2021 were tested by the 5-plex PCR to investigate the presence of SADS-CoV in U.S. swine and the frequency of detecting swine enteric CoVs. All 1807 samples tested negative for SADS-CoV. Among the samples positive for swine enteric CoVs, there was a low frequency of detecting TGEV, an intermediate frequency of detecting PDCoV, and a high frequency of detecting PEDV. Although there is no evidence of SADS-CoV presence in the U.S. at present, the availability of the 5-plex PCR will enable us to conduct ongoing surveillance to detect and differentiate these viruses in swine samples and other host species samples as some of these coronaviruses can cause cross-species infection.

Association between blood pressure categories and cardiovascular disease mortality in China.

BACKGROUND: Blood pressure (BP) categories are useful to simplify preventions in public health, and diagnostic and treatment approaches in clinical practice. Updated evidence about the associations of BP categories with cardiovascular diseases (CVDs) and its subtypes is warranted. METHODS AND FINDINGS: About 0.5 million adults aged 30 to 79 years were recruited from 10 areas in China during 2004-2008. The present study included 430 977 participants without antihypertension treatment, cancer, or CVD at baseline. BP was measured at least twice in a single visit at baseline and CVD deaths during follow-up were collected via registries and the national health insurance databases. Multivariable Cox regression was used to estimate the associations between BP categories and CVD mortality. Overall, 16.3% had prehypertension-low, 25.1% had prehypertension-high, 14.1% had isolated systolic hypertension (ISH), 1.9% had isolated diastolic hypertension (IDH), and 9.1% had systolic-diastolic hypertension (SDH). During a median 10-year follow-up, 9660 CVD deaths were documented. Compared with normal, the hazard ratios (95% CI) of prehypertension-low, prehypertension-high, ISH, IDH, SDH for CVD were 1.10 (1.01-1.19), 1.32 (1.23-1.42), 2.04 (1.91-2.19), 2.20 (1.85-2.61), and 3.81 (3.54-4.09), respectively. All hypertension subtypes were related to the increased risk of CVD subtypes, with a stronger association for hemorrhagic stroke than for ischemic heart disease. The associations were stronger in younger than older adults. CONCLUSIONS: Prehypertension-high should be considered in CVD primary prevention given its high prevalence and increased CVD risk. All hypertension subtypes were independently associated with CVD and its subtypes mortality, though the strength of associations varied substantially.

Effects of medium chain fatty acids as a mitigation or prevention strategy against porcine epidemic diarrhea virus in swine feed.

Feed has been shown to be a vector for viral transmission. Four experiments were conducted to: 1) determine if medium chain fatty acids (MCFA) are effective mitigants when applied to feed both pre- and post-porcine epidemic diarrhea virus (PEDV) inoculation measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR), 2) evaluate varying levels and combinations of MCFA measured by qRT-PCR, and 3) evaluate selected treatments in bioassay to determine infectivity. In exp. 1, treatments were arranged in a 2 × 2 + 1 factorial with main effects of treatment (0.3% commercial formaldehyde [CF] product, Sal CURB [Kemin Industries, Inc.; Des Moines, IA], or 1% MCFA blend (Blend) of 1:1:1 C6:C8:C10 [PMI, Arden Hills, MN]) and timing of application (pre- or post-inoculation with PEDV) plus a positive control (PC; feed inoculated with PEDV and no treatment). All combinations of treatment and timing decreased detectable PEDV compared with the PC (P < 0.05). Pre-inoculation treatment elicited decreased magnitude of PEDV detection (cycle threshold value) compared with post-inoculation (P = 0.009). Magnitude of PEDV detection was decreased for CF compared with Blend (P < 0.0001). In exp. 2, pre-inoculation treatments consisted of: 1) PC, 2) 0.3% CF, 3 to 5) 0.125% to 0.33% C6:0, 6 to 8) 0.125% to 0.33% C8:0, 9 to 11) 0.125% to 0.33% C10:0, and 12 to 15) 0.125% to 0.66% C5:0. Treating feed with 0.33% C8:0 resulted in decreased (P < 0.05) PEDV detection compared with all other treatments. Increasing concentration of each individual MCFA decreased PEDV detectability (P < 0.042). In exp. 3, pre-inoculation treatments consisted of: 1) PC, 2) 0.3% CF, 3 to 7) 0.25% to 1% Blend, 8 to 10) 0.125% to 0.33% C6:0 + C8:0, 11 to 13) 0.125% to 0.33% C6:0 + C10:0, and 14 to 16) 0.125% to 0.33% C8:0 + C10:0. Treating feed with CF, 0.5% Blend, 0.75% Blend, 1% Blend, all levels of C6:0+C8:0, 0.25% C6:0 + 0.25% C10:0, 0.33% C6:0 + 0.33% C10:0, 0.25% C8:0 + 0.25% C10:0, or 0.33% C8:0 + 0.33% C10:0 elicited decreased detection of PEDV compared with PC (P < 0.05). Increasing concentration of each MCFA combination decreased PEDV detectability (linear, P < 0.012). In exp. 4, feed was treated pre-inoculation with: 1) no treatment (PC), 2) 0.3% CF, 3) 0.5% Blend, or 4) 0.3% C8:0 and analyzed via qRT-PCR and bioassay. Adding 0.5% Blend or 0.3% C8:0 resulted in decreased PEDV compared with PC and only PC resulted in a positive bioassay. Therefore, MCFA can decrease detection of PEDV in feed. Further, inclusion of lower levels of MCFA than previously evaluated are effective against PEDV.