Refining PRRSV-2 genetic classification based on global ORF5 sequences and investigation of their geographic distributions and temporal changes.

IMPORTANCE: In this study, comprehensive analysis of 82,237 global porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) open reading frame 5 sequences spanning from 1989 to 2021 refined PRRSV-2 genetic classification system, which defines 11 lineages and 21 sublineages and provides flexibility for growth if additional lineages, sublineages, or more granular classifications are needed in the future. Geographic distribution and temporal changes of PRRSV-2 were investigated in detail. This is a thorough study describing the molecular epidemiology of global PRRSV-2. In addition, the reference sequences based on the refined genetic classification system are made available to the public for future epidemiological and diagnostic applications worldwide. The data from this study will facilitate global standardization and application of PRRSV-2 genetic classification.

Development, Evaluation, and Clinical Application of PRRSV-2 Vaccine-like Real-Time RT-PCR Assays.

In this study, we developed and validated (1) singleplex real-time RT-PCR assays for specific detection of five PRRSV-2 MLV vaccine viruses (Ingelvac MLV, Ingelvac ATP, Fostera, Prime Pac, and Prevacent) and (2) a four-plex real-time RT-PCR assay (IngelvacMLV/Fostera/Prevacent/XIPC) including the internal positive control XIPC for detecting and distinguishing the three most commonly used vaccines in the USA (Prevacent, Ingelvac MLV, and Fostera). The singleplex and 4-plex vaccine-like PCRs and the reference PCR (VetMAXTM PRRSV NA&EU, Thermo Fisher Scientific, Waltham, MA, USA) did not cross-react with non-PRRSV swine viral and bacterial pathogens. The limits of detection of vaccine-like PCRs ranged from 25 to 50 genomic copies/reactions. The vaccine-like PCRs all had excellent intra-assay and inter-assay repeatability. Based on the testing of 531 clinical samples and in comparison to the reference PCR, the diagnostic sensitivity, specificity, and agreement were in the respective range of 94.67-100%, 100%, and 97.78-100% for singleplex PCRs and 94.94-100%, 100%, and 97.78-100% for the 4-plex PCR, with a CT cutoff of 37. In addition, 45 PRRSV-2 isolates representing different genetic lineages/sublineages were tested with the vaccine-like PCRs and the results were verified with sequencing. In summary, the vaccine-like PCRs specifically detect the respective vaccine-like viruses with comparable performances to the reference PCR, and the 4-plex PCR allows to simultaneously detect and differentiate the three most commonly used vaccine viruses in the same sample. PRRSV-2 vaccine-like PCRs provide an additional tool for detecting and characterizing PRRSV-2.

Porcine Respiratory Coronavirus (PRCV): Isolation and Characterization of a Variant PRCV from USA Pigs.

Porcine respiratory coronavirus (PRCV), a mutant of the transmissible gastroenteritis virus (TGEV), was first reported in Belgium in 1984. PRCV typically replicates and induces mild lesions in the respiratory tract, distinct from the enteric tropism of TGEV. In the past 30 years, PRCV has rarely been studied, and most cited information is on traditional isolates obtained during the 1980s and 1990s. Little is known about the genetic makeup and pathogenicity of recent PRCV isolates. The objective of this study was to obtain a contemporary PRCV isolate from US pigs for genetic characterization. In total, 1245 lung homogenate samples from pigs in various US states were tested via real-time PCR targeting PRCV and TGEV RNA. Overall, PRCV RNA was detected in five samples, and a single isolate (ISU20-92330) was successfully cultured and sequenced for its full-length genome. The isolate clustered with a new group of variant TGEVs and differed in various genomic regions compared to traditional PRCV isolates. Pathogens, such as PRCV, commonly circulate in pig herds without causing major disease. There may be value in tracking genomic changes and regularly updating the diagnostic methods for such viruses to be better prepared for the emergence of variants in ecology and pathogenicity.

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.

Characterization of PRRSV in clinical samples and the corresponding cell culture isolates.

Isolation of porcine reproductive and respiratory syndrome virus (PRRSV) in cell culture is a primary means of obtaining virus isolates for autogenous vaccine production and other applications. However, it has not been well characterized whether cell culture isolate and the virus in clinical sample are equivalent. This study compared PRRSV ORF5 sequences from 1024 clinical samples (995 PRRSV-2, 26 PRRSV-1, and three PRRSV-1 and PRRSV-2 PCR-positive) and their isolates in MARC-145 and/or ZMAC cells. For three PRRSV-1 and PRRSV-2 PCR-positive clinical samples, both PRRSV-1 and PRRSV-2 were isolated in ZMAC cells, whereas either PRRSV-1 or PRRSV-2, but not both, was isolated in MARC-145 cells, with isolate sequences matching the respective viruses in clinical samples. Twenty-six PRRSV-1 and most of 995 PRRSV-2 PCR-positive clinical samples had matching viral ORF5 sequences with their cell culture isolates. However, 14 out of 995 PRRSV-2 cases (1.4%) had nonmatching viral sequences between clinical samples and MARC-145 isolates, although viral sequences from clinical samples and ZMAC isolates matched. This is concerning because, if the MARC-145 isolate is directly used for autogenous vaccine production without sequencing confirmation against the virus in the clinical sample, it is possible that the produced autogenous vaccine does not include the desired wild-type virus strain found on the farm and instead contains vaccine-like virus. Vaccine-specific PCR and next-generation sequencing performed on six selected cases indicated presence of ≥2 PRRSV-2 strains (mixed infection) in such clinical samples. In summary, PRRSV ORF5 sequences from clinical samples and cell culture isolates matched each other for majority of the cases. However, PRRSV sequences between clinical sample and MARC-145 cell culture isolate could occasionally be different when the clinical sample contains ≥2 PRRSV-2 strains. Characterizing PRRSV sequences from clinical samples and cell culture isolates should be conducted before using isolates for producing autogenous vaccines or other applications.

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.

Molecular characterization of emerging variants of PRRSV in the United States: new features of the -2/-1 programmed ribosomal frameshifting signal in the nsp2 region.

In this study, we characterized an emerging porcine reproductive and respiratory syndrome virus (PRRSV) isolate UIL21-0712, which is a lineage 1C variant with ORF5 restriction fragment length polymorphism (RFLP) cutting pattern of 1-4-4. The UIL21-0712 genome sequence has 85.3% nucleotide identity with the prototypic PRRSV-2 strain VR2332. The nsp2 region is the most variable, and the -2/-1 programmed ribosome frameshifting (PRF) signal therein is distinct from historical PRRSV strains. Analysis of PRRSV sequences in GenBank revealed that the majority of the emerging PRRSV variants contain substitutions that disrupt the -1 PRF stop codon to generate a nsp2N protein with a C-terminal extension. Two of the -1 PRF stop codon variant patterns were identified to be predominantly circulating in the field. They demonstrated higher growth kinetics than the other variants, suggesting that the most dominant -1 PRF stop codon variant patterns may provide enhanced growth fitness for the virus.

Development and validation of a reverse transcription real-time PCR assay for specific detection of PRRSGard vaccine-like virus.

Increasing use of modified live virus (MLV) vaccines presents challenges to interpret positive results of porcine reproductive and respiratory syndrome virus (PRRSV) screening PCR that can detect both wild-type and vaccine strains. Instead, vaccine-specific PCR provides a convenient tool to detect vaccine-like virus from a sample. Here we report the development and validation of a real-time RT-PCR specific for PRRSGard® , a newly available commercial PRRSV-2 MLV vaccine. Analytical specificity, sensitivity and diagnostic performance of PRRSGard PCR were evaluated and compared to a commercial PRRSV screening PCR (reference PCR). PRRSGard and reference PCRs did not cross-react with any of the 27 non-PRRSV swine pathogens. PRRSGard PCR did not cross-react with other PRRSV-2 vaccine viruses and 31 laboratory and field PRRSV-2 isolates representing various genetic lineages of PRRSV-2. PRRSGard and reference PCRs consistently detected up to 10-6 and 10-5 dilutions of PRRSGard vaccine virus, respectively. Based on testing serial dilutions of in vitro transcribed RNA, the 95% limit of detection of PRRSGard PCR was 16 genomic copies/reaction with CT cut-off value of 36 and 7 genomic copies/reaction with CT cut-off value of 37. Diagnostic performance of PRRSGard PCR was evaluated using 846 clinical samples (684 serum and 162 oral fluid samples). Compared to the reference screening PCR, diagnostic sensitivity, specificity and agreement of PRRSGard PCR were 95.34%, 98.85% and 97.52% with cut-off CT value of 36 and 98.14%, 96.56% and 97.16% with cut-off CT value of 37. In addition, PRRSGard PCR was able to detect PRRSGard vaccine virus in a sample even with the co-presence of another PRRSV strain. In summary, in contrast to a reference screening PCR that detects both vaccine and field PRRSV strains, PRRSGard PCR provides a convenient tool to specifically detect PRRSGard vaccine-like virus and to inform PRRSV vaccination protocols.

Designing and Testing of a System for Aerosolization and Recovery of Viable Porcine Reproductive and Respiratory Syndrome Virus (PRRSV): Theoretical and Engineering Considerations.

Porcine reproductive and respiratory syndrome virus (PRRSV) infections cause significant economic losses to swine producers every year. Aerosols containing infectious PRRSV are an important route of transmission, and proper treatment of air could mitigate the airborne spread of the virus within and between barns. Previous bioaerosol studies focused on the microbiology of PRRSV aerosols; thus, the current study addressed the engineering aspects of virus aerosolization and collection. Specific objectives were to (1) build and test a virus aerosolization system, (2) achieve a uniform and repeatable aerosol generation and collection throughout all replicates, (3) identify and minimize sources of variation, and (4) verify that the collection system (impingers) performed similarly. The system for virus aerosolization was built and tested (Obj. 1). The uniform airflow distribution was confirmed using a physical tracer (<12% relative standard deviation) for all treatments and sound engineering control of flow rates (Obj. 2). Theoretical uncertainty analyses and mass balance calculations showed <3% loss of air mass flow rate between the inlet and outlet (Obj. 3). A comparison of TCID50 values among impinger fluids showed no statistical difference between any two of the three trials (p-value = 0.148, 0.357, 0.846) (Obj. 4). These results showed that the readiness of the system for research on virus aerosolization and treatment (e.g., by ultraviolet light), as well as its potential use for research on other types of airborne pathogens and their mitigation on a laboratory scale.

Comparison of ZMAC and MARC-145 Cell Lines for Improving Porcine Reproductive and Respiratory Syndrome Virus Isolation from Clinical Samples.

The MARC-145 cell line is commonly used to isolate porcine reproductive and respiratory syndrome virus (PRRSV) for diagnostics, research, and vaccine production, but it yields frustratingly low success rates of virus isolation (VI). The ZMAC cell line, derived from porcine alveolar macrophages, has become available, but its utilization for PRRSV VI from clinical samples has not been evaluated. This study compared PRRSV VI results in ZMAC and MARC-145 cells from 375 clinical samples (including 104 lung, 140 serum, 90 oral fluid, and 41 processing fluid samples). The PRRSV VI success rate was very low in oral fluids and processing fluids regardless of whether ZMAC cells or MARC-145 cells were used. Success rates of PRRSV VI from lung and serum samples were significantly higher in ZMAC than in MARC-145 cells. Lung and serum samples with threshold cycle (CT ) values of <30 had better VI success. PRRSV-2 in genetic lineages 1 and 8 was isolated more successfully in ZMAC cells than in MARC-145 cells, whereas PRRSV-2 in genetic lineage 5 was isolated in the two cell lines with similar success rates. For samples with positive VI in both ZMAC and MARC-145 cells, 14 of 23 PRRSV-2 isolates had similar titers in the two cell lines. A total of 51 of 95 (53.7%) ZMAC-obtained PRRSV-2 or PRRSV-1 isolates grew in MARC-145 cells, and all 46 (100%) MARC-145-obtained isolates grew in ZMAC cells. In summary, ZMAC cells allow better isolation of a wide range of PRRSV field strains; however, not all of the ZMAC-obtained PRRSV isolates grow in MARC-145 cells. This report provides important guidelines to improve isolation of PRRSV from clinical samples for further characterization and/or for producing autogenous vaccines.

Development of a bead-based assay for detection and differentiation of field strains and four vaccine strains of type 2 porcine reproductive and respiratory syndrome virus (PRRSV-2) in the USA.

Porcine reproductive and respiratory syndrome (PRRS) remains one of the most economically devastating diseases in swine population in the United States of America. Due to high mutation rate of the PRRS virus (PRRSV) genome, it is difficult to develop an accurate diagnostic assay with high strain coverage. Differentiation of field strains from the four vaccines that have been used in the USA, namely Ingelvac PRRS MLV, Ingelvac ATP, Fostera PRRS and Prime Pac PRRS, adds an additional challenge. It is difficult to use current real-time PCR systems to detect and differentiate the field strains from the vaccine strains. Luminex xTAG technology allows us to detect more molecular targets in a single reaction with a cost similar to a single real-time PCR reaction. By analysing all available 678 type 2 PRRSV (PRRSV-2) complete genome sequences, including the 4 vaccine strains, two pairs of detection primers were designed targeting the conserved regions of ORF4-ORF7, with strain coverage of 98.8% (670/678) based on in silico analysis. The virus strains sharing ≥98% identity of the complete genomes with the vaccine strains were considered vaccine or vaccine-like strains. One pair of primers for each vaccine strain were designed targeting the nsp2 region. In silico analysis showed the assay matched 94.7% (54/57) of Ingelvac PRRS® MLV (MLV) strain and the MLV-like strains, and 100% of the other three vaccine strains. Analytical sensitivity of the Luminex assay was one to two logs lower than that of the reverse transcription real-time PCR assay. Evaluated with 417 PRRSV-2 positive clinical samples, 95% were detected by the Luminex assay. Compared to ORF5 sequencing results, the Luminex assay detected 92.4% (73/79) of MLV strains, 78.3% (18/23) of Fostera strains and 50% (2/4) of ATP strains. None of the 472 samples were the Prime Pac strain tested by either ORF5 sequencing or the Luminex assay.

IDENTIFICATION AND CORRELATION OF A NOVEL SIADENOVIRUS IN A FLOCK OF BUDGERIGARS (MELOPSITTACUS UNDULATES) INFECTED WITH SALMONELLA TYPHIMURIUM IN THE UNITED STATES.

A flock of budgerigars (Melopsittacus undulates) was purchased from a licensed breeder and quarantined at a zoologic facility within the United States in 2016. Following 82 deaths within the flock, the remaining 66 birds were depopulated because of ongoing clinical salmonellosis despite treatment. Gross necropsy was performed on all 66 birds. Histopathologic examination was performed on 10 birds identified with gross lesions and 10 birds without. Pathologic findings were most often observed in the liver, kidney, and spleen. Lesions noted in the livers and spleens were consistent with published reports of salmonellosis in psittacine species. Multisystemic changes associated with septicemia were not noted, most likely because of antibiotic intervention before euthanasia. Of the 20 budgerigars evaluated by histopathology, six had large basophilic intranuclear inclusion bodies within tubular epithelia in a portion of the kidneys. Electronic microscopy, next-generation sequencing, Sanger sequencing, and phylogenetic analyses were used to identify and categorize the identified virus as a novel siadenovirus strain BuAdV-1 USA-IA43444-2016. The strain was 99% similar to budgerigar adenovirus 1 (BuAdV-1), previously reported in Japan, and to a psittacine adenovirus 5 recently identified in a U.S. cockatiel. Salmonella typhimurium carriers were identified via polymerase chain reaction (PCR) and bacterial culture and compared with viral carriers identified via PCR. Inclusion bodies and Salmonella detection were significant in birds with gross lesions versus those without; however, there was no correlation between budgerigars positive with siadenovirus by PCR and concurrent Salmonella infection. Identifying subclinical siadenovirus strain BuAdV-1 USA-IA43444-2016 infection in this flock significantly differs from a previous report of clinical illness in five budgerigars resulting in death caused by BuAdV-1 in Japan. S. typhimurium remains a significant pathogen in budgerigars, and zoonotic concerns prompted depopulation to mitigate the public health risks of this flock.

Genetic diversity of porcine reproductive and respiratory syndrome virus 1 in the United States of America from 2010 to 2018.

Porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) was first detected in the United States of America (USA) in 1999, several strains were also recognized soon later, and these isolates are typically called North American (NA) PRRSV-1. However, few reports have characterized PRRSV-1 viruses in the USA. We explored the genetic characteristics and diversity of PRRSV-1 viruses circulating in the USA. PRRSV-1 PCR-positive samples collected from seven states in 2010-2018 (n = 27) were subjected to next-generation sequencing. The 27 PRRSV-1 viruses had 88.4-91.3% nucleotide identity to the PRRSV-1 Lelystad-virus strain (the type 1 prototype strain) and 87.4-89.8% to the previously reported NA PRRSV-1 viruses. Individual proteins had several unique genetic characteristics and only one of the 27 tested samples had the characteristic 17-amino acid (aa) deletion in Nsp2, a genetic marker of NA PRRSV-1 viruses described previously. Fourteen isolates displayed a 3-aa C-terminal truncation in the highly conserved Nsp12 gene; 16 samples had a 21- or 18-aa C-terminal truncation in GP3 gene; and one was observed with a 1-aa deletion at the overlapping region of GP3 and GP4. In addition, the GP5 protein in most isolates, excluding one exception, demonstrated similar genetic variation as other reported NA PRRSV-1 isolates. All tested isolates clustered within subtype 1 together with other available NA PRRSV-1 viruses. Collectively, our results provide up-to-date information on PRRSV-1 viruses circulating in the USA in the past 9 years although the number of PRRSV-1 isolates included in this study is limited. These PRRSV-1 viruses have undergone gradual genetic variation and exhibited some previously undescribed genetic characteristics and diversity, which complicates the diagnosis and control of NA PRRSV-1.

Identification of porcine epidemic diarrhea virus variant with a large spike gene deletion from a clinical swine sample in the United States.

Two genetically different porcine epidemic diarrhea virus (PEDV) strains have been identified in the USA: US prototype (also called non-S INDEL) and S INDEL PEDVs. In February 2017, a PEDV variant (USA/OK10240-8/2017) was identified in a rectal swab from a sow farm in Oklahoma, USA. Complete genome sequence analyses indicated this PEDV variant was genetically similar to US non-S INDEL strain but had a continuous 600-nt (200-aa) deletion in the N-terminal domain of the spike gene compared to non-S INDEL PEDVs. This is the first report of detecting PEDV bearing large spike gene deletion in clinical swine samples in the USA.

Computational analyses of curcuminoid analogs against kinase domain of HER2.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) has an important role in cancer aggressiveness and poor prognosis. HER2 has been used as a drug target for cancers. In particular, to effectively treat HER2-positive cancer, small molecule inhibitors were developed to target HER2 kinase. Knowing that curcumin has been used as food to inhibit cancer activity, this study evaluated the efficacy of natural curcumins and curcumin analogs as HER2 inhibitors using in vitro and in silico studies. The curcumin analogs considered in this study composed of 4 groups classified by their core structure, ß-diketone, monoketone, pyrazole, and isoxazole. RESULTS: In the present study, both computational and experimental studies were performed. The specificity of curcumin analogs selected from the docked results was examined against human breast cancer cell lines. The screened curcumin compounds were then subjected to molecular dynamics simulation study. By modifying curcumin analogs, we found that protein-ligand affinity increases. The benzene ring with a hydroxyl group could enhance affinity by forming hydrophobic interactions and the hydrogen bond with the hydrophobic pocket. Hydroxyl, carbonyl or methoxy group also formed hydrogen bonds with residues in the adenine pocket and sugar pocket of HER2-TK. These modifications could suggest the new drug design for potentially effective HER2-TK inhibitors. Two outstanding compounds, bisdemethylcurcumin (AS-KTC006) and 3,5-bis((E)-3,4-dimethoxystyryl)isoxazole (AS-KTC021 ),were well oriented in the binding pocket almost in the simulation time, 30 ns. This evidence confirmed the results of cell-based assays and the docking studies. They possessed more distinguished interactions than known HER2-TK inhibitors, considering them as a promising drug in the near future. CONCLUSIONS: The series of curcumin compounds were screened using a computational molecular docking and followed by human breast cancer cell lines assay. Both AS-KTC006 and AS-KTC021 could inhibit breast cancer cell lines though inhibiting of HER2-TK. The intermolecular interactions were confirmed by molecular dynamics simulation studies. This information would explore more understanding of curcuminoid structures and HER2-TK.