Evaluating the dynamics and efficacy of a live, attenuated Mycoplasma anserisalpingitidis vaccine candidate under farm conditions.

The aim of the present study was to monitor the dynamics and to measure the safety and efficacy of a live, attenuated, thermosensitive Mycoplasma anserisalpingitidis vaccine candidate, namely MA271, in geese breeder flocks under field conditions. Two rearing flocks were vaccinated with MA271 at 4 weeks of age and boosted at 24 weeks of age by cloaca inoculation (1 ml) and eye-dropping (60 µl). The geese then were transported to multi-aged breeding farms. Two breeding flocks served as controls. Colonization of the cloaca by MA271 showed 75% maximum prevalence between 4 and 6 weeks after the first vaccination. Then the prevalence decreased to 25% until the cooler, humid fall months which coincided with the booster vaccination. Boosting raised cloacal colonization to 100%. No clinical signs were observed in the vaccinated birds. After transportation to five multi-aged breeding farms, the wild-type strain appeared as well as MA271 in three flocks. In one flock, the wild-type strain completely displaced MA271, while in one flock only MA271 was detected. Only wild-type strains were detected in the control flocks; however, due to an HPAI outbreak, both flocks were exterminated before the end of the study. Based on the available data, the median percentage of infertile eggs was 3.7-5.1% in the MA271 vaccinated flocks, and 7.7% in the non-vaccinated flock. In conclusion, MA271 can colonize the cloaca of geese under field conditions. MA271 proved to be safe and presumably protects against M. anserisalpingitidis-induced reproduction losses.

Polarization- and Chaos-Game-Based Fingerprinting of Molecular Targets of Listeria Monocytogenes Vaccine and Fully Virulent Strains.

Two approaches to the synthesis of 2D binary identifiers ("fingerprints") of DNA-associated symbol sequences are considered in this paper. One of these approaches is based on the simulation of polarization-dependent diffraction patterns formed by reading the modeled DNA-associated 2D phase-modulating structures with a coherent light beam. In this case, 2D binarized distributions of close-to-circular extreme polarization states are applied as fingerprints of analyzed nucleotide sequences. The second approach is based on the transformation of the DNA-associated chaos game representation (CGR) maps into finite-dimensional binary matrices. In both cases, the differences between the structures of the analyzed and reference symbol sequences are quantified by calculating the correlation coefficient of the synthesized binary matrices. A comparison of the approaches under consideration is carried out using symbol sequences corresponding to nucleotide sequences of the hly gene from the vaccine and wild-type strains of Listeria monocytogenes as the analyzed objects. These strains differ in terms of the number of substituted nucleotides in relation to the vaccine strain selected as a reference. The results of the performed analysis allow us to conclude that the identification of structural differences in the DNA-associated symbolic sequences is significantly more efficient when using the binary distributions of close-to-circular extreme polarization states. The approach given can be applicable for genetic differentiation immunized from vaccinated animals (DIVA).

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.

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.

Evaluation of the Deletion of MGF110-5L-6L on Swine Virulence from the Pandemic Strain of African Swine Fever Virus and Use as a DIVA Marker in Vaccine Candidate ASFV-G-ΔI177L.

African swine fever virus (ASFV) is responsible for an ongoing pandemic that is affecting central Europe, Asia, and recently the Dominican Republic, the first report of the disease in the Western Hemisphere in over 40 years. ASFV is a large, complex virus with a double-stranded DNA (dsDNA) genome that carries more than 150 genes, most of which have not been studied. Here, we assessed the role of the MGF110-5L-6L gene during virus replication in cell cultures and experimental infection in swine. A recombinant virus with MGF110-5L-6L deleted (ASFV-G-ΔMGF110-5L-6L) was developed using the highly virulent ASFV Georgia (ASFV-G) isolate as a template. ASFV-G-ΔMGF110-5L-6L replicates in swine macrophage cultures as efficiently as the parental virus ASFV-G, indicating that the MGF110-5L-6L gene is nonessential for virus replication. Similarly, domestic pigs inoculated with ASFV-G-ΔMGF110-5L-6L presented with a clinical disease undistinguishable from that caused by the parental ASFV-G, confirming that the MGF110-5L-6L gene is not involved in producing disease in swine. Sera from animals inoculated with an efficacious vaccine candidate, ASFV-G-ΔMGF, strongly recognized the protein encoded by the MGF110-5L-6L gene as a potential target for the development of an antigenic marker differentiation of infected from vaccinated animals (DIVA) vaccine. To test this hypothesis, the MGF110-5L-6L gene was deleted from the highly efficacious ASFV vaccine candidate ASFV-G-ΔI177L, generating the recombinant ASFV-G-ΔI177L/ΔMGF110-5L-6L. Animals inoculated with ASFV-G-ΔI177L/ΔMGF110-5L-6L developed an ASFV-specific antibody response detected by enzyme-linked immunosorbent assay (ELISA). The sera strongly recognized ASFV p30 expressed in eukaryotic cells but did not recognize ASFV MGF110-5L-6L protein, demonstrating that deletion of the MGF110-5L-6L gene can enable DIVA capabilities in preexisting vaccine candidates. IMPORTANCE Currently, there are no African swine fever (ASF) commercial vaccines that can be used to prevent or control the spread of ASF. The only effective experimental vaccines against ASF are live-attenuated vaccines. However, these experimental vaccines, which rely on a deletion of a specific gene of the current circulating strain of ASF, make it hard to tell the difference between a vaccinated and an infected animal. In our search for a serological marker, we identified that the virus protein encoded by the MGF110-5L-6L gene induced an immune response, making a virus lacking this gene a vaccine candidate that allows the differentiation of infected from vaccinated animals (DIVA). Here, we show that deletion of MGF110-5L-6L does not affect virulence or virus replication. However, when the deletion of MGF110-5L-6L was added to vaccine candidate ASFV-G-ΔI177L, a reduction in the effectiveness of the vaccine occurred.

Phylogeny and historical biogeography of neotropical catfishes Trichomycterus (Siluriformes: Trichomycteridae) from eastern Brazil.

The catfish subfamily Trichomycterinae is widely distributed in South America inhabiting several habitats, but specially mountain streams. Trichomycterus is the most speciose trichomycterid genus and recently due to his paraphyletic condition has been restricted to a clade from eastern Brazil called Trichomycterus sensu stricto comprising around 80 valid species distributed in seven areas of endemism of eastern Brazil. This paper aims to analyse the biogeographical events responsible for the distribution of Trichomycterus s.s., by reconstructing the ancestral data based on a time-calibrated multigene phylogeny. A multi-gene phylogeny was generated using 61 species of Trichomycterus s.s. and 30 outgroups, with divergence events calculated based on the estimated origin of Trichomycteridae. Two event-based analyses were applied to investigate the biogeographical events responsible the present distribution of Trichomycterus s.s. and suggest that the modern distribution of the group is a result of different vicariance and dispersal events. The diversification of Trichomycterus s.s. subgenera occurred in the Miocene, except for Megacambeva, with different biogeographical events shaping its distribution in eastern Brazil. An initial vicariant event split up the Fluminense ecoregion from the Northeastern Mata Atlantica + Paraíba do Sul + Fluminense + Ribeira do Iguape + Upper Paraná ecoregions. Dispersal events occurred mainly between Paraíba do Sul and neighboring river basins, with additional dispersal events from Northeastern Mata Atlantica to Paraíba do Sul, from São Francisco to Northeastern Mata Atlântica, and from Upper Paraná to São Francisco.

A live attenuated DIVA vaccine affords protection against Listeria monocytogenes challenge in sheep.

Listeria monocytogenes (Lm) is a deadly foodborne pathogen that comprises 14 serotypes, among which, serotype 4b Lm is the primary cause of listeriosis outbreaks in humans and animals. Here, we evaluated the safety, immunogenicity, and protective efficacy of a serotype 4b vaccine candidate Lm NTSNΔactA/plcB/orfX in sheep. The infection dynamics, clinical features, and pathological observation verified that the triple genes deletion strain has adequate safety for sheep. Moreover, NTSNΔactA/plcB/orfX significantly stimulated humoral immune response and provided 78% immune protection to sheep against lethal wild-type strain challenge. Notably, the attenuated vaccine candidate could differentiate infected and vaccinated animals (DIVA) via serology determination of the antibody against listeriolysin O (LLO, encoded by hly) and phosphatidylinositol-specific phospholipase C (PI-PLC, encoded by plcB). These data suggest that the serotype 4b vaccine candidate has high efficacy, safety, and DIVA characteristics, and may be used to prevent Lm infection in sheep. Our study provides a theoretical basis for its future application in livestock and poultry breeding.

A review of three decades of use of the cattle brucellosis rough vaccine Brucella abortus RB51: myths and facts.

Cattle brucellosis is a severe zoonosis of worldwide distribution caused by Brucella abortus and B. melitensis. In some countries with appropriate infrastructure, animal tagging and movement control, eradication was possible through efficient diagnosis and vaccination with B. abortus S19, usually combined with test-and-slaughter (T/S). Although S19 elicits anti-smooth lipopolysaccharide antibodies that may interfere in the differentiation of infected and vaccinated animals (DIVA), this issue is minimized using appropriate S19 vaccination protocols and irrelevant when high-prevalence makes mass vaccination necessary or when eradication requisites are not met. However, S19 has been broadly replaced by vaccine RB51 (a rifampin-resistant rough mutant) as it is widely accepted that is DIVA, safe and as protective as S19. These RB51 properties are critically reviewed here using the evidence accumulated in the last 35 years. Controlled experiments and field evidence shows that RB51 interferes in immunosorbent assays (iELISA, cELISA and others) and in complement fixation, issues accentuated by revaccinating animals previously immunized with RB51 or S19. Moreover, contacts with virulent brucellae elicit anti-smooth lipopolysaccharide antibodies in RB51 vaccinated animals. Thus, accepting that RB51 is truly DIVA results in extended diagnostic confusions and, when combined with T/S, unnecessary over-culling. Studies supporting the safety of RB51 are flawed and, on the contrary, there is solid evidence that RB51 is excreted in milk and abortifacient in pregnant animals, thus being released in abortions and vaginal fluids. These problems are accentuated by the RB51 virulence in humans, lack diagnostic serological tests detecting these infections and RB51 rifampicin resistance. In controlled experiments, protection by RB51 compares unfavorably with S19 and lasts less than four years with no evidence that RB51-revaccination bolsters immunity, and field studies reporting its usefulness are flawed. There is no evidence that RB51 protects cattle against B. melitensis, infection common when raised together with small ruminants. Finally, data acumulated during cattle brucellosis eradication in Spain shows that S19-T/S is far more efficacious than RB51-T/S, which does not differ from T/S alone. We conclude that the assumption that RB51 is DIVA, safe, and efficaceous results from the uncritical repetition of imperfectly examined evidence, and advise against its use.

Epidemiology-driven approaches to surveillance in HPAI-vaccinated poultry flocks aiming to demonstrate freedom from circulating HPAIV.

Incursion pressure of high pathogenicity avian influenza viruses (HPAIV) by secondary spread among poultry holdings and/or from infected migratory wild bird populations increases worldwide. Vaccination as an additional layer of protection of poultry holdings using appropriately matched vaccines aims at reducing clinical sequelae of HPAIV infection, disrupting HPAIV transmission, curtailing economic losses and animal welfare problems and cutting exposure risks of zoonotic HPAIV at the avian-human interface. Products derived from HPAIV-vaccinated poultry should not impose any risk of virus spread or exposure. Vaccination can be carried out with zero-tolerance for infection in vaccinated herds and must then be flanked by appropriate surveillance which requires tailoring at several levels: (i) Controlling appropriate vaccination coverage and adequate population immunity in individual flocks and across vaccinated populations; (ii) assessing HPAI-infection trends in unvaccinated and vaccinated parts of the poultry population to provide early detection of new/re-emerged HPAIV outbreaks; and (iii) proving absence of HPAIV circulation in vaccinated flocks ideally by real time-monitoring. Surveillance strategies, i.e. selecting targets, tools and random sample sizes, must be accommodated to the specific epidemiologic and socio-economic background. Methodological approaches and practical examples from three countries or territories applying AI vaccination under different circumstances are reviewed here.

Implementation of a midline catheter service in a regional setting.

Introduction: Midline catheters have been reported to be an effective and safe means of providing patients with intravenous access within the hospital and community setting. With minimal experience in the introduction of a midline service across the local health network, a regional hospital pursued this task. This observational study assesses the provision of a safe clinical framework for midline insertion, and the improvement of patient care and experiences by avoiding treatment interruptions and unnecessary cannulation attempts from failed traditional peripheral vascular access devices. Methods: From the introduction of the midline service in June 2018, outcome measures of all patients who received a midline over the following two-year period were documented including rate of line success, complication rates, dwell time, and the number of insertion attempts. Results: The midline service provided 207 lines over a two-year period with a total dwell time of 1,585 days. Project goals were achieved with 85% (Aim > 85%) of all lines completing treatment prior to removal. First attempt insertion was 86% (Aim > 80%) with a maximum insertion attempt of two. Rates of line-related complications were less than 8%, with five documented cases of phlebitis (2.5%) and one deep vein thrombosis with no infections documented. Conclusion: Despite limited resources, a successful midline service was introduced. Future expansion will see an increase in insertor numbers providing improved access to the service.

Development of Differentiating Infected from Vaccinated Animals (DIVA) Real-Time PCR for African Horse Sickness Virus Serotype 1.

African horse sickness (AHS) is a highly infectious and often fatal disease caused by 9 serotypes of the orbivirus African horse sickness virus (AHSV). In March 2020, an AHS outbreak was reported in Thailand in which AHSV serotype 1 was identified as the causative agent. Trivalent live attenuated vaccines serotype 1, 3, and 4 were used in a targeted vaccination campaign within a 50-km radius surrounding the infected cases, which promptly controlled the spread of the disease. However, AHS-like symptoms in vaccinated horses required laboratory diagnostic methods to differentiate infected horses from vaccinated horses, especially for postvaccination surveillance. We describe a real-time reverse transcription PCR-based assay for rapid characterization of the affecting field strain. The development and validation of this assay should imbue confidence in differentiating AHS-vaccinated horses from nonvaccinated horses. This method should be applied to determining the epidemiology of AHSV in future outbreaks.

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.

Infrared augmented reality device versus standard procedure for peripheral venous catheterisation in children less than 3 years old: A quasi-experimental cluster randomised controlled trial.

AIM AND OBJECTIVE: The objective of this study was to evaluate the AccuVeinAV400® viewing device for peripheral venous catheter insertion in children on the first try. BACKGROUND: Inserting a peripheral venous catheter is the most frequent invasive procedure carried out by healthcare professionals in hospitalised children. Several attempts are sometimes necessary, and veins can be damaged. DESIGN: A quasi-experimental cluster randomised controlled trial based on the CONSORT 2010 guidelines. METHODS: This randomised study comparing standard practice to the use of AccuVein400® was carried out on children who were less than 3 years old, with difficult intravenous access (DIVA Score), hospitalised in three paediatric units and who needed cannulation. RESULT: A total of 304 children were included (156 in the AccuVein arm and 148 in the standard arm). There was no significant difference between AccuVein and standard groups in age (respectively, 2.5 ± 0.9 years vs. 2.5 ± 0.8), or mean DIVA score (respectively 5.9 ± 1.3 vs. 5.5 ± 1.2). The success of cannulation on the first attempt was 40.38% in the AccuVein arm vs. 41.2% in the standard arm (p = .6). The caregiver's assessment of pain on the Face Legs Activity Cry Consolability scale was 4.8 ± 0.2 in the AccuVein arm vs. 5.0 ± 0.2 (p = .4). CONCLUSIONS: The use of AccuVein400® did not lead to greater success in intravenous insertion at the first attempt in children under 3 years of age with difficult intravenous access. RELEVANCE TO CLINICAL PRACTICE: This device can therefore be used according to the healthcare professionals' situation and needs. It is widely used in paediatric wards, and our study shows that it offers a support tool that reassures healthcare professionals and helps validate their choice of vein easiest to catheterise.

Differences in alcohol and cannabis use amongst substance use disorder patients with and without comorbid attention-deficit/hyperactivity disorder.

Background: Substance use disorders (SUDs) continue to be a public health problem. Attention-deficit/hyperactivity disorder (ADHD) is seen as a risk factor for SUD. Prevalence of alcohol and cannabis use amongst adults with SUD and comorbid ADHD impacts both disorders cognitively and behaviourally. Aim: Our study aimed to compare alcohol and cannabis use between treatment-seeking SUD patients with ADHD and SUD patients without ADHD symptomatology. Setting: Various rehabilitation centres, including the South African National Council on Alcoholism and Drug Dependence (SANCA) Centres, and Private and Provincial Government Substance Abuse Treatment Centres. Methods: A cross-sectional study of adults on drug rehabilitation was conducted. Data on socio-demographic information and alcohol and cannabis use from 185 post-detox inpatients were collected. Diagnoses were based on DSM-IV criteria. Attention-deficit/hyperactivity disorder diagnosis was confirmed by the Diagnostic-Interview for ADHD in Adults (DIVA 2.0). SUD+ADHD (n = 52) and SUD-ADHD (n = 128) groups were compared on alcohol and cannabis use as a function of gender. Results: No significant differences in the use of alcohol between the SUD+ADHD and SUD-ADHD groups were found. However, the SUD+ADHD group showed increased cannabis consumption. Especially, the SUD+ADHD females showed an earlier age of onset of cannabis use than the SUD-ADHD females and revealed that they use cannabis for a longer period compared with the SUD-ADHD females and SUD+ADHD and SUD-ADHD males. Conclusion: The results revealed the relationship between ADHD and cannabis use, especially amongst females with ADHD and reinforce the need to consider ADHD in cannabis use SUD in clinical interventions.

The next-generation coronavirus diagnostic techniques with particular emphasis on the SARS-CoV-2.

The potential zoonotic coronaviruses (SARS-CoV, MERS-CoV, and SARS-CoV-2) are of global health concerns. Early diagnosis is the milestone in their mitigation, control, and eradication. Many diagnostic techniques are showing great success and have many advantages, such as the rapid turnover of the results, high accuracy, and high specificity and sensitivity. However, some of these techniques have several pitfalls if samples were not collected, processed, and transported in the standard ways and if these techniques were not practiced with extreme caution and precision. This may lead to false-negative/positive results. This may affect the downstream management of the affected cases. These techniques require regular fine-tuning, upgrading, and optimization. The continuous evolution of new strains and viruses belong to the coronaviruses is hampering the success of many classical techniques. There are urgent needs for next generations of coronaviruses diagnostic assays that overcome these pitfalls. This new generation of diagnostic tests should be able to do simultaneous, multiplex, and high-throughput detection of various coronavirus in one reaction. Furthermore, the development of novel assays and techniques that enable the in situ detection of the virus on the environmental samples, especially air, water, and surfaces, should be given considerable attention in the future. These approaches will have a substantial positive impact on the mitigation and eradication of coronaviruses, including the current SARS-CoV-2 pandemic.

Application of portable real-time recombinase-aided amplification (rt-RAA) assay in the clinical diagnosis of ASFV and prospective DIVA diagnosis.

African swine fever, a serious infectious disease, has been found in many countries around the world over the last nearly 100 years, and causes untold damage to the economy wherever it occurs. Diagnosis is currently performed by real-time PCR, which is highly sensitive but can only be carried out in a diagnostic laboratory environment with sophisticated equipment and expertise. A sensitive, rapid diagnostic method that can be implemented in agricultural settings is thus urgently needed for the detection and control of African swine fever virus (ASFV) infection. In this study, we developed an isothermal amplification technology to achieve molecular diagnosis of ASFV in clinical samples, using recombinase-aided amplification (RAA) assay combined with a portable instrument. This assay method avoids the limitations of traditional real-time PCR and offers detection times within 20 min, enabling detection of as few as 10 copies of ASFV DNA molecules per reaction without cross-reaction with other common swine viruses. We evaluated clinical performance using 200 clinical blood samples. The coincidence rate of the detection results between rt-RAA and RT-qPCR was 96.94% positive, 100% negative, and 97.50% total. We have also developed an rt-RAA system for the detection of ASFV targeting the EP402R gene, with detection of as few as 10 copies of DNA per reaction; this offers the possibility of DIVA (differentiating infected from vaccinated animals) diagnosis, because CD2V gene-deleted ASFV could soon be approved to be the leading candidate for live attenuated vaccine in China. The rt-RAA assay is a reliable, rapid, highly sensitive method, and it offers a reasonable alternative to RT-qPCR for point-of-care detection of ASFV. KEY POINTS: • The RT-RAA assay can detect as few as 10 copies of ASFV genome per reaction within 20 min. • The rt-RAA assay system targeting different genes can achieve differentiating infected from vaccinated diagnosis.

Vulvovaginal atrophy of menopause and its impact on sexual function in an Italian clinical cohort of post-menopausal women.

The aim of current study was to estimate the impact of vulvovaginal atrophy (VVA) on sexual function in a clinical population of Italian postmenopausal women. Women aged 45-75 years with at least one VVA symptom completed three questionnaires: Day-to-Day Impact of Vaginal Aging (DIVA), Female Sexual Function Index (FSFI) and Female Sexual Distress Scale revised (FSDS-R). A gynaecological examination was performed for VVA confirmation. Among the 1,066 evaluable patients, VVA was confirmed in around 90% of the sample. Sexual function impairment was significantly higher in patients with confirmed VVA as observed by significant differences in the sexual function component of the DIVA questionnaire (p = .014), the FSDS-R (p < .0005), and the FSFI (p < .0005), as well as for all the FSFI subdomains: desire (p < .0005), arousal (p < .0005), lubrication (p < .0005), orgasm (p < .0005), satisfaction (p < .0005) and pain (p < .0005). Significant impairment of sexual function was demonstrated in Italian postmenopausal women who were clinically confirmed with signs of VVA through gynaecological examination.IMPACT STATEMENTWhat is already known on this subject: At least half of postmenopausal women report VVA associated symptoms with significant impact on sexual function and ultimately on sexual activity.What the results of this study add: As compared with patients without confirmed VVA, the negative impact on sexual function was significantly higher in patients with confirmed VVA. This difference was observed for the sexual function component (DIVA-C) of the DIVA questionnaire, for the overall FSDS-R result, and for the overall FSFI score, as well as for all the FSFI subdomains (desire, arousal, lubrication, orgasm, satisfaction and pain).What the implications are of these findings for clinical practice and/or further research: An impairment of sexual function is significantly associated with VVA diagnosis in Italian post-menopausal women, especially when diagnosis was objectively confirmed by clinical signs of VVA visible in the gynaecological examination. In addition, this study demonstrates that inquiring about VVA using a structured questionnaire may increase the diagnosis of VVA related changes in sexual function.

Immunoproteomics of Brucella abortus reveals potential of recombinant antigens for discriminating vaccinated from naturally infected cattle.

Brucellosis serodiagnosis is still a challenge and vaccination is the main measure used to control bovine brucellosis, being S19 and RB51 the most currently used vaccines. So, in order to contribute to brucellosis control, a bidimensional (2D) immunoblot-based approach was used to find immunogenic proteins to be used in serodiagnosis, particularly with ability to be employed in DIVA (Differentiating Infected from Vaccinated Animals) strategy. Immunoproteomic profile of Brucella abortus 2308 was analyzed in 2D western blotting using pooled sera from S19 vaccinated animals, RB51 vaccinated animals, B. abortus naturally infected animals and non-vaccinated seronegative animals. Evaluation of the antigens differentially immunoreactive against the groups of sera showed three proteins of particular importance: MDH (malate dehydrogenase) immunoreactive for S19-vaccinated animals, SOD (superoxide dismutase) reactive for infected animals and ABC transporter (multispecies sugar ABC transporter) reactive against sera from vaccinated animals (S19 and RB51). These three proteins were produced in E. coli and tested in an indirect ELISA (I-ELISA). For MDH, comparison between the vaccinated animals (independent of the vaccine used) and the seropositive and seronegative animals in I-ELISA showed significant differences. Data on the I-ELISA using SOD showed that sera from non-vaccinated naturally infected animals exhibited significant difference in comparison with all other groups. Otherwise, sera from vaccinated animals (S19 and RB51) and from non-vaccinated naturally infected animals did not show significant difference in OD values, but they were all significant different from non-vaccinated seronegative animals using ABC transporter as antigen in I-ELISA. In conclusion, together the 2D western blot analysis and the preliminary I-ELISA results suggest that the combined use of MDH and SOD could be successful employed in a LPS-free protein based serodiagnosis approach to detect bovine brucellosis and to discriminate vaccinated from naturally infected animals, in early post-vaccination stages.

Analysis of antibody response to an epitope in the haemagglutinin subunit 2 of avian influenza virus H5N1 for differentiation of infected and vaccinated chickens.

The H5N1 subtype of highly pathogenic avian influenza virus has been circulating in poultry in Indonesia since 2003 and vaccination has been used as a strategy to eradicate the disease. However, monitoring of vaccinated poultry flocks for H5N1 infection by serological means has been difficult, as vaccine antibodies are not readily distinguishable from those induced by field viruses. Therefore, a test that differentiates infected and vaccinated animals (DIVA) would be essential. Currently, no simple and specific DIVA test is available for screening of a large number of vaccinated chickens. Several epitopes on E29 domain of the haemagglutinin H5N1 subunit 2 (HA2) have recently been examined for their antigenicity and potential as possible markers for DIVA in chicken. In this study, the potential of E29 as an antigen for DIVA was evaluated in detail. Three different forms of full-length E29 peptide, a truncated E29 peptide (E15), and a recombinant E29 were compared for their ability to detect anti-E29 antibodies. Preliminary ELISA experiments using mono-specific chicken and rabbit E29 sera, and a mouse monoclonal antibody revealed that the linear E29 peptide was the most antigenic. Further examination of the E29 antigenicity in ELISA, using several sera from experimentally infected or vaccinated chickens, revealed that the full-length E29 peptide had the greatest discrimination power between infected and vaccinated chicken sera while providing the least non-specific reaction. This study demonstrates the usefulness of the HPAI H5N1 HA2 E29 epitope as a DIVA antigen in HPAI H5N1-vaccinated and -infected chickens.RESEARCH HIGHLIGHTS E29 (HA2 positions 488-516) epitope is antigenic in chickens.Antibodies to E29 are elicited following live H5N1 virus infection in chickens.E29 epitope is a potential DIVA antigen for use in ELISA.

Multiplexed DIVA tests for rapid detection of FMDV infection/circulation in endemic countries.

Foot-and-mouth disease (FMD) is an important transboundary disease affecting domestic and wild ruminants. Due to FMD outbreaks, the annual economic losses in endemic countries range from USD 6.1 billion to 200 billion. It also restricts the export of animals/animal by-products to FMD-free countries. FMD-free countries can experience a more severe economic loss due to the culling of infected animals as experienced by the UK in 2001 outbreaks. In endemic countries outbreaks occur mainly due to unrestricted animal movements. This creates a difficult situation in an endemic setting for controlling FMD spread to nearby areas. During post-vaccination surveillance, testing of serum samples using single test may not be able to substantiate complete freedom from infection. Thus, there is a requirement of more sensitive, robust, and accurate diagnostic tests to detect the FMDV infection/virus circulation in the vaccinated population with more accuracy than the available diagnostic tests. This can be achieved by using multiple antigens and setting the criteria for the positivity/negativity of the samples. Thus, this review emphasizes the comparison and the practical utility of the available diagnostic tests which detect antibodies against single antigen with those which detect antibodies against multiple antigens in single testing. It also emphasizes the utility of these tests in PCP-FMD (Progressive Control Pathway for Foot-and-Mouth Disease) going on in endemic countries.