Drastic evolution of point defects in vertically grown ZnO nanorods induced by lithium ion implantation.

The evolution of various point defects in 100 keV lithium (Li) ion-implanted ZnO nanorods (NRs) by varying the fluences from 1 × 1014 to 7 × 1015 ions per cm2 has been investigated experimentally and using a simulation by stopping and range of ions in matter (SRIM). The X-ray photoelectron spectroscopy results indicate that the Li1+ ions have been incorporated at Zn2+ sites, which forms LiZn acceptors in the implanted NRs. The structural disorder and the number of oxygen vacancies in the implanted ZnO NRs increase drastically with an increase in the Li fluence as indicated by the X-ray diffractometry and Raman scattering analyses. Both the formation of acceptors and implantation-induced defects make the Li-implanted NRs electrically highly resistive. The yellow-orange photoluminescence (PL) emission of the as-grown ZnO NRs has evolved into green emission in the implanted NRs. A suppression of the green PL at higher fluences is possibly due to an apparent decrease in the zinc vacancy concentration. The SRIM results explain the quantitative energy loss, the distributions of the implanted Li ions and the point defects along the target ZnO NRs. The consistency between the experimental and theoretical simulations validates our analyses on the formation and evolution of various point defects in highly resistive Li-implanted ZnO NRs.

Assessment and verification of chemical inactivation of peste des petits ruminants virus by virus isolation following virus capture using Nanotrap magnetic virus particles.

This study reports development and optimization of a new method for the assessment and verification of the inactivation of peste des petits ruminants virus (PPRV) by chemical agents, including Triton X-100 and commercially available viral lysis buffers. Virus inactivation was confirmed by virus isolation (VI) on Vero cells following capture of the potential residual viruses from treated samples using Nanotrap magnetic virus particles (NMVPs). Since chemical agents are cytotoxic, treated PPRV samples could not be used directly for VI on Vero cell monolayers; instead, they were diluted in Eagle's Minimum Essential Medium (EMEM) to neutralize cytotoxicity and then subjected to virus capture using NMVPs. The NMVPs and the captured viruses were then clarified on a magnetic stand, reconstituted in EMEM, and inoculated onto Vero cells that were examined for cytopathic effect (CPE). No CPE was observed on cells inoculated with treated viruses captured by NMVPs; but CPE was observed on cells inoculated with untreated viruses, including those captured by NMVPs. For further verification, the supernatants of the VI cultures (treated or untreated) were subjected to RNA extraction and PPRV-specific real-time RT-PCR (RT-qPCR). The cycle threshold values were undetectable for the supernatants of VI cultures inoculated with NMVPs reconstituted from treated PPRV but detectable for the supernatants of VI cultures inoculated with untreated PPRV or the NMVPs reconstituted from untreated PPRV, indicating complete inactivation of PPRV. This new method of verification of virus inactivation using NMVPs can be applied to other high impact viruses of agricultural or public health importance. IMPORTANCE Research including diagnosis on highly contagious viruses at the molecular level such as PCR and next-generation sequencing requires complete inactivation of the virus to ensure biosafety and biosecurity so that any accidental release of the virus does not compromise the safety of the susceptible population and the environment. In this work, peste des petits ruminants virus (PPRV) was inactivated with chemical agents, and the virus inactivation was confirmed by virus isolation (VI) using Vero cells. Since the chemical agents are cytotoxic, inactivated virus (PPRV) was diluted 1:100 to neutralize cytotoxicity, and the residual viruses (if any) were captured using Nanotrap magnetic virus particles (NMVPs). The NMVPs and the captured viruses were subjected to VI. No CPE was observed, indicating complete inactivation, and the results were further supported by real-time RT-PCR. This new protocol to verify virus inactivation can be applicable to other viruses.

Development of a loop-mediated isothermal amplification assay for rapid detection of capripoxviruses.

Sheep pox (SP), goat pox (GP), and lumpy skin disease (LSD), caused by capripoxviruses (CaPVs), are economically important diseases of sheep, goats, and cattle, respectively. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of CaPVs. LAMP primers were designed to target a conserved gene encoding the poly(A) polymerase small subunit (VP39) of CaPVs. Hydroxynaphthol blue (HNB) was incorporated to monitor assay progress by color change from violet when negative to sky blue when positive, and results were verified by agarose gel electrophoresis. The LAMP assay was shown to be highly specific for CaPVs, with no apparent cross-reactivity to other related viruses (near neighbors) or viruses that cause similar clinical signs (look-a-like viruses). The performance of LAMP was compared to that of a highly sensitive quantitative real-time PCR (qPCR) assay. LAMP and qPCR exhibited similar analytical sensitivities, with limits of detection of 3 and 8 viral genome copies, respectively. Diagnostic specificity was assessed on 36 negative specimens, including swabs and EDTA blood from control sheep, goats, and cattle. Diagnostic sensitivity was assessed on 275 specimens, including EDTA blood, swabs, and tissues from experimentally infected sheep, goats, and cattle. Overall agreement on diagnostic test results between the two assays was 90 to 95% for specificity and 89 to 100% for sensitivity. The LAMP assay described in this report is simple to use, inexpensive, highly sensitive, and particularly well suited for the diagnosis of capripox in less well equipped laboratories and in rural settings where resources are limited.

Development of multiplex real-time PCR assays for differential detection of capripoxvirus, parapoxvirus and foot-and-mouth disease virus.

This study reports the development of multiplex real-time PCR assays for differential detection of capripoxvirus (CaPV), parapoxvirus (PaPV) and foot-and-mouth disease virus (FMDV) in sheep, goats and cattle. Three multiplex assays were developed, a capripox (CaP) rule-out assay for simultaneous detection and differentiation of CaPV and PaPV, a FMD rule-out assay for simultaneous detection and differentiation of FMDV and PaPV, and a FMD/CaP rule-out assay for simultaneous detection and differentiation of CaPV, PaPV and FMDV. All multiplex assays included ß-actin gene ACTB as an internal positive control to monitor PCR inhibition and accuracy of nucleic acid extractions. The optimized assays were highly specific to the target viruses (CaPV, PaPV and FMDV) with no cross-reactivity against other viruses that cause similar clinical signs. Using positive control plasmids as template, the limit of detection (LOD) of the multiplex assays were estimated as 2 CaPV, 7 PaPV and 15 FMDV copies per assay. The amplification efficiency (AE) and correlation coefficient (R2 ), estimated from the standard curves (Ct vs. log10 template dilution), were 94%-106% and >0.99, respectively, for CaP and FMD rule-out assays, 96%-116% (AE) and >0.98 (R2 ), respectively, for CaP/FMD rule-out assays and 91%-102% and >0.99, respectively, for the corresponding singleplex assays. The diagnostic sensitivity (DSe) of the multiplex assays was assessed on 35 CaPV and 39 FMDV clinical specimens from experimentally infected (CS-E) animals, and 29 CaPV (LSDV), 28 FMDV and 36 PaPV clinical specimens from naturally infected (CS-N) animals; all tested positive (DSe 100%) except two CS-E FMDV specimens that were tested negative by FMD rule-out and the corresponding singleplex (FMDV) assays (37/39; DSe 95%). The newly developed multiplex assays offer a valuable tool for differential detection of clinically indistinguishable CaPV, PaPV and FMDV in suspected animals and animals with mixed infections.

Development and evaluation of multiplex real-time RT-PCR assays for the detection and differentiation of foot-and-mouth disease virus and Seneca Valley virus 1.

Foot-and-mouth disease virus (FMDV) causes a highly contagious and economically important vesicular disease in cloven-hoofed animals that is clinically indistinguishable from symptoms caused by Seneca Valley virus 1 (SVV-1). To differentiate SVV-1 from FMDV infections, we developed a SVV-1 real-time RT-PCR (RT-qPCR) assay and multiplexed with published FMDV assays. Two published FMDV assays (Journal of the American Veterinary Medical Association, 220, 2002, 1636; Journal of Virological Methods, 236, 2016, 258) targeting the 3D polymerase (3D) region were selected and multiplexed with the SVV-1 assay that has two targets, one in the 5' untranslated region (5' UTR, this study) and the other in the 3D region (Journal of Virological Methods, 239, 2017, 34). In silico analysis showed that the primers and probes of SVV-1 assay matched 98.3% of the strain sequences (113/115). The primer and probe sequences of the Shi FMDV assay matched 85.4% (806/944), and that of the Callahan FMDV assay matched 62.7% (592/944) of the sequences. The limit of detection (LOD) for the two multiplex RT-qPCR assays for SVV-1 was both 9 copies per reaction by cloned positive plasmids and 0.16 TCID50 per reaction by cell culture. The LOD for FMDV by both multiplex assays was 11 copies per reaction using cloned positive plasmids. With cell cultures of the seven serotypes of FMDV, the Shi assay (Journal of Virological Methods, 236, 2016, 258) had LODs between 0.04 and 0.18 TCID50 per reaction that were either the same or lower than the Callahan assay. Interestingly, multiplexing with SVV-1 increased the amplification efficiencies of the Callahan assay (Journal of the American Veterinary Medical Association, 220, 2002, 1636) from 51.5%-66.7% to 89.5%-96.6%. Both assays specifically detected the target viruses without cross-reacting to SVV-1 or to other common porcine viruses. An 18S rRNA housekeeping gene that was amplified from multiple cloven-hoofed animal species was used as an internal control. The prevalence study did not detect any FMDV, but SVV-1 was detected from multiple types of swine samples with an overall positive rate of 10.5% for non-serum samples.

Removal of real-time reverse transcription polymerase chain reaction (RT-PCR) inhibitors associated with cloacal swab samples and tissues for improved diagnosis of Avian influenza virus by RT-PCR.

Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) is routinely used for the rapid detection of Avian influenza virus (AIV) in clinical samples, but inhibitory substances present in some clinical specimens can reduce or block PCR amplification. Most commercial RNA extraction kits have limited capacity to remove inhibitors from clinical samples, but using a modified commercial protocol (Ambion MagMAX, Applied Biosystems, Foster City, CA) with an added high-salt wash of 2 M NaCl and 2 mM ethylenediamine tetra-acetic acid was shown to improve the ability of the kit to remove inhibitors from cloacal swabs and some tissues. Real-time RT-PCR was carried out in the presence of an internal positive control to detect inhibitors present in the purified RNA. Cloacal swabs from wild birds were analyzed by real-time RT-PCR comparing RNA extracted with the standard (MagMAX-S) and modified (MagMAX-M) protocols. Using the standard protocol on 2,668 samples, 18.4% of the samples had evidence of inhibitor(s) in the samples, but the modified protocol removed inhibitors from all but 21 (4.8%) of the problem samples. The modified protocol was also tested for RNA extraction from tissues using a TRIzol-MagMAX-M hybrid protocol. Tissues from chickens and ducks experimentally infected with high-pathogenicity Asian H5N1 AIV were analyzed by real-time RT-PCR, and the limit of detection of the virus was improved by 0.5-3.0 threshold cycle units with the RNA extracted by the MagMAX-M protocol. The MagMAX-M protocol reported in the present study can be useful in extracting high-quality RNA for accurate detection of AIV from cloacal swabs and tissues by real-time RT-PCR.

Development of conventional and real time PCR assays for rapid species authentication of mammalian cell lines commonly used in veterinary diagnostic laboratories.

Mammalian cell lines are valuable tools in biomedical fields, with applications ranging from disease diagnosis to the production of biological reagents and vaccines. Here we report the development of new conventional (cPCR) and real time PCR (qPCR) assays for species identification of several mammalian kidney cell lines originated from swine, green monkey, hamster and bovine tissues that are extensively used in veterinary diagnostic laboratories. The PCR primers and probes were selected from highly conserved mitochondrial genes and analyzed in silico by nucleotide BLAST in the National Center for Biotechnology Information (NCBI) website to ensure target specificity. The assays were highly species-specific and had no cross-reactivity against other tested cell lines originated from different mammalian species. Assay sensitivity (limit of detection; LOD) was determined using serial dilutions of cell line DNA as template. The estimated LODs were between 2.95 and 48 pg (picogram) DNA/assay for cPCR, and between 1.5 × 10-3 and 4.8 × 10-2 pg DNA/assay for qPCR. Multiplex qPCR assays were developed for simultaneous detection of up to three species in a single assay. The multiplex qPCR assays exhibited the same sensitivity as the corresponding singleplex assays with the exception of the green monkey species that demonstrated a 10-100 fold decline in the sensitivity. Contamination of swine cells was detected in one of the rabbit cell lines. The contamination was further confirmed by Sanger and Next-Generation sequencing.

Defect-Assisted Broad-Band Photosensitivity with High Responsivity in Au/Self-Seeded TiO2 NR/Au-Based Back-to-Back Schottky Junctions.

TiO2 nanorods (NRs) have generated much interest for both fundamental understanding of defect formation and technological applications in energy harvesting, optoelectronics, and catalysis. Herein, we have grown TiO2 NR films on glass substrates using a self-seeded approach and annealed them in H2 ambient to modify their surface defects. It has been shown that broad-band photosensing properties of Au/self-seeded TiO2 NR/Au-based two back-to-back Schottky junctions (SJs) for a broad wavelength of light are much superior as compared to those of the pristine and the control samples. Photoresponsivity values for the H2-annealed sample are 0.42, 0.71, 0.07, and 0.08 A/W for detecting, respectively, 350, 400, 470, and 570 nm lights. Very low dark current and high photocurrent lead to a gain value as high as 1.85 × 104 for 400 nm light. Unprecedentedly modified NR-based SJs show excellent photoresponsivity for detecting as low as 25, 36, 48, and 28 µW/cm2 power densities of 350, 400, 470, and 570 nm lights, respectively. It is found that Ti3+ defects play a key role in an efficient photoelectron transfer from TiO2 to Au. Our work, for the first time, highlights the simplicity and reveals the rationale behind the excellent properties of Au/self-seeded TiO2 NR film/Au back-to-back SJs.

The complete genome sequence of Moorella thermoacetica (f. Clostridium thermoaceticum).

This paper describes the genome sequence of Moorella thermoacetica (f. Clostridium thermoaceticum), which is the model acetogenic bacterium that has been widely used for elucidating the Wood-Ljungdahl pathway of CO and CO(2) fixation. This pathway, which is also known as the reductive acetyl-CoA pathway, allows acetogenic (often called homoacetogenic) bacteria to convert glucose stoichiometrically into 3 mol of acetate and to grow autotrophically using H(2) and CO as electron donors and CO(2) as an electron acceptor. Methanogenic archaea use this pathway in reverse to grow by converting acetate into methane and CO(2). Acetogenic bacteria also couple the Wood-Ljungdahl pathway to a variety of other pathways to allow the metabolism of a wide variety of carbon sources and electron donors (sugars, carboxylic acids, alcohols and aromatic compounds) and electron acceptors (CO(2), nitrate, nitrite, thiosulfate, dimethylsulfoxide and aromatic carboxyl groups). The genome consists of a single circular 2 628 784 bp chromosome encoding 2615 open reading frames (ORFs), which includes 2523 predicted protein-encoding genes. Of these, 1834 genes (70.13%) have been assigned tentative functions, 665 (25.43%) matched genes of unknown function, and the remaining 24 (0.92%) had no database match. A total of 2384 (91.17%) of the ORFs in the M. thermoacetica genome can be grouped in orthologue clusters. This first genome sequence of an acetogenic bacterium provides important information related to how acetogens engage their extreme metabolic diversity by switching among different carbon substrates and electron donors/acceptors and how they conserve energy by anaerobic respiration. Our genome analysis indicates that the key genetic trait for homoacetogenesis is the core acs gene cluster of the Wood-Ljungdahl pathway.

Detection of H5N1 high-pathogenicity avian influenza virus in meat and tracheal samples from experimentally infected chickens.

The Asian H5N1 highly pathogenic avian influenza (HPAI) virus causes a systemic disease with high mortality of poultry and is potentially zoonotic. In both chickens and ducks, the virus has been demonstrated to replicate in both cardiac and skeletal muscle cells. Experimentally, H5N1 HPAI virus has been transmitted to chickens through the consumption of raw infected meat. In this study, we investigated virus replication in cardiac and skeletal muscle and in the trachea of chickens after experimental intranasal inoculation with the H5N1 HPAI virus. The virus was detected in tissues by real-time reverse transcription-polymerase chain reaction (RRT-PCR) and virus isolation, and in the trachea by RRT-PCR and a commercial avian influenza (AI) viral antigen detection test. A modified RNA extraction protocol was developed for rapid detection of the virus in tissues by RRT-PCR. The H5N1 HPAI virus was sporadically detected in meat and the tracheas of infected birds without any clinical sign of disease as early as 6 hr postinfection (PI), and was detected in all samples tested at 24 hr PI and later. No differences in sensitivity were seen between virus isolation and RRT-PCR in meat samples. The AI viral antigen detection test on tracheal swabs was a useful method for identifying infected chickens when they were sick or dead, but was less sensitive in detecting infected birds when they were preclinical. This study provides data indicating that preslaughter tracheal swab testing can identify birds infected with HPAI among the daily mortality and prevent infected flocks from being sent to processing plants. In addition, the modified RNA extraction and RRT-PCR test on meat samples provide a rapid and sensitive method of identifying HPAI virus in illegal contraband or domestic meat samples.

Detection of foot-and-mouth disease virus in milk samples by real-time reverse transcription polymerase chain reaction: Optimisation and evaluation of a high-throughput screening method with potential for disease surveillance.

This study aimed to evaluate the utility of milk as a non-invasive sample type for the surveillance of foot-and-mouth disease (FMD), a highly contagious viral disease of cloven-hooved animals. Four milking Jersey cows were infected via direct-contact with two non-milking Jersey cows that had been previously inoculated with FMD virus (FMDV: isolate O/UKG/34/2001). Milk and blood were collected throughout the course of infection to compare two high-throughput real-time reverse transcription polymerase chain reaction (rRT-PCR) protocols with different RT-PCR chemistries. Using both methods, FMDV was detected in milk by rRT-PCR one to two days before the presentation of characteristic foot lesions, similar to detection by virus isolation. Furthermore, rRT-PCR detection from milk was extended, up to 28 days post contact (dpc), compared to detection by virus isolation (up to 14 dpc). Additionally, the detection of FMDV in milk by rRT-PCR was possible for 18 days longer than detection by the same method in serum samples. FMDV was also detected with both rRT-PCR methods in milk samples collected during the UK 2007 outbreak. Dilution studies were undertaken using milk from the field and experimentally-infected animals, where for one sample it was possible to detect FMDV at 10-7. Based on the peak CT values detected in this study, these findings indicate that it could be possible to identify one acutely-infected milking cow in a typical-sized dairy herd (100-1000 individuals) using milk from bulk tanks or milk tankers. These results motivate further studies using milk in FMD-endemic countries for FMD surveillance.

Characterization of a corrinoid protein involved in the C1 metabolism of strict anaerobic bacterium Moorella thermoacetica.

The strict anaerobic, thermophilic bacterium Moorella thermoacetica metabolizes C1 compounds for example CO(2)/H(2), CO, formate, and methanol into acetate via the Wood/Ljungdahl pathway. Some of the key steps in this pathway include the metabolism of the C1 compounds into the methyl group of methylenetetrahydrofolate (MTHF) and the transfer of the methyl group from MTHF to the methyl group of acetyl-CoA catalyzed by methyltransferase, corrinoid protein and CO dehydrogenase/acetyl CoA synthase. Recently, we reported the crystallization of a 25 kDa methanol-induced corrinoid protein from M. thermoacetica (Zhou et al., Acta Crystallogr F 2005; 61:537-540). In this study we analyzed the crystal structure of the 25 kDa protein and provide genetic and biochemical evidences supporting its role in the methanol metabolism of M. thermoacetia. The 25 kDa protein was encoded by orf1948 of contig 303 in the M. thermoacetica genome. It resembles similarity to MtaC the corrinoid protein of the methanol:CoM methyltransferase system of methane producing archaea. The latter enzyme system also contains two additional enzymes MtaA and MtaB. Homologs of MtaA and MtaB were found to be encoded by orf2632 of contig 303 and orf1949 of contig 309, respectively, in the M. thermoacetica genome. The orf1948 and orf1949 were co-transcribed from a single polycistronic operon. Metal analysis and spectroscopic data confirmed the presence of cobalt and the corrinoid in the purified 25 kDa protein. High resolution X-ray crystal structure of the purified 25 kDa protein revealed corrinoid as methylcobalamin with the imidazole of histidine as the alpha-axial ligand replacing benziimidazole, suggesting base-off configuration for the corrinoid. Methanol significantly activated the expression of the 25 kDa protein. Cyanide and nitrate inhibited methanol metabolism and suppressed the level of the 25 kDa protein. The results suggest a role of the 25 kDa protein in the methanol metabolism of M. thermoacetica.

Development and bench validation of real-time reverse transcription polymerase chain reaction protocols for rapid detection of the subtypes H6, H9, and H11 of avian influenza viruses in experimental samples.

Real-time reverse transcription polymerase chain reaction (RRT-PCR) is commonly used for the rapid detection, as well as to determine the subtype, of avian influenza viruses (AIVs). There are 16 known serologically distinct hemagglutinin (HA) subtypes of AIV described. Currently, determination of the subtypes of AIVs by RRT-PCR tests has been limited to the H5 and H7 subtypes. In this study, RRT-PCR assays were developed in simplex formats for rapid detection of AIV subtypes H6, H9, and H11. The primers and probes for RRT-PCR were designed from nucleotide sequences of the HA genes, which were either downloaded from GenBank (for H6 and H9) or sequenced for this study. The specificity and sensitivity of the RRT-PCR assays were determined based on the detection of the virus from a proficiency panel consisting of 15 different HA subtypes of AIVs and from serial dilutions of target viral RNA. The subtype-specific RRT-PCR assays were used to detect the virus in cloacal and oropharyngeal swabs of experimental chickens inoculated with H6, H9, and H11 AIVs, and the test results were compared with validated RRT-PCR assays based on the amplification of AI matrix (MA) gene. A high correlation of the matrix test and the specific H6, H9, and H11 by the RRT-PCR assays was observed; kappa coefficients for the agreement of test results in cloacal and oropharyngeal swabs combined were 0.927, 0.962, and 0.981, respectively.

Review of rapid molecular diagnostic tools for avian influenza virus.

Molecular diagnostic tests are commonly used to diagnose avian influenza virus because they are sensitive and can be performed rapidly, with high throughput, and at a moderate cost. Molecular diagnostic tests recently have proven themselves to be invaluable in controlling disease outbreaks around the world. Several different methods, including traditional reverse transcription-polymerase chain reaction (PCR), real-time reverse transcription-polymerase chain reaction, and nucleic acid sequence-based amplification among others, have been described for the diagnosis of avian influenza in poultry with many different variations of primers, probes, enzymes, etc. Few of these tests have been validated, with the understanding that validation should be described as a level of comparison testing to show "fitness for purpose." None of the molecular diagnostic tests are validated for all species or specimen types that might be presented to a diagnostic laboratory. The sensitivity and specificity for all the molecular tests are governed by three critical control points, including RNA extraction, enzymes used for amplification, and the sequence of primers and probes. The RNA extraction step is of particular concern, since high-quality RNA is needed for any of the molecular tests. Some sample types, including cloacal (fecal) swabs and tissues, are difficult to process, with issues of poor RNA extraction or PCR inhibitors being common. The development of internal controls, robotics, and bead reagents are providing improved performance of existing tests, and new technologies will likely provide better tests for the future. With any molecular test, assay assurance must be performed on an ongoing basis, which includes the use of proficiency panels to measure test performance.

Modification of two capripoxvirus quantitative real-time PCR assays to improve diagnostic sensitivity and include beta-actin as an internal positive control.

Capripoxviruses (CaPVs), consisting of Sheeppox virus (SPV), Goatpox virus (GPV), and Lumpy skin disease virus (LSDV) species, cause economically significant diseases in sheep, goats, and cattle, respectively. Quantitative real-time polymerase chain reaction (qPCR) assays are routinely used for rapid detection of CaPVs in surveillance and outbreak management programs. We further modified and optimized 2 previously published CaPV qPCR assays, referred to as the Balinsky and Bowden assays, by changing commercial PCR reagents used in the tests. The modified assays displayed 100% analytical specificity and showed no apparent changes in analytical sensitivities for detection of CaPVs compared with the original assays. Diagnostic sensitivities, assessed using 50 clinical reference samples from experimentally infected sheep, goats, and cattle, improved from 82% to 92% for the modified Balinsky assay and from 58% to 82% for the modified Bowden assay. The modified qPCR assays were multiplexed for detection of beta-actin as an indicator for potential false-negative results. The multiplex modified qPCR assays exhibited the same diagnostic sensitivities as the singleplex assays suggesting their utility in the detection of CaPVs.

Development and validation of a highly sensitive real-time PCR assay for rapid detection of parapoxviruses.

Parapoxviruses (PaPVs) cause widespread infections in ruminants worldwide. All PaPVs are zoonotic and may infect humans after direct or indirect contact with infected animals. Herein we report the development and validation of a highly sensitive real-time PCR assay for rapid detection of PaPVs. The new assay (referred to as the RVSS assay) was specific for PaPVs only and had no cross-reactivity against other pox viruses. Using a recombinant plasmid as positive control, the analytical sensitivity of the assay was determined to be 16 genome copies of PaPV per assay. The amplification efficiency estimate (91-99%), the intra- and interassay variability estimate (standard deviation [SD]: 0.28-1.06 and 0.01-0.14, respectively), and the operator variability estimate (SD: 0.78 between laboratories and 0.28 between operators within a laboratory) were within the acceptable range. The diagnostic specificity was assessed on 100 specimens from healthy normal animals and all but 1 tested negative (99%). The diagnostic sensitivity (DSe) was assessed on 77 clinical specimens (skin/scab) from infected sheep, goats, and cattle, and all tested positive (100%). The assay was multiplexed with beta-actin as an internal positive control, and the multiplex assay exhibited the same DSe as the singleplex assay. Further characterization of the PaPV specimens by species-specific real-time PCR and nucleotide sequencing of the PCR products following conventional PCR showed the presence of Orf virus not only in sheep and goats but also in 1 bovid. The validated RVSS assay demonstrated high specificity, sensitivity, reproducibility, and ruggedness, which are critical for laboratory detection of PaPVs.

A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses.

Mixed infection with different pathogens is common in swine production systems especially under intensive production conditions. Quick and accurate detection and differentiation of different pathogens are necessary for epidemiological surveillance, disease management and import and export controls. In this study, we developed and validated a panel of multiplex real-time PCR/RT-PCR assays composed of four subpanels, each detects three common swine pathogens. The panel detects 12 viruses or viral serotypes, namely, VSV-IN, VSV-NJ, SVDV, CSFV, ASFV, FMDV, PCV2, PPV, PRV, PRRSV-NA, PRRSV-EU and SIV. Correlation coefficients (R(2)) and PCR amplification efficiencies of all singular and triplex real-time PCR reactions are within the acceptable range. Comparison between singular and triplex real-time PCR assays of each subpanel indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 226 target clinical samples or 4 viral strains and 91 non-target clinical samples revealed that the real-time PCR panel is 100% specific, and there is no cross amplification observed. The limit of detection of each triplex real-time PCR is less than 10 copies per reaction for DNA, and less than 16 copies per reaction for RNA viruses. The newly developed multiplex real-time PCR panel also detected different combinations of co-infections as confirmed by other means of detections.

Isolation, crystallization and preliminary X-ray analysis of a methanol-induced corrinoid protein from Moorella thermoacetica.

A corrinoid protein was induced and overexpressed in methanol-grown cells of the thermophilic anaerobic bacterium Moorella thermoacetica. The protein was purified from cytosolic extracts. After screening for crystallization conditions and optimization, crystals were obtained that diffracted strongly on a rotating-anode X-ray source. A diffraction data set was collected and processed including reflections to 1.9 A resolution. Reflections were indexed in a primitive orthorhombic cell with unit-cell parameters a = 55.69, b = 62.74, c = 34.54 A. N-terminal amino-acid sequencing indicates that the crystals contain a C-terminal fragment of the protein.

Comparison of methods for improved RNA extraction from blood for early detection of Classical swine fever virus by real-time reverse transcription polymerase chain reaction.

Classical swine fever (CSF) is a highly contagious disease of pigs. Early detection of the Classical swine fever virus (CSFV) in infected animals and routine surveillance is important for a rapid response and control of an outbreak of CSF. The current study investigated whole blood as a clinical specimen for the detection of CSFV by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) in experimentally infected pigs. The virus was detectable in pre-clinical animals in whole blood and in different fractions of blood, including white blood cells, red blood cells (RBC), and serum. Based on an in-vitro binding assay, CSFV is retained in the RBC fraction. Naturally occurring PCR inhibitors of whole blood were shown to inhibit detection, and several commercial RNA extraction kits failed to remove these inhibitors. The commercial blood RNA extraction protocols were modified to achieve optimized single tube and high-throughput 96-well plate RNA extraction that efficiently removed PCR inhibitors from whole blood and enhanced detection of CSFV in experimentally inoculated pigs. This enabled detection 1-2 days earlier than observed using unmodified RNA extraction protocols. The results show effective use of whole blood as a clinical specimen for diagnosis and surveillance of CSF in pre-clinical animals.

Development of an internal positive control for rapid diagnosis of avian influenza virus infections by real-time reverse transcription-PCR with lyophilized reagents.

We developed an internal positive control (IPC) RNA to help ensure the accuracy of the detection of avian influenza virus (AIV) RNA by reverse transcription (RT)-PCR and real-time RT-PCR (RRT-PCR). The IPC was designed to have the same binding sites for the forward and reverse primers of the AIV matrix gene as the target amplicon, but it had a unique internal sequence used for the probe site. The amplification of the viral RNA and the IPC by RRT-PCR were monitored with two different fluorescent probes in a multiplex format, one specific for the AIV matrix gene and the other for the IPC. The RRT-PCR test was further simplified with the use of lyophilized bead reagents for the detection of AIV RNA. The RRT-PCR with the bead reagents was more sensitive than the conventional wet reagents for the detection of AIV RNA. The IPC-based RRT-PCR detected inhibitors in blood, kidney, lungs, spleen, intestine, and cloacal swabs, but not allantoic fluid, serum, or tracheal swabs The accuracy of RRT-PCR test results with the lyophilized beads was tested on cloacal and tracheal swabs from experimental birds inoculated with AIV and compared with virus isolation (VI) on embryonating chicken eggs. There was 97 to 100% agreement of the RRT-PCR test results with VI for tracheal swabs and 81% agreement with VI for cloacal swabs, indicating a high level of accuracy of the RRT-PCR assay. The same IPC in the form of armored RNA was also used to monitor the extraction of viral RNA and subsequent detection by RRT-PCR.