Ricardo Madrid

A prompt and accurate diagnosis of respiratory viral diseases in intensive poultry production is essential to safeguard animal health and ensure the economic sustainability of farms. Currently, much effort is being devoted to preventing the spread of the avian influenza virus in farms. However, the diagnosis of other relevant respiratory viruses, as infectious laryngotracheitis virus (ILTV), is also crucial. Indeed, infection by ILTV does lead to substantial economic losses due to high morbidity, reduced growth, and decreased productivity, making rapid detection a critical aspect of disease control. Conventional diagnostics, including PCR and qPCR, while sensitive and specific, require expensive laboratory infrastructure and well-trained personnel, limiting their deployment in field settings where immediate intervention is most valuable. To address these limitations, this protocol describes a portable molecular diagnostic workflow based on loop-mediated isothermal amplification (LAMP) combined with gold nanoparticle–DNA nanoprobes for specific and visual detection of ILTV directly at the point of need. Gold nanoparticles synthesized via the Turkevich method are functionalized with thiolated DNA probes, which undergo full-length, sequence-specific hybridization to LAMP amplicons, enabling a naked-eye colorimetric readout. The procedure integrates streamlined steps for DNA probe preparation, nanoparticle synthesis and assembly, and minimal sample processing, compatible with on-farm deployment. Results obtained with this workflow on field samples demonstrated 100% sensitivity and specificity, matching the performance of gold-standard assays. This approach offers a rapid, cost-effective, and equipment-free detection system of viral pathogens, enabling timely decision-making for disease containment and biosecurity. By overcoming the barriers of conventional diagnostics, this protocol enables producers with powerful tools for efficient monitoring and response to respiratory outbreaks in poultry farms.

Infectious laryngotracheitis virus (ILTV), a DNA virus classified as Gallid alphaherpesvirus 1, causes a highly contagious respiratory disease in chickens, leading to significant economic losses and health risks for the poultry industry. The rapid detection of ILTV is essential to control its spread and prevent outbreaks. Traditional diagnostic methods like PCR are costly, require specialized personnel, and delay response efforts. To address this, we developed a point-of-care diagnostic test combining loop-mediated isothermal amplification (LAMP) with DNA nanoprobes on respiratory swabs. LAMP targets the ILTV-glycoprotein E (gE) gene, enabling rapid nucleic acid amplification at 65 °C without extraction, making it suitable for on-site detection. DNA nanoprobes provide a colorimetric readout visible to the naked eye. Gold nanoparticles drive this readout, as their red color, based on localized surface plasmon resonance, persists in the presence of ILTV DNA through DNA-DNA hybridization, ensuring reliable detection. The assay achieved 100% sensitivity and specificity for ILTV-gE, with a detection limit of 200 copies per reaction, allowing for the early identification of infections. The results are available within 45 min, enabling prompt measures to control ILTV spread. Cost-effective and user-friendly, this method enhances disease management and biosecurity in poultry farms.

Airborne animal viral pathogens can rapidly spread and become a global threat, resulting in substantial socioeconomic and health consequences. To prevent and control potential epidemic outbreaks, accurate, fast, and affordable point-of-care (POC) tests are essential. As a proof-of-concept, we have developed a molecular system based on the loop-mediated isothermal amplification (LAMP) technique for avian metapneumovirus (aMPV) detection, an airborne communicable agent mainly infecting turkeys and chickens. For this purpose, a colorimetric system was obtained by coupling the LAMP technique with specific DNA-functionalized AuNPs (gold nanoparticles). The system was validated using 50 different samples (pharyngeal swabs and tracheal tissue) collected from aMPV-infected and non-infected chickens and turkeys. Viral detection can be achieved in about 60 min with the naked eye, with 100% specificity and 87.88% sensitivity for aMPV. In summary, this novel molecular detection system allows suitable virus testing in the field, with accuracy and limit of detection (LOD) values highly close to qRT-PCR-based diagnosis. Furthermore, this system can be easily scalable to a platform for the detection of other viruses, addressing the current gap in the availability of POC tests for viral detection in poultry farming. Key points: •aMPV diagnosis using RT-LAMP is achieved with high sensitivity and specificity. •Fifty field samples have been visualized using DNA-nanoprobe validation. •The developed system is a reliable, fast, and cost-effective option for POCT. Graphical

Background: Both SARS-CoV-2 and HIV infection exhibit alterations in the senescence profile and immune checkpoint (IC) molecules. However, the midterm impact of SARS-CoV-2 on these profiles in people with HIV (PWH) remains unclear. This study aimed to evaluate differences in plasma biomarker levels related to ICs, the senescence-associated secretory phenotype (SASP), and pro- and anti-inflammatory cytokines in PWH following recovery from SARS-CoV-2 infection. Methods: We conducted a cross-sectional study of 95 PWH receiving antiretroviral therapy, stratified by SARS-CoV-2 infection status: a) 48 previously infected (HIV/SARS) and b) 47 controls without previous infection (HIV). Plasma biomarkers (n=44) were assessed using Procartaplex Multiplex Immunoassays. Differences were analyzed using a generalized linear model adjusted for sex and ethnicity and corrected for the false discovery rate. Significant values were defined as an adjusted arithmetic mean ratio ≥1.2 or ≤0.8 and a qvalue<0.1. Spearman correlation evaluated relationships between plasma biomarkers (significant correlations, rho≥0.3 and q value<0.1). Results: The median age of the PWH was 45 years, and 80% were men. All SARS-CoV-2-infected PWH experienced symptomatic infection; 83.3% had mild symptomatic infection, and sample collection occurred at a median of 12 weeks postdiagnosis. The HIV/SARS group showed higher levels of ICs (CD80, PDCD1LG2, CD276, PDCD1, CD47, HAVCR2, TIMD4, TNFRSF9, TNFRSF18, and TNFRSF14), SASP (LTA, CXCL8, and IL13), and inflammatory plasma biomarkers (IL4, IL12B, IL17A, CCL3, CCL4, and INF1A) than did the HIV group. Conclusions: SARS-CoV-2 infection in PWH causes significant midterm disruptions in plasma ICs and inflammatory cytokine levels, highlighting SASP-related factors, which could be risk factors for the emergence of complications in PWH.

The mid-term impact of SARS-CoV-2 infection on senescence profile and immune checkpoints biomarkers in people living with HIV (PLWH) was assessed. Cross-sectional study in 95 PLWH on ART stratified by SARS-CoV-2 infection: a) 48 PLWH previously infected (PCR+) (HIV/SARS); b) 47 PLWH controls without previous infection (HIV). Plasma biomarkers (n=44) related to cell immune checkpoint molecules, associated with the Senescence-Associated Secretory Phenotype (SASP), and related to pro and anti-inflammatory cytokines were assessed by Procartaplex Multiplex Immunoassays (Xmap-Luminex technology). Differences between groups were analyzed using a generalized linear model, adjusted by sex and ethnicity and corrected by false discovery rate. Significant values were defined as the adjusted arithmetic mean ratio ≥1.2 or ≤0.8; q-value<0.1. The relationship between plasma biomarkers was evaluated by Spearman correlation (significant correlations rho≥0.3 and q-value<0.1). PLWH had a median age of 45 years and 80% were men. All PLWH infected by SARS-CoV-2 had a symptomatic infection, 83.3% as mild, and with a median of 12 weeks after diagnosis of SARS-CoV-2 infection. HIV/SARS group showed higher levels of the cell immune checkpoint plasma biomarkers CD80, PDCD1LG2, CD276, PDCD1, CD47, HAVCR2, TIMD4, TNFRSF9, TNFRSF18, and TNFRSF14 respect to HIV group. The SASP biomarkers LTA, CXCL8, and IL13 and the inflammatory biomarkers IL4, IL12B, IL17A, CCL3, CCL4, and INF1A showed significantly higher levels in the HIV/SARS group. SARS-CoV-2 infection in PLWH leads to significant medium-term disruption in plasma immune checkpoint molecules and inflammatory cytokines, highlighting SASP-related. This could be a risk factor for the emergence of complications in PLWH.

There is growing interest in emerging viruses that can cause serious or lethal disease in humans and animals. The proliferation of cloacal virome studies, mainly focused on poultry and other domestic birds, reveals a wide variety of viruses, although their pathogenic significance is currently uncertain. Analysis of viruses detected in wild birds is complex and often biased towards waterfowl because of the obvious interest in avian influenza or other zoonotic viruses. Less is known about the viruses present in the order Passeriformes, which comprises approximately 60% of extant bird species. This review aims to compile the most significant contributions on the DNA/RNA viruses affecting passerines, from traditional and metagenomic studies. It highlights that most passerine species have never been sampled. Especially the RNA viruses from Flaviviridae, Orthomyxoviridae and Togaviridae are considered emerging because of increased incidence or avian mortality/morbidity, spread to new geographical areas or hosts and their zoonotic risk. Arguably poxvirus, and perhaps other virus groups, could also be considered “emerging viruses”. However, many of these viruses have only recently been described in passerines using metagenomics and their role in the ecosystem is unknown. Finally, it is noteworthy that only one third of the viruses affecting passerines have been officially recognized.

Airbone viral pathogens can rapidly spread and become a global menace, including both human and animal viruses which can trigger important socioeconomical and health effects. Therefore, to prevent and contain potential epidemic outbreaks, accurate, fast, and affordable diagnostic point-of-care tests (POCT) are required. As a proof of concept, we have developed a molecular detection system based in Loop-mediated isothermal AMPlification technique (LAMP) for two different RNA airborne viruses: the well-known human SARS-CoV-2, and the avian metapneumovirus (aMPV), the aetiologic agent of a communicable disease infecting mainly turkeys and chickens. To obtain a POC diagnostic system, we coupled the LAMP technique to DNA-functionalized gold nanoparticles detection. Validation of this system was carried out in 140 pharyngeal swabs from COVID-19 symptomatic and asymptomatic patients, and in 50 different samples (pharyngeal swabs and tracheal tissue samples) collected from aMPV infected chickens and turkeys. The system allows viral detection in about 60 minutes by the naked eye with 100% specificity, and 97.22% and 87.88% sensitivity for SARS-CoV-2 and aMPV, respectively. In summary, this novel detection system based on the coupling of RT-LAMP to DNA-nanoprobes allows suitable virus testing in the field, with accurate levels very close to conventional qRT-PCR based diagnosis.