Breaking the unvirtuous cycle: barriers and opportunities for research and development in Paraguay. A case study.

Introduction: Medical research and development (R&D) is an undoubtedly relevant activity to drive innovation, improve healthcare policies and bring patients treatment opportunities for common and rare diseases. Equity and inclusion are matters of concern in research. High-income countries' research teams are more likely to have more impactful publications, grant funding, and clinical trials than middle or low-income countries. Low budget allocations to R&D and existing gaps in regulatory frameworks are some obstacles to growth. This unvirtuous cycle results in scarce advances in common endemic diseases and the underrepresentation of specific populations in innovative therapeutics research. Materials and methods: We conducted a policy review and qualitative research to determine the principal characteristics of basic and clinical medical research in Paraguay, as well as barriers and facilitators to improve innovative R&D strategies in this country. To this aim, we examined published articles from 2005 to 2020, the organizational structure of national research agencies, the current regulation framework, and the composition and experience of local research groups and ethical review boards (ERBs). In addition, we performed semi-structured interviews to evaluate perceptions and expectations from different stakeholders, including investigators, ERBs members, sponsor associates, and Regulatory Agency executive staff. Results: In 2018, Paraguay ranked 10th out of 12 South American countries in total number of publications and cumulative h-index score. Total Gross Domestic Product (GDP) allocation for R&D was 0.15%, ranking eighth out of 12 in the region. In 2021, the number of trials registered on ClinicalTrials.gov was 52, with only 16 ongoing recruiting studies at that time.Some of the main barriers identified included low incentives for academic careers and lack of experience in pharmaceutical research. An emergent necessity to develop a straight- forward normative framework was detected. Main facilitators included the development of two research initiative programs (PRONII and PROCIENCIA) from CONACYT (National Council of Science and Technology) which were associated with higher budget allocation and total number of publications in the 2011 to 2017 period. A total of six stakeholders participated in the semi-structured surveys. Interviewees highlighted the necessity of a centralized policy to promote R&D, which incorporates investigators and ERBs training, the development of standardized procedures, and the dissemination of research activities. Sponsor associates underlined that real-world evidence may represent a distinctive opportunity to enhance local research. Conclusion: Coordinated efforts are needed to break the unvirtuous cycle. There is an increasing interest in enhancing health research in Paraguay, materialized in the creation of specific programs that encourage the collaborative work of healthcare providers, basic scientists, and private investors. Nonetheless, a comprehensive approach is needed also to strengthen regulatory agencies and attract external sponsorship. While modern and currently popular topics, including artificial intelligence, real-world data, and translational research may represent key opportunities to seek investment, special policies should be adopted to prioritize research on the determinants of health in the Paraguayan population.

Seroconversion of a Swine Herd in a Free-Range Rural Multi-Species Farm against HPAI H5N1 2.3.4.4b Clade Virus.

Starting from October 2021, several outbreaks of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 were reported in wild and domestic birds in Italy. Following the detection of an HPAIV in a free-ranging poultry farm in Ostia, province of Rome, despite the lack of clinical signs, additional virological and serological analyses were conducted on samples collected from free-ranging pigs, reared in the same holding, due to their direct contact with the infected poultry. While the swine nasal swabs were all RT-PCR negative for the influenza type A matrix (M) gene, the majority (%) of the tested pigs resulted serologically positive for the hemagglutination inhibition test and microneutralization assay, using an H5N1 strain considered to be homologous to the virus detected in the farm. These results provide further evidence of the worrisome replicative fitness that HPAI H5Nx viruses of the 2.3.4.4b clade have in mammalian species. Moreover, our report calls for additional active surveillance, to promptly intercept occasional spillover transmissions to domestic mammals in close contact with HPAI affected birds. Strengthened biosecurity measures and efficient separation should be prioritized in mixed-species farms in areas at risk of HPAI introduction.

The Evolution of Highly Pathogenic Avian Influenza A (H5) in Poultry in Nigeria, 2021-2022.

In 2021, amidst the COVID-19 pandemic and global food insecurity, the Nigerian poultry sector was exposed to the highly pathogenic avian influenza (HPAI) virus and its economic challenges. Between 2021 and 2022, HPAI caused 467 outbreaks reported in 31 of the 37 administrative regions in Nigeria. In this study, we characterized the genomes of 97 influenza A viruses of the subtypes H5N1, H5N2, and H5N8, which were identified in different agro-ecological zones and farms during the 2021-2022 epidemic. The phylogenetic analysis of the HA genes showed a widespread distribution of the H5Nx clade 2.3.4.4b and similarity with the HPAI H5Nx viruses that have been detected in Europe since late 2020. The topology of the phylogenetic trees indicated the occurrence of several independent introductions of the virus into the country, followed by a regional evolution of the virus that was most probably linked to its persistent circulation in West African territories. Additional evidence of the evolutionary potential of the HPAI viruses circulating in this region is the identification in this study of a putative H5N1/H9N2 reassortant virus in a mixed-species commercial poultry farm. Our data confirm Nigeria as a crucial hotspot for HPAI virus introduction from the Eurasian territories and reveal a dynamic pattern of avian influenza virus evolution within the Nigerian poultry population.

Protective Efficacy of H9N2 Avian Influenza Vaccines Inactivated by Ionizing Radiation Methods Administered by the Parenteral or Mucosal Routes.

H9N2 viruses have become, over the last 20 years, one of the most diffused poultry pathogens and have reached a level of endemicity in several countries. Attempts to control the spread and reduce the circulation of H9N2 have relied mainly on vaccination in endemic countries. However, the high level of adaptation to poultry, testified by low minimum infectious doses, replication to high titers, and high transmissibility, has severely hampered the results of vaccination campaigns. Commercially available vaccines have demonstrated high efficacy in protecting against clinical disease, but variable results have also been observed in reducing the level of replication and viral shedding in domestic poultry species. Antigenic drift and increased chances of zoonotic infections are the results of incomplete protection offered by the currently available vaccines, of which the vast majority are based on formalin-inactivated whole virus antigens. In our work, we evaluated experimental vaccines based on an H9N2 virus, inactivated by irradiation treatment, in reducing viral shedding upon different challenge doses and compared their efficacy with formalin-inactivated vaccines. Moreover, we evaluated mucosal delivery of inactivated antigens as an alternative route to subcutaneous and intramuscular vaccination. The results showed complete protection and prevention of replication in subcutaneously vaccinated Specific Pathogen Free White Leghorn chickens at low-to-intermediate challenge doses but a limited reduction of shedding at a high challenge dose. Mucosally vaccinated chickens showed a more variable response to experimental infection at all tested challenge doses and the main effect of vaccination attained the reduction of infected birds in the early phase of infection. Concerning mucosal vaccination, the irradiated vaccine was the only one affording complete protection from infection at the lowest challenge dose. Vaccine formulations based on H9N2 inactivated by irradiation demonstrated a potential for better performances than vaccines based on the formalin-inactivated antigen in terms of reduction of shedding and prevention of infection.

Replication of Influenza D Viruses of Bovine and Swine Origin in Ovine Respiratory Explants and Their Attachment to the Respiratory Tract of Bovine, Sheep, Goat, Horse, and Swine.

Bovine is considered the main reservoir of influenza D virus (IDV), however, low levels of seropositivity in other farmed species suggest a wide range of potential hosts. Nevertheless, it is not clear whether this scenario is the result of rare spillover events upon contact with bovines, or a lack of adaptation of IDV to these hosts. Among these species, sheep represents a crucial component of the rural economy in many developing countries, but little is known about its role in the ecology of the disease. To evaluate the susceptibility of sheep to IDV viruses of different origin, we used ovine respiratory tissues as an ex vivo model and investigated the infective phenotype of two IDV strains isolated from either bovine (IDV-BOV) or swine (IDV-SW). For translatability purposes, we included a parainfluenza type 3 virus, as positive control, given its known respiratory tropism in sheep. We performed a timed evaluation of the viral infectivity, cell tropism and the associated histopathology, by means of tissue culture infectious dose assays on supernatants and histological/immunohistochemical analyses on explanted tissues, respectively. To further investigate differences in the phenotype of these two strains and to identify the potential targets of replication in the most commonly land-based farmed mammalian species, we carried out virus binding assays on histological sections of the respiratory tract of bovine, caprine, ovine, horse and swine. Our results demonstrated that IDV successfully replicates in nasal, tracheal and lung ovine tissues, suggesting a moderate susceptibility of this species to IDV infection. Interestingly, despite the high genetic identity of these strains, IDV- BOV consistently replicated to higher titers than IDV-SW in all respiratory tracts, suggesting IDV viruses might display considerable levels of variability in their phenotype when crossing the species barrier. Virus binding assays confirmed a superior affinity of the IDV viruses for the bovine upper respiratory tract, and a preference for the pharyngeal epithelium of small ruminants, indicating possible targets to improve the sensitivity of virological sampling for diagnostic and post-mortem purposes. Further pathogenesis and cross-species transmission studies will be necessary to elucidate the ecology of IDV and eventually allow the design of cost-effective surveillance strategies.

A G1-lineage H9N2 virus with oviduct tropism causes chronic pathological changes in the infundibulum and a long-lasting drop in egg production.

Since 1997, G1-lineage H9N2 avian influenza viruses have been circulating in Asia and later on in the Middle East, and they have been associated to mild respiratory disease, drops in egg production and moderate mortality in chickens, in particular in the presence of concurrent infections. In this study, we investigated the importance of the G1-lineage H9N2 A/chicken/Israel/1163/2011 virus as a primary pathogen in layers, analyzing its tropism and binding affinity for the oviduct tissues, and investigating the long-term impact on egg production. Besides causing a mild respiratory infection, the virus replicated in the oviduct of 60% of the hens causing different degrees of salpingitis throughout the organ, in particular at the level of the infundibulum, where the detection of the virus was associated with severe heterophilic infiltrate, and necrosis of the epithelium. Binding affinity assays confirmed that the infundibulum was the most receptive region of the oviduct. The drop in egg production was at its peek at 2 weeks post-infection (pi) (60% decrease) and continued up to 80 days pi (35% decrease). On day 80 pi, non-laying birds showed egg yolk peritonitis, and histopathological analyses described profound alteration of the infundibulum architecture, duct ectasia and thinning of the epithelium, while the rest of the oviduct and ovary appeared normal. Our results show that this H9N2 virus is a primary pathogen in layer hens, and that its replication in the infundibulum is responsible for acute and chronic lesions that limits the effective functionality of the oviduct, compromising the commercial life of birds.

Spillback transmission of European H1N1 avian-like swine influenza viruses to turkeys: A strain-dependent possibility?

In 1979, an avian influenza virus of the H1N1 subtype began to circulate in European swine herds, rapidly replacing classical swine H1N1 viruses. Spill-back transmissions to turkeys were recorded occasionally, but they might have been underreported due to the asymptomatic nature of the infection and the lack of specific surveillance. In our study, we evaluated the infectivity and transmissibility in turkeys of seven strains of H1N1 avian-like swine viruses isolated from 1979 to 2006, and compared them with their closest progenitor A/duck/Bavaria/1/77 (H1N1), to establish whether the adaptation to pigs has gradually decreased their fitness in turkeys. Our data indicate that the circulation of European H1N1 in pigs might have impaired the possibility of infecting turkeys. Nevertheless, the two swine-origin strains, which showed the ability to replicate and transmit in turkeys, possess typical swine-like genetic traits, not different from the rest of the tested isolates, suggesting replication of avian-like swine H1N1 viruses in turkeys as a strain-dependent polygenic feature.

Lethal nephrotropism of an H10N1 avian influenza virus stands out as an atypical pathotype.

A nephrotropic H10N1 avian influenza virus (AIV) with an intravenous pathogenicity index (IVPI) of 1.9 and a haemagglutinin monobasic amino acid cleavage site motif, was genetically and phenotypically characterized. Specific pathogen free chickens of 3 or 6 weeks of age were challenged with a 10(6)EID50/0.1mL dose by either oro-nasal or intravenous route, to study the distribution, tissue tropism and virulence of the virus. Direct transmission was tested by introducing sentinel birds on day 4 post infection. Virus shedding and viremia were investigated by means of type A influenza real-time RT-PCR. Dead birds were necropsied and selected organs were collected for histology, immunohistochemistry, and to detect and re-isolate the virus. Serological analyses were carried out to evaluate seroconversion, three weeks from challenge. The oro-nasal challenge of the 6-week-old birds elicited 47% mortality as a result of viremia and massive replication of the virus in the kidneys. Unexpectedly, among birds of 3 weeks of age the same challenge caused 5% mortality and few clinical signs. Surprisingly the intravenous administration of the virus in the 3-week-old birds recorded an IVPI of 2.4. A full genome characterization of the virus could not identify any molecular determinant underlying the observed phenotype. Our findings describe the complex pathobiology of an AIV of the H10 subtype that stands out for its peculiar pathogenicity and tissue tropism in chickens.

Differences in the detection of highly pathogenic avian influenza H5N1 virus in feather samples from 4-week-old and 24-week-old infected Pekin ducks (Anas platyrhynchos var. domestica).

Previous studies have reported the detection of H5N1 HPAI virus in feathers from ducks naturally and experimentally infected and suggested that feather calami (FC) could be used as diagnostic samples for the early detection of H5N1 HPAI infections. Ducks are readily infected with H5N1 HPAI viruses although the development of clinical signs and deaths were reported as age-related with younger birds being more susceptible. The correlation between age and virus localisation in FC of infected ducks has not been studied to date. In the present study juvenile (4-week-old) and adult (24-week-old) Pekin ducks (Anas platyrhynchos var. domestica) were infected experimentally with a clade 2.2 H5N1 HPAI virus (A/duck/Nigeria/1071-23/2007). Tracheal (Tr) and cloacal (Cl) swabs and FC were collected at 3, 5, 7 and 10 days post infection and tested by RRT-PCR and a double antibody sandwich-ELISA (DAS-ELISA) developed in house. Virus was detected in swabs and FC of challenged ducks with a higher rate of detection in juvenile ducks. In this age group virus was detected over a longer period of time in FC compared to swabs. Our study showed that FC samples collected from young ducks are a valid diagnostic specimen for H5N1 HPAI virus detection. The DAS-ELISA on FC proved to be a suitable alternative diagnostic test when molecular and/or virus isolation techniques are not available therefore it could be useful in the diagnosis of H5N1 HPAI infections in under-resourced countries.

Antigenic characterization of recent H5N1 highly pathogenic avian influenza viruses circulating in Egyptian poultry.

The extensive circulation of Highly Pathogenic (HP) H5N1 Avian Influenza in Egypt in poultry since 2006 resulted in the emergence of distinct clades with the recent identification of a further clade: 2.2.1.1. The aim of this study was to characterize for the first time the antigenic profile of an extensive collection of genetically diverse Egyptian H5N1 HP viruses isolated between 2007 and 2010 applying antigenic cartography and principal component analysis to serological data. We identified that Egyptian H5N1 viruses have undergone significant antigenic diversification between 2007 and 2010 and two distinct antigenic clusters co-circulated in 2010. Such clusters correlated with 2.2.1 and 2.2.1.1 clades, showing for the first time that the new emerging clade 2.2.1.1 is antigenically distinct. This study highlights that the antigenic diversity of H5N1 HP Egyptian viruses may represent a potential challenge for the development of an effective vaccination programme for animal and human health in Egypt.

Improving heat stability of haemagglutinating antigens for avian influenza.

The increasing demand for avian influenza diagnostic reagents worldwide, has included requests for significant supplies of product to developing countries. Difficulties in dispatching to remote areas and tropical countries are a major concern to suppliers, international organisations and donors as delays in forwarding parcels often result in storage at non-optimal or inadequate temperatures results in loss in titre and thus wastage of resources. In this study we demonstrate that the heat stability of avian influenza haemagglutination inhibition antigens of the H5, H7 and H9 subtype following 14 days of exposure to 37°C and 45°C is significantly increased by adding D-(+)-Trehalose to the freshly prepared antigen. Increased stability was detected both for freeze-dried antigens over an extended period of 6 months and also in heat exposed antigens that were then stored at +4C for up to 35 days post reconstitution.

Appearance of serum antibodies against the avian influenza nonstructural 1 protein in experimentally infected chickens and turkeys.

In order to support eradication efforts of avian influenza (AI) infections in poultry, the implementation of "differentiation of infected from vaccinated animals" (DIVA) vaccination strategies has been recommended by international organizations. These systems enable the detection of field exposure in vaccinated flocks, and through this detection, infected flocks may be properly managed, thus interrupting the perpetuation of the infectious cycle. A promising system, based on the detection of antibodies to the nonstructural 1 (NS1) protein of AI, has been deemed a good candidate. However, there are presently no data available, in support of this DIVA system, with regard to the kinetics of antibody production against the NS1 proteins in poultry following infection. The present investigation was undertaken to establish the dynamics of the appearance of anti-NS1 antibodies in a naïve population. Following experimental infection of turkeys, antibodies to a peptide spanning the c-terminal of the NS1 protein were detected by enzyme-linked immunosorbent assay (ELISA) starting between day 3 and day 5 postinfection. In contrast, no antibodies to the NS1 peptide could be detected in chickens over the test period. In addition, the turkeys and chickens reacted differently at a clinical level to the infection by the H9N2 challenge virus. Taken together, these findings indicate that there is a significant difference in the viral replication in turkeys and chickens, resulting in a variation in the production of antibodies to NS1, as detected by the peptide-based ELISA used. This fact must be taken into consideration when using a DIVA system based on the identification of antibodies to the NS1 protein.

Effect of different temperatures on the stability of avian influenza reference reagents.

The production and supply of reference reagents for the diagnosis of avian influenza (AI) is one of the duties of the World Organization for Animal Health reference laboratories. The lyophilized reagents are routinely shipped on dry ice to both national and international clients. In order to determine the effect of different short-term storage temperatures on the activity of AI reference reagents, vials containing lyophilized avian influenza A antigens and antisera preparations were maintained at 4 C, 22 C, and 37 C over a 21-day period. At days 0, 3, 7, 14, and 21 the reagents were titrated using the hemagglutination test, the hemagglutination inhibition test, or the agar gel immunodiffusion test (AGID). All of the AI antigens that were kept at 4 C and 22 C retained hemagglutinating activity for at least 21 days, but when they were stored at 37 C several lost their hemagglutinating activity. All of the reference antisera tested were still able to inhibit hemagglutination after 21 days, and the antigen used in AGID also gave clear results after 21 days even after incubation at 37 C. Our results therefore indicate that lyophilized avian influenza antigens maintain their hemagglutinating activity at temperatures between 4 C and 22 C for at least 21 days, and both antisera and antigens prepared for AGID remain stable for 21 days between 4 C and 37 C. This information will allow for alternative shipping temperatures than those presently recommended, in addition to the short-term storage of these reagents at nonrefrigerated temperatures.