Transcriptome Analysis in Air-Liquid Interface Porcine Respiratory Epithelial Cell Cultures Reveals That the Betacoronavirus Porcine Encephalomyelitis Hemagglutinating Virus Induces a Robust Interferon Response to Infection.

Porcine hemagglutinating encephalomyelitis virus (PHEV) replicates in the upper respiratory tract and tonsils of pigs. Using an air-liquid interface porcine respiratory epithelial cells (ALI-PRECs) culture system, we demonstrated that PHEV disrupts respiratory epithelia homeostasis by impairing ciliary function and inducing antiviral, pro-inflammatory cytokine, and chemokine responses. This study explores the mechanisms driving early innate immune responses during PHEV infection through host transcriptome analysis. Total RNA was collected from ALI-PRECs at 24, 36, and 48 h post inoculation (hpi). RNA-seq analysis was performed using an Illumina Hiseq 600 to generate 100 bp paired-end reads. Differential gene expression was analyzed using DeSeq2. PHEV replicated actively in ALI-PRECs, causing cytopathic changes and progressive mucociliary disruption. Transcriptome analysis revealed downregulation of cilia-associated genes such as CILK1, DNAH11, LRRC-23, -49, and -51, and acidic sialomucin CD164L2. PHEV also activated antiviral signaling pathways, significantly increasing the expression of interferon-stimulated genes (RSAD2, MX1, IFIT, and ISG15) and chemokine genes (CCL5 and CXCL10), highlighting inflammatory regulation. This study contributes to elucidating the molecular mechanisms of the innate immune response to PHEV infection of the airway epithelium, emphasizing the critical roles of the mucociliary, interferon, and chemokine responses.

The porcine circovirus 3 humoral response: characterization of maternally derived antibodies and dynamic following experimental infection.

Since Porcine Circovirus 3 (PCV3) was first identified in 2016, our understanding of the humoral response is still relatively scarce. Current knowledge of the PCV3 humoral response is primarily based on field studies identifying the seroprevalence of PCV3 Cap-induced antibodies. Studies on the humoral response following experimental PCV3 infection have conflicting results where one study reports the development of the Cap IgG response 7 days postinfection with no concurrent Cap IgM response, while a second study shows a Cap IgM response at the same time point with no detection of Cap IgG. The dynamics of the PCV3 Cap and Rep IgG following maternal antibody transfer and experimental infection have not been well characterized. Additionally, the cross-reactivity of convalescent serum from PCV2 and PCV3 experimentally infected animals to serologic methods of the alternate PCV has limited evaluation. Here, we show that maternally derived antibodies were detectable in piglet serum 7-9 weeks postfarrowing for the Cap IgG and 5-weeks-post farrowing for the Rep IgG using Cap- and Rep-specific enzyme linked immunosorbent assays (ELISA) and immunofluorescent assays (IFA) methods. Following experimental inoculation, Cap IgG was detected at 2-weeks-post inoculation and Rep IgG detection was delayed until 4-weeks-post inoculation. Furthermore, convalescent serum from either PCV2 or PCV3 methods displayed no cross-reactivity by serological methods against the other PCV. The information gained in this study highlights the development of both the Cap- and Rep-specific antibodies following experimental infection and through the transfer of maternal antibodies. The increased understanding of the dynamics of maternal antibody transfer and development of the humoral response following infection gained in the present study may aid in the establishment of husbandry practices and potential application of prophylactics to control PCV3 clinical disease. IMPORTANCE: Research on Porcine Circovirus 3 (PCV3) immunology is vital for understanding and controlling this virus. Previous studies primarily relied on field observations, but they have shown conflicting results about the immunological response against PCV3. This study helps fill those gaps by looking at how antibodies develop in pigs, especially those maternal-derived, and their impact in neonatal pigs preventing PCV3-associated disease in piglets. In addition, we look at the dynamics of antibodies in experimental infections mimicking infection in pigs in the grower-phase condition. Understanding this process can help to develop better strategies to prevent PCV3 infection. Also, this research found that PCV2 and PCV3 do not cross-react, which is crucial for serological test development and results interpretation. Overall, this work is essential for improving swine health and farming practices in the face of PCV3 infections.

Differential antigenicity of individual Mycoplasma hyorhinis variable lipoproteins.

The Mycoplasma hyorhinis (Mhr) variable lipoprotein (Vlp) family, comprising Vlps A, B, C, D, E, F, and G, are highly variable in expression, size, and cytoadhesion capabilities across Mhr strains. The 'Vlp system' plays a crucial role in cytoadhesion, immune evasion, and in eliciting a host immunologic response. This pilot study described the development of Vlp peptide-based ELISAs to evaluate the antigenic reactivity of individual Vlps against Mhr antisera collected throughout a longitudinal study focused on Mhr strain 38983, reproducing Mhr-associated disease under experimental conditions. Specifically, serum samples were collected at day post-inoculation 0, 7, 10, 14, 17, 21, 24, 28, 35, 42, 49, and 56 from Mhr- and mock (Friis medium)-inoculated cesarean-derived, colostrum-deprived pigs. Significant Mhr-specific IgG responses were detected at specific time points throughout the infection, with some variations for each Vlp. Overall, individual Vlp ELISAs showed consistently high accuracy rates, except for VlpD, which would likely be associated with its expression levels or the anti-Vlp humoral immune response specific to the Mhr strain used in this study. This study provides the basis and tools for a more refined understanding of these Vlp- and Mhr strain-specific variations, which is foundational in understanding the host immune response to Mhr.

Pen-Based Swine Oral Fluid Samples Contain Both Environmental and Pig-Derived Targets.

Laboratory methods for detecting specific pathogens in oral fluids are widely reported, but there is little research on the oral fluid sampling process itself. In this study, a fluorescent tracer (diluted red food coloring) was used to test the transfer of a target directly from pigs or indirectly from the environment to pen-based oral fluid samples. Pens of ~30, ~60, and ~125 14-week-old pigs (32 pens/size) on commercial swine farms received one of two treatments: (1) pig exposure, i.e., ~3.5 mL of tracer solution sprayed into the mouth of 10% of the pigs in the pen; (2) environmental exposure, i.e., 20 mL of tracer solution was poured on the floor in the center of the pen. Oral fluids collected one day prior to treatment (baseline fluorescence control) and immediately after treatment were tested for fluorescence. Data were evaluated by receiver operating characteristic (ROC) analysis, with Youden's J statistic used to set a threshold. Pretreatment oral fluid samples with fluorescence responses above the ROC threshold were removed from further analysis (7 of 96 samples). Based on the ROC analyses, oral fluid samples from 78 of 89 pens (87.6%), contained red food coloring, including 43 of 47 (91.5%) pens receiving pig exposure and 35 of 42 (83.3%) pens receiving environmental exposure. Thus, oral fluid samples contain both pig-derived and environmental targets. This methodology provides a safe and quantifiable method to evaluate oral fluid sampling vis-à-vis pen behavior, pen size, sampling protocol, and target distribution in the pen.

Assessment of Strategies for Preserving Swine Viral RNA Targets in Diagnostic Specimens.

Successful downstream molecular analyses of viral ribonucleic acid (RNA) in diagnostic laboratories, e.g., reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or next-generation sequencing, are dependent on the quality of the RNA in the specimen. In swine specimens, preserving the integrity of RNA requires proper sample handling at the time the sample is collected on the farm, during transport, and in the laboratory until RNA extraction is performed. Options for proper handling are limited to maintaining the cold chain or using commercial specimen storage matrices. Herein, we reviewed the refereed literature for evidence that commercial specimen storage matrices can play a role in preserving swine viral RNA in clinical specimens. Refereed publications were included if they compared RNA detection in matrix-treated vs. untreated samples. At present, the small number of refereed studies and the inconsistency in reported results preclude the routine use of commercial specimen storage matrices. For example, specimen storage matrices may be useful under specific circumstances, e.g., where it is mandatory to render the virus inactive. In a broader view, statistically sound side-by-side comparisons between specimens, viral RNA targets, and storage conditions are needed to establish if, when, and how commercial specimen storage matrices could be used in diagnostic medicine.

Dynamics of antibody response and bacterial shedding of Mycoplasma hyorhinis and M. hyosynoviae in oral fluids from experimentally inoculated pigs.

Mycoplasma hyorhinis (Mhr) and M. hyosynoviae (Mhs) are commensal organisms of the upper respiratory tract and tonsils but may also cause arthritis in pigs. In this study, 8-week-old cesarean-derived colostrum-deprived (CDCD) pigs (n = 30; 3 groups, 10 pigs per group, 2 pigs per pen) were inoculated with Mhr, Mhs, or mock-inoculated with culture medium and then pen-based oral fluids were collected at different time points over the 56 days of the experimental study. Oral fluids tested by Mhr and Mhs quantitative real-time PCRs revealed Mhr DNA between day post inoculation (DPI) 5-52 and Mhs DNA between DPI 5-15. Oral fluids were likewise tested for antibody using isotype-specific (IgG, IgA, IgM) indirect ELISAs based on a recombinant chimeric polypeptide of variable lipoproteins (A-G) for Mhr and Tween 20-extracted surface proteins for Mhs. Mhr IgA was detected at DPI 7 and, relative to the control group, significant (p < 0.05) antibody responses were detected in the Mhr group between DPI 12-15 for IgM and DPI 36-56 for both IgA and IgG. In the Mhs group, IgM was detected at DPI 10 and significant (p < 0.05) IgG and IgA responses were detected at DPI 32-56 and DPI 44-56, respectively. This study demonstrated that oral fluid could serve as an effective and convenient antemortem sample for monitoring Mhr and Mhs in swine populations.

How do deer respiratory epithelial cells weather the initial storm of SARS-CoV-2 WA1/2020 strain?

The potential infectivity of severe acute respiratory syndrome associated coronavirus-2 (SARS-CoV-2) in animals raises a public health and economic concern, particularly the high susceptibility of white-tailed deer (WTD) to SARS-CoV-2. The disparity in the disease outcome between humans and WTD is very intriguing, as the latter are often asymptomatic, subclinical carriers of SARS-CoV-2. To date, no studies have evaluated the innate immune factors responsible for the contrasting SARS-CoV-2-associated disease outcomes in these mammalian species. A comparative transcriptomic analysis in primary respiratory epithelial cells of human (HRECs) and WTD (Deer-RECs) infected with the SARS-CoV-2 WA1/2020 strain was assessed throughout 48 h post inoculation (hpi). Both HRECs and Deer-RECs were susceptible to virus infection, with significantly (P < 0.001) lower virus replication in Deer-RECs. The number of differentially expressed genes (DEG) gradually increased in Deer-RECs but decreased in HRECs throughout the infection. The ingenuity pathway analysis of DEGs further identified that genes commonly altered during SARS-CoV-2 infection mainly belong to cytokine and chemokine response pathways mediated via interleukin-17 (IL-17) and nuclear factor-κB (NF-κB) signaling pathways. Inhibition of the NF-κB signaling in the Deer-RECs pathway was predicted as early as 6 hpi. The findings from this study could explain the lack of clinical signs reported in WTD in response to SARS-CoV-2 infection as opposed to the severe clinical outcomes reported in humans.IMPORTANCEThis study demonstrated that human and white-tailed deer primary respiratory epithelial cells are susceptible to the SARS-CoV-2 WA1/2020 strain infection. However, the comparative transcriptomic analysis revealed that deer cells could limit viral replication without causing hypercytokinemia by downregulating IL-17 and NF-κB signaling pathways. Identifying differentially expressed genes in human and deer cells that modulate key innate immunity pathways during the early infection will lead to developing targeted therapies toward preventing or mitigating the "cytokine storm" often associated with severe cases of coronavirus disease 19 (COVID-19). Moreover, results from this study will aid in identifying novel prognostic biomarkers in predicting SARS-CoV-2 adaption and transmission in deer and associated cervids.

Normalizing real-time PCR results in routine testing.

Normalization, the process of controlling for normal variation in sampling and testing, can be achieved in real-time PCR assays by converting sample quantification cycles (Cqs) to "efficiency standardized Cqs" (ECqs). We calculated ECqs as E-ΔCq, where E is amplification efficiency and ΔCq is the difference between sample and reference standard Cqs. To apply this approach to a commercial porcine reproductive and respiratory syndrome virus (PRRSV) RT-qPCR assay, we created reference standards by rehydrating and then diluting (1 × 10-4) a PRRSV modified-live vaccine (PRRS MLV; Ingelvac) with serum or oral fluid (OF) to match the sample matrix to be tested. Sample ECqs were calculated using the mean E and reference standard Cq calculated from the 4 reference standards on each plate. Serum (n = 132) and OF (n = 130) samples were collected from each of 12 pigs vaccinated with a PRRSV MLV from -7 to 42 d post-vaccination, tested, and sample Cqs converted to ECqs. Mean plate Es were 1.75-2.6 for serum and 1.7-2.3 for OF. Mean plate reference standard Cqs were 29.1-31.3 for serum and 29.2-31.5 for OFs. Receiver operating characteristic analysis calculated the area under the curve for serum and OF sample ECqs as 0.999 (95% CI: 0.997, 1.000) and 0.947 (0.890, 1.000), respectively. For serum, diagnostic sensitivity and specificity of the commercial PRRSV RT-qPCR assay were estimated as 97.9% and 100% at an ECq cutoff ≥ 0.20, and for OF, 82.6% and 100%, respectively, at an ECq cutoff ≥ 0.45.

Detection of Porcine Circovirus Type 3 in Serum, Semen, Oral Fluid, and Preputial Fluid Samples of Boars.

Porcine circovirus type 3 (PCV3) is commonly associated with clinical symptoms such as porcine dermatitis and nephropathy syndrome (PDNS)-like lesions, respiratory signs, and reproductive disorders. This study aimed to investigate the epidemiology of PCV3 in a boar stud. The objectives were to detect PCV3 in semen, as well as matched serum, oral fluid, and preputial fluid samples from adult boars using quantitative polymerase chain reaction (qPCR), analyze PCV3-IgG antibody data, and genetically characterize a positive sample. A total of 112 samples from 28 boars were collected from a large-scale pig farm in Guangxi, China. The qPCR results showed that the PCV3 DNA was not detected in semen, with a positive rate of 0% (0/28), while it was detected in serum (3.57%-1/28), oral fluid (64.28%-18/28), and preputial fluid (46.4%-13/28). The seropositivity rate of PCV3-IgG in serum was 82.14% (23/28) according to the indirect enzyme-linked immunosorbent serologic assay (ELISA) results. Phylogenetic analysis revealed that one of the PCV3 isolates belonged to the PCV3c clades. This is the first report of PCV3 detection in preputial fluid from boars. The results suggest that PCV3 is transmitted among boars on pig farms and exhibits epidemic characteristics.

Performance of a Differentiation of Infected from Vaccinated Animals (DIVA) Classical Swine Fever Virus (CSFV) Serum and Oral Fluid Erns Antibody AlphaLISA Assay.

Classical swine fever virus (CSFV) is an OIE-listed disease that requires effective surveillance tools for its detection and control. The aim of this study was to develop and evaluate the diagnostic performance of a novel CSFV Erns IgG AlphaLISA for both serum and oral fluid specimens that would likewise be compatible with the use of CSFV E2 DIVA vaccines. Test performance was evaluated using a panel of well-characterized serum (n = 760) and individual (n = 528) or pen-based (n = 30) oral fluid samples from four groups of animals: (1) negative controls (n = 60 pigs); (2) inoculated with ALD strain wild-type CSFV (n = 30 pigs); (3) vaccinated with LOM strain live CSFV vaccine (n = 30 pigs); and (4) vaccinated with live CSFV marker vaccine on commercial farms (n = 120 pigs). At a cutoff of S/P ≥ 0.7, the aggregate estimated diagnostic sensitivities and specificities of the assay were, respectively, 97.4% (95% CI 95.9%, 98.3%) and 100% for serum and 95.4% (95% CI 92.9%, 97.0%) and 100% for oral fluid. The Erns IgG antibody AlphaLISA combined DIVA capability with solid diagnostic performance, rapid turnaround, ease of use, and compatibility with both serum and oral fluid specimens.

A Retrospective Analysis of Porcine Circovirus Type 3 in Samples Collected from 2008 to 2021 in Mexico.

Porcine circovirus type 3 (PCV3) is a nonenveloped virus of the Circoviridae family. This virus has been identified in pigs of different ages and pigs with several clinical manifestations of the disease or even in apparently healthy pigs. While PCV3 was first reported in 2015, several retrospective studies have reported the virus before that year. The earliest report indicates that PCV3 has been circulated in swine farms since 1996. In this study, we evaluated the presence of PCV3 in samples collected in Mexico in 2008, 2015, 2020, and 2021. This study assessed PCV3 DNA by qPCR and antibodies against CAP protein by indirect ELISA. The results showed that PCV3 (DNA and anti-CAP antibodies) was detected in the samples collected from 2008 to 2021. The highest prevalence was in 2008 (100%), and the lowest was in 2015 (negative). Genetic analysis of ORF2 showed that the virus identified belonged to genotype a, as most of the viruses identified thus far. PCV3 was detected in samples from piglets with respiratory signs and growth retardation, sows with reproductive failure, or asymptomatic piglets and sows. Pigs with respiratory signs, growth retardation, or reproductive failure had a higher prevalence of antibodies and qPCR-positive samples. In conclusion, this study showed that PCV3 has been circulating in Mexico since 2008 and that PCV3 DNA and antibodies were more prevalent in samples from pigs with clinical manifestations of diseases.

Genetic and In Vitro Characteristics of a Porcine Circovirus Type 3 Isolate from Northeast China.

Porcine circovirus 3 (PCV3) is an emerging virus first discovered in the United States in 2015, and since then, PCV3 has been found in many regions of the world, including America, Asia, and Europe. Although several PCV3 investigations have been carried out, there is a lack of knowledge regarding the pathogenicity of PCV3, mostly due to the limited number of PCV3 isolates that are readily available. In this study, PCV3-DB-1 was isolated in PK-15 cells and characterized in vitro. Electron microscopy revealed the presence of PCV-like particles, and in situ hybridization RNA analysis demonstrated the replication of PCV3 in PK-15 cell culture. Based on phylogenetic analysis of PCV3 isolates from the Heilongjiang province of China, PCV3-DB-1 with 24 alanine and 27 lysine in the Cap protein was originally isolated and determined to belong to the clade PCV3a.

Dynamics of Infection of Atypical Porcine Pestivirus in Commercial Pigs from Birth to Market: A Longitudinal Study.

Atypical porcine pestivirus (APPV) was found to be associated with pigs demonstrating congenital tremors (CT), and clinical signs in pigs have been reproduced after experimental challenge. Subsequently, APPV has been identified in both symptomatic and asymptomatic swine of all ages globally. The objective of this research was to perform a longitudinal study following two cohorts of pigs, those born in litters with pigs exhibiting CT and those born in litters without CT, to analyze the virus and antibody dynamics of APPV infection in serum from birth to market. There was a wide range in the percentage of affected pigs (8-75%) within CT-positive litters. After co-mingling with CT-positive litters at weaning, pigs from CT-negative litters developed viremia that was cleared after approximately 2 months, with the majority seroconverting by the end of the study. In contrast, a greater percentage of pigs exhibiting CT remained PCR positive throughout the growing phase, with less than one-third of these animals seroconverting. APPV RNA was present in multiple tissues from pigs in both groups at the time of marketing. This study improved our understanding of the infection dynamics of APPV in swine and the impact that the immune status and timing of infection have on the persistence of APPV in serum and tissues.

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.

Evaluation of a Porcine Endogenous Reference Gene (Internal Sample Control) in a Porcine Reproductive and Respiratory Syndrome Virus RT-qPCR.

Endogenous reference genes are used in gene-expression studies to "normalize" the results and, increasingly, as internal sample controls (ISC) in diagnostic quantitative polymerase chain reaction (qPCR). Three studies were conducted to evaluate the performance of a porcine-specific ISC in a commercial porcine reproductive and respiratory syndrome virus (PRRSV) reverse transcription-qPCR. Study 1 evaluated the species specificity of the ISC by testing serum from seven non-porcine domestic species (n = 34). In Study 2, the constancy of ISC detection over time (≥42 days) was assessed in oral fluid (n = 130), serum (n = 215), and feces (n = 132) collected from individual pigs of known PRRSV status. In Study 3, serum (n = 150), oral fluid (n = 150), and fecal samples (n = 75 feces, 75 fecal swabs) from commercial herds were used to establish ISC reference limits. Study 1 showed that the ISC was porcine-specific, i.e., all samples from non-porcine species were ISC negative (n = 34). In Study 2, the ISC was detected in all oral fluid, serum, and fecal samples, but differed in concentration between specimens (p < 0.05; mixed-effects regression model). The results of Study 3 were used to establish ISC reference limits for the 5th, 2.5th and 1.25th percentiles. Overall, the ISC response was consistent to the point that failure in detection is sufficient justification for re-testing and/or re-sampling.

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.

Modeling the accuracy of a novel PCR and antibody ELISA for African swine fever virus detection using Bayesian latent class analysis.

Introduction: Diagnostic test evaluation for African swine fever (ASF) in field settings like Vietnam is critical to understanding test application in intended populations for surveillance and control strategies. Bayesian latent class analysis (BLCA) uses the results of multiple imperfect tests applied to an individual of unknown disease status to estimate the diagnostic sensitivity and specificity of each test, forgoing the need for a reference test. Methods: Here, we estimated and compared the diagnostic sensitivity and specificity of a novel indirect ELISA (iELISA) for ASF virus p30 antibody (Innoceleris LLC.) and the VetAlert™ ASF virus DNA Test Kit (qPCR, Tetracore Inc.) in field samples from Vietnam by assuming that disease status 1) is known and 2) is unknown using a BLCA model. In this cross-sectional study, 398 paired, individual swine serum/oral fluid (OF) samples were collected from 30 acutely ASF-affected farms, 37 chronically ASF-affected farms, and 20 ASF-unaffected farms in Vietnam. Samples were tested using both diagnostic assays. Diagnostic sensitivity was calculated assuming samples from ASF-affected farms were true positives and diagnostic sensitivity by assuming samples from unaffected farms were true negatives. ROC curves were plotted and AUC calculated for each test/sample combination. For comparison, a conditionally dependent, four test/sample combination, three population BLCA model was fit. Results: When considering all assumed ASF-affected samples, qPCR sensitivity was higher for serum (65.2%, 95% Confidence Interval [CI] 58.1-71.8) and OF (52%, 95%CI 44.8-59.2) compared to the iELISA (serum: 42.9%, 95%CI 35.9-50.1; OF: 33.3%, 95%CI 26.8-40.4). qPCR-serum had the highest AUC (0.895, 95%CI 0.863-0.928). BLCA estimates were nearly identical to those obtained when assuming disease status and were robust to changes in priors. qPCR sensitivity was considerably higher than ELISA in the acutely-affected population, while ELISA sensitivity was higher in the chronically-affected population. Specificity was nearly perfect for all test/sample types. Discussion: The effect of disease chronicity on sensitivity and specificity could not be well characterized here due to limited data, but future studies should aim to elucidate these trends to understand the best use of virus and antibody detection methods for ASF. Results presented here will help the design of surveillance and control strategies in Vietnam and other countries affected by ASF.

Outbreak of Pathogenic Streptococcus equi subsp. zooepidemicus in Guinea Pigs Farms of The Andean Region.

Streptococcus zooepidemicus is an emerging zoonotic pathogen involved in septicemic infections in humans and livestock. Raising guinea pigs in South America is an important economic activity compared to raising them as pets in other countries. An outbreak of severe lymphadenitis was reported in guinea pigs from farms in the Andean region. S. zooepidemicus was isolated from multiple cervical and mandibular abscesses. Isolate was characterized by multilocus sequence typing and phylogenetic analysis. This is the first molecular characterization of a highly pathogenic strain, showing major important virulence factors such as the M-like protein genes szP and mlpZ, the fimbrial subunit protein gene fszF, and the protective antigen-like protein gene spaZ. Additionally, this guinea pig strain was phylogenetically related to equines but distant from zoonotic and pig isolates reported in other countries.

SARS-CoV-2 Is More Efficient than HCoV-NL63 in Infecting a Small Subpopulation of ACE2+ Human Respiratory Epithelial Cells.

Human coronavirus (HCoV)-NL63 is an important contributor to upper and lower respiratory tract infections, mainly in children, while severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, can cause lower respiratory tract infections, and more severe, respiratory and systemic disease, which leads to fatal consequences in many cases. Using microscopy, immunohistochemistry (IHC), virus-binding assay, reverse transcriptase qPCR (RT-qPCR) assay, and flow cytometry, we compared the characteristics of the susceptibility, replication dynamics, and morphogenesis of HCoV-NL63 and SARS-CoV-2 in monolayer cultures of primary human respiratory epithelial cells (HRECs). Less than 10% HRECs expressed ACE2, and SARS-CoV-2 seemed much more efficient than HCoV-NL63 at infecting the very small proportion of HRECs expressing the ACE2 receptors. Furthermore, SARS-CoV-2 replicated more efficiently than HCoV-NL63 in HREC, which correlates with the cumulative evidence of the differences in their transmissibility.