Co-circulation of Hepatitis E Virus (HEV) Genotype 3 and Moose-HEV-Like Strains in Free-Ranging-Spotted Deer (Axis axis) in Uruguay.

Hepatitis E caused by hepatitis E virus (HEV) is considered an emerging foodborne zoonosis in industrialized, non-endemic countries. Domestic pigs and wild boars are considered the main reservoir of HEV. However, HEV can also infect an ever-expanding host range of animals, but they exact role in transmitting the virus to other species or humans is mostly unknown. In this work, we investigated the spread of HEV in free-living and captive spotted deer (Axis axis) from Uruguay in a 2-year period (2020-2022) and examined the role of this invasive species as a new potential reservoir of the virus. In addition, with the aim to gain new insights into viral ecology in the context of One Health, by using camera trapping, we identified and quantified temporal and spatial coexistence of spotted deer, wild boars, and cattle. In free-living animals, we detected an anti-HEV seropositivity of 11.1% (6/54). HEV infection and viral excretion in feces were assessed by RT-PCR. Thirteen of 19 samples (68.4%) had HEV RNA. Six samples were amplified using a broadly reactive RT-PCR and sequenced. No captive animal showed evidence of HEV infection. Additionally, HEV RNA was detected in a freshwater pond shared by these species. Phylogenetic and p-distance analysis revealed that zoonotic HEV genotype 3 strains circulate together with unclassified variants related to moose HEV whose potential risk of transmission to humans and other domestic and wild animals is unknown. The data presented here suggest that spotted deer (A. axis) may be a novel host for zoonotic HEV strains.

A highly sensitive nanobody-based immunoassay detecting SARS-CoV-2 nucleocapsid protein using all-recombinant reagents.

Antigen tests have been crucial for managing the COVID-19 pandemic by identifying individuals infected with SARS-CoV-2. This remains true even after immunity has been widely attained through natural infection and vaccination, since it only provides moderate protection against transmission and is highly permeable to the emergence of new virus variants. For this reason, the widespread availability of diagnostic methods is essential for health systems to manage outbreaks effectively. In this work, we generated nanobodies to the virus nucleocapsid protein (NP) and after an affinity-guided selection identified a nanobody pair that allowed the detection of NP at sub-ng/mL levels in a colorimetric two-site ELISA, demonstrating high diagnostic value with clinical samples. We further modified the assay by using a nanobody-NanoLuc luciferase chimeric tracer, resulting in increased sensitivity (detection limit = 61 pg/mL) and remarkable improvement in diagnostic performance. The luminescent assay was finally evaluated using 115 nasopharyngeal swab samples. Receiver Operating Characteristic (ROC) curve analysis revealed a sensitivity of 78.7% (95% confidence interval: 64.3%-89.3%) and specificity of 100.0% (95% confidence interval: 94.7%-100.0%). The test allows the parallel analysis of a large number of untreated samples, and fulfills our goal of producing a recombinant reagent-based test that can be reproduced at low cost by other laboratories with recombinant expression capabilities, aiding to build diagnostic capacity.

The environment matters: season and female contact affect the behavior of captive Addax nasomaculatus male antelope.

The addax antelope (Addax nasomaculatus) is a species under serious threat of extinction, as it is more abundant in captivity than in the wild. However, little is known about its basic biology. The aims of this study were to determine how locomotor, feeding, aggressive, marking, and sexual behavior of male addax allocated in all-male groups vary with season and with female contact (i.e., biostimulation). The study was conducted in captive conditions, in two groups of adult males: one with no-physical contact with females, aside from visual and olfactory interactions (CF group, n = 4), and another group completely isolated from females (IF group, n = 4). The frequency of behaviors was recorded during the daytime, 4 days per season (total time of observation = 256 h). Lying, standing, walking, aggressive, marking, grazing, and ruminating behaviors as well as water and supplement consumptions varied with season (all p < 0.05). The lying, walking, marking, grazing, and ruminating behaviors were more frequently observed for CF than IF males (all p < 0.05). Also, all behaviors, except for marking, varied with the interaction between the group and seasons (all p < 0.05). Sexual behavior was extremely scarce, so it was not possible to analyze how it varied with seasons and the group. The present study suggests that management program and housing conditions, especially in ex situ breeding plans, should consider the influence of the season and the sociosexual context on the behavior of addax males.

Nanobody-Based Blocking of Binding ELISA for the Detection of Anti-NS1 Zika-Virus-Specific Antibodies in Convalescent Patients.

Zika virus has spread around the world with rapid pace in the last five years. Although symptoms are typically mild and unspecific, Zika's major impact occurs during pregnancy, generating a congenital syndrome. Serology plays a key role in its diagnosis. However, its use is limited due to the uncertainty caused by the cross-reaction of antibodies elicited in response to other flavivirus infections when tested in direct immunoassays. Using a panel of previously generated anti-Zika non-structural protein 1 (NS1) nanobodies, a set was selected that only recognizes epitopes present in Zika and is immunogenic to humans. A proper arrangement of these nanobodies was made and conditions were optimized in order to develop a novel serology assay. This new ELISA relies on the inhibition of the binding of a set of selected nanobodies to Zika-immobilized NS1 when previously incubated with Zika convalescent sera. Using the developed blocking of binding assay, it was possible to discriminate between Zika-specific and cross-reactive antibodies in serum samples from infections with Zika and other flaviviruses.

Bispecific Single-Domain Antibodies as Highly Standardized Synthetic Calibrators for Immunodiagnosis.

Commonly, serological immunoassays and diagnostic kits include reference standard reagents (calibrators) that contain specific antibodies to be measured, which are used for the quantification of unknown antibodies present in the sample. However, in some cases, such as the diagnosis of allergies or autoimmune diseases, it is often difficult to have sufficient quantities of these reference standards, and there are limitations to their lot-to-lot reproducibility and standardization over time. To overcome this difficulty, this study introduces the use of surrogate recombinant calibrators formulated on the basis of two single-domain antibodies (nanobodies) combined through a short peptide linker to produce a recombinant bispecific construct. One of the nanobodies binds to the cognate analyte of the target antibody and the second is specific for the paratope of the secondary detecting antibody. The bispecific nanobody inherits the outstanding properties of stability and low-cost production by bacterial fermentation of the parent nanobodies, and once calibrated against the biological reference standard, it can be reproduced indefinitely from its sequence in a highly standardized manner. As a proof of concept, we present the generation and characterization of two bispecific calibrators with potential application for the diagnosis of allergy against the antibiotics aztreonam and amoxicillin in humans.

Hepatitis E Virus (HEV) infection in captive white-collared peccaries (Pecari tajacu) from Uruguay.

Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide. Clinical presentation of hepatitis E mainly occurs as an acute and self-limited disease, though chronic cases are now being commonly reported in immunocompromised individuals. In high-income developed areas and non-endemic regions, HEV is mainly transmitted by the zoonotic route through direct contact with infected animals or by consumption of contaminated meat products. Although pigs and wild boars are the main reservoirs of the disease, HEV can also infect deer, camels, and rats and seems to have an ever-expanding host range. Peccaries (Tayassuidae family, superfamily Suoidea), the 'new world pigs', share susceptibility to several pathogens with domestic pigs and wild boars. Herein, we performed a serological and molecular survey of two captive populations of white-collared peccaries (Pecari tajacu) from Uruguay, with the aim to assess the role of the species as an HEV reservoir. One-hundred and one serum samples were analysed for anti-HEV antibodies. Further evidences of active HEV infection were investigated in stool by RT-nested PCR. Animals from both wildlife reserves were exposed to HEV with an overall prevalence of 24.7%. Moreover, HEV RNA could be detected in peccaries' stool samples from one of the reserves. Phylogenetic analysis clustered the strains within HEV-3, closely related to both human and swine isolates. Our work provides the first evidences supporting the notion that white-collared peccaries are susceptible to HEV. However, these data should not be overinterpreted. Further research is needed concerning the role of peccaries in the transmission of HEV.

A nanobody-based test for highly sensitive detection of hemoglobin in fecal samples.

Colon cancer has a high prevalence worldwide and is a serious public health problem. Early diagnosis greatly improves its prognosis and, among the existing methods, the detection of fecal occult blood is the only noninvasive test recommended for screening of the disease. To promote its massive application as a screening tool for asymptomatic populations in low-resource settings, the availability of a reliable and cost-effective method is imperative. Here, we describe the development and validation of a sensitive nanobody-based immunoassay for the detection of hemoglobin in human fecal samples. The nanobodies were selected from a library generated from a llama immunized with human hemoglobin, using a high-throughput platform that enabled the identification of the best nanobody pair. The assay allowed a sub-ng/mL limit of detection to be reached in phosphate-buffered saline, and was validated with stool samples, showing excellent reproducibility (CV% < 15 inter-day precision) and accuracy at 2 and 4 µg of hemoglobin per gram of feces, which are well below the recommended cutoff for this test (10-20 µg/g). Moreover, no cross-reactivity was observed with a panel of dietary non-human hemoglobins removing the need for pre-test dietary restrictions. Considering that the monodomain nature of nanobodies facilitates their straightforward and low-cost production by bacterial fermentation, with their provided sequences and using synthetic genes, the assay reported here could be replicated in any laboratory to perform thousands of tests for early detection of colorectal cancer at almost no cost. Graphical abstract.