Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors.

Beta human papillomavirus (ß-HPV) are hypothesized to make DNA damage more mutagenic and potentially more carcinogenic. Double strand breaks (DSBs) are the most deleterious DNA lesion. They are typically repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs after DNA replication while NHEJ can occur at any point in the cell cycle. HR and NHEJ are not thought to occur in the same cell at the same time. HR is restricted to cells in phases of the cell cycle where homologous templates are available, while NHEJ occurs primarily during G1. ß-HPV type 8 protein E6 (8E6) attenuates both repair pathways. We use a series of immunofluorescence microscopy and flow cytometry experiments to better define the impact of this attenuation. We found that 8E6 causes colocalization of HR factors (RPA70 and RAD51) with an NHEJ factor (activated DNA-PKcs or pDNA-PKcs) at persistent DSBs. 8E6 also causes RAD51 foci to form during G1. The initiation of NHEJ and HR at the same lesion could lead to antagonistic DNA end processing. Further, HR cannot be readily completed in an error-free manner during G1. Both aberrant repair events would cause deletions. To determine if these mutations were occurring, we used next generation sequencing of the 200kb surrounding a CAS9-induced DSB. 8E6 caused a 21-fold increase in deletions. Chemical and genetic inhibition of p300 as well as an 8E6 mutant that is incapable of destabilizing p300 demonstrates that 8E6 is acting via p300 destabilization. More specific chemical inhibitors of DNA repair provided mechanistic insight by mimicking 8E6-induced dysregulation of DNA repair in a virus-free system. Specifically, inhibition of NHEJ causes RAD51 foci to form in G1 and colocalization of RAD51 with pDNA-PKcs.

Beta Human Papillomavirus 8 E6 Induces Micronucleus Formation and Promotes Chromothripsis.

Cutaneous beta genus human papillomaviruses (ß-HPVs) are suspected to promote the development of nonmelanoma skin cancer (NMSC) by destabilizing the host genome. Multiple studies have established the genome destabilizing capacities of ß-HPV proteins E6 and E7 as a cofactor with UV. However, the E6 protein from ß-HPV8 (HPV8 E6) induces tumors in mice without UV exposure. Here, we examined a UV-independent mechanism of HPV8 E6-induced genome destabilization. We showed that HPV8 E6 reduced the abundance of anaphase bridge resolving helicase, Bloom syndrome protein (BLM). The diminished BLM was associated with increased segregation errors and micronuclei. These HPV8 E6-induced micronuclei had disordered micronuclear envelopes but retained replication and transcription competence. HPV8 E6 decreased antiproliferative responses to micronuclei and time-lapse imaging revealed HPV8 E6 promoted cells with micronuclei to complete mitosis. Finally, whole-genome sequencing revealed that HPV8 E6 induced chromothripsis in nine chromosomes. These data provide insight into mechanisms by which HPV8 E6 induces genome instability independent of UV exposure. IMPORTANCE Some beta genus human papillomaviruses (ß-HPVs) may promote skin carcinogenesis by inducing mutations in the host genome. Supporting this, the E6 protein from ß-HPV8 (8 E6) promotes skin cancer in mice with or without UV exposure. Many mechanisms by which 8 E6 increases mutations caused by UV have been elucidated, but less is known about how 8 E6 induces mutations without UV. We address that knowledge gap by showing that 8 E6 causes mutations stemming from mitotic errors. Specifically, 8 E6 reduces the abundance of BLM, a helicase that resolves and prevents anaphase bridges. This hinders anaphase bridge resolution and increases their frequency. 8 E6 makes the micronuclei that can result from anaphase bridges more common. These micronuclei often have disrupted envelopes yet retain localization of nuclear-trafficked proteins. 8 E6 promotes the growth of cells with micronuclei and causes chromothripsis, a mutagenic process where hundreds to thousands of mutations occur in a chromosome.

Bovine rhinitis B virus is highly prevalent in acute bovine respiratory disease and causes upper respiratory tract infection in calves.

Bovine respiratory disease (BRD) is the most significant cause of cattle morbidity and mortality worldwide. This multifactorial disease has a complex aetiology. Dogma posits a primary viral infection followed by secondary bacterial pneumonia. Bovine rhinitis B virus (BRBV) is an established aetiological agent of BRD, but little is known regarding its pathogenesis. Here, a BRD PCR panel identified 18/153 (11.8 %) lung samples and 20/49 (40.8 %) nasal swabs collected from cattle with respiratory signs as positive for BRBV, which was the most prevalent virus in nasal swabs. Primary bovine tracheal epithelial cells were used to isolate BRBV that was phylogenetically related to contemporary sequences from the USA and Mexico and genetically divergent from the previous sole BRBV isolate. To investigate virus pathogenesis, 1-week-old colostrum-deprived dairy calves were inoculated intranasally with 7.0 log10 TCID50 BRBV. Virus was isolated from nasal swabs, nasal turbinates, trachea and the brain of the challenged animals. Neutralizing antibodies were detected beginning 7 days post-inoculation and peaked at day 14. In situ hybridization (ISH) localized BRBV infection in the upper respiratory ciliated epithelial and goblet cells, occasionally associated with small defects of the superficial cilia lining. Sporadically, pinpoint ISH signals were also detected in cells resembling glial cells in the cerebrum in one calf. Together, these results demonstrate the BRBV infection is highly prevalent in acute BRD samples and while the pathogenicity of BRBV is minimal with infection largely limited to the upper respiratory tract, further research is needed to elucidate a possible initiatory role in BRD.

Whole-genome classification of rotavirus C and genetic diversity of porcine strains in the USA.

Rotavirus C (RVC) is associated with acute diarrhoea in both children and young animals. Because of its frequent occurrence, additional sequences have recently been generated. In this study, we sequenced 21 complete genomes from porcine diarrhoea samples and analysed them together with all available reference sequences collected from the GenBank database [National Center for Biotechnology Information (NCBI)]. Based on phylogenetic analysis and genetic distance calculation, the number of each segment was identified as 31G, 26P, 13I, 5R, 5C, 5M, 12A, 10 N, 9T, 8E and 4 H for genotypes encoding VP7, VP4, VP6, VP1, VP2, VP3 and NSP1, NSP2, NSP3, NSP4 and NSP5, respectively. From the analysis, genotypes G19-G31, P[22]-P[26], R5, A9-A12, N9-N10, T7-T9 and E6-E8 were defined as newly identified genotypes, and genotype C6 was combined with C5, and M6 was combined with M1, due to their closely related nature. Estimated with the identity frequency ratio between the intergenotype and intragenotype, the nucleotide identity cutoff values for different genotypes were determined as 85, 85, 86, 84, 83, 84, 82, 87, 84, 81 and 79 % for VP7, VP4, VP6, VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4 and NSP5, respectively. Genotyping of the 49 US strains indicated possible segment reassortment in 9 of the 11 segments, with the exceptions being VP1 and NSP5, and the most prevalent genotypes for each segment genes in the USA were G6/G5/G21/G9-P5/P4-I6/I5-R1-C5-M1-A8-N1/N10-T1-E1-H1. Our study updated the genotypes of RVC strains and provided more evidence of RVC strain diversity that may be relevant to better understand genetic diversity, and the distribution and evolution of RVC strains.

A Novel Porcine Circovirus Distantly Related to Known Circoviruses Is Associated with Porcine Dermatitis and Nephropathy Syndrome and Reproductive Failure.

Porcine circovirus-associated disease (PCVAD) is clinically manifested by postweaning multisystemic wasting syndrome (PMWS), respiratory and enteric disease, reproductive failure, and porcine dermatitis and nephropathy syndrome (PDNS). Porcine circovirus 2 (PCV2) is an essential component of PCVAD, although an etiologic role in PDNS is not well established. Here, a novel circovirus, designated porcine circovirus 3 (PCV3), was identified in sows that died acutely with PDNS-like clinical signs. The capsid and replicase proteins of PCV3 are only 37% and 55% identical to PCV2 and bat circoviruses, respectively. Aborted fetuses from sows with PDNS contained high levels of PCV3 (7.57 × 107 genome copies/ml), and no other viruses were detected by PCR and metagenomic sequencing. Immunohistochemistry (IHC) analysis of sow tissue samples identified PCV3 antigen in skin, kidney, lung, and lymph node samples localized in typical PDNS lesions, including necrotizing vasculitis, glomerulonephritis, granulomatous lymphadenitis, and bronchointerstitial pneumonia. Further study of archived PDNS tissue samples that were negative for PCV2 by IHC analysis identified 45 of 48 that were PCV3 positive by quantitative PCR (qPCR), with 60% of a subset also testing positive for PCV3 by IHC analysis. Analysis by qPCR of 271 porcine respiratory disease diagnostic submission samples identified 34 PCV3-positive cases (12.5%), and enzyme-linked immunosorbent assay detection of anti-PCV3 capsid antibodies in serum samples found that 46 (55%) of 83 samples tested were positive. These results suggest that PCV3 commonly circulates within U.S. swine and may play an etiologic role in reproductive failure and PDNS. Because of the high economic impact of PCV2, this novel circovirus warrants further studies to elucidate its significance and role in PCVAD. IMPORTANCE: While porcine circovirus 2 (PCV2) was first identified in sporadic cases of postweaning multisystemic wasting syndrome in Canada in the early 1990s, an epidemic of severe systemic disease due to PCV2 spread worldwide in the ensuing decade. Despite being effectively controlled by commercial vaccines, PCV2 remains one of the most economically significant viruses of swine. Here, a novel porcine circovirus (PCV3) that is distantly related to known circoviruses was identified in sows with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure. PCV2, which has previously been associated with these clinical presentations, was not identified. High levels of PCV3 nucleic acid were observed in aborted fetuses by quantitative PCR, and PCV3 antigen was localized in histologic lesions typical of PDNS in sows by immunohistochemistry (IHC) analysis. PCV3 was also identified in archival PDNS diagnostic samples that previously tested negative for PCV2 by IHC analysis. The emergence of PCV3 warrants further investigation.

Highly diverse posaviruses in swine faeces are aquatic in origin.

Posaviruses are a group of highly divergent viruses identified in swine faeces that are distantly related to other members of the order Picornavirales. Eighteen posavirus genomes were assembled from 10 out of 25 (40 %) faecal-swab pools collected from healthy adult swine. Phylogenetic analysis of the conserved RNA-dependent RNA polymerase (Pol) domain found that posaviruses form a large, highly diverse, monophyletic clade, which includes similar viruses identified in human (husavirus) and fish (fisavirus) faeces or intestinal contents, respectively. Quantitative reverse transcription PCR analysis of water samples collected from commercial swine barns identified four out of 19 (21 %) samples were positive using a 5'-nuclease assay targeting the Pol region of posavirus 1. ICPD (immunoprecipitation coupled to PCR detection) assays to explore serological evidence of posavirus infection found only a single positive sample, suggesting posaviruses do not commonly infect swine, and together these results suggests a likely aquatic host.

Widespread detection and characterization of porcine parainfluenza virus 1 in pigs in the USA.

Porcine parainfluenza virus 1 (PPIV1) was first identified in 2013 in slaughterhouse pigs in Hong Kong, China. Here, two near-complete genomes were assembled from swine exhibiting acute respiratory disease that were 90.0-95.3% identical to Chinese PPIV1. Analysis of the HN gene from ten additional PPIV1-positive samples found 85.0-95.5% identity, suggesting genetic diversity between strains. Molecular analysis identified 17 out of 279 (6.1%) positive samples from pigs with respiratory disease. Eleven nursery pigs from a naturally infected herd were asymptomatic; however, nasal swabs from six pigs and the lungs of a single pig were quantitative reverse transcriptase (qRT)-PCR positive. Histopathology identified PPIV1 RNA in the nasal respiratory epithelium and trachea. Two serological assays demonstrated seroconversion of infected pigs and further analysis of 59 swine serum samples found 52.5% and 66.1% seropositivity, respectively. Taken together, the results confirm the widespread presence of PPIV1 in the US swine herd.

Discovery of a novel Parvovirinae virus, porcine parvovirus 7, by metagenomic sequencing of porcine rectal swabs.

Parvoviruses are a diverse group of viruses containing some of the smallest known species that are capable of infecting a wide range of animals. Metagenomic sequencing of pooled rectal swabs from adult pigs identified a 4103-bp contig consisting of two major open reading frames encoding proteins of 672 and 469 amino acids (aa) in length. BLASTP analysis of the 672-aa protein found 42.4 % identity to fruit bat (Eidolon helvum) parvovirus 2 (EhPV2) and 37.9 % to turkey parvovirus (TuPV) TP1-2012/HUN NS1 proteins. The 469-aa protein had no significant similarity to known proteins. Genetic and phylogenetic analyses suggest that PPV7, EhPV2, and TuPV represent a novel genus in the family Parvoviridae. Quantitative PCR screening of 182 porcine diagnostic samples found a total of 16 positives (8.6 %). Together, these data suggest that PPV7 is a highly divergent novel parvovirus prevalent within the US swine.

Inactivation of highly transmissible livestock and avian viruses including influenza A and Newcastle disease virus for molecular diagnostics.

There is a critical need for an inactivation method that completely inactivates pathogens at the time of sample collection while maintaining the nucleic acid quality required for diagnostic PCR testing. This inactivation method is required to alleviate concerns about transmission potential, minimize shipping complications and cost, and enable testing in lower containment laboratories, thereby enhancing disease diagnostics through improved turn-around time. This study evaluated a panel of 10 surrogate viruses that represent highly pathogenic animal diseases. These results showed that a commercial PrimeStore® molecular transport media (PSMTM) completely inactivated all viruses tested by >99.99%, as determined by infectivity and serial passage assays. However, the detection of viral nucleic acid by qRT-PCR was comparable in PSMTM and control-treated conditions. These results were consistent when viruses were evaluated in the presence of biological material such as sera and cloacal swabs to mimic diagnostic sample conditions for non-avian and avian viruses, respectively. The results of this study may be utilized by diagnostic testing laboratories for highly pathogenic agents affecting animal and human populations. These results may be used to revise guidance for select agent diagnostic testing and the shipment of infectious substances.

Comparison of the Effect of Corn-fermented Protein and Traditional Ingredients on the Fecal Microbiota of Dogs.

Corn-fermented protein (CFP), a co-product from the ethanol industry, is produced using post-fermentation technology to split the protein and yeast from fiber prior to drying. The objective of this study was to determine the effect of CFP compared to traditional ingredients on the fecal microbiota of dogs. The four experimental diets included a control with no yeast and diets containing either 3.5% brewer's dried yeast, 2.5% brewer's dried yeast plus 17.5% distiller's dried grains with solubles, or 17.5% CFP. The experimental diets were fed to adult dogs (n = 12) in a 4 × 4 replicated Latin square design. Fresh fecal samples (n = 48) were analyzed by 16S metagenomic sequencing. Raw sequences were processed through mothur. Community diversity was evaluated in R. Relative abundance data were analyzed within the 50 most abundant operational taxonomic units using a mixed model of SAS. Alpha and beta diversity were similar for all treatments. Predominant phyla among all samples were Firmicutes (73%), Bacteroidetes (15%), Fusobacteria (8%), and Actinobacteria (4%). There were no quantifiable (p > 0.05) shifts in the predominant phyla among the treatments. However, nine genera resulted in differences in relative abundance among the treatments. These data indicate that compared to traditional ingredients, CFP did not alter the overall diversity of the fecal microbiota of healthy adult dogs over 14 days.

Beta human papillomavirus 8E6 promotes alternative end joining.

Double strand breaks (DSBs) are one of the most lethal DNA lesions in cells. The E6 protein of beta-human papillomavirus (HPV8 E6) impairs two critical DSB repair pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). However, HPV8 E6 only delays DSB repair. How DSBs are repaired in cells with HPV8 E6 remains to be studied. We hypothesize that HPV8 E6 promotes a less commonly used DSB repair pathway, alternative end joining (Alt-EJ). Using CAS9-based Alt-EJ reporters, we show that HPV8 E6 promotes Alt-EJ. Further, using small molecule inhibitors, CRISPR/CAS9 gene knockout, and HPV8 E6 mutant, we find that HPV8 E6 promotes Alt-EJ by binding p300, an acetyltransferase that facilitates DSB repair by HR and NHEJ. At least some of this repair occurs through a subset of Alt-EJ known as polymerase theta dependent end joining. Finally, whole genome sequencing analysis showed HPV8 E6 caused an increased frequency of deletions bearing the microhomology signatures of Alt-EJ. This study fills the knowledge gap of how DSB is repaired in cells with HPV8 E6 and the mutagenic consequences of HPV8 E6 mediated p300 destabilization. Broadly, this study supports the hypothesis that beta-HPV promotes cancer formation by increasing genomic instability.

Inhibition of p300 increases cytotoxicity of cisplatin in pancreatic cancer cells.

Pancreatic cancer is a notoriously deadly disease with a five-year survival rate around 10 percent. Since early detection of these tumors is difficult, pancreatic cancers are often diagnosed at advanced stages. At this point, genotoxic chemotherapeutics can be used to manage tumor growth. However, side effects of these drugs are severe, limiting the amount of treatment that can be given and resulting in sub-optimal dosing. Thus, there is an urgent need to identify chemo-sensitizing agents that can lower the effective dose of genotoxic agents and as a result reduce the side effects. Here, we use transformed and non-transformed pancreatic cell lines to evaluate DNA repair inhibitors as chemo-sensitizing agents. We used a novel next generation sequencing approach to demonstrate that pancreatic cancer cells have a reduced ability to faithfully repair DNA damage. We then determine the extent that two DNA repair inhibitors (CCS1477, a small molecule inhibitor of p300, and ART558, a small molecule inhibitor of polymerase theta) can exploit this repair deficiency to make pancreatic cancer cells more sensitive to cisplatin, a commonly used genotoxic chemotherapeutic. Immunofluorescence microscopy and cell viability assays show that CCS1477 delayed repair and significantly sensitized pancreatic cancer cells to cisplatin. The increased toxicity was not seen in a non-transformed pancreatic cell line. We also found that while ART558 sensitizes pancreatic cancer cells to cisplatin, it also sensitized non-transformed pancreatic cancer cells.

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter.

Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication stress, ionizing radiation, unrepaired UV damage), DSBs occur in most cells each day. In addition to cell death, unrepaired DSBs reduce genome integrity and the resulting mutations can drive tumorigenesis. These risks and the prevalence of DSBs motivate investigations into the mechanisms by which cells repair these lesions. Next generation sequencing can be paired with the induction of DSBs by ionizing radiation to provide a powerful tool to precisely define the mutations associated with DSB repair defects. However, this approach requires computationally challenging and cost prohibitive whole genome sequencing to detect the repair of the randomly occurring DSBs associated with ionizing radiation. Rare cutting endonucleases, such as I-Sce1, provide the ability to generate a single DSB, but their recognition sites must be inserted into the genome of interest. As a result, the site of repair is inherently artificial. Recent advances allow guide RNA (sgRNA) to direct a Cas9 endonuclease to any genome locus of interest. This could be applied to the study of DSB repair making next generation sequencing more cost effective by allowing it to be focused on the DNA flanking the Cas9-induced DSB. The goal of the manuscript is to demonstrate the feasibility of this approach by presenting a protocol that can define mutations that stem from the repair of a DSB upstream of the CD4 gene. The protocol can be adapted to determine changes in the mutagenic potential of DSB associated with exogenous factors, such as repair inhibitors, viral protein expression, mutations, and environmental exposures with relatively limited computation requirements. Once an organism's genome has been sequenced, this method can be theoretically employed at any genomic locus and in any cell culture model of that organism that can be transfected. Similar adaptations of the approach could allow comparisons of repair fidelity between different loci in the same genetic background.

Bovine Respiratory Syncytial Virus Genome Sequences from Cattle with Clinical Respiratory Disease in Kansas, 2021.

We report two near-complete bovine respiratory syncytial virus genome sequences collected from 10-month-old cattle with respiratory disease in Kansas in December 2021. No other respiratory pathogens were confirmed in the samples. These genome sequences update the currently circulating BRSV field strains in the United States.

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.

Detection and investigation of atypical porcine pestivirus in a swine production system.

A commercial farrow-to-finish farm was suspicious of atypical porcine pestivirus (APPV) after observing clinical signs of congenital tremors (CT) and splay leg (SL) of newborn pigs. If introduced onto the farrow-to-finish, the two potential routes of introduction could be through replacement gilts or incoming semen doses. Therefore, this study aimed to determine the prevalence of clinical APPV within the sampled population, identify the route of APPV introduction to this system, and determine prevalence of detectable APPV RNA within a population of gilt multiplication farm offspring through an isolation nursery and finisher barn. Farrowing records were analyzed for the presence of CT or SL and corresponding parity of the dam. Overall, prevalence of clinically affected litters within batch farrowing groups ranged from 0 to 31%. Phylogenetic analysis was conducted on a serum sample from a gilt at the isolation nursery, semen dose for the farrow-to-finish farm, and serum of a CT piglet. Results indicated that the virus circulating in clinically affected piglets was most similar to an incoming semen dose (98.9% nucleotide identity). Blood samples were collected at four time points and revealed APPV clinical prevalence was 37.5-77.5% during the nursery phase and 0-26% during the finisher phase. Oral fluids were also collected during the finisher phase and APPV clinical prevalence was 100% for all sampling time points. In summary, introduction of APPV into naïve herds is associated with increased clinical CT and SL cases and is detectable in asymptomatic pigs during the nursery and finisher production phases. This study found that potential screening tests for APPV could include oral fluids or qRT-PCR analysis of semen doses especially when trying to identify prevalence levels on naïve farm.

Effect of the host genotype at a Porcine Reproductive and Respiratory Syndrome (PRRS) resistance marker on evolution of the modified-live PRRS vaccine virus in pigs.

Porcine reproductive and respiratory syndrome; Porcine reproductive and respiratory syndrome virus; PRRS; Whole genome sequencing; Quasispecies; WUR allele.

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.

Genome Sequences of Foot-and-Mouth Disease Virus SAT1 Strains Purified from Coinfected Cape Buffalo in Kenya.

Nearly complete genomes of 49 novel foot-and-mouth disease virus (FMDV) SAT1 strains acquired from oropharyngeal fluid samples from asymptomatic African Cape buffalo in Kenya in 2016 were determined. Sequences were from primary passage or plaque-purified dually SAT1/SAT2-infected samples. These sequences are important for elucidation of the molecular epidemiology of persistent and subclinical FMDV infections.

Genome Sequences of Foot-and-Mouth Disease Virus SAT2 Strains Purified from Coinfected Cape Buffalo in Kenya.

Foot-and-mouth disease virus (FMDV) SAT2 sequences were acquired from Cape buffalo in Kenya in 2016, from either primary passage (n = 38) or plaque purification of dually SAT1/SAT2-infected samples (n = 61). All samples were derived from asymptomatic animals. These sequences contribute to our understanding of FMDV diversity in reservoirs and during subclinical FMDV infections.