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.

A Phosphoproteomics Study of the Soybean root necrosis 1 Mutant Revealed Type II Metacaspases Involved in Cell Death Pathway.

The soybean root necrosis 1 (rn1) mutation causes progressive browning of the roots soon after germination and provides increased tolerance to the soil-borne oomycete pathogen Phytophthora sojae in soybean. Toward understanding the molecular basis of the rn1 mutant phenotypes, we conducted tandem mass tag (TMT)-labeling proteomics and phosphoproteomics analyses of the root tissues of the rn1 mutant and progenitor T322 line to identify potential proteins involved in manifestation of the mutant phenotype. We identified 3,160 proteins. When the p-value was set at ≤0.05 and the fold change of protein accumulation between rn1 and T322 at ≥1.5 or ≤0.67, we detected 118 proteins that showed increased levels and 32 proteins decreased levels in rn1 as compared to that in T322. The differentially accumulated proteins (DAPs) are involved in several pathways including cellular processes for processing environmental and genetic information, metabolism and organismal systems. Five pathogenesis-related proteins were accumulated to higher levels in the mutant as compared to that in T322. Several of the DAPs are involved in hormone signaling, redox reaction, signal transduction, and cell wall modification processes activated in plant-pathogen interactions. The phosphoproteomics analysis identified 22 phosphopeptides, the levels of phosphorylation of which were significantly different between rn1 and T322 lines. The phosphorylation levels of two type II metacaspases were reduced in rn1 as compared to T322. Type II metacaspase has been shown to be a negative regulator of hypersensitive cell death. In absence of the functional Rn1 protein, two type II metacaspases exhibited reduced phosphorylation levels and failed to show negative regulatory cell death function in the soybean rn1 mutant. We hypothesize that Rn1 directly or indirectly phosphorylates type II metacaspases to negatively regulate the cell death process in soybean roots.

Interaction of Phytophthora sojae Effector Avr1b With E3 Ubiquitin Ligase GmPUB1 Is Required for Recognition by Soybeans Carrying Phytophthora Resistance Rps1-b and Rps1-k Genes.

Phytophthora sojae is an oomycete that causes stem and root rot disease in soybean. P. sojae delivers many RxLR effector proteins, including Avr1b, into host cells to promote infection. We show here that Avr1b interacts with the soybean U-box protein, GmPUB1-1, in yeast two-hybrid, pull down, and bimolecular fluorescence complementation (BIFC) assays. GmPUB1-1, and a homeologous copy GmPUB1-2, are induced by infection and encode 403 amino acid proteins with U-Box domains at their N-termini. Non-synonymous mutations in the Avr1b C-terminus that abolish suppression of cell death also abolished the interaction of Avr1b with GmPUB1-1, while deletion of the GmPUB1-1 C-terminus, but not the U box, abolished the interaction. BIFC experiments suggested that the GmPUB1-1-Avr1b complex is targeted to the nucleus. In vitro ubiquitination assays demonstrated that GmPUB1-1 possesses E3 ligase activity. Silencing of the GmPUB1 genes in soybean cotyledons resulted in loss of recognition of Avr1b by gene products encoded by Rps1-b and Rps1-k. The recognition of Avr1k (which did not interact with GmPUB1-1) by Rps1-k plants was not, however, affected following GmPUB1-1 silencing. Furthermore, over-expression of GmPUB1-1 in particle bombardment experiments triggered cell death suggesting that GmPUB1 may be a positive regulator of effector-triggered immunity. In a yeast two-hybrid system, GmPUB1-1 also interacted with a number of other RxLR effectors including Avr1d, while Avr1b and Avr1d interacted with a number of other infection-induced GmPUB proteins, suggesting that the pathogen uses a multiplex of interactions of RxLR effectors with GmPUB proteins to modulate host immunity.

First report of quinoa anthracnose caused by Colletotrichum nigrum and C. truncatum in the United States.

Quinoa (Chenopodium quinoa Willd.) is increasingly produced outside its native Andean range. In September 2019, stem lesions were observed on all six plants of quinoa accessions PI 510547 (25% severity) and PI 596293 (75% severity) in a demonstration plot in Ames, IA. Lesions were bleached, silvery-white to dark gray, slightly sunken, oval to linear with slightly tapered tips and contained setose acervuli. Fungi were isolated from both accessions following disinfestation with 70% ethanol and plating onto ½ acidified potato dextrose agar (APDA) and V8 medium. Isolates were examined morphologically. On V8 medium, isolate CQ1 produced sparse, flat, gray mycelia with profuse sclerotia and hyaline, aseptate, cylindrical conidia (n= 50, mean: 21.0 (range: 19.2-24) by 4.3 (2.4-4.8) µm); isolate CQ2 produced fluffy, gray to dark gray mycelia with profuse sclerotia and acervuli and hyaline, aseptate, falcate conidia (n =50, 26.8 (24-31.2) by 2.4 µm). Direct hyphal PCR was used to amplify ITS (ITS1/ITS4), ACT (ACT-512F/ACT-783R), GAPDH (GDF1/GDR1), CHS-1 (CHS-79F/CHS-234R), and TUB2 (T1/Bt-2b, CQ1 only) (Fu et al. 2019, Liu et al. 2013), and products were sequenced bidirectionally (MT772082-3, MT786524-30). A maximum likelihood tree was generated in MEGA X (Kumar et al. 2018) from a multiple sequence alignment of vouchered CBS isolates (Liu et al. 2013) and CQ1 was identified as Colletotrichum nigrum. CQ2 sequences showed 99-100% similarity to Colletotrichum truncatum sequences in Genbank (MN581860, MK675238, MF682518, MK118057). Koch's postulates were completed once with two isolates of each species grown on V8 medium under 12 hours of near UV light for two weeks. Greenhouse conditions were a 12 hr day/night cycle and temperature range of 26-30° C. Approximately 5 mL of mycelium on agar medium was sterilely removed and macerated in 6 mL of sterile distilled water. Non-inoculated medium was macerated in sterile water as a control. Forty-day old quinoa PI 634920 were inoculated by making three, 2-3 mm incisions between the cotyledons and first true leaves with a sterile razor blade. Next, 500 µL of slurry was placed on 2.54 cm2 of sterile cheesecloth and placed against the wound and wrapped with Parafilm. Six plants were inoculated per isolate and control. After two weeks, sunken, bleached to tan areas extended past wound sites of inoculated plants. No discoloration or sunken tissue was observed on control plants. Plants were tented with plastic film for one week. Acervuli were observed on C. truncatum- and C. nigrum-inoculated stems, and sclerotia were observed on C. nigrum-inoculated stems. Stems were surface disinfested with 10% bleach and plated onto ½ APDA. Colony morphologies of isolated fungi matched those of original inoculum for inoculated plants. Colletotrichum spp. were never isolated from control plants. When stems were inoculated, approximately 100 µL of slurry was also placed on 3-5 detached quinoa leaves in Petri dishes with moistened blotter paper and incubated for 48 hours at 25° C. Brownish, circular lesions developed on leaves inoculated with either species, but no lesions developed on control-slurry leaves. Colletotrichum spp. cause disease in quinoa relatives including spinach (Kurt 2015), beets (Gourley 1966) and amaranth (Wu 2001). This is the first description of Colletotrichum spp. causing stem lesions on quinoa in the United States. This disease may emerge in new quinoa production regions and may cause yield losses due to lodging.

A Real-Time PCR Differentiating Pantoea stewartii subsp. stewartii From P. stewartii subsp. indologenes in Corn Seed.

Stewart's wilt of corn caused by the bacterium Pantoea stewartii subsp. stewartii is a seed-borne disease of major phytosanitary importance. Many countries have imposed restrictions on corn seed imports from regions where the disease occurs to prevent the potential introduction of the pathogen. Current laboratory testing methods (enzyme-linked immunosorbent assay [ELISA] and polymerase chain reaction [PCR]) cannot readily distinguish P. stewartii subsp. stewartii from the closely related subspecies Pantoea stewartii subsp. indologenes. However, P. stewartii subsp. indologenes, a nonpathogen on corn, is occasionally found on corn seed as part of the resident bacterial population and can yield false positive test results. A real-time PCR targeting the cpsAB intergenic sequence was developed to specifically detect P. stewartii subsp. stewartii from corn seeds and distinguish it from P. stewartii subsp. indologenes. The assay successfully detected P. stewartii subsp. stewartii from corn seed, and P. stewartii subsp. indologenes-contaminated seed lots, which previously yielded false positives by ELISA and published PCR methods, were negative. The absence of P. stewartii subsp. stewartii and the presence of P. stewartii subsp. indologenes in this seed were confirmed by size differentiation of the cpsAB amplicons in a conventional PCR. By distinguishing the two subspecies, the assays described would avoid false positive results and help prevent unnecessary restrictions on international movement of corn seed.

Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment.

BACKGROUND: Motility and the coordination of moving food through the gastrointestinal tract rely on a complex network of neurons known as the enteric nervous system (ENS). Despite its critical function, many of the molecular mechanisms that direct the development of the ENS and the elaboration of neural network connections remain unknown. The goal of this study was to transcriptionally identify molecular pathways and candidate genes that drive specification, differentiation and the neural circuitry of specific neural progenitors, the phox2b expressing ENS cell lineage, during normal enteric nervous system development. Because ENS development is tightly linked to its environment, the transcriptional landscape of the cellular environment of the intestine was also analyzed. RESULTS: Thousands of zebrafish intestines were manually dissected from a transgenic line expressing green fluorescent protein under the phox2b regulatory elements [Tg(phox2b:EGFP) w37 ]. Fluorescence-activated cell sorting was used to separate GFP-positive phox2b expressing ENS progenitor and derivatives from GFP-negative intestinal cells. RNA-seq was performed to obtain accurate, reproducible transcriptional profiles and the unbiased detection of low level transcripts. Analysis revealed genes and pathways that may function in ENS cell determination, genes that may be identifiers of different ENS subtypes, and genes that define the non-neural cellular microenvironment of the ENS. Differential expression analysis between the two cell populations revealed the expected neuronal nature of the phox2b expressing lineage including the enrichment for genes required for neurogenesis and synaptogenesis, and identified many novel genes not previously associated with ENS development. Pathway analysis pointed to a high level of G-protein coupled pathway activation, and identified novel roles for candidate pathways such as the Nogo/Reticulon axon guidance pathway in ENS development. CONCLUSION: We report the comprehensive gene expression profiles of a lineage-specific population of enteric progenitors, their derivatives, and their microenvironment during normal enteric nervous system development. Our results confirm previously implicated genes and pathways required for ENS development, and also identify scores of novel candidate genes and pathways. Thus, our dataset suggests various potential mechanisms that drive ENS development facilitating characterization and discovery of novel therapeutic strategies to improve gastrointestinal disorders.

Toxin delivery by the coat protein of an aphid-vectored plant virus provides plant resistance to aphids.

The sap-sucking insects (order Hemiptera), including aphids, planthoppers, whiteflies and stink bugs, present one of the greatest challenges for pest management in global agriculture. Insect neurotoxins offer an alternative to chemical insecticides for controlling these pests, but require delivery into the insect hemocoel. Here we use the coat protein of a luteovirus, an aphid-vectored plant virus, to deliver a spider-derived, insect-specific toxin that acts within the hemocoel. The luteovirid coat protein is sufficient for delivery of fused proteins into the hemocoel of pea aphids, Acyrthosiphon pisum, without virion assembly. We show that when four aphid pest species-A. pisum, Rhopalosiphum padi, Aphis glycines and Myzus persicae-feed on a recombinant coat protein-toxin fusion, either in an experimental membrane sachet or in transgenic Arabidopsis plants, they experience significant mortality. Aphids fed on these fusion proteins showed signs of neurotoxin-induced paralysis. Luteovirid coat protein-insect neurotoxin fusions represent a promising strategy for transgenic control of aphids and potentially other hemipteran pests.

Aphicidal efficacy of scorpion- and spider-derived neurotoxins.

Insect-specific neurotoxins that act within the insect hemocoel (body cavity) represent an untapped resource for insect pest management. On the basis of recent advances made in development of appropriate delivery systems for transport of these toxins from the insect gut, across the gut epithelium to their target site, we screened neurotoxins derived from scorpion or spider venom for efficacy against the pea aphid, Acyrthosiphon pisum, and the green peach aphid, Myzus persicae. Toxins were selected to represent different modes of electrophysiological action, including activity on voltage-gated calcium channels (ω-TRTX-Gr1a, ω-agatoxin Aa4a, ω-hexatoxin-Hv1a), calcium- and voltage-activated potassium channels (charybdotoxin, maurotoxin), chloride channels (chlorotoxin) and voltage-gated sodium channels (LqhαIT). The Bacillus thuringiensis-derived toxin Cyt1Aa was also tested as a positive control for toxicity. In per os bioassays with both aphid species, toxicity was only seen for ω-TRTX-Gr1a and Cyt1Aa. On injection into the hemocoel of A. pisum, LD50 values ranged from 1 to 8 ng/mg body weight, with ω-hexatoxin-Hv1a being the most toxic (1.02 ng/mg body weight). All neurotoxins caused rapid paralysis, with charybdotoxin, maurotoxin and chlorotoxin also causing melanization of injected aphids. These data represent the first comprehensive screen of neurotoxins against aphids, and highlight the potential for practical use of the insect-specific toxin ω-hexatoxin-Hv1a in aphid management.

Low-grade fibromyxoid sarcoma of anterior abdominal wall.

Low grade fibromyxoid sarcoma (LGFMS) is a rare soft tissue tumor, having metastatic potential and high local recurrence rate despite its low grade histologic findings. This tumor of deep and subcutaneous soft tissues occurs in 3rd and 5th decade of life. Diagnosis of LGFMS remains problematic because of its bland looking histologic features that can be potentially confused with other benign or low grade soft tissue tumors. We report here a rare case report of low grade fibromyxoid sarcoma of anterior abdominal wall.

Comparison of the effectiveness of passive (dam) versus active (piglet) immunization against porcine circovirus type 2 (PCV2) and impact of passively derived PCV2 vaccine-induced immunity on vaccination.

The objectives of this study were (1) to compare the efficacy of two different PCV2 vaccination protocols (colostrum-derived immunity versus piglet vaccination) in a conventional PCV2 growing pig challenge model and (2) to evaluate the efficacy of vaccinating piglets with the same vaccine used in the dams. Two different commercially available vaccines (VAC1; VAC2) were used in the same experiment. Seventy-eight piglets born to vaccinated or non-vaccinated sows were divided into 8 groups. A proportion of the pigs with and a proportion of the pigs without passively acquired immunity were vaccinated at 21 days of age. All pigs except negative controls were challenged with PCV2b at 35 days post-vaccination and necropsied at 21 days post-challenge (dpc). The data indicates that both dam vaccination and piglet vaccination had similar efficacies in reducing PCV2 viral loads and antigen levels in the growing pigs. Interestingly, dam vaccination alone did result in significantly (P<0.05) lower anti-PCV2-antibodies levels at challenge in piglets from dams immunized with VAC2 compared to piglets from VAC1 immunized dams. When data obtained from the growing piglets that were vaccinated with VAC1 or VAC2 were compared, antibody levels and reduction of incidence of PCV2-antigen were not different; however, piglets vaccinated with VAC2 had reduced PCV2-DNA genomic copies in serum by 21 dpc. Vaccination of piglets with the same vaccine as was used on their dams did not appear to affect vaccine efficacy as piglets in these groups had anti-PCV2-antibody levels and PCV2 genomic copies similar to the groups where vaccine was administered to the piglets only.

Infectivity of porcine circovirus type 2 DNA in semen from experimentally-infected boars.

Porcine circovirus type 2 (PCV2) is an economically important pathogen. It has been demonstrated that PCV2 DNA can be detected in boar semen by PCR; however, the biological relevance of this is unknown. The objectives of this study were to determine if semen positive for PCV2 DNA is infectious (1) in a swine bioassay, or (2) when used for artificial insemination. For the first objective, 4-week-old pigs were inoculated intraperitoneally with PCV2 DNA-negative (bioassay-control; n = 3), PCV2a DNA-positive (bioassay-PCV2a; n = 3), or PCV2b DNA-positive (bioassay-PCV2b; n = 3) raw semen, or PCV2 live virus (bioassay-positive; n = 3), respectively. Pigs inoculated with PCV2 DNA-positive semen and PCV2 live virus became viremic and developed anti-PCV2 antibodies indicating that the PCV2 DNA present in semen was infectious. For the second objective, three Landrace gilts were inseminated with PCV2 DNA-negative semen (gilts-controls) from experimentally-infected boars, and six gilts were artificially inseminated with semen positive for PCV2a DNA (gilts-PCV2a; n = 3) or PCV2b DNA (gilts-PCV2b; n = 3). Serum samples collected from the gilts in all groups remained negative for anti-PCV2 antibodies for the duration of the experiment. In addition, fetal serum samples from all 105-day-gestation fetuses were negative for anti-PCV2 antibodies or PCV2 DNA. Under the conditions of this study, PCV2 DNA-positive semen was not infectious when used to artificially inseminate gilts; however, it was demonstrated to be infectious in a swine bioassay model and therefore is a potential means of PCV2 transmission amongst swine herds.

Characterization of shedding patterns of Porcine circovirus types 2a and 2b in experimentally inoculated mature boars.

Porcine circovirus-2 (PCV-2) is an economically important swine pathogen and causes PCV-associated disease (PCVAD) in pigs worldwide. Currently, 2 genotypes of PCV-2, PCV-2a and -2b, are circulating in U.S. swine herds. The objectives of the current study were to evaluate the amount of PCV-2 DNA present in semen over time, compare and correlate incidence and amount of PCV-2 present in semen samples to that present in serum samples and blood swabs, and determine if there are differences in shedding patterns between PCV-2a and -2b. Fifteen 7-month-old PCV-2-naïve Landrace boars (Sus scrofa) were randomly allocated to 3 treatment groups. The boars in group 1 (n = 3) served as negative controls, and those in groups 2 (n = 6) and 3 (n = 6) were intranasally and intramuscularly inoculated with PCV-2a and -2b, respectively. Semen, serum, and blood swab samples were collected up to 90 days postinoculation (DPI), and necropsies were performed on DPI 23, 48, and 90. Larger quantities of both PCV-2a and -2b DNA were detected earlier in serum and blood swab samples than in raw semen of experimentally inoculated boars. The incidence and duration of presence of PCV-2 DNA in semen varied among boars; however, intermittent shedding was not observed. In all sex glands, PCV-2 DNA was detected by polymerase chain reaction; however, PCV-2 antigen was not detected by immunohistochemistry, and PCV-2 had no effect on sperm morphology. Differences in shedding patterns between PCV-2a and -2b were not observed under the study conditions.

Infectious genomic RNA of Rhopalosiphum padi virus transcribed in vitro from a full-length cDNA clone.

Availability of a cloned genome from which infectious RNA can be transcribed is essential for investigating RNA virus molecular mechanisms. To date, no such clones have been reported for the Dicistroviridae, an emerging family of invertebrate viruses. Previously we demonstrated baculovirus-driven expression of a cloned Rhopalosiphum padi virus (RhPV; Dicistroviridae) genome that was infectious to aphids, and we identified a cell line (GWSS-Z10) from the glassy-winged sharpshooter, that supports RhPV replication. Here we report that RNA transcribed from a full-length cDNA clone is infectious. Transfection of GWSS-Z10 cells with the RhPV transcript resulted in cytopathic effects, ultrastructural changes, and accumulation of progeny virions, consistent with virus infection. Virions from transcript-infected cells were infectious in aphids. This infectious transcript of a cloned RhPV genome provides a valuable tool, and a more tractable system without interference from baculovirus infection, for investigating replication and pathogenesis of dicistroviruses.

Potential ligands of DmP29, a putative juvenile hormone esterase binding protein of Drosophila melanogaster.

We previously reported the identification of a putative juvenile hormone esterase (JHE) binding protein DmP29 in Drosophila melanogaster and its primary localization to the mitochondria [Liu, Z., Ho, L., Bonning, B.C., 2007. Localization of a Drosophila melanogaster homolog of the putative juvenile hormone esterase binding protein of Manduca sexta. Insect Biochem. Mol. Biol. 37(2), 155-163]. To further characterize DmP29, we identified potential ligands of this protein. Recombinant DmP29 was shown by ligand blot and co-immunoprecipitation analyses to bind recombinant JHE as well as to larval serum proteins (LSP). The possible biological relevance of the in vitro DmP29-JHE interaction is provided by detection of JHE activity in D. melanogaster mitochondrial fractions; 0.48 nmol JH hydrolyzed/min/mg mitochondrial protein, 97% of which was inhibited by the JHE-specific inhibitor OTFP. However, the DmP29-LSP interactions may not be biologically relevant. Given the high abundance, and "sticky" nature of these proteins, interaction of DmP29 with LSP may result from non-specific associations. No DmP29 interactions with non-specific esterases were detected by co-immunoprecipitation analyses. The potential role of DmP29 as a chaperone of JHE is discussed.

A baculovirus-expressed dicistrovirus that is infectious to aphids.

Detailed investigation of virus replication is facilitated by the construction of a full-length infectious clone of the viral genome. To date, this has not been achieved for members of the family Dicistroviridae. Here we demonstrate the construction of a baculovirus that expresses a dicistrovirus that is infectious in its natural host. We inserted a full-length cDNA clone of the genomic RNA of the dicistrovirus Rhopalosiphum padi virus (RhPV) into a baculovirus expression vector. Virus particles containing RhPV RNA accumulated in the nuclei of baculovirus-infected Sf21 cells expressing the recombinant RhPV clone. These virus particles were infectious in R. padi, a ubiquitous aphid vector of major cereal viruses. The recombinant virus was transmitted efficiently between aphids, despite the presence of 119 and 210 vector-derived bases that were stably maintained at the 5' and 3' ends, respectively, of the RhPV genome. The maintenance of such a nonviral sequence was surprising considering that most RNA viruses tolerate few nonviral bases beyond their natural termini. The use of a baculovirus to express a small RNA virus opens avenues for investigating replication of dicistroviruses and may allow large-scale production of these viruses for use as biopesticides.

A glassy-winged sharpshooter cell line supports replication of Rhopalosiphum padi virus (Dicistroviridae).

Rhopalosiphum padi virus (RhPV) (family Dicistroviridae; genus Cripavirus) is an icosahedral aphid virus with a 10kb positive-sense RNA genome. To study the molecular biology of RhPV, identification of a cell line that supports replication of the virus is essential. We screened nine cell lines derived from species within the Lepidoptera, Diptera and Hemiptera for susceptibility to RhPV following RNA transfection. We observed cytopathic effects (CPE) only in cell lines derived from hemipterans, specifically GWSS-Z10 cells derived from the glassy winged sharp shooter, Homalodisca coagulata and DMII-AM cells derived from the corn leaf hopper, Dalbulus maidis. Translation and appropriate processing of viral gene products, RNA replication and packaging of virus particles in the cytoplasm of GWSS-Z10 cells were examined by Western blot analysis, Northern blot hybridization and electron microscopy. Infectivity of the GWSS-Z10 cell derived-virus particles to the bird cherry-oat aphid, R. padi, was confirmed by RT-PCR and Western blot. The GWSS-Z10 cell line provides a valuable tool to investigate replication, structure and assembly of RhPV.