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Similar to influenza A and B, ICV infections are seen associated with bacterial and viral co-infections which complicates the assessment of its real clinical significance. Further, the antivirals against influenza A and B viruses are ineffective against ICV which mandates the need to study the pathobiological aspects of this virus.

Every day, thousands of samples from diverse populations of animals are submitted to veterinary diagnostic laboratories (VDLs) for testing. Each VDL has its own laboratory information management system (LIMS), with processes and procedures to capture submission information, perform laboratory tests, define the boundaries of test results (i.e., positive or negative), and report results, in addition to internal business and accounting applications. Enormous quantities of data are accumulated and stored within VDL LIMSs. There is a need for platforms that allow VDLs to exchange and share portions of laboratory data using standardized, reliable, and sustainable information technology processes. Here we report concepts and applications for standardization and aggregation of data from swine submissions to multiple VDLs to detect and monitor porcine enteric coronaviruses by RT-PCR. Oral fluids, feces, and fecal swabs were the specimens submitted most frequently for enteric coronavirus testing. Statistical algorithms were used successfully to scan and monitor the overall and state-specific percentage of positive submissions. Major findings revealed a consistently recurrent seasonal pattern, with the highest percentage of positive submissions detected during December–February for porcine epidemic diarrhea virus, porcine deltacoronavirus, and transmissible gastroenteritis virus (TGEV). After 2014, very few submissions tested positive for TGEV. Monitoring VDL data proactively has the potential to signal and alert stakeholders early of significant changes from expected detection. We demonstrate the importance of, and applications for, data organized and aggregated by using LOINC and SNOMED CTs, as well as the use of customized messaging to allow inter-VDL exchange of information.

In 2014, the hypothesis that feed ingredients could serve as vehicles for the transport and transmission of viral pathogens was proposed and evaluated by multiple investigators under laboratory conditions. In an attempt to validate these data, we used a demonstration project to test whether three significant viruses of swine could survive in feed ingredients under real‐world shipping conditions. Samples of soya bean meal (organic and conventional), lysine, choline and vitamin A were spiked with a mixture of PRRSV 174, PEDV and SVA and transported for 21 days in the trailer of a commercial transport vehicle, encompassing 14 states and 9,741 km. Samples were tested for viral genome and viability at the end of the transit period. Regarding viability, PRRSV, PEDV and SVA were all detected as infectious in bioassays following inoculation with both soy products. In addition, viable PRRSV and SVA were detected by bioassay pigs inoculated with samples of vitamin A, and infectious SVA was detected in pigs inoculated with samples of lysine and choline. These results provide further evidence that select viral pathogens of pigs can survive in certain feed ingredients during commercial transit.

We developed a model to predict the cyclic pattern of porcine reproductive and respiratory syndrome virus (PRRSV) RNA detection by reverse-transcription real-time PCR (RT-rtPCR) from 4 major swine-centric veterinary diagnostic laboratories (VDLs) in the United States and to use historical data to forecast the upcoming year’s weekly percentage of positive submissions and issue outbreak signals when the pattern of detection was not as expected. Standardized submission data and test results were used. Historical data (2015–2017) composed of the weekly percentage of PCR-positive submissions were used to fit a cyclic robust regression model. The findings were used to forecast the expected weekly percentage of PCR-positive submissions, with a 95% confidence interval (CI), for 2018. During 2018, the proportion of PRRSV-positive submissions crossed 95% CI boundaries at week 2, 14–25, and 48. The relatively higher detection on week 2 and 48 were mostly from submissions containing samples from wean-to-market pigs, and for week 14–25 originated mostly from samples from adult/sow farms. There was a recurring yearly pattern of detection, wherein an increased proportion of PRRSV RNA detection in submissions originating from wean-to-finish farms was followed by increased detection in samples from adult/sow farms. Results from the model described herein confirm the seasonal cyclic pattern of PRRSV detection using test results consolidated from 4 VDLs. Wave crests occurred consistently during winter, and wave troughs occurred consistently during the summer months. Our model was able to correctly identify statistically significant outbreak signals in PRRSV RNA detection at 3 instances during 2018.

Veterinary diagnostic laboratories annually derive thousands of nucleotide sequences from clinical samples of swine pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV), Senecavirus A, and swine enteric coronaviruses. In addition, next generation sequencing has resulted in the rapid production of full-length genomes. Presently, sequence data are released to diagnostic clients for the purposes of informing control measures, but are not publicly available as data may be associated with sensitive information. However, public sequence data can be used to objectively design field-relevant vaccines; determine when and how pathogens are spreading across the landscape; identify virus transmission hotspots; and are a critical component in genomic surveillance for pandemic preparedness. We have developed a centralized sequence database that integrates a selected set of previously private clinical data, using PRRSV data as an exemplar, alongside publicly available genomic information. We implemented the Tripal toolkit, using the open source Drupal content management system and the Chado database schema. Tripal consists of a collection of Drupal modules that are used to manage, visualize, and disseminate biological data stored within Chado. Hosting is provided by Amazon Web Services (AWS) EC2 cloud instance with resource scaling. New sequences sourced from diagnostic labs contain at a minimum four data items: genomic information; date of collection; collection location (state or province level); and a unique identifier. Users can download annotated genomic sequences from the database using a customized search interface that incorporates data mined from published literature; search for similar sequences using BLAST-based tools; and explore annotated reference genomes. Additionally, because the bulk of data presently are PRRSV sequences, custom curation and annotation pipelines have determined PRRSV genotype (Type 1 or 2), the location of open reading frames and nonstructural proteins, generated amino acid sequences, the occurrence of putative frame shifts, and restriction fragment length polymorphism (RFLP) classification of GP5 genes. Genomic data from seven major swine pathogens have been curated and annotated. The resource provides researchers timely access to sequences discovered by veterinary diagnosticians, allowing for epidemiological and comparative virology studies. The result will be a better understanding on the emergence of novel swine viruses in the United States (US), and how these novel strains are disseminated in the US and abroad.Database URLhttps://swinepathogendb.org

Bovine viral diarrhea viruses (BVDV) are significant pathogens of cattle, leading to losses associated with reproductive failure, respiratory disease and immune dysregulation. While cattle are the reservoir for BVDV, a wide range of domestic and wild ruminants are susceptible to infection and disease caused by BVDV. Samples from four American bison (Bison bison) from a captive herd were submitted for diagnostic testing due to their general unthriftiness. Metagenomic sequencing on pooled nasal swabs and serum identified co-infection with a BVDV and a bovine bosavirus. The BVDV genome was more similar to the vaccine strain Oregon C24 V than to other BVDV sequences in GenBank, with 92.7 % nucleotide identity in the open reading frame. The conserved 5'-untranslated region was 96.3 % identical to Oregon C24 V. Bosavirus has been previously identified in pooled fetal bovine serum but its clinical significance is unknown. Sequencing results were confirmed by virus isolation and PCR detection of both viruses in serum and nasal swab samples from two of the four bison. One animal was co-infected with both BVDV and bosavirus while separate individuals were positive solely for BVDV or bosavirus. Serum and nasal swabs from these same animals collected 51 days later remained positive for BVDV and bosavirus. These results suggest that both viruses can persistently infect bison. While the etiological significance of bosavirus infection is unknown, the ability of BVDV to persistently infect bison has implications for BVDV control and eradication programs. Possible synergy between BVDV and bosavirus persistent infection warrants further study.

Influenza remains a global health risk and challenge. Currently, neuraminidase (NA) inhibitors are extensively used to treat influenza, but their efficacy is compromised by the emergence of drug-resistant variants. Neutralizing antibodies targeting influenza A virus surface glycoproteins are critical components of influenza therapeutic agents and may provide alternative strategies to the existing countermeasures. However, the major hurdle for the extensive application of antibody therapies lies in the difficulty of generating nonimmunogenic antibodies in large quantities rapidly. Here, we report that one human monoclonal antibody (MAb), 53C10, isolated from transchromosomic (Tc) cattle exhibits potent neutralization and hemagglutination inhibition titers against different clades of H1N1 subtype influenza A viruses. In vitro selection of antibody escape mutants revealed that 53C10 recognizes a novel noncontinuous epitope in the hemagglutinin (HA) head domain involving three amino acid residues, glycine (G), serine (S), and glutamic acid (E) at positions 172, 207, and 212, respectively. The results of our experiments supported a critical role for substitution of arginine at position 207 (S207R) in mediating resistance to 53C10, while substitutions at either G172E or E212A did not alter antibody recognition and neutralization. The E212A mutation may provide structural stability for the epitope, while the substitution G172E probably compensates for loss of fitness introduced by S207R. Our results offer novel insights into the mechanism of action of MAb 53C10 and indicate its potential role in therapeutic treatment of H1 influenza virus infection in humans. IMPORTANCE Respiratory diseases caused by influenza viruses still pose a serious concern to global health, and neutralizing antibodies constitute a promising area of antiviral therapeutics. However, the potential application of antibodies is often hampered by the challenge in generating nonimmunogenic antibodies in large scale. In the present study, transchromosomic (Tc) cattle were used for the generation of nonimmunogenic monoclonal antibodies (MAbs), and characterization of such MAbs revealed one monoclonal antibody, 53C10, exhibiting a potent neutralization activity against H1N1 influenza viruses. Further characterization of the neutralization escape mutant generated using this MAb showed that three amino acid substitutions in the HA head domain contributed to the resistance. These findings emphasize the importance of Tc cattle in the production of nonimmunogenic MAbs and highlight the potential of MAb 53C10 in the therapeutic application against H1 influenza virus infection in humans.

Genetic sequencing, or DNA sequencing, using the Sanger technique has become widely used in the veterinary diagnostic community. This technology plays a role in verification of PCR results and is used to provide the genetic sequence data needed for phylogenetic analysis, epidemiologic studies, and forensic investigations. The Laboratory Technology Committee of the American Association of Veterinary Laboratory Diagnosticians has prepared guidelines for sample preparation, submission to sequencing facilities or instrumentation, quality assessment of nucleic acid sequence data performed, and for generating basic sequencing data and phylogenetic analysis for diagnostic applications. This guidance is aimed at assisting laboratories in providing consistent, high-quality, and reliable sequence data when using Sanger-based genetic sequencing as a component of their laboratory services.

The goals of this study were to compare the pathogenicity and infection dynamics of a historical and a contemporary SVA strains (SVV 001 and SD15-26) and to assess cross-neutralizing and cross-reactive T cell responses following experimental infection in pigs. Both SVA strains successfully infected all inoculated animals, resulting in viremia and robust antibody and cellular immune responses. SVA SD15-26 infection resulted in characteristic clinical signs and vesicular lesions, however, SVA SVV 001 did not cause overt clinical disease with inoculated animals remaining clinically normal during the experiment. Notably, neutralization- and -recall IFN-γ expression-assays revealed marked cross-neutralizing antibody and cross-reactive T cell responses between the two viral strains. Together these results demonstrate that the historical SVA SVV 001 strain presents low virulence in pigs when compared to the contemporary SVA SD15-26 strain. Additionally, immunological assays indicate that SVA SVV 001 and SD15-26 are antigenically related and share conserved antigenic determinants.

Bovine herpesvirus 1 (BoHV-1) has long been associated with reproductive failure in cattle following infection of the ovary and/or fetus. Vaccination prior to breeding has been an effective approach to lessen the impact of BoHV-1 on reproduction. Prior studies in the 1980s and 1990s established the susceptibility of the ovary and particularly the corpus luteum (CL) to BoHV-1 infection. A series of studies at breeding time established that: (1) in naïve animals, the CL was the major target of BoHV-1 pathology; (2) CL lesions occurred within 4-9 days after estrus; (3) similar lesions was seen with BoHV-1 MLV vaccines; (4) ovarian lesions varied by the vaccine strain used; (5) progesterone decreased with or without CL lesions; and (6) following reactivation of BoHV-1 latent infection, ovaries could become reinfected in the face of BoHV-1 immunity. Large scale field studies demonstrated that conception was highest in animals previously vaccinated and boostered with inactivated vaccine compared to animals revaccinated with MLV. In the early 2000s, to get a label claim to vaccinate calves nursing pregnant cows, safety study outlines were approved by USDA-APHIS CVB. These studies were designed to determine the effect of revaccination with MLV during pregnancy on previously vaccinated cows and were not rigorous enough to confirm complete fetal safety. As designed these studies showed no difference in reproductive loss between the previously vaccinated animals and the animals revaccinated ∼4, 7 and 9 months later, leading to the label approval for MLV vaccination in pregnant cows. Subsequent investigations by diagnostic laboratories found an increase in BoHV-1 reproductive loss after the approval for use in pregnant animals. A method was developed to differentiate IBR vaccine strains from field strains. Analysis of viruses from 31 cases from 2009-2016 indicated that all 31 isolates matched with vaccine strains. Going forward, it will be necessary to develop vaccine approaches that use non-abortifacient, nonlatent BoHV-1 vaccines that develop lifelong immunity, protecting the animal while doing no harm to the fetus.

The use of indium tin oxide (ITO) and focused monomode microwave heating for the ultra-rapid crystallization of L-alanine (a model amino acid) is reported. Commercially available ITO dots (< 5 mm) attached to blank poly(methyl)methacrylate (PMMA, 5 cm in diameter with 21-well silicon isolators: referred to as the iCrystal plates) were found to withstand prolonged microwave heating during crystallization experiments. Crystallization of L-alanine was performed at room temperature (a control experiment), with the use of two microwave sources: a 2.45 GHz conventional microwave (900 W, power level 1, a control experiment) and 8 GHz (20 W) solid state, monomode microwave source with an applicator tip that focuses the microwave field to a 5-mm cavity. Initial appearance of L-alanine crystals and on iCrystal plates with ITO dots took 47 ± 2.9 min, 12 ± 7.6 min and 1.5 ± 0.5 min at room temperature, using a conventional microwave and focused monomode microwave heating, respectively. Complete evaporation of the solvent using the focused microwaves was achieved in 3.2 ± 0.5 min, which is ~52-fold and ~172-fold faster than that observed at room temperature and using conventional microwave heating, respectively. The size and number of L-alanine crystals was dependent on the type of the 21-well iCrystal plates and the microwave heating method: 33 crystals of 585 ± 137 μm in size at room temperature > 37 crystals of 542 ± 100 μm in size with conventional microwave heating > 331 crystals of 311 ± 190 μm in size with focused monomode microwave. FTIR, optical microscopy and powder X-ray diffraction analysis showed that the chemical composition and crystallinity of the L-alanine crystals did not change when exposed to microwave heating and ITO surfaces. In addition, theoretical simulations for the binding of L-alanine molecules to ITO and other metals showed the predicted nature of hydrogen bonds formed between L-alanine and these surfaces.

This project investigates the macroepidemiological aspects of porcine reproductive and respiratory syndrome virus (PRRSV) RNA detection by veterinary diagnostic laboratories (VDLs) for the period 2007 through 2018. Standardized submission data and PRRSV real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) test results from porcine samples were retrieved from four VDLs representing 95% of all swine samples tested in NAHLN laboratories in the US. Anonymized data were retrieved and organized at the case level using SAS (SAS® Version 9.4, SAS® Institute, Inc., Cary, NC) with the use of PROC DATA, PROC MERGE, and PROC SQL scripts. The final aggregated and anonymized dataset comprised of 547,873 unique cases was uploaded to Power Business Intelligence-Power BI® (Microsoft Corporation, Redmond, Washington) to construct dynamic charts. The number of cases tested for PRRSV doubled from 2010 to 2018, with that increase mainly driven by samples typically used for monitoring purposes rather than diagnosis of disease. Apparent seasonal trends for the frequency of PRRSV detection were consistently observed with a higher percentage of positive cases occurring during fall or winter months and lower during summer months, perhaps due to increased testing associated with well-known seasonal occurrence of swine respiratory disease. PRRSV type 2, also known as North American genotype, accounted for 94.76% of all positive cases and was distributed across the US. PRRSV type 1, also known as European genotype, was geographically restricted and accounted for 2.15% of all positive cases. Co-detection of both strains accounted for 3.09% of the positive cases. Both oral fluid and processing fluid samples, had a rapid increase in the number of submissions soon after they were described in 2008 and 2017, respectively, suggesting rapid adoption of these specimens by the US swine industry for PRRSV monitoring in swine populations. As part of this project, a bio-informatics tool defined as Swine Disease Reporting System (SDRS) was developed. This tool has real-time capability to inform the US swine industry on the macroepidemiological aspects of PRRSV detection, and is easily adaptable for other analytes relevant to the swine industry.

Physical stability of synthetic skin samples during their exposure to microwave heating was investigated to demonstrate the use of the metal-assisted and microwave-accelerated decrystallization (MAMAD) technique for potential biomedical applications. In this regard, optical microscopy and temperature measurements were employed for the qualitative and quantitative assessment of damage to synthetic skin samples during 20 s intermittent microwave heating using a monomode microwave source (at 8 GHz, 2–20 W) up to 120 s. The extent of damage to synthetic skin samples, assessed by the change in the surface area of skin samples, was negligible for microwave power of ≤7 W and more extensive damage (>50%) to skin samples occurred when exposed to >7 W at initial temperature range of 20–39 °C. The initial temperature of synthetic skin samples significantly affected the extent of change in temperature of synthetic skin samples during their exposure to microwave heating. The proof of principle use of the MAMAD technique was demonstrated for the decrystallization of a model biological crystal (l-alanine) placed under synthetic skin samples in the presence of gold nanoparticles. Our results showed that the size (initial size ∼850 μm) of l-alanine crystals can be reduced up to 60% in 120 s without damage to synthetic skin samples using the MAMAD technique. Finite-difference time-domain-based simulations of the electric field distribution of an 8 GHz monomode microwave radiation showed that synthetic skin samples are predicted to absorb ∼92.2% of the microwave radiation.

The crystal morphology of amino acids can be altered in a controlled manner through inclusion of tailor-made additives in their structure, in order to widen their scope for applications in drug design and targeted delivery. In this study, the effect of multiadditive combinations of hydrophobic and hydrophilic amino acids on the growth and morphology of l-alanine was investigated. Theoretical calculations were performed using two crystal growth models in Materials Studio software: (1) build-in model; (2) surface docking model. Crystallization experiments were carried out using the metal-assisted and microwave accelerated evaporative crystallization (MA-MAEC) technique with multiple hydrophobic and hydrophilic amino acids added in stoichiometric amounts to l-alanine solution. The crystal morphology was established and compared with predicted crystal morphology. The use of hydrophilic and hydrophobic additives was predicted to have significant changes in the morphology of l-alanine crystals. Multiadditive co...

Senecavirus A (SVA) is an emerging picornavirus that has been recently associated with an increased number of outbreaks of vesicular disease and neonatal mortality in swine. Many aspects of SVA infection biology and epidemiology remain unknown. Here, we present a diagnostic investigation conducted in swine herds affected by vesicular disease and increased neonatal mortality. Clinical and environmental samples were collected from affected and unaffected herds and were screened for the presence of SVA by real-time reverse transcriptase PCR and virus isolation. Notably, SVA was detected and isolated from vesicular lesions and tissues of affected pigs, environmental samples, mouse feces, and mouse small intestine. SVA nucleic acid was also detected in houseflies collected from affected farms and from a farm with no history of vesicular disease. Detection of SVA in mice and housefly samples and recovery of viable virus from mouse feces and small intestine suggest that these pests may play a role on the epidemiology of SVA. These results provide important information that may allow the development of improved prevention and control strategies for SVA.

This study investigated viruses in bovine respiratory disease (BRD) cases in feedlots, including bovine herpesvirus-1 (BoHV-1), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine coronaviruses (BoCV) and parainfluenza-3 virus (PI3V). Nasal swabs were collected from 114 cattle on initial BRD treatment. Processing included modified live virus (MLV) vaccination. Seven BRD necropsy cases were included for 121 total cases. Mean number of days on feed before first sample was 14.9 days. Swabs and tissue homogenates were tested by gel based PCR (G-PCR), quantitative-PCR (qPCR) and quantitative real time reverse transcriptase PCR (qRT-PCR) and viral culture. There were 87/114 (76.3%) swabs positive for at least one virus by at least one test. All necropsy cases were positive for at least one virus. Of 121 cases, positives included 18/121 (14.9%) BoHV-1; 19/121 (15.7%) BVDV; 76/121 (62.8%) BoCV; 11/121 (9.1%) BRSV; and 10/121 (8.3%) PI3V. For nasal swabs, G-PCR (5 viruses) detected 44/114 (38.6%); q-PCR and qRT-PCR (4 viruses) detected 81/114 (71.6%); and virus isolation detected 40/114 (35.1%). Most were positive for only one or two tests, but not all three tests. Necropsy cases had positives: 5/7 G-PCR, 5/7 q-PCR and qRT-PCR, and all were positive by cell culture. In some cases, G-PCR and both real time PCR were negative for BoHV-1, BVDV, and PI3V in samples positive by culture. PCR did not differentiate field from vaccines strains of BoHV-1, BVDV, and PI3V. However based on sequencing and analysis, field and vaccine strains of culture positive BoHV-1, BoCV, BVDV, and PI3V, 11/18 (61.1%) of BoHV-1 isolates, 6/17 (35.3%) BVDV isolates, and 1/10 (10.0%) PI3V identified as vaccine. BRSV was only identified by PCR testing. Interpretation of laboratory tests is appropriate as molecular based tests and virus isolation cannot separate field from vaccine strains. Additional testing using sequencing appears appropriate for identifying vaccine strains.

Highlights • Molecular assays such as rRT-PCR are the method of choice for PEDV diagnosis. • Multiplex rRT-PCR allow simultaneous testing for PEDV, TGEV and PDCoV. • Serological assays provide valuable information on previous exposure to PEDV and population immunity., Porcine epidemic diarrhea virus (PEDV) is the causative agent of an acute, highly contagious, and severe enteric disease that leads to high mortality rates in suckling piglets. Therefore, accurate diagnosis of PEDV infection is critical for the implementation of control measures for the virus. Many diagnostic tests have been recently developed and are currently available for the detection of PEDV, its proteins or nucleic acid, including virus isolation, immunofluorescence (IF) or immunohistochemistry (IHC), polymerase chain reaction (PCR) and isothermal amplification assays. Additionally, several serological assays have been developed and are currently used for the detection of antibodies against PEDV. Molecular assays such as real-time reverse transcriptase-PCR (rRT-PCR) became the methods of choice for the diagnosis of PEDV infection, providing sensitive, specific and rapid detection of the virus RNA in clinical samples. Whereas serological assays have been widely used to monitor prior exposure to the virus and to evaluate the efficacy of novel vaccine candidates or vaccination strategies. Here we discuss the properties of current PEDV diagnostic assays and prospects for improving diagnostic strategies in the future.