Evaluating dry vs. wet disinfection in boot baths on detection of porcine epidemic diarrhea virus and porcine reproductive and respiratory virus RNA.

Maintaining biosecurity between swine barns is challenging, and boot baths are an easily implementable option some utilize to limit pathogen spread. However, there are concerns regarding their efficacy, especially when comparing wet or dry disinfectants. The objective of this study was to evaluate the efficacy of boot baths in reducing the quantity of detectable porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) genetic material using wet or dry disinfectants. Treatments included 1) control, 2) dry chlorine powder (Traffic C.O.P., PSP, LLC, Rainsville, AL), and 3) wet quaternary ammonium/glutaraldehyde liquid (1:256 Synergize, Neogen, Lexington, KY). Prior to disinfection, rubber boots were inoculated with 1 mL of a co-inoculants of PRRSV (1 × 105 TCID50 per mL) and PEDV (1 × 105 TCID50 per mL) and dried for 15 min. After the drying period, a researcher placed the boot on the right foot and stepped directly on a stainless steel coupon (control). Alternatively, the researcher stepped first into a boot bath containing either the wet or dry sanitizer, stood for 3 s, and then stepped onto a steel coupon. After one minute, an environmental swab was then collected and processed from each boot and steel coupon. The procedure was replicated 12 times per disinfectant treatment. Samples were analyzed using a duplex qPCR at the Kansas State Veterinary Diagnostic Laboratory. Cycle threshold values were analyzed using SAS GLIMMIX v 9.4 (SAS, Inc., Cary, NC). There was no evidence of a disinfectant × surface × virus interaction (P > 0.10). An interaction between disinfectant × surface impacted (P < 0.05) the quantity of detectable viral RNA. As expected, the quantity of the viruses on the coupon was greatest in the control, indicating that a contaminated boot has the ability to transfer viruses from a contaminated surface to a clean surface. Comparatively, the dry disinfectant treatment resulted in no detectable viral RNA on either the boot or subsequent coupon. The wet disinfectant treatment had statistically similar (P > 0.05) viral contamination to the control on the boot, but less viral contamination compared to the control on the metal coupon. In this experiment, a boot bath with dry powder was the most efficacious in reducing the detectable viral RNA on both boots and subsequent surfaces.

Development of a three-panel multiplex real-time PCR assay for simultaneous detection of nine canine respiratory pathogens.

Infectious respiratory disease is one of the most common diseases in dogs worldwide. Several bacterial and viral pathogens can serve as causative agents of canine infectious respiratory disease (CIRD), including Mycoplasma cynos, Mycoplasma canis, Bordetella bronchiseptica, canine adenovirus type 2 (CAdV-2), canine herpesvirus 1 (CHV-1), canine parainfluenza virus (CPIV), canine distemper virus (CDV), canine influenza virus (CIA) and canine respiratory coronavirus (CRCoV). Since these organisms cause similar clinical symptoms, disease diagnosis based on symptoms alone can be difficult. Therefore, a quick and accurate test is necessary to rapidly identify the presence and relative concentrations of causative CIRD agents. In this study, a multiplex real-time PCR panel assay was developed and composed of three subpanels for detection of the aforementioned pathogens. Correlation coefficients (R2) were >0.993 for all singleplex and multiplex real-time PCR assays with the exception of one that was 0.988; PCR amplification efficiencies (E) were between 92.1% and 107.8% for plasmid DNA, and 90.6-103.9% for RNA templates. In comparing singular and multiplex PCR assays, the three multiplex reactions generated similar R2 and E values to those by corresponding singular reactions, suggesting that multiplexing did not interfere with the detection sensitivities. The limit of detection (LOD) of the multiplex real-time PCR for DNA templates was 5, 2, 3, 1, 1, 1, 4, 24 and 10 copies per microliter for M. cynos, M. canis, B. brochiseptica, CAdV-2, CHV-1, CPIV, CDV, CIA and CRCoV, respectively; and 3, 2, 6, 17, 4 and 8 copies per microliter for CAdV-2, CHV-1, CPIV, CDV, CIA and CRCoV, respectively, when RNA templates were used for the four RNA viruses. No cross-detection was observed among the nine pathogens. For the 740 clinical samples tested, the newly designed PCR assay showed higher diagnostic sensitivity compared to an older panel assay; pathogen identities from selected samples positive by the new assay but undetected by the older assay were confirmed by Sanger sequencing. Our data showed that the new assay has higher diagnostic sensitivity while maintaining the assay's specificity, as compared to the older version of the panel assay.

Assessment of porcine Rotavirus-associated virome variations in pigs with enteric disease.

Enteric disease is the predominant cause of morbidity and mortality in young mammals including pigs. Viral species involved in porcine enteric disease complex (PEDC) include rotaviruses, coronaviruses, picornaviruses, astroviruses and pestiviruses among others. The virome of three groups of swine samples submitted to the Kansas State University Veterinary Diagnostic Laboratory for routine testing were assessed, namely, a Rotavirus A positive (RVA) group, a Rotavirus co-infection (RV) group and a Rotavirus Negative (RV Neg) group. All groups were designated by qRT-PCR test results for Porcine Rotavirus A, B, C and H such that samples positive for RVA only went in the RVA group, samples positive for > 1 rotavirus went in the RV group and samples negative for all were grouped in the RVNeg group. All of the animals had clinical enteric disease resulting in scours and swollen joints/lameness, enlarged heart and/or a cough. All samples were metagenomic sequenced and analyzed for viral species composition that identified 14 viral species and eight bacterial viruses/phages. Sapovirus and Escherichia coli phages were found at a high prevalence in RVA and RV samples but were found at low or no prevalence in the RVNeg samples. Picobirnavirus was identified at a high proportion and prevalence in RVNeg and RV samples but at a low prevalence in the RVA group. Non-rotaviral diversity was highest in RVA samples followed by RV then RV Neg samples. A sequence analysis of the possible host of Picobirnaviruses revealed fungi as the most likely host. Various sequences were extracted from the sample reads and a phylogenetic update was provided showing a high prevalence of G9 and P[23] RVA genotypes. These data are important for pathogen surveillance and control measures.

Molecular detection of SARS-CoV-2 and differentiation of Omicron and Delta variant strains.

The SARS-CoV-2 virus is the causative agent of COVID-19 and has undergone continuous mutations throughout the pandemic. The more transmissible Omicron variant has quickly spread and is replacing the Delta variant as the most prevalent strain globally, including in the United States. A new molecular assay that can detect and differentiate both the Delta and Omicron variants was developed. A collection of 660,035 SARS-CoV-2 full- or near-full genomes, including 169,454 Delta variant and 24,202 Omicron variant strains, were used for primer and probe designs. In silico data analysis predicted an assay coverage of >99% of all strains, including >99% of the Delta and >99% of Omicron strains. The Omicron variant differential test was designed based on the Δ31-33 aa deletion in the N-gene, which is present in the original B.1.1.529 main genotype, BA.1, as well as in BA.2 and BA.3 subtypes. Therefore, the assay should detect the majority of all Omicron variant strains. Standard curves generated with human clinical samples indicated that the PCR amplification efficiencies were 104%, 90.7% and 90.4% for the Omicron, Delta, and non-Delta/non-Omicron wild-type genotypes, respectively. Correlation coefficients of the standard curves were all >0.99. The detection limit of the assay was 14.3, 32.0, and 21.5 copies per PCR reaction for Omicron, Delta, and wild-type genotypes, respectively. The assay was designed to specifically detect SAR-CoV-2 strains. Selected samples with Omicron, Delta and wild-type genotypes identified by the RT-qPCR assay were also confirmed by sequencing. The assay did not detect any animal coronavirus-positive samples that were tested. Human nasal swab samples that previously tested positive (n = 182) or negative (n = 42) for SARS-CoV-2 by the ThermoFisher TaqPath COVID-19 Combo Kit, produced the same result with the new assay. Among positive samples, 55.5% (101/182), 23.1% (42/182), and 21.4% (39/182) were identified as Omicron, Delta, and non-Omicron/non-Delta wild-type genotypes, respectively.

Molecular detection of SARS-CoV-2 strains and differentiation of Delta variant strains.

The Delta variant of SARS-CoV-2 has now become the predominant strain in the global COVID-19 pandemic. Strain coverage of some detection assays developed during the early pandemic stages has declined due to periodic mutations in the viral genome. We have developed a real-time RT-PCR (RT-qPCR) for SARS-CoV-2 detection that provides nearly 100% strain coverage, and differentiation of highly transmissible Delta variant strains. All full or nearly full (≥28 kb) SARS-CoV-2 genomes (n = 403,812), including 6422 Delta and 280 Omicron variant strains, were collected from public databases at the time of analysis and used for assay design. The two amino acid deletions in the spike gene (S-gene, Δ156-157) that is characteristic of the Delta variant were targeted during the assay design. Although strain coverage for the Delta variant was very high (99.7%), detection coverage for non-Delta wild-type strains was 93.9%, mainly due to the confined region of design. To increase strain coverage of the assay, the design for CDC N1 target was added to the assay. In silico analysis of 403,812 genomes indicated a 95.4% strain coverage for the CDC N1 target, however, in combination with our new non-Delta S-gene target, total coverage for non-Delta wild-type strains increased to 99.8%. A human 18S rRNA gene was also analyzed and used as an internal control. The final four-plex RT-qPCR assay generated PCR amplification efficiencies between 95.4% and 102.0% with correlation coefficients (R2 ) of >0.99 for cloned positive controls; Delta and non-Delta human clinical samples generated PCR efficiencies of 93.4%-97.0% and R2  > 0.99. The assay also detects 98.6% of 280 Omicron sequences. Assay primers and probes have no match to other closely related human coronaviruses, and did not produce a signal from samples positive to selected animal coronaviruses. Genotypes of selected clinical samples identified by the RT-qPCR were confirmed by Sanger sequencing.

Identification of the SARS-CoV-2 Delta variant C22995A using a high-resolution melting curve RT-FRET-PCR.

Knowledge of SARS-CoV-2 variants is essential for formulating effective control policies. Currently, variants are only identified in relatively small percentages of cases as the required genome sequencing is expensive, time-consuming, and not always available. In countries with facilities to sequence the SARS-CoV-2, the Delta variant currently predominates. Elsewhere, the prevalence of the Delta variant is unclear. To avoid the need for sequencing, we investigated a RT-FRET-PCR that could detect all SARS-CoV-2 strains and simultaneously identify the Delta variant. The established Delta RT-FRET-PCR was performed on reference SARS-CoV-2 strains, and human nasal swab samples positive for the Delta and non-Delta strains. The Delta RT-FRET-PCR established in this study detected as few as ten copies of the DNA target and 100 copies of RNA target per reaction. Melting points of products obtained with SARS-CoV-2 Delta variants (around 56.1°C) were consistently higher than products obtained with non-Delta strains (around 52.5°C). The Delta RT-FRET-PCR can be used to diagnose COVID-19 patients and simultaneously identify if they are infected with the Delta variant. The Delta RT-FRET-PCR can be performed with all major thermocycler brands meaning data on Delta variant can now be readily generated in diagnostic laboratories worldwide.

Near-Complete Genome of SARS-CoV-2 Delta (AY.3) Variant Identified in a Dog in Kansas, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) descriptions of infection and transmission have been increasing in companion animals in the past year. Although canine susceptibility is generally considered low, their role in the COVID-19 disease cycle remains unknown. In this study, we detected and sequenced a delta variant (AY.3) from a 12-year-old Collie living with owners that previously tested positive for SARS-CoV-2. It is unclear if the dogs' symptoms were related to SARS-CoV-2 infection or underlying conditions. The whole genome sequence obtained from the dog sample had several unique consensus level changes not previously identified in a SARS-CoV-2 genome that may play a role in the rapid adaptation from humans to dogs. Within the spike coding region, 5/7 of the subconsensus variants identified in the dog sequence were also identified in the closest in-house human reference case. Taken together, the whole genome sequence, and phylogenetic and subconsensus variant analyses indicate the virus infecting the animal originated from a local outbreak cluster. The results of these analyses emphasize the importance of rapid detection and characterization of SARS-CoV-2 variants of concern in companion animals.

Cover Image, Volume 93, Number 9, September 2021

Front Cover Caption: The cover image is based on the Research Article High-resolution melting curve FRET-PCR rapidly identifies SARS-CoV-2 mutations by Subarna Barua et al., https://doi.org/10.1002/jmv.27139.

High-resolution melting curve FRET-PCR rapidly identifies SARS-CoV-2 mutations.

Reverse transcription fluorescence resonance energy transfer-polymerase chain reaction (FRET-PCRs) were designed against the two most common mutations in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) (A23403G in the spike protein; C14408T in the RNA-dependent RNA polymerase). Based on high-resolution melting curve analysis, the reverse transcription (RT) FRET-PCRs identified the mutations in american type culture collection control viruses, and feline and human clinical samples. All major makes of PCR machines can perform melting curve analysis and thus further specifically designed FRET-PCRs could enable active surveillance for mutations and variants in countries where genome sequencing is not readily available.

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.

Development of a real-time PCR assay for detection of African swine fever virus with an endogenous internal control.

Real-time PCR assays are highly sensitive, specific and rapid techniques for the identification of ASF virus (ASFV) (Section 3.8, OIE Terrestrial Manual, 2019). Although an ASFV p72 gene-based real-time PCR assay (a.k.a. the Zsak assay) (Journal of Clinical Microbiology, 2005, 43, 112) has been widely used for ASFV detection, several more ASFV whole genome sequences have become available in the 15 years since the design of the Zsak assay. In this study, we developed a new ASFV p72 gene-based real-time PCR after analysis of all currently available sequences of the p72 gene and multiplexed the new assay with a modified Zsak assay aiming to have a broader coverage of ASFV strain/isolates. To reduce false-negative detections, porcine house-keeping gene, beta actin (ACTB), was applied as an internal control. Eight ACTB sequences from the GenBank and 61 partial ACTB sequences generated in this study, and 1,012 p72 sequences from the GenBank and 23 p72 sequences generated at FADDL, were used for ACTB and ASFV primer and probe designs, respectively, to ensure broader host and ASFV coverage. Multiplexing ACTB in the reaction did not affect ASFV amplification. The multiplex assay was evaluated for strain/isolate coverage, sensitivity and specificity. The in silico analysis showed high ASFV strain/isolate coverage: 98.4% (978/994) of all p72 sequences currently available. The limit of detection (LOD) was 6 plasmid copies or 0.1-1 TCID50 /ml of ASFV isolates per reaction. Only targeted ASFV isolates and the viruses in the positive clinical samples were detected, indicating that the assay is highly specific (100% specificity). The test results of 26 ASFV isolates with different country origins showed that this newly developed multiplex assay performed better than the Zsak assay that has been widely accepted and used worldwide, indicating that it may be used as an alternative assay for ASFV detection.