Advancement in SARS-CoV-2 diagnosis: A new and stable electrochemical biosensor for genomic RNA detection.

Coronavirus disease (COVID-19) is caused by infection with the SARS-CoV-2 virus, having already caused more than seven million deaths worldwide. Conventional techniques for SARS-CoV-2 detection have limitations, as high cost, low specificity, and longer analysis time, among others. Biosensors emerge as a necessary alternative to overcome the difficulties of current diagnostics. This paper reports a sensor platform where silver-doped zinc oxide nanomaterial (Ag:ZnONp) was used onto carbon screen-printed electrode and ethidium bromide as indicator for development of a specific electrochemical genosensor for COVID-19. This genosensor demonstrated good linearity between the concentrations of 5.62 × 104-5.62 copies/mL and a detection limit of 5 copies/mL with gRNA in patient's samples, with a response time within 30 min. Molecular modeling and morphological analysis are in agreement with obtained electrochemical results. Additional techniques such as AFM, SEM, and EIS were conducted to characterize the morphological and electrochemical properties of the biosensor's surface. The biosensor was also capable of detecting the target presence in spiked samples and demonstrated a stability of 60 days, higher than other similar biosensors for SARS-CoV-2.

Epstein-Barr virus-associated post-transplant smooth muscle tumours in a kidney transplant patient.

We report on a 14-year-old girl who developed post-transplantation smooth muscle tumours (PTSMT) located in the spleen, lungs, liver, and central nervous system (CNS), 4 years after kidney transplantation. She was asymptomatic, and the disease was detected during the work-up for a urinary tract infection. Diagnosis was performed by the analysis of a tissue specimen, through the biopsy of a lung tumour, which revealed a proliferation of spindle-shaped cells which were positive for actin and vimentin. In situ hybridization studies were positive for Epstein-Barr virus, and her serologic status was negative prior to transplantation. We reduced immunosuppression by stopping mycophenolate and switching tacrolimus for sirolimus. After 18 months of follow-up, she remains asymptomatic, and the CNS tumour reduced its diameter from 24 × 21 mm to 14 × 13 mm. PTSMT should be considered in the differential diagnosis of transplanted patients who develop neoplastic complications associated with immunosuppression.

Exposure Practices to Animal-Origin Influenza A Virus at the Animal-Human Interface in Poultry and Swine Backyard Farms.

AIM: Backyard production systems (BPS) represent an interface of contact between people, domestic and wild animals. Studies conducted in Chile during the last decade have provided extensive evidence of influenza A virus (IAV) circulation in backyard poultry and swine. The aim of this study was to investigate exposure practices of humans to animal-origin IAV within backyards. METHODS AND RESULTS: Backyard farmers and household members of a total of 101 BPS in the proximity of wetlands located throughout Chile were interviewed between 2021 and 2022. Data were collected on the nature of human-animal contacts through participation in productive activities conducted within backyards, which was used to estimate participants' exposure risk to animal-origin IAV. Additionally, RT-qPCR and serologic IAV active surveillance was carried out in backyard animals. Multilinear regression was used to identify factors associated with exposure risk. Overall, IAV prevalence was 10.1% (95% CI: 4.7%-15.5%) and seroprevalence was 43.5% (95% CI: 29.7%-54.2%), both at the BPS level. Of 180 interviewees, 86% reported participating regularly in poultry or swine exposure activities within the backyard. A greater participation of male participants was observed when evaluating swine exposure activities, while female participation was greater for some activities related to poultry handling. Handwashing was a very extended hygiene practice; however, the use of personal protective equipment was uncommon. Different factors related to participants, households and backyards were associated with an increased exposure risk of participants to animal-origin IAV: (i) older age, (ii) less years of education, (iii) no off-farm work, (iv) greater backyard production value and (v) greater household consumption of backyard products. CONCLUSION: These results indicate the circulation of IAV in BPS and the frequent human-animal contact at this interface, highlighting the need for awareness campaigns and educational programmes aimed at backyard farmers on prevention and biosecurity measures in the management of backyard animals.

Chemometrics and analytical blank on the at-line monitoring of Zika-VLP production using near-infrared spectroscopy.

The Zika disease caused by the Zika virus was declared a Public Health Emergency by the World Health Union (WHO), with microcephaly as the most critical consequence. Aiming to reduce the spread of the virus, biopharmaceutical organizations invest in vaccine research and production, based on multiple platforms. A crescent vaccine production approach is based on virus-like particles (VLP), for not having genetic material in its composition, hypoallergenic and non-mutant character. For bioprocess, it is essential to have means of real-time monitoring, which can be assessed using process analysis techniques such as Near-infrared (NIR) spectroscopy, that can be combined with chemometric methods, like Partial-Least Squares (PLS) and Artificial Neural Networks (ANN) for prediction of biochemical variables. This work proposes a biochemical Zika VLP upstream production at-line monitoring model using NIR spectroscopy comparing sampling conditions (with or without cells), analytical blank (air, ultrapure water), and spectra pre-processing approaches. Seven experiments in a benchtop bioreactor using recombinant baculovirus/Sf9 insect cell platform in serum-free medium were performed to obtain biochemical and spectral data for chemometrics modeling (PLS and ANN), composed by a random data split (80 % calibration, 20 % validation) for cross-validation of the PLS models and 70 % training, 15 % testing, 15 % validation for ANN. The best models generated in the present work presented an average absolute error of 1.59 × 105 cell/mL for density of viable cells, 2.37 % for cell viability, 0.25 g/L for glucose, 0.007 g/L for lactate, 0.138 g/L for glutamine, 0.18 g/L for glutamate, 0,003 g/L for ammonium, and 0.014 g/L for potassium.

Genomics and evolutionary analysis of Chlorella variabilis-infecting viruses demarcate criteria for defining species of giant viruses.

Chloroviruses exhibit a close relationship with their hosts with the phenotypic aspect of their ability to form lytic plaques having primarily guided the taxonomy. However, with the isolation of viruses that are only able to complete their replication cycle in one strain of Chlorella variabilis, systematic challenges emerged. In this study, we described the genomic features of 53 new chlorovirus isolates and used them to elucidate part of the evolutionary history and taxonomy of this clade. Our analysis revealed new chloroviruses with the largest genomes to date (>400 kbp) and indicated that four genomic features are statistically different in the viruses that only infect the Syngen 2-3 strain of C. variabilis (OSy viruses). We found large regions of dissimilarity in the genomes of viruses PBCV-1 and OSy-NE5 when compared with the other genomes. These regions contained genes related to the interaction with the host cell machinery and viral capsid proteins, which provided insights into the evolution of the replicative and structural modules in these giant viruses. Phylogenetic analysis using hallmark genes of Nucleocytoviricota revealed that OSy-viruses evolved from the NC64A-viruses, possibly emerging as a result of the strict relationship with their hosts. Merging phylogenetics and nucleotide identity analyses, we propose strategies to demarcate viral species, resulting in seven new species of chloroviruses. Collectively, our results show how genomic data can be used as lines of evidence to demarcate viral species. Using the chloroviruses as a case study, we expect that similar initiatives will emerge using the basis exhibited here.IMPORTANCEChloroviruses are a group of giant viruses with long dsDNA genomes that infect different species of Chlorella-like green algae. They are host-specific, and some isolates can only replicate within a single strain of Chlorella variabilis. The genomics of these viruses is still poorly explored, and the characterization of new isolates provides important data on their genetic diversity and evolution. In this work, we describe 53 new chlorovirus genomes, including many isolated from alkaline lakes for the first time. Through comparative genomics and molecular phylogeny, we provide evidence of genomic gigantism in chloroviruses and show that a subset of viruses became highly specific for their hosts at a particular point in evolutionary history. We propose criteria to demarcate species of chloroviruses, paving the way for an update in the taxonomy of other groups of viruses. This study is a new and important piece in the complex puzzle of giant algal viruses.

Unveiling ancestral threads: Exploring CCR5 ∆32 mutation frequencies in Colombian populations for HIV/AIDS therapeutics.

AIDS remains a significant global health challenge since its emergence in 1981, with millions of deaths and new cases every year. The CCR5 ∆32 genetic deletion confers immunity to HIV infection by altering a cell membrane protein crucial for viral entry. Stem cell transplants from homozygous carriers of this mutation to HIV-infected individuals have resulted in viral load reduction and disease remission, suggesting a potential therapeutic avenue. This study aims to investigate the relationship between genetic ancestry and the frequency of the CCR5 ∆32 mutation in Colombian populations, exploring the feasibility of targeted donor searches based on ancestry composition. Utilizing genomic data from the CÓDIGO-Colombia consortium, comprising 532 individuals, the study assessed the presence of the CCR5 ∆32 mutation and examined if the population was on Hardy-Weinberg equilibrium. Individuals were stratified into clusters based on African, American, and European ancestry percentages, with logistic regression analysis performed to evaluate the association between ancestry and mutation frequency. Additionally, global genomic databases were utilized to visualize the worldwide distribution of the mutation. The findings revealed a significant positive association between European ancestry and the CCR5 ∆32 mutation frequency, underscoring its relevance in donor selection. African and American ancestry showed negative but non-significant associations with CCR5 ∆32 frequency, which may be attributed to the study's limitations. These results emphasize the potential importance of considering ancestry in donor selection strategies, reveal the scarcity of potential donors in Colombia, and underscore the need to consider donors from other populations with mainly European ancestry if the CCR5 ∆32 stem cell transplant becomes a routine treatment for HIV/AIDS in Colombia.

Erythema nodosum as an atypical dermatological manifestation of HTLV-1 infection: A case report and literature review.

Human T-lymphotropic virus 1 (HTLV-1) is the etiological agent of several pathologies, and some of them are not investigated, resulting in a lack of literature that impacts the correct diagnosis. Skin manifestations, such as HTLV-1-associated infectious dermatitis (IDH), are common in patients living with HTLV-1 but could not be the only one. Here, we report for the first time a patient infected with HTLV-1, without previous diagnosis of HTLV-1-related diseases, presenting erythema nodosum (EN). Given the patient's long-term asymptomatic carrier status, the emergence of EN underscores the importance of considering HTLV-1 in the differential diagnosis when encountering EN, especially in endemic regions.

Co-Circulation of 2 Oropouche Virus Lineages, Amazon Basin, Colombia, 2024.

In early 2024, explosive outbreaks of Oropouche virus (OROV) linked to a novel lineage were documented in the Amazon Region of Brazil. We report the introduction of this lineage into Colombia and its co-circulation with another OROV lineage. Continued surveillance is needed to prevent further spread of OROV in the Americas.

Orthohantavirus diversity in Central-East Argentina: Insights from complete genomic sequencing on phylogenetics, Geographic patterns and transmission scenarios.

Hantavirus Pulmonary Syndrome (HPS), characterized by its high fatality rate, poses a significant public health concern in Argentina due to the increasing evidence of person-to-person transmission of Andes virus. Several orthohantaviruses were described in the country, but their phylogenetic relationships were inferred from partial genomic sequences. The objectives of this work were to assess the viral diversity of the most prevalent orthohantaviruses associated with HPS cases in the Central-East (CE) region of Argentina, elucidate the geographic patterns of distribution of each variant and reconstruct comprehensive phylogenetic relationships utilizing complete genomic sequencing. To accomplish this, a detailed analysis was conducted of the geographic distribution of reported cases within the most impacted province of the region. A representative sample of cases was then selected to generate a geographic map illustrating the distribution of viral variants. Complete viral genomes were obtained from HPS cases reported in the region, including some from epidemiologically linked cases. The phylogenetic analysis based on complete genomes defined two separate clades in Argentina: Andes virus in the Southwestern region and Andes-like viruses in other parts of the country. In the CE region, Buenos Aires virus and Lechiguanas virus clearly segregate in two subclades. Complete genomes were useful to distinguish person-to-person transmission from environmental co-exposure to rodent population. This study enhances the understanding of the genetic diversity, geographical spread, and transmission dynamics of orthohantaviruses in Central Argentina and prompt to consider the inclusion of Buenos Aires virus and Lechiguanas virus in the species Orthohantavirus andesense, as named viruses.

PD-L1 immunohistochemical expression considering HPV status in oropharyngeal squamous cell carcinoma.

This systematic review aims to determine whether the presence of human papillomavirus (HPV) influences the immunohistochemical expression of programmed cell death-1 ligand (PD-L1) in oropharyngeal squamous cell carcinoma (OPSCC). PD-L1 immunohistochemical expression varies in OPSCC, and the presence of HPV is a plausible explanation for this variability. Comprehending these findings is crucial, as high PD-L1 expression in the tumor microenvironment of OPSCC can help identify patient subgroups that could be suitable for immunotherapy. Therefore, a systematic review was conducted following PRISMA guidelines (CRD42023437800). An electronic literature search was performed without time or language restrictions. The search included PubMed/MEDLINE, Embase, Scopus, Web of Science, https://clinictrials.gov, and relevant journals. A meta-analysis was performed using RStudio. Fourteen studies involving 1,629 participants were included. The sample consisted predominantly of males (81.26%) with a mean age of 58.3 years. Concerning clinical and pathological characteristics, the most frequently described anatomical location was the tonsils (68.54%), and most participants were either current or former smokers (78%) and alcohol users (79%). Advanced TNM IV was the most common stage. Regarding histopathological characteristics, HPV 16 was the only type mentioned, and half of the cases were detected through immunohistochemistry. The SP142 clone (35.7%) and the pattern of membrane immunostaining in tumor cells (71%) were the most commonly employed methods. The most prevalent findings were positive expression of PD-L1 (64.28%) and negative HPV status (57.14%). The association between PD-L1 positivity and HPV positivity (78.57%) was confirmed by meta-analysis. The conclusion was that HPV-positive status has an impact on immunohistochemical expression of PD-L1 in OPSCC.

Jamaican fruit bats mount a stable and highly neutralizing antibody response after bat influenza virus infection.

Antibodies are an essential component of the antiviral response in many species, but to date, there is no compelling evidence that bats are capable of eliciting a robust humoral immunity, including neutralizing antibodies. Here, we report that infection of Jamaican fruit bats with the bat influenza A virus H18N11 elicits a rapid and stable humoral immune response with a strong neutralizing capacity, associated with no detectable viral shedding after repeat challenge infection. Thus, the neutralizing antibody response of bats might play an important role in the bat immunity.

Hepatitis C virus infection and Parkinson's disease: insights from a joint sex-stratified BioOptimatics meta-analysis.

Hepatitis C virus (HCV) infection poses a significant public health challenge and often leads to long-term health complications and even death. Parkinson's disease (PD) is a progressive neurodegenerative disorder with a proposed viral etiology. HCV infection and PD have been previously suggested to be related. This work aimed to identify potential biomarkers and pathways that may play a role in the joint development of PD and HCV infection. Using BioOptimatics-bioinformatics driven by mathematical global optimization-, 22 publicly available microarray and RNAseq datasets for both diseases were analyzed, focusing on sex-specific differences. Our results revealed that 19 genes, including MT1H, MYOM2, and RPL18, exhibited significant changes in expression in both diseases. Pathway and network analyses stratified by sex indicated that these gene expression changes were enriched in processes related to immune response regulation in females and immune cell activation in males. These findings suggest a potential link between HCV infection and PD, highlighting the importance of further investigation into the underlying mechanisms and potential therapeutic targets involved.

Oropouche Virus: An Emerging Orthobunyavirus.

On 2 February 2024, the Pan American Health Organization/World Health Organization issued an epidemiological alert on rising Oropouche virus (OROV) infections in South America. By 3 August 2024, this alert level had escalated from medium to high. OROV has been a public health concern in Central and South America since its emergence in Brazil in the 1960s. However, the 2024 outbreak marks a turning point, with the sustained transmission in non-endemic regions of Brazil, local transmission in Cuba, two fatalities and several cases of vertical transmission. As of the end of August 2024, 9852 OROV cases have been confirmed. The 2024 OROV outbreak underscores critical gaps in our understanding of OROV pathogenesis and highlights the urgent need for antivirals and vaccines. This review aims to provide a concise overview of OROV, a neglected orthobunyavirus.

Wolbachia infection in natural mosquito populations from Argentina.

The increasing spread of mosquito vectors has made mosquito-borne arboviral diseases a global threat to public health, leading to the urgent need for effective population control methods. Strategies based in the intracellular bacterium Wolbachia Hertig, 1936 are considered environmentally friendly, safe for humans, and potentially cost-effective for controlling arboviral diseases. To minimize undesirable side effects, it is relevant to assess whether Wolbachia is present in the area and understand the diversity associated with native infections before implementing these strategies. With this purpose, we investigated Wolbachia infection status, diversity, and prevalence in populations of Aedes albifasciatus (Macquart, 1838), Aedes fluviatilis (Lutz, 1904), and hybrids of the Culex pipiens (Linnaeus, 1758) complex from Argentina. Aedes albifasciatus and C. pipiens complex samples were collected in the province of Buenos Aires, and A. fluviatilis in the province of Misiones. Aedes albifasciatus was found to be uninfected, while infections with strains wFlu and wPip were detected in A. fluviatilis and hybrids of the C. pipiens complex, respectively. All strains were fixed or close to fixation and clustered within supergroup B. These finding provides valuable information on Wolbachia strains found in natural mosquito populations in Argentina that might be used in heterologous infections in the future or be considered when designing control strategies based on Wolbachia infection.

Eggplant latent viroid is located in the chloroplasts and nuclei of eggplant infected cells.

Viroids that belong to genera Avsunviroid and Pelamovirod (family Avsunviroidae) replicate and accumulate in the chloroplasts of infected cells. In this report, we confirmed by RNA in situ hybridization using digoxigenin-UTP-labelled riboprobes that the positive strands of eggplant latent viroid (ELVd), the only member of genus Elaviroid within the family Avsunviroidae, also accumulate in the chloroplasts of infected cells. However, comparison of ELVd in situ hybridization signals with those from bona fide chloroplastic and nuclear non-coding RNAs, such as chloroplast 5S rRNA and U1 small nuclear RNA, supports the notion that this viroid is also present in the nuclei of infected cells. These results suggest that the subcellular localization of viroids within the family Avsunviroidae may be more complex than previously assumed with dynamic presence in several compartments during the infectious cycle.

Molecular Epidemiology and Phyloevolutionary Analysis of Porcine Parvoviruses (PPV1 through PPV7) Detected in Replacement Gilts from Colombia.

Eight porcine parvovirus (PPV) species, designated as PPV1 through PPV8, have been identified in swine. Despite their similarities, knowledge about their distribution and genetic differences remains limited, resulting in a gap in the genetic classification of these viruses. In this study, we conducted a comprehensive analysis using PPV1 to PPV7 genome sequences from Colombia and others available in the GenBank database to propose a classification scheme for all PPVs. Sera from 234 gilts aged 180 to 200 days were collected from 40 herds in Colombia. Individual detection of each PPV (PPV1 through PPV7) was performed using end-point PCR. Complete nucleotide (nt) sequencing was performed on the PPV1 viral protein (VP), and near-complete genome (NCG) sequencing was carried out for novel porcine parvoviruses (nPPVs) (PPV2 through PPV7). Phylogenetic analyses were conducted by comparing PPV1-VP sequences to 94 available sequences and nPPVs with 565 NCG, 846 nPPV-VP, and 667 nPPV-nonstructural protein (NS) sequences. Bayesian phylogenetic analysis was used to estimate substitution rates and the time to the most recent common ancestor for each PPV. The highest prevalence was detected for PPV3 (40.1%), followed by PPV5 (20.5%), PPV6 (17%), PPV1 (14.5%), PPV2 (9.8%), PPV4 (4.2%), and PPV7 (1.3%). Notably, all tested sera were negative for PPV8 genomes. An analysis of the PPV1-VP sequences revealed two main clades (PPV1-I and PPV1-II), with the sequences recovered in this study grouped in the PPV1-II clade. Comparative analysis showed significant genetic distances for PPV2 to PPV7 at the NCG (>6.5%), NS (>6.3%), and VP (>7.5%) regions, particularly when compared to equivalent regions of PPV genomes recovered worldwide. This study highlights the endemic circulation of nPPVs in Colombian pig herds, specifically among gilts. Additionally, it contributes to the phylogenetic classification and evolutionary studies of these viruses. The proposed method aims to categorize and divide subtypes based on current knowledge and the genomes available in databanks.

An integrative analysis of SARS-CoV 2 during the first and second waves of COVID-19 in Salta, Argentina.

Wastewater surveillance has been extensively applied to provide information about SARS-CoV-2 circulation in the community. However, its applicability is limited in regions lacking adequate sewerage infrastructure, without wastewater treatment plants (WWTP) or with insufficient coverage. During the COVID-19 pandemic, from July 2020 to September 2021, comprehensive epidemiological data encompassing positive, recovered, and deceased cases were collected alongside precipitation records. Additionally, wastewater samples from 13 main sewersheds and river water from two points (up- and downstream the main WWTP), in the city of Salta, were gathered. A total of 452 water samples were analyzed for quantitative detection of SARS-CoV-2 using reverse transcription real-time PCR. Across the 62-week study period, two distinct waves of COVID-19 were identified. The dynamics of deceased cases showed peaks 10 and 28 days after the peaks of positive cases in the first and second waves, respectively. Downstream river water exhibited higher fecal contamination than the upstream samples, evincing the impact of the WWTP discharges. Viral concentration in river waters mirrored those from wastewater, reflecting the progression of cases. Despite the lower reported number of cases during the first wave in comparison to the second (5420 vs. 8516 cases at the respective peaks), higher viral concentrations were detected in water samples (1.97 × 107 vs. 2.36 × 106 gc/L, respectively), suggesting underreporting during the first wave, and highlighting the positive effect of vaccination during the second. To the best of our knowledge, this is the first study that simultaneously and systematically analyzed surface water and wastewater over a prolonged period, the effect of precipitations were considered for the variations in the concentrations, and the findings compared with epidemiological information. Environmental surveillance was demonstrated to be a great tool to obtain valuable information about the circulation patterns of SARS-CoV-2, especially under resource constraints to massively test the population, thus, underreporting cases. Furthermore, the methodology employed herein can be easily expanded to the community-level surveillance of other pathogens excreted in urine and feces, encompassing viruses, bacteria, and protozoa.

Human pegivirus -1 (HPgV-1) RNA frequency and genotype distribution in pediatric oncology patients with febrile neutropenia.

Human Pegivirus-1, typically regarded as a commensal virus, exhibits high prevalence in humans. Its frequency and impact on oncologic pediatric patients with febrile neutropenia (FN), a frequent chemotherapy complication, remains unexplored. In this study, we assessed HPgV-1 RNA prevalence in pediatric patients experiencing FN. Blood samples were collected from 30 children, 15 presenting FN and 15 comprising a control group of either undergoing treatment or in remission. Overall, HPgV-1 RNA was detected in 23.3 % of samples (26.7 % among FN patients and 20.0 % among those under treatment or in remission). Phylogenetic analysis unveiled HPgV-1 genotype 2 predominance among these samples, the most prevalent strain circulating in Brazil. Our findings prompt crucial inquiries into the role of HPgV-1 RNA in FN: is it an incidental finding and if it can influences this clinical entity? Further investigation is imperative to elucidate HPgV-1 implications in vulnerable patients cohorts, potentially informing new approaches and understanding viral dynamics in immunocompromised populations.

Edited eukaryotic translation initiation factors confer resistance against maize lethal necrosis.

Maize lethal necrosis (MLN), which is caused by maize chlorotic mottle virus along with a potyvirus, has threatened the food security of smallholders in sub-Saharan Africa. Mutations in eukaryotic translation initiation factors (eIFs), which also facilitate virus genome translation, are known to confer variable resistance against viruses. Following phylogenetic analysis, we selected two eIF4E proteins from maize as the most likely candidates to facilitate MLN infection. A knockout (KO) of each of the corresponding genes in elite but MLN-susceptible maize lines conferred only partial protection. Our inability to knockout both the genes together suggested that at least one was required for survival. When we edited (ED) the eIF4E genes in Mini Maize, however, the plants with the eif4e1-KO became highly resistant, whereas those with the eif4e2-KO remained susceptible. Neither of the causal viruses could be detected in the MLN-inoculated eif4e1-KO plants. The eIF4E2 cDNA in Mini Maize lacked the entire 4th exon, causing a 22-amino acid in-frame deletion, which shortened the protein to 198 amino acids. When we introduced mutations in the 4th exon of the eIF4E2 gene in two elite, MLN-susceptible lines pre-edited for an eif4e1-KO, we obtained as strong resistance against MLN as in eif4e1-KO Mini Maize. The MLN-inoculated lines with eif4e1-KO/eIF4E2-exon-4ED performed as well as the uninoculated wild-type lines. We demonstrate that the C-terminal 38 amino acids of eIF4E2 are dispensable for normal plant growth but are required for the multiplication of MLN viruses. Our discovery has wide applications across plant species for developing virus-resistant varieties.

Development of an indirect ELISA for the serologic detection of bovine viral diarrhea virus based on E2 antigen sub-genotypes 1b, 1e, and 1d.

Bovine viral diarrhea virus (BVDV) causes ongoing economic losses to cattle industries, directly through reduced herd performance or indirectly through control program costs. ELISA assays, one of the most widely used techniques due to their ease of implementation, have been a valuable tool for mass surveillance and detection of BVDV. In this study, we developed a new indirect ELISA (rE2-ELISA) for serologic detection of BVDV. The assay considers three recombinant E2 protein subtypes as antigens, allowing serologic diagnosis of BVDV-1b (high prevalence worldwide), BVDV-1d and 1e (high prevalence in southern Chile) sub-genotypes. Recombinant E2 (rE2) proteins were successfully expressed in stably transfected CHO cells. Conditions for rE2 ELISAs were established after determining appropriate concentrations of antigen, blocking agent, secondary antibody, and serum dilutions to achieve maximum discrimination between positive and negative serum samples. The developed rE2-ELISA showed a sensitivity of 92.86% and a specificity of 98.33%. Clinical testing of 180 serum samples from herds in southern Chile showed high accuracy (kappa > 0.8) compared to the commercial BVDV Total Ab kit (IDEXX), with 95.37% positive and 87.5% negative predictive value. In addition, the rE2 ELISA has shown the capability to detect anti-BVDV antibodies from naturally infected animals with sub-genotypes 1b, 1e, or undetermined. These results indicate that the developed indirect ELISA could serve as a valid, and efficient alternative for identifying BVDV-infected animals, thus contributing to the success of disease control and eradication programs.