Fast-track virtual reality software to facilitate 3-dimensional reconstruction in congenital heart disease.

OBJECTIVES: Two limitations of the clinical use of 3-dimensional (3D) reconstruction and virtual reality systems are the relatively high cost and the amount of experience required to use hardware and software to effectively explore medical images. We have tried to simplify the process and validate a new tool developed for this purpose with a novel software package. METHODS: Five patients with right partial anomalous pulmonary venous return with adequate preoperative images acquired with magnetic resonance imaging were enrolled. Five volunteers with no previous experience in the field of 3D reconstruction were instructed to use the software after viewing a short video tutorial. Users were then asked to create a 3D model of each patient's heart using DIVA software. Their results were compared quantitatively and qualitatively with a benchmark reconstruction performed by an experienced user. RESULTS: All our participants recreated 3D models in a relatively short time, maintaining a good overall quality (average quality score ≥ 3 on a scale of 1-5). The overall trend of all the parameters analysed showed a statistical improvement between case 1 and case 5, as users became more and more experienced. CONCLUSIONS: DIVA is a simple software program that allows accurate 3D reconstruction in a relatively short time ("fast-track" virtual reality). In this study, we demonstrated the potential use of DIVA by inexperienced users, with a significant improvement in quality and time after a few cases were performed. Further studies are needed to confirm the potential application of this technology on a larger scale.

Construction and efficacy of a new live chimeric C-strain vaccine with DIVA characteristics against classical swine fever.

To develop the new classical swine fever (CSF) vaccine candidate with differentiating infected vaccinated animals (DIVA) characteristics, a chimeric CSF virus (CSFV) was constructed based on an infectious cDNA clone of the CSF vaccine C-strain. The 5'- and 3'-untranslated regions (UTRs) and partial E2 region (residues 690-860) of the C-strain were substituted with the corresponding regions of bovine viral diarrhoea virus (BVDV) to construct the chimeric cDNA clone pC/bUTRs-tE2. The chimeric virus rC/bUTRs-tE2 was generated by several passages of pC/bUTRs-tE2-transfected PK15 cells. Stable growth and genetic properties of rC/bUTRs-tE2 were obtained after 30 serial passages. Compared to parental rC/bUTRs-tE2 (1st passage), two residue mutations (M834K and M979K) located in E2 in rC/bUTRs-tE2 P30 were observed. Compared to the C-strain, rC/bUTRs-tE2 exhibited unchanged cell tropism and decreased plaque-forming ability. Substituting the C-strain UTRs with the BVDV UTRs resulted in significantly increased viral replication in PK15 cells. Compared to CSFV Erns-positive and BVDV tE2-negative antibody responses induced by the CSF vaccine C-strain, immunization of rabbits and piglets with rC/bUTRs-tE2 resulted in serological profiles of CSFV Erns- and BVDV tE2-positive antibodies, which are used to serologically discriminate pigs that are clinically infected and vaccinated. Vaccination of piglets with rC/bUTRs-tE2 conferred complete protection against lethal CSFV challenge. Our results suggest that rC/bUTRs-tE2 is a promising new CSF marker vaccine candidate.

Comparison of LED-Based Transillumination Device with Traditional Vein Viewing Methods for Difficult Intravenous Cannulation in Indian Children: A Nonrandomized Controlled Trial.

OBJECTIVES: To compare the utility of transillumination device with traditional vein viewing in situations with difficult peripheral venous access in pediatric patients. METHODS: This was a nonrandomized, controlled trial. All the children aged between 3 to 36 mo admitted in tertiary care referral hospital, who satisfied difficult intravenous access (DIVA) score of equal to or more than 4 were included in the study. The children were assigned to transillumination device group (intervention) and traditional vein viewing group (traditional). The proportion of successful cannulation in the first attempt and median number attempts required to successfully cannulate in each group were estimated. RESULTS: A total of 509 children were included in the study. The proportion of single attempt cannulation was significantly higher in the intervention group as compared to traditional group (p value = 0.001). The median number of attempts to successfully cannulate was found to be significantly less in the interventional group (median 1 vs. 2; p value = 0.001). On bivariate analysis, use of transillumination device was found to have a 2.64 times higher likelihood to successfully cannulate in the first attempt. CONCLUSION: The use of transillumination device significantly improves the first attempt success rate and number of attempts for successful cannulation.

Evaluation of a Point-of-Care Feline Immunodeficiency Virus (FIV) Test Kit (RapidSTATUS™ FIV) to Determine the FIV Status of FIV-Vaccinated and FIV-Unvaccinated Pet Cats in Australia.

Feline immunodeficiency virus (FIV) is a retrovirus that can cause immunosuppression, co-morbidities, and neoplasia in infected cats, and is commonly tested for in veterinary clinics and animal shelters in Australia. FIV diagnosis using point-of-care (PoC) kits to detect FIV antibodies in Australia is complicated by the commercial availability of an inactivated whole-FIV vaccine. The aim of this study was to determine the accuracy of the RapidSTATUS™ FIV antibody test kit in FIV-vaccinated and FIV-unvaccinated cats in Australia. Plasma from pet cats of known FIV vaccination and FIV infection statuses (n = 361), comprised of 57 FIV-uninfected cats annually vaccinated against FIV, 10 FIV-uninfected cats with lapsed FIV vaccination histories, 259 FIV-unvaccinated/FIV-uninfected cats, and 35 FIV-infected cats, was tested. RapidSTATUS™ FIV testing had sensitivity of 97.1% (34/35) and specificity of 100% (326/326), with an overall accuracy of 99.7% (360/361). Additional testing was undertaken using plasma from FIV-uninfected cats recently administered a primary FIV vaccination course (n = 12) or an annual booster FIV vaccination (n = 10). RapidSTATUS™ FIV was 98.8% (81/82) accurate and 100% (32/32) accurate in cats recently administered primary or annual FIV vaccinations, respectively. The high level of accuracy of RapidSTATUS™ FIV (98.8-100%) therefore establishes this PoC kit as a DIVA (differentiating infected from vaccinated animals) test. RapidSTATUS™ FIV is recommended to aid animal shelters, veterinarians, and researchers in Australia to accurately determine FIV infection status, irrespective of FIV vaccination history.

Prospective lipid-A altered live attenuated Salmonella Gallinarum confers protectivity, DIVA capability, safety and low endotoxicity against fowl typhoid.

The present study describes creating an attenuated Salmonella Gallinarum (SG) strain with reduced endotoxicity to prevent fowl typhoid. The strain was attenuated by deleting the lon, cpxR, and rfaL virulence-related genes. Endotoxicity was reduced by deleting the pagL open reading frame and replacing it with the lpxE gene derived from Francisella tularencis. Both events, (1) deletion of the pagL and (2) introduction of the lpxE genes, conferred reduced endotoxicity by detoxifying the lipid A structure. The detoxified SG strain (SGVSdt) was well tolerated in 7-day-old chicks when administered orally at 1 × 108 CFU/bird and in 14-day-old birds administered 1 × 107 CFU/bird subcutaneously. Parenteral immunization of detoxified vaccine strain was completely safe in birds and free of environmental contamination. Subcutaneous immunization conferred disease protection and induced humoral and cell-mediated immune responses marked by Th1-skewed patterns similar to those produced by the commercial SG9R vaccine strain. Compared with the SG9R-based vaccine, the SGVSdt construct generated significantly fewer inflammatory TNF-α responses while significantly inducing IFN-γ cytokine levels as an indication of an adaptive antibacterial response. The differentiating infected from vaccinated animals (DIVA) capability was on par with the predecessor SGVS. This study presents an appealing biological strategy to minimize lipid A-mediated endotoxicity without compromising protective efficacy against the SG challenge. Reduced endotoxicity permits the utilization of higher inoculation doses to maximize protection against fowl typhoid.

Transiently Transfected Mammalian Cell Cultures: An Adaptable and Effective Platform for Virus-like Particle-Based Vaccines against Foot-and-Mouth Disease Virus.

RNA viruses, such as foot-and-mouth disease virus (FMDV), have error-prone replication resulting in the continuous emergence of new viral strains capable of evading current vaccine coverage. Vaccine formulations must be regularly updated, which is both costly and technically challenging for many vaccine platforms. In this report, we describe a plasmid-based virus-like particle (VLP) production platform utilizing transiently transfected mammalian cell cultures that combines both the rapid response adaptability of nucleic-acid-based vaccines with the ability to produce intact capsid epitopes required for immunity. Formulated vaccines which employed this platform conferred complete protection from clinical foot-and-mouth disease in both swine and cattle. This novel platform can be quickly adapted to new viral strains and serotypes through targeted exchanges of only the FMDV capsid polypeptide nucleic acid sequences, from which processed structural capsid proteins are derived. This platform obviates the need for high biocontainment manufacturing facilities to produce inactivated whole-virus vaccines from infected mammalian cell cultures, which requires upstream expansion and downstream concentration of large quantities of live virulent viruses.

Characterization of antibody response to an epitope spanning the haemagglutinin cleavage site of H7N9 subtype avian influenza virus for the differentiation of infected and vaccinated chickens.

H7N9 subtype avian influenza virus (AIV) is endemic in poultry in China, and vaccination is used as the primary strategy for disease control. However, by current serological tests, monitoring H7N9 virus infection in vaccinated poultry is difficult because vaccine-induced antibodies are not readily distinguishable from field viruses. Therefore, a test differentiating infected and vaccinated animals (DIVA) is critical for monitoring H7N9 virus. However, no DIVA test is available for the H7N9 subtype AIV. This study investigated the potential of an epitope (peptide 11) spanning the haemagglutinin (HA) cleavage site as a DIVA antigen for the H7N9 virus. The results showed that the H7N9 virus infection sera and post-challenge sera obtained from H7N9-vaccinated chickens reacted with peptide 11, whereas the sera elicited by inactivated and viral-vectored H7N9 vaccines had no reactivity with this peptide. Peptide 11 was further split into two peptides at the HA cleavage site, and the truncated peptides failed to discriminate H7N9 infected and vaccinated chickens. Peptide 11 is located in a main surface loop in the HA protein, and contains highly conserved residues in the HA cleavage site among the H7N9 subtype and different subtypes of groups 1 and 2, suggesting the potential of this peptide as a broad DIVA antigen for influenza viruses. Our study highlighted that peptide 11 is a promising DIVA antigen, and serological tests based on this peptide may serve as useful tools for monitoring H7N9 virus infection in vaccinated poultry. RESEARCH HIGHLIGHTSThe epitope spanning the HA cleavage site is a potential DIVA antigen for H7N9 AIV.The epitope reacted with LP and HP H7N9 viruses.The epitope has potential as a broad DIVA antigen for influenza viruses.

Generation of an avian influenza DIVA vaccine with a H3-peptide replacement located at HA2 against both highly and low pathogenic H7N9 virus.

A differentiating infected from vaccinated animals (DIVA) vaccine is an ideal strategy for viral eradication in poultry. Here, according to the emerging highly pathogenic H7N9 avian influenza virus (AIV), a DIVA vaccine strain, named rGD4HALo-mH3-TX, was successfully developed, based on a substituted 12 peptide of H3 virus located at HA2. In order to meet with the safety requirement of vaccine production, the multi-basic amino acid located at the HA cleavage site was modified. Meanwhile, six inner viral genes from a H9N2 AIV TX strainwere introduced for increasing viral production. The rGD4HALo-mH3-TX strain displayed a similar reproductive ability with rGD4 and low pathogenicity in chickens, suggesting a good productivity and safety. In immuned chickens, rGD4HALo-mH3-TX induced a similar antibody level with rGD4 and provided 100% clinical protection and 90% shedding protection against highly pathogenic virus challenge. rGD4HALo-mH3-TX strain also produced a good cross-protection against low pathogenic AIV JD/17. Moreover, serological DIVA characteristics were evaluated by a successfully established competitive inhibition ELISA based on a 3G10 monoclonal antibody, and the result showed a strong reactivity with antisera of chickens vaccinated with H7 subtype strains but not rGD4HALo-mH3-TX. Collectedly, rGD4HALo-mH3-TX is a promising DIVA vaccine candidate against both high and low pathogenic H7N9 subtype AIV.

The Combined Expression of the Nonstructural Protein NS1 and the N-Terminal Half of NS2 (NS21-180) by ChAdOx1 and MVA Confers Protection against Clinical Disease in Sheep upon Bluetongue Virus Challenge.

Bluetongue, caused by bluetongue virus (BTV), is a widespread arthropod-borne disease of ruminants that entails a recurrent threat to the primary sector of developed and developing countries. In this work, we report modified vaccinia virus Ankara (MVA) and ChAdOx1-vectored vaccines designed to simultaneously express the immunogenic NS1 protein and/or NS2-Nt, the N-terminal half of protein NS2 (NS21-180). A single dose of MVA or ChAdOx1 expressing NS1-NS2-Nt improved the protection conferred by NS1 alone in IFNAR(-/-) mice. Moreover, mice immunized with ChAdOx1/MVA-NS1, ChAdOx1/MVA-NS2-Nt, or ChAdOx1/MVA-NS1-NS2-Nt developed strong cytotoxic CD8+ T-cell responses against NS1, NS2-Nt, or both proteins and were fully protected against a lethal infection with BTV serotypes 1, 4, and 8. Furthermore, although a single immunization with ChAdOx1-NS1-NS2-Nt partially protected sheep against BTV-4, the administration of a booster dose of MVA-NS1-NS2-Nt promoted a faster viral clearance, reduction of the period and level of viremia and also protected from the pathology produced by BTV infection. IMPORTANCE Current BTV vaccines are effective but they do not allow to distinguish between vaccinated and infected animals (DIVA strategy) and are serotype specific. In this work we have develop a DIVA multiserotype vaccination strategy based on adenoviral (ChAdOx1) and MVA vaccine vectors, the most widely used in current phase I and II clinical trials, and the conserved nonstructural BTV proteins NS1 and NS2. This immunization strategy solves the major drawbacks of the current marketed vaccines.

Deletion of E184L, a Putative DIVA Target from the Pandemic Strain of African Swine Fever Virus, Produces a Reduction in Virulence and Protection against Virulent Challenge.

African swine fever (ASF) is currently causing a major pandemic affecting the swine industry and protein availability from Central Europe to East and South Asia. No commercial vaccines are available, making disease control dependent on the elimination of affected animals. Here, we show that the deletion of the African swine fever virus (ASFV) E184L gene from the highly virulent ASFV Georgia 2010 (ASFV-G) isolate produces a reduction in virus virulence during the infection in swine. Of domestic pigs intramuscularly inoculated with a recombinant virus lacking the E184L gene (ASFV-G-ΔE184L), 40% experienced a significantly (5 days) delayed presentation of clinical disease and, overall, had a 60% rate of survival compared to animals inoculated with the virulent parental ASFV-G. Importantly, all animals surviving ASFV-G-ΔE184L infection developed a strong antibody response and were protected when challenged with ASFV-G. As expected, a pool of sera from ASFV-G-ΔE184L-inoculated animals lacked any detectable antibody response to peptides partially representing the E184L protein, while sera from animals inoculated with an efficacious vaccine candidate, ASFV-G-ΔMGF, strongly recognize the same set of peptides. These results support the potential use of the E184L deletion for the development of vaccines able to differentiate infected from vaccinated animals (DIVA). Therefore, it is shown here that the E184L gene is a novel ASFV determinant of virulence that can potentially be used to increase safety in preexisting vaccine candidates, as well as to provide them with DIVA capabilities. To our knowledge, E184L is the first ASFV gene product experimentally shown to be a functional DIVA antigenic marker. IMPORTANCE No commercial vaccines are available to prevent African swine fever (ASF). The ASF pandemic caused by the ASF virus Georgia 2010 (ASFV-G) strain is seriously affecting pork production in a contiguous geographical area from Central Europe to East Asia. The only effective experimental vaccines are viruses attenuated by deleting ASFV genes associated with virus virulence. Therefore, identification of such genes is of critical importance for vaccine development. Here, we report the discovery of a novel determinant of ASFV virulence, the E184L gene. Deletion of the E184L gene from the ASFV-G genome (ASFV-G-ΔE184L) produced a reduction in virus virulence, and importantly, animals surviving infection with ASFV-G-ΔE184L were protected from developing ASF after challenge with the virulent parental virus ASFV-G. Importantly, the virus protein encoded by E184L is highly immunogenic, making a virus lacking this gene a vaccine candidate that allows the differentiation of infected from vaccinated animals (DIVA). Here, we show that unlike what is observed in animals inoculated with the vaccine candidate ASFV-G-ΔMGF, ASFV-G-ΔE184L-inoculated animals do not mount a E184L-specific antibody response, indicating the feasibility of using the E184L deletion as the antigenic marker for the development of a DIVA vaccine in ASFV.

Development of a Novel Assay Based on Plant-Produced Infectious Bursal Disease Virus VP3 for the Differentiation of Infected From Vaccinated Animals.

Infectious bursal disease virus is the causative agent of Gumboro disease, a severe infection that affects young chickens and is associated with lymphoid depletion in the bursa of Fabricius. Traditional containment strategies are based either on inactivated or live-attenuated vaccines. These approaches have several limitations such as residual virulence or low efficacy in the presence of maternally derived antibodies (MDA) but, most importantly, the impossibility to detect the occurrence of natural infections in vaccinated flocks. Therefore, the development of novel vaccination strategies allowing the differentiation of infected from vaccinated animals (DIVA) is a priority. Recently, commercial vectored and experimental subunit vaccines based on VP2 have been proved effective in protecting from clinical disease and posed the basis for the development of novel DIVA strategies. In this study, an engineered version of the VP3 protein of IBDV (His-VP3) was produced in plants, successfully purified from Nicotiana benthamiana leaves, and used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-VP3 antibodies. The His-VP3 ELISA was validated with a panel of 180 reference sera and demonstrated to have 100% sensitivity (95% CI: 94.7-100.0) and 94.17% specificity (95% CI: 88.4-97.6). To evaluate the application of His-VP3 ELISA as a DIVA test, the novel assay was used to monitor, in combination with a commercial kit, detecting anti-VP2 antibodies, the immune response of chickens previously immunized with an inactivated IBDV vaccine, a recombinant Turkey herpes virus carrying the VP2 of IBDV (HVT-ND-IBD) or with plant-produced VP2 particles. The combined tests correctly identified the immune status of the vaccinated specific pathogen free white-leghorn chickens. Moreover, the His-VP3 ELISA correctly detected MDA against VP3 in commercial broiler chicks and showed that antibody titers fade with time, consistent with the natural decrease of maternally derived immunity. Finally, the novel assay, in combination with a VP2-specific ELISA, demonstrated its potential application as a DIVA test in chickens inoculated with VP2-based vaccines, being able to detect the seroconversion after challenge with a very virulent IBDV strain.

Pathotyping of Newcastle Disease Virus: a Novel Single BsaHI Digestion Method of Detection and Differentiation of Avirulent Strains (Lentogenic and Mesogenic Vaccine Strains) from Virulent Virus.

We provide a novel single restriction enzyme (RE; BsaHI) digestion approach for detecting distinct pathotypes of Newcastle disease virus (NDV). After scanning 4,000 F gene nucleotide sequences in the NCBI database, we discovered a single RE (BsaHI) digestion site in the cleavage site. APMV-I "F gene" class II-specific primer-based reverse transcriptase PCR was utilized to amplify a 535-bp fragment, which was then digested with the RE (BsaHI) for pathotyping avian NDV field isolates and pigeon paramyxovirus-1 isolates. The avirulent (lentogenic and mesogenic strains) produced 189- and 346-bp fragments, respectively, but the result in velogenic strains remained undigested with 535-bp fragments. In addition, 45 field NDV isolates and 8 vaccine strains were used to confirm the approach. The sequence-based analysis also agrees with the data obtained utilizing the single RE (BsaHI) digestion approach. The proposed technique has the potential to distinguish between avirulent and virulent strains in a short time span, making it valuable in NDV surveillance and monitoring research. IMPORTANCE The extensive use of the NDV vaccine strain and the existence of avirulent NDV strains in wild birds makes it difficult to diagnose Newcastle Disease virus (NDV). The intracerebral pathogenicity index (ICPI) and/or sequencing-based identification, which are required to determine virulent NDV, are time-consuming, costly, difficult, and cruel techniques. We evaluated 4,000 F gene nucleotide sequences and discovered a restriction enzyme (RE; BsaHI) digestion technique for detecting NDV and vaccine pathotypes in a short time span, which is cost-effective and useful for field cases as well as for large-scale NDV monitoring and surveillance. The data acquired using the single RE BsaHI digestion technique agree with the sequence-based analysis.

Development of a Dendrimeric Peptide-Based Approach for the Differentiation of Animals Vaccinated with FlagT4G against Classical Swine Fever from Infected Pigs.

Classical swine fever virus (CSFV) causes a viral disease of high epidemiological and economical significance that affects domestic and wild swine. Control of the disease in endemic countries is based on live-attenuated vaccines (LAVs) that induce an early protective immune response against highly virulent CSFV strains. The main disadvantage of these currently available LAVs is the lack of serological techniques to differentiate between vaccinated and infected animals (DIVA concept). Here, we describe the development of the FlagDIVA test, a serological diagnostic tool allowing for the differentiation between animals vaccinated with the FlagT4G candidate and those infected with CSFV field strains. The FlagDIVA test is a direct ELISA based on a dendrimeric peptide construct displaying a conserved epitope of CSFV structural protein E2. Although FlagDIVA detected anti-CSFV anti-bodies in infected animals, it did not recognize the antibody response of FlagT4G-vaccinated animals. Therefore, the FlagDIVA test constitutes a valuable accessory DIVA tool in implementing vaccination with the FlagT4G candidate.

Multimodality Management in bAVM Surgery - Recent Experience from International Cerebrovascular Fellowship.

BACKGROUND: Arteriovenous malformation (bAVM) presents maldevelopment of the brain's vessels with a direct connection between cerebral arteries and veins. By current data, patients from Spetzler Ponce A (SP) are found to benefit from the treatment. Considering the outcome, most of SP C and some of the SP B are the most debatable. OBJECTIVE: Arteriovenous malformation presents maldevelopment of the brain's vessels with a consequent direct connection between cerebral arteries and veins. The annual risk of hemorrhage in adults is reported for 2-3 %. They usually present with unilateral headaches seizures and intracranial hemorrhage. By current data, patients from Spetzler Ponce A (SP) are found to benefit from the treatment. Considering the outcome, most of SP C and some of the SP B are the most debatable. METHODS: The study included a cohort of bAVM patients referred to Fujita Health University Bantane Hotokukai Hospital, Nagoya, Aichi, Japan where the main author (AA) has completed an international cerebrovascular fellowship under the mentorship of Professor Yoko Kato. Japanese Stroke Guidelines (JSG) were used for the treatment decision. Patients were graded according to the Spetzler Ponce (SP) system. Considering American Heart Association criteria (AHA), embolization was used as a part of multimodal treatment. Intraoperative microscopic video tools included Indocyanine green ICG, FLOW 800 and dual image video angiography DIVA. Clinical outcomes were measured using Modified Ranking Score (mRs). RESULTS: A total of eleven patients with brain bAVM were studied with a median age of 32 years [IQR = 22-52]. There were ten patients presented with supratentorial and a single patient with infratentorial AVM. Patients were graded according to the Spetzler Ponce (SP) system. There were eight patients in SP A (72,7%), one in group B (9 %) while the rest of them were in C (18 %). Two patients had associated aneurysms that required treatment. The median size of the AVM nidus was 3,50 cm [IQR= 2-5]. Deep venous drainage was found in six patients while three were located in eloquent zones. Clinical outcomes were considered good by mRs <2 in eight patients, seven from the surgically treated group (72,7 % respectively). Surgery median length time was 427, 5 minutes; [IQR =320 - 463] with complete AVM resection in all patients and no mortality recorded in this cohort with the median follow up of 39,5 months [IQR = 19-59]. CONCLUSION: Ideal management of bAVM is still controversial. Those complex vascular lesions require multimodal treatment in a majority of cases in highly specialized centers. In SP A patients, surgery provides the best results with a positive outcome and a small number of complications. With the improvement of endovascular feeder occlusion SP B patients become prone to a more positive outcome. Nowadays, intraoperative microscopic tools such as FLOW 800, ICG and DIVA are irreplaceable while improving safety to deal with bAVM. For SP C patients, a combination of endovascular and stereotactic radiosurgery was found to be a good option in the present time.

Development and Validation of Confirmatory Foot-and-Mouth Disease Virus Antibody ELISAs to Identify Infected Animals in Vaccinated Populations.

In foot-and-mouth disease (FMD)-endemic countries, vaccination is commonly used to control the disease, whilst in FMD-free countries, vaccination is considered as an option, in addition to culling the infected and in contact animals. FMD vaccines are mainly comprised of inactivated virions and stimulate protective antibodies to virus structural proteins. In contrast, infection with FMD virus leads to virus replication and additional antibody responses to viral nonstructural proteins (NSP). Therefore, antibodies against NSPs are used to differentiate infection in vaccinated animals (DIVA), in order to estimate the prevalence of infection or its absence. Another advantage of NSP antibody tests is that they detect FMD infection in the field, irrespective of the serotypes of virus in circulation. In cattle, the NSP tests that target the 3ABC polyprotein provides the highest sensitivity, detecting up to 90% of vaccinated animals that become carriers after exposure to infection, with a specificity of around 99%. Due to insufficient diagnostic sensitivity and specificity, detection of a low level of infection is difficult at the population level with a high degree of confidence. The low level of non-specific responses can be overcome by retesting samples scored positive using a second confirmatory test, which should have at least comparable sensitivity to the first test. In this study, six in-house tests were developed incorporating different NSP antigens, and validated using bovine sera from naïve animals, field cases and experimentally vaccinated and/or infected animals. In addition, two (short and long incubation) new commercial NSP tests based on 3ABC competitive blocking ELISAs (ID Screen® FMD NSP Competition, IDvet, France) were validated in this study. The two commercial ELISAs had very similar sensitivities and specificities that were not improved by lengthening the incubation period. Several of the new in-house tests had performance characteristics that were nearly as good as the commercial ELISAs. Finally, the in-house tests were evaluated for use as confirmatory tests following screening with the PrioCHECK® and ID Screen® FMDV NS commercial kits, to assess the diagnostic performance produced by a multiple testing strategy. The in-house tests could be used in series (to confirm) or in parallel (to augment) with the PrioCHECK® and IDvet® FMDV NS commercial kits, in order to improve either the specificity or sensitivity of the overall test system, although this comes at the cost of a reduction in the counterpart (sensitivity/specificity) parameter.

Effect of a strutted intradiscal spacer (DIVA®) on disc reherniation following lumbar discectomy: A 2-year retrospective matched cohort study.

INTRODUCTION: Discectomy for lumbar disc herniation has a high rate of reoperation and recurrent herniation. METHODS: Retrospectively matched cohort of patients undergoing lumbar discectomy alone or with a strutted intradiscal spacer. RESULTS: 133 discectomy and 112 patients with discectomy plus spacer were included. Pain and disability scores were significantly lower for both groups at 2 years. Patients receiving a strutted intradiscal spacer following discectomy had a reduced rate of all-cause reoperations and operations for recurrent herniations compared to discectomy alone. CONCLUSION: Use of a strutted intradiscal spacer following discectomy improves surgical outcomes following surgery for lumbar herniation versus discectomy alone.

An efficient peptide-based ELISA for differentiating fowl adenovirus 4-infected chickens from vaccinated chickens.

Fowl adenovirus serotype 4 (FAdV4), the causative agent of hepatitis-hydropericardium syndrome (HPS), has caused major economic losses to the poultry industry worldwide. Although inactivated vaccines have been deployed widely against FAdV4, a DIVA (differentiating infected from vaccinated animals) test specific for FAdV4 has not been available. We synthesized an immunogenic peptide, corresponding to regions 66-88 aa of the 22K nonstructural protein of FAdV4, and used the peptide as coating antigen to develop an indirect ELISA for a DIVA test specific to FAdV4. Specificity analysis showed that the ELISA only reacted with sera against FAdV4, and not with sera against other pathogens tested. Moreover, the ELISA could effectively differentiate FAdV4-infected chickens from vaccinated chickens. In a test of sera from experimentally infected chickens, the ELISA had 95% and 85% concordance with an indirect immunofluorescence assay (indirect IFA) and a commercial ELISA, respectively, and the concordance was 80.5% between the ELISA and the indirect IFA in detecting clinical infection samples. Our peptide-based ELISA provides an efficient DIVA test for FAdV4 in clinical samples.

Comparative Analysis of the Productivity and Immunogenicity of an Attenuated Classical Swine Fever Vaccine (LOM) and an Attenuated Live Marker Classical Swine Fever Vaccine (Flc-LOM-BErns) from Laboratory to Pig Farm.

Herein, we compared the productivity of pigs inoculated with one of two classical swine fever (CSF) vaccines (low virulent of Miyagi (LOM) or Flc-LOM-BErns) plus the swine erysipelothrix rhusiopathiae (SE) vaccine. The feed intake and weight increase of the pigs inoculated with Flc-LOM-BErns + SE were normal. However, the feed intake of the pigs inoculated with LOM + SE dropped sharply from four days post-vaccination (dpv). In addition, the slaughter date was an average of eight days later than that of the pigs inoculated with Flc-LOM-BErns + SE. All pigs inoculated with the Flc-LOM-BErns + SE vaccine were completely differentiated at 14 days against CSF Erns antibody and at approximately 45 days against the bovine viral diarrhea virus (BVDV) Erns antibody; the titers were maintained until slaughter. Leucopenia occurred temporarily in the LOM + SE group, but not in the Flc-LOM-BErns + SE group. Expression of tumor necrosis factor (TNF)-α and IFN-γ was significantly (p < 0.05) higher in the LOM + SE group than in the mock (no vaccine) group. When conducting the same experiment on a breeding farm, the results were similar to those of the laboratory experiments. In conclusion, the biggest advantage of replacing the CSF LOM vaccine with the Flc-LOM-BErns vaccine is improved productivity.

Auditory-vocal integration impairment: New challenges and opportunities for voice assessment and therapy / Alteración de la integración auditivo-vocal: nuevos retos y oportunidades para la evaluación y la terapia de la voz

This reflection paper addresses the importance of the interaction between voice perception and voice production, emphasizing the processes of auditory-vocal in-tegration that are not yet widely reported in the context of voice clinicians. Given the above, this article seeks to 1) highlight the important link between voice pro-duction and voice perception and 2) consider whether this relationship might be exploited clinically for diagnostic purposes and therapeutic benefit. Existing theories on speech production and its interaction with auditory perception provide context for discussing why the evaluation of auditory-vocal processes could help identify associ-ated origins of dysphonia and inform the clinician around appropriate management strategies. Incorporating auditory-vocal integration assessment through sensorimotor adaptation paradigm testing could prove to be an important addition to voice assess-ment protocols at the clinical level. Further, if future studies can specify the means to manipulate and enhance a person's auditory-vocal integration, the efficiency of voice therapy could be increased, leading to improved quality of life for people with voice disorders

Este artículo de reflexión aborda la importancia de la interacción entre la percepción y la producción de la voz, haciendo hincapié en los procesos de integración auditivo-vocal, los cuales aún no han sido muy divulgados en el contexto de los clínicos de voz. Dado lo anterior, este articulo busca: 1) destacar la importante relación entre la producción y la percepción de la voz y 2) considerar si esta relación pudiese explotarse clínicamente con fines diagnósticos y terapéuticos. Las teorías existentes sobre la producción de la voz y su interacción con la percepción auditiva proporcionan el contexto para discutir por qué la evaluación de los procesos auditivo-vocales podría ayudar a identificar los orígenes asociados a cierto tipo de disfonías e informar al clínico sobre las estrategias de abordaje adecuadas. La incorporación de la evaluación de la integración auditivo-vocal a través de la prueba del paradigma de adaptación sensoriomotora podría ser una importante adición a los protocolos de evaluación de la voz a nivel clínico. Además, si los estudios futuros pueden especificar los medios para manipular y mejorar la integración auditivo-vocal de una persona, la eficacia de la terapia de la voz podría aumentar, lo que llevaría a mejorar la calidad de vida de las personas con trastornos de la voz

Genetically distinct pestiviruses pave the way to improved classical swine fever marker vaccine candidates based on the chimeric pestivirus concept.

Classical swine fever (CSF) is one of the most important viral diseases of pigs. In many countries, the use of vaccines is restricted due to limitations of subunit vaccines with regard to efficacy and onset of protection as well as failure of live vaccines to differentiate infected from vaccinated animals (DIVA principle). Chimeric pestiviruses based on CSF virus (CSFV) and the related bovine viral diarrhea virus (BVDV) have been licensed as live marker vaccines in Europe and Asia, but cross-reactive antibodies can cause problems in DIVA application due to close antigenic relationship. To develop marker vaccine candidates with improved DIVA properties, three chimeric viruses were generated by replacing Erns of CSFV Alfort-Tübingen with homologue proteins of only distantly related pestiviruses. The chimeric viruses "Ra", "Pro", and "RaPro" contained Erns sequences of Norway rat and Pronghorn pestiviruses or a combination of both, respectively. In porcine cells, the "Pro" chimera replicated to high titers, while replication of the "Ra" chimera was limited. The "RaPro" chimera showed an intermediate phenotype. All vaccine candidates were attenuated in a vaccination/ challenge trial in pigs, but to different extents. Inoculation induced moderate to high levels of neutralizing antibodies that protected against infection with a genetically heterologous, highly virulent CSFV. Importantly, serum samples of vaccinated animals did not show any cross-reactivity in a CSFV Erns antibody ELISA. In conclusion, the Erns antigen from distantly related pestiviruses can provide a robust serological negative marker for a new generation of improved CSFV marker vaccines based on the chimeric pestivirus concept.