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.

Intranasal Delivery of Quillaja brasiliensis Saponin-Based Nanoadjuvants Improve Humoral Immune Response of Influenza Vaccine in Aged Mice.

Increasing the effectiveness of vaccines against respiratory viruses is particularly relevant for the elderly, since they are prone to develop serious infections due to comorbidities and the senescence of the immune system. The addition of saponin-based adjuvants is an interesting strategy to increase the effectiveness of vaccines. We have previously shown that ISCOM matrices from Q. brasiliensis (IMXQB) are a safe and potent adjuvant. In this study, we evaluated the use of IMXQB as an adjuvant for the seasonal trivalent influenza vaccine (TIV) in an aged mice model. Herein, we show that subcutaneous injection of the adjuvanted vaccine promoted higher titers of IgM, IgG (and isotypes), and serum hemagglutination inhibition titers (HAI). Notably, aged mice immunized by intranasal route also produced higher IgG (and isotypes) and IgA titers up to 120 days after priming, as well as demonstrating an improvement in the HAI antibodies against the TIV. Further, experimental infected aged mice treated once with sera from adult naïve mice previously immunized with TIV-IMXQB subcutaneously successfully controlled the infection. Overall, TIV-IMXQB improved the immunogenicity compared to TIV by enhancing systemic and mucosal immunity in old mice conferring a faster recovery after the H1N1pdm09-like virus challenge. Thus, IMXQB nanoparticles may be a promising platform for next-generation viral vaccines.

Phylodynamics of avian influenza A(H5N1) viruses from outbreaks in Brazil.

Our study identified strains of the A/H5N1 virus in analyzed samples of subsistence poultry, wild birds, and mammals, belonging to clade 2.3.4.4b, genotype B3.2, with very high genetic similarity to strains from Chile, Uruguay, and Argentina. This suggests a migratory route for wild birds across the Pacific, explaining the phylogenetic relatedness. The Brazilian samples displayed similarity to strains that had already been previously detected in South America. Phylogeographic analysis suggests transmission of US viruses from Europe and Asia, co-circulating with other lineages in the American continent. As mutations can influence virulence and host specificity, genomic surveillance is essential to detect those changes, especially in critical regions, such as hot spots in the HA, NA, and PB2 sequences. Mutations in the PB2 gene (D701N and Q591K) associated with adaptation and transmission in mammals were detected suggesting a potential zoonotic risk. Nonetheless, resistance to neuraminidase inhibitors (NAIs) was not identified, however, continued surveillance is crucial to detect potential resistance. Our study also mapped the spread of the virus in the Southern hemisphere, identifying possible entry routes and highlighting the importance of surveillance to prevent outbreaks and protect both human and animal populations.

Low antiviral resistance in Influenza A and B viruses isolated in Mexico from 2010 to 2023.

The most widely used class of antivirals available for Influenza treatment are the neuraminidase inhibitors (NAI) Oseltamivir and Zanamivir. However, amino acid (AA) substitutions in the neuraminidase may cause reduced inhibition or high antiviral resistance. In Mexico, the current state of knowledge about NAI susceptibility is scarce, in this study we report the results of 14 years of Influenza surveillance by phenotypic and genotypic methods. A total of 255 isolates were assessed with the NAI assay, including Influenza A(H1N1)pdm09, A(H3N2) and Influenza B (IBV). Furthermore, 827 sequences contained in the GISAID platform were analyzed in search of relevant mutations.Overall, five isolates showed highly reduced inhibition or reduced inhibition to Oseltamivir, and two showed reduced inhibition to Zanamivir in the NAI assays. Additionally, five A(H1N1)pdm09 sequences from the GISAID possessed AA substitutions associated to reduced inhibition to Oseltamivir and none to Zanamivir. Oseltamivir resistant A(H1N1)pdm09 harbored the H275Y mutation. No genetic mutations were identified in Influenza A(H3N2) and IBV. Overall, these results show that in Mexico the rate of NAI resistance is low (0.6%), but it is essential to continue the Influenza surveillance in order to understand the drug susceptibility of circulating strains.

Highly Pathogenic Avian Influenza A(H5N1) Viruses from Multispecies Outbreak, Argentina, August 2023.

We report full-genome characterization of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus from an outbreak among sea lions (August 2023) in Argentina and possible spillover to fur seals and terns. Mammalian adaptation mutations in virus isolated from marine mammals and a human in Chile were detected in mammalian and avian hosts.

Incursion of Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus, Brazil, 2023.

We report 4 highly pathogenic avian influenza A(H5N1) clade 2.3.4.4.b viruses in samples collected during June 2023 from Royal terns and Cabot's terns in Brazil. Phylodynamic analysis revealed viral movement from Peru to Brazil, indicating a concerning spread of this clade along the Atlantic Americas migratory bird flyway.

Using guinea pig as a model for evaluation of equine influenza vaccine

Equine influenza is a highly contagious viral disease, specially among 1-5 years old naive horses. Vaccination is considered the best way to control the disease spread and outbreaks. Although foals are the main animal used for evaluation of equine influenza vaccines, guinea pigs were chosen as an alternative model in the present work, as they have a negligible antibody titer against equine influenza virus and are cheaper and easier to handle than foals. Five equine influenza vaccine batches were evaluated in two animal models, foals and guinea pigs, by injection of two doses/animal with 4 weeks apart using 2 mL/animal/dose and evaluation of immune responses by hemagglutination inhibition test and enzyme-linked immunosorbent assay. On the 7th week post vaccination, equine influenza antibodies titers reached maximum values of 9-10.2 and 8.7-10 hemagglutination inhibition units for foals and guinea pigs, respectively; sample/negative ratios were 0.126-0.464 and 0.128-0.445 for both animals, respectively. The use of guinea pigs as an animal model for the evaluation of equine influenza vaccines could be recommended instead of foals(AU)

La gripe equina es una enfermedad viral muy contagiosa, especialmente entre los caballos jóvenes de 1 a 5 años de edad. La vacunación se considera la mejor forma de controlar la propagación y los brotes de la enfermedad. Aunque los potros son el principal animal utilizado para la evaluación de vacunas contra la gripe equina, en el presente trabajo se eligieron cobayos como modelo alternativo, ya que tienen un título insignificante de anticuerpos contra el virus de la gripe equina y son más baratos y fáciles de manejar que los potros. Se evaluaron cinco lotes de vacunas contra la gripe equina en dos modelos animales, potros y cobayos, mediante la inyección de dos dosis/animal con 4 semanas de intervalo utilizando 2 mL/animal/dosis y la evaluación de las respuestas inmunitarias mediante la prueba de inhibición de la hemaglutinación y el ensayo inmunoenzimático. En la 7ª semana posvacunación, los títulos de anticuerpos contra la gripe equina alcanzaron valores máximos de 9-10,2 y 8,7-10 unidades de inhibición de la hemaglutinación para potros y cobayos, respectivamente; las relaciones muestras/negativos fueron de 0,126-0,464 y 0,128-0,445 para ambos animales, respectivamente. Podría recomendarse el uso de cobayos como modelo animal para la evaluación de vacunas contra la gripe equina, en lugar de potros(AU)

First report and genetic characterization of the highly pathogenic avian influenza A(H5N1) virus in Cabot's tern (Thalasseus acuflavidus), Brazil.

In 2021, the H5N1 virus lineage 2.3.4.4b spread to the Americas, causing high mortality in wild and domestic avian populations. South American countries along the Pacific migratory route have reported wild bird deaths due to A/H5Nx virus since October 2022. However, limited genomic data resulted in no cases reported in Brazil until May 2023. Brazil reported its first case of highly pathogenic avian influenza virus (HPAI A/H5N1) in May 2023. The virus was detected in Cabot's tern specimen in Marataízes, Espírito Santo. Cases were also found in backyard poultry and other wild birds, but no human or commercial poultry cases occurred. HPAI poses risks to the poultry industry, food security, and public health. Researchers used next-gen sequencing and phylogenetic analysis to study the Brazilian sample. It confirmed its affiliation with the 2.3.4.4b clade and proximity to sequences from Chile and Peru. This sheds light on the spread and evolution of HPAI A/H5N1 in the Americas, emphasizing continuous monitoring to mitigate risks for both avian and human populations. Understanding the virus's genetics and transmission allows implementing effective control measures to protect public health and the poultry industry.

Ex Vivo Experiments Shed Light on the Innate Immune Response from Influenza Virus.

The innate immune response is recognized as a key driver in controlling an influenza virus infection in a host. However, the mechanistic action of such innate response is not fully understood. Infection experiments on ex vivo explants from swine trachea represent an efficient alternative to animal experiments, as the explants conserved key characteristics of an organ from an animal. In the present work we compare three cellular automata models of influenza virus dynamics. The models are fitted to free virus and infected cells data from ex vivo swine trachea experiments. Our findings suggest that the presence of an immune response is necessary to explain the observed dynamics in ex vivo organ culture. Moreover, such immune response should include a refractory state for epithelial cells, and not just a reduced infection rate. Our results may shed light on how the immune system responds to an infection event.

Evolution and Current Status of Influenza A Virus in Chile: A Review.

The influenza A virus (IAV) poses a significant global threat to public health and food security. Particularly concerning is the avian influenza virus (AIV) subtype H5N1, which has spread from Europe to North and Central/South America. This review presents recent developments in IAV evolution in birds, mammals, and humans in Chile. Chile's encounter with IAV began in 2002, with the highly pathogenic avian influenza (HPAI) H7N3 virus, derived from a unique South American low pathogenic avian influenza (LPAI) virus. In 2016-2017, LPAI H7N6 caused outbreaks in turkey, linked to wild birds in Chile and Bolivia. The pandemic influenza A (H1N1) 2009 (H1N1pdm09) virus in 2009 decreased egg production in turkeys. Since 2012, diverse IAV subtypes have emerged in backyard poultry and pigs. Reassortant AIVs, incorporating genes from both North and South American isolates, have been found in wild birds since 2007. Notably, from December 2022, HPAI H5N1 was detected in wild birds, sea lions, and a human, along Chile's north coast. It was introduced through Atlantic migratory flyways from North America. These findings emphasize the need for enhanced biosecurity on poultry farms and ongoing genomic surveillance to understand and manage AIVs in both wild and domestic bird populations in Chile.

Seasonal respiratory virus trends in pediatric patients during the COVID-19 pandemic in Brazil.

Acute respiratory infections are a constant public health problem causing childhood morbidity and mortality worldwide. Reported cases of major respiratory infections decreased in 2020 after restrictive measures were adopted to contain the COVID-19 pandemic, but there is little data on the impact after these measures were relaxed in the subsequent years. This study conducted molecular analysis to identify rhinovirus, respiratory syncytial virus, influenza A virus, and adenovirus in SARS-CoV-2-negative samples taken from symptomatic pediatric patients during 2021 and 2022 to ascertain the impact of pandemic response measures within the broader epidemiological scenario. The positivity rates found were 28.3% and 50.8%, in 2021 and 2022, respectively, representing a significant increase (1.8 times) in the circulation of non-SARS-CoV-2 viruses after the reduction of non-pharmacological measures to contain the COVID-19 pandemic. Within the positive samples, rhinovirus and respiratory syncytial virus were most frequent (44.4 and 18% in 2021; 44.5 and 22.5% in 2022), whereas influenza A and adenovirus were found in lower frequency (12.5 and 5.5% in 2021; 13.4 and 4.9% in 2022, respectively). Because these different respiratory virus diseases produce similar symptoms, diagnosis based on clinical condition alone can be inaccurate, and more reliable testing is required to select the best therapeutic approach for each case. The loosening of restrictive measures to contain the COVID-19 pandemic led to higher numbers of other respiratory infections in pediatric patients. Ongoing surveillance and differential diagnosis of respiratory viruses are required to better understand their seasonal patterns after the COVID-19 pandemic to guide prevention and control strategies.

Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus in Wild Birds, Chile.

In December 2022, highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus emerged in Chile. We detected H5N1 virus in 93 samples and obtained 9 whole-genome sequences of strains from wild birds. Phylogenetic analysis suggests multiple viral introductions into South America. Continued surveillance is needed to assess risks to humans and domestic poultry.

Quillaja brasiliensis nanoparticle adjuvant formulation improves the efficacy of an inactivated trivalent influenza vaccine in mice.

The threat of viral influenza infections has sparked research efforts to develop vaccines that can induce broadly protective immunity with safe adjuvants that trigger robust immune responses. Here, we demonstrate that subcutaneous or intranasal delivery of a seasonal trivalent influenza vaccine (TIV) adjuvanted with the Quillaja brasiliensis saponin-based nanoparticle (IMXQB) increases the potency of TIV. The adjuvanted vaccine (TIV-IMXQB) elicited high levels of IgG2a and IgG1 antibodies with virus-neutralizing capacity and improved serum hemagglutination inhibition titers. The cellular immune response induced by TIV-IMXQB suggests the presence of a mixed Th1/Th2 cytokine profile, antibody-secreting cells (ASCs) skewed toward an IgG2a phenotype, a positive delayed-type hypersensitivity (DTH) response, and effector CD4+ and CD8+ T cells. After challenge, viral titers in the lungs were significantly lower in animals receiving TIV-IMXQB than in those inoculated with TIV alone. Most notably, mice vaccinated intranasally with TIV-IMXQB and challenged with a lethal dose of influenza virus were fully protected against weight loss and lung virus replication, with no mortality, whereas, among animals vaccinated with TIV alone, the mortality rate was 75%. These findings demonstrate that TIV-IMXQB improved the immune responses to TIV, and, unlike the commercial vaccine, conferred full protection against influenza challenge.

Burden of Influenza and Respiratory Syncytial Viruses in Suspected COVID-19 Patients: A Cross-Sectional and Meta-Analysis Study.

Non-SARS-CoV-2 respiratory viral infections, such as influenza virus (FluV) and human respiratory syncytial virus (RSV), have contributed considerably to the burden of infectious diseases in the non-COVID-19 era. While the rates of co-infection in SARS-CoV-2-positive group (SCPG) patients have been determined, the burden of other respiratory viruses in the SARS-CoV-2-negative group (SCNG) remains unclear. Here, we conducted a cross-sectional study (São José do Rio Preto county, Brazil), and we collected our data using a meta-analysis to evaluate the pooled prevalence of FluV and RSV among SCNG patients. Out of the 901 patients suspected of COVID-19, our molecular results showed positivity of FluV and RSV in the SCNG was 2% (15/733) and 0.27% (2/733), respectively. Co-infection with SARS-CoV-2 and FluV, or RSV, was identified in 1.7% of the patients (3/168). Following our meta-analysis, 28 studies were selected (n = 114,318 suspected COVID-19 patients), with a pooled prevalence of 4% (95% CI: 3-6) for FluV and 2% (95% CI: 1-3) for RSV among SCNG patients were observed. Interestingly, FluV positivity in the SCNG was four times higher (OR = 4, 95% CI: 3.6-5.4, p < 0.01) than in the SCPG. Similarly, RSV positivity was significantly associated with SCNG patients (OR = 2.9, 95% CI: 2-4, p < 0.01). For subgroup analysis, cold-like symptoms, including fever, cough, sore throat, headache, myalgia, diarrhea, and nausea/vomiting, were positively associated (p < 0.05) with the SCPG. In conclusion, these results show that the pooled prevalence of FluV and RSV were significantly higher in the SCNG than in the SCPG during the early phase of the COVID-19 pandemic.

Severe influenza infection is associated with inflammatory programmed cell death in infected macrophages.

Introduction: Influenza A virus (IAV) is one of the leading causes of respiratory tract infections in humans, representing a major public health concern. The various types of cell death have a crucial role in IAV pathogenesis because this virus may trigger both apoptosis and necroptosis in airway epithelial cells in parallel. Macrophages play an important role in the clearance of virus particles, priming the adaptive immune response in influenza. However, the contribution of macrophage death to pathogenesis of IAV infection remains unclear. Methods: In this work, we investigated IAV-induced macrophage death, along with potential therapeutic intervention. We conducted in vitro and in vivo experiments to evaluate the mechanism and the contribution of macrophages death to the inflammatory response induced by IAV infection. Results: We found that IAV or its surface glycoprotein hemagglutinin (HA) triggers inflammatory programmed cell death in human and murine macrophages in a Toll-like receptor-4 (TLR4)- and TNF-dependent manner. Anti-TNF treatment in vivo with the clinically approved drug etanercept prevented the engagement of the necroptotic loop and mouse mortality. Etanercept impaired the IAV-induced proinflammatory cytokine storm and lung injury. Conclusion: In summary, we demonstrated a positive feedback loop of events that led to necroptosis and exacerbated inflammation in IAV-infected macrophages. Our results highlight an additional mechanism involved in severe influenza that could be attenuated with clinically available therapies.

Clinical and Economic Impact of COVID-19 on Agricultural Workers, Guatemala1.

We evaluated clinical and socioeconomic burdens of respiratory disease in banana farm workers in Guatemala. We offered all eligible workers enrollment during June 15-December 30, 2020, and annually, then tracked them for influenza-like illnesses (ILI) through self-reporting to study nurses, sentinel surveillance at health posts, and absenteeism. Workers who had ILI submitted nasopharyngeal swab specimens for testing for influenza virus, respiratory syncytial virus, and SARS-CoV-2, then completed surveys at days 0, 7, and 28. Through October 10, 2021, a total of 1,833 workers reported 169 ILIs (12.0 cases/100 person-years), and 43 (25.4%) were laboratory-confirmed infections with SARS-CoV-2 (3.1 cases/100 person-years). Workers who had SARS-CoV-2‒positive ILIs reported more frequent anosmia, dysgeusia, difficulty concentrating, and irritability and worse clinical and well-being severity scores than workers who had test result‒negative ILIs. Workers who had positive results also had greater absenteeism and lost income. These results support prioritization of farm workers in Guatemala for COVID-19 vaccination.

Phylogenetic Inference of the 2022 Highly Pathogenic H7N3 Avian Influenza Outbreak in Northern Mexico.

The Mexican lineage H7N3 highly pathogenic avian influenza virus (HPAIV) has persisted in Mexican poultry since its first isolation in 2012. To date, the detection of this virus has gradually expanded from the initial one state to 18 states in Mexico. Despite the HPAIV H7N3 outbreak occurring yearly, the transmission pathways have never been studied, disallowing the establishment of effective control measures. We used a phylogenetic approach to unravel the transmission pathways of 2022 H7N3 HPAIVs in the new outbreak areas in Northern Mexico. We present genetic data of H7N3 viruses produced from 18 poultry farms infected in the spring of 2022. Our results indicate that the virus responsible for the current outbreak in Northern Mexico evolved from the Mexican lineage H7N3 HPAIV discovered in 2012. In the current outbreak, we identified five clusters of infection with four noticeably different genetic backgrounds. It is a cluster IV-like virus that was transmitted into one northern state causing an outbreak, then spreading to another neighboring northern state, possibly via a human-mediated mechanical transmission mechanism. The long-distance transmission event highlights the necessity for the more rigorous enforcement of biosafety measures in outbreaks. Additionally, we examined the evolutionary processes shaping the viral genetic and antigenic diversities. It is imperative to enhance active surveillance to include birds, the environment, and humans to detect HPAI in domestic poultry at an earlier point and eliminate it.

A(H3N2) antigenic variation of influenza is associated with low vaccine efficacy in the early 2018 influenza season in Mexico City.

OBJECTIVES: We evaluated the VE and the mutations of the viruses present in the Mexican population at the beginning of 2018. METHODS: We diagnosed influenza in outpatients with a high-performance Rapid Influenza Diagnostic Test (RIDT) qRT-PCR. Descriptive statistics were used to describe the study population, while the chi-square test was used to determine clinical variables. VE was analyzed through a negative test design. We sequenced the hemagglutinin (HA) gene, performed a phylogenetic analysis, and analyzed the nonsynonymous substitutions both in and outside antigenic sites. RESULTS: Of the 240 patients analyzed, 42.5% received the trivalent vaccine, and 37.5% were positive for influenza. The VE for the general population for any influenza virus type or subtype was 37.0%, while the VE for the predominant influenza A(H3N2) subtype was the lowest (19.7%). The phylogenetic analysis of HA showed the co-circulation of clades and subclades 3C.2a1, 3C.2a1b, 3C.2a2, 3C.2a2re, 3C.2a3, and 3C.3a with identities approximately 97-98% similar to the vaccine composition. CONCLUSION: Low VE was related to the co-circulation of multiple clades and subclades of influenza A(H3N2), with sufficient genetic and phenotypic distance to allow for the infection of vaccinated individuals.

Design of Hydrogel Silk-Based Microarrays and Molecular Beacons for Reagentless Point-of-Care Diagnostics.

We have developed a novel microarray system based on three technologies: 1) molecular beacons designed to interact with DNA targets at room temperature (25-27°C), 2) tridimensional silk-based microarrays containing the molecular beacons immersed in the silk hydrogel, and 3) shallow angle illumination, which uses separated optical pathways for excitation and emission. Unlike conventional microarrays that exhibit reduced signal-to-background ratio, require several stages of incubation, rinsing, and stringency control, and measure only end-point results, our microarray technology provides enhanced signal-to-background ratio (achieved by separating the optical pathways for excitation and emission, resulting in reduced stray light), performs analysis rapidly in one step without the need for labeling DNA targets, and measures the entire course of association kinetics between target DNA and the molecular beacons. To illustrate the benefits of our technology, we conducted microarray assays designed for the identification of influenza viruses. We show that in a single microarray slide, we can identify the virus subtype according to the molecular beacons designed for hemagglutinin (H1, H2, and H3) and neuraminidase (N1, N2). We also show the identification of human and swine influenza using sequence-specific molecular beacons. This microarray technology can be easily implemented for reagentless point-of-care diagnostics of several contagious diseases, including coronavirus variants responsible for the current pandemic.