Persistence of low pathogenic avian influenza virus in artificial streams mimicking natural conditions of waterfowl habitats in the Mediterranean climate.

Avian influenza viruses (AIVs) can affect wildlife, poultry, and humans, so a One Health perspective is needed to optimize mitigation strategies. Migratory waterfowl globally spread AIVs over long distances. Therefore, the study of AIV persistence in waterfowl staging and breeding areas is key to understanding their transmission dynamics and optimizing management strategies. Here, we used artificial streams mimicking natural conditions of waterfowl habitats in the Mediterranean climate (day/night cycles of photosynthetic active radiation and temperature, low water velocity, and similar microbiome to lowland rivers and stagnant water bodies) and then manipulated temperature and sediment presence (i.e., 10-13 °C vs. 16-18 °C, and presence vs. absence of sediments). An H1N1 low pathogenic AIV (LPAIV) strain was spiked in the streams, and water and sediment samples were collected at different time points until 14 days post-spike to quantify viral RNA and detect infectious particles. Viral RNA was detected until the end of the experiment in both water and sediment samples. In water samples, we observed a significant combined effect of temperature and sediments in viral decay, with higher viral genome loads in colder streams without sediments. In sediment samples, we didn't observe any significant effect of temperature. In contrast to prior laboratory-controlled studies that detect longer persistence times, infectious H1N1 LPAIV was isolated in water samples till 2 days post-spike, and none beyond. Infectious H1N1 LPAIV wasn't isolated from any sediment sample. Our results suggest that slow flowing freshwater surface waters may provide conditions facilitating bird-to-bird transmission for a short period when water temperature are between 10 and 18 °C, though persistence for extended periods (e.g., weeks or months) may be less likely. We hypothesize that experiments simulating real environments, like the one described here, provide a more realistic approach for assessing environmental persistence of AIVs.

Infravec2 guidelines for the design and operation of containment level 2 and 3 insectaries in Europe.

With the current expansion of vector-based research and an increasing number of facilities rearing arthropod vectors and infecting them with pathogens, common measures for containment of arthropods as well as manipulation of pathogens are becoming essential for the design and running of such research facilities to ensure safe work and reproducibility, without compromising experimental feasibility. These guidelines and comments were written by experts of the Infravec2 consortium, a Horizon 2020-funded consortium integrating the most sophisticated European infrastructures for research on arthropod vectors of human and animal diseases. They reflect current good practice across European laboratories with experience of safely handling different mosquito species and the pathogens they transmit. As such, they provide experience-based advice to assess and manage the risks to work safely with mosquitoes and the pathogens they transmit. This document can also form the basis for research with other arthropods, for example, midges, ticks or sandflies, with some modification to reflect specific requirements.

Ecological factors driving avian influenza virus dynamics in Spanish wetland ecosystems.

Studies exploring the ecological interactions between avian influenza viruses (AIV), natural hosts and the environment are scarce. Most work has focused on viral survival and transmission under laboratory conditions and through mathematical modelling. However, more integrated studies performed under field conditions are required to validate these results. In this study, we combined information on bird community, environmental factors and viral epidemiology to assess the contribution of biotic and abiotic factors in the occurrence of low pathogenic AIV in Spanish wetlands. For that purpose, seven locations in five different wetlands were studied during two years (2007-2009), including seven sampling visits by location. In each survey, fresh faeces (n = 4578) of wild birds and water samples were collected for viral detection. Also, the vegetation structure, water physical properties of wetlands, climatic conditions and wild bird community composition were determined. An overall AIV prevalence of 1.7%±0.4 was detected in faecal samples with important fluctuations among seasons and locations. Twenty-six AIV were isolated from the 78 RRT-PCR positive samples and eight different haemagglutinines and five neuraminidases were identified, being the combination H3N8 the most frequent. Variation partitioning procedures identified the combination of space and time variables as the most important pure factor - independently to other factors - explaining the variation in AIV prevalence (36.8%), followed by meteorological factor (21.5%) and wild bird community composition/vegetation structure (21.1%). These results contribute to the understanding of AIV ecological drivers in Spanish ecosystems and provide useful guidelines for AIV risk assessment identifying potential hotspots of AIV activity.

Highly (H5N1) and low (H7N2) pathogenic avian influenza virus infection in falcons via nasochoanal route and ingestion of experimentally infected prey.

An experimental infection with highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) viruses was carried out on falcons in order to examine the effects of these viruses in terms of pathogenesis, viral distribution in tissues and viral shedding. The distribution pattern of influenza virus receptors was also assessed. Captive-reared gyr-saker (Falco rusticolus x Falco cherrug) hybrid falcons were challenged with a HPAI H5N1 virus (A/Great crested grebe/Basque Country/06.03249/2006) or a LPAI H7N2 virus (A/Anas plathyrhynchos/Spain/1877/2009), both via the nasochoanal route and by ingestion of previously infected specific pathogen free chicks. Infected falcons exhibited similar infection dynamics despite the different routes of exposure, demonstrating the effectiveness of in vivo feeding route. H5N1 infected falcons died, or were euthanized, between 5-7 days post-infection (dpi) after showing acute severe neurological signs. Presence of viral antigen in several tissues was confirmed by immunohistochemistry and real time RT-PCR (RRT-PCR), which were generally associated with significant microscopical lesions, mostly in the brain. Neither clinical signs, nor histopathological findings were observed in any of the H7N2 LPAI infected falcons, although all of them had seroconverted by 11 dpi. Avian receptors were strongly present in the upper respiratory tract of the falcons, in accordance with the consistent oral viral shedding detected by RRT-PCR in both H5N1 HPAI and H7N2 LPAI infected falcons. The present study demonstrates that gyr-saker hybrid falcons are highly susceptible to H5N1 HPAI virus infection, as previously observed, and that they may play a major role in the spreading of both HPAI and LPAI viruses. For the first time in raptors, natural infection by feeding on infected prey was successfully reproduced. The use of avian prey species in falconry husbandry and wildlife rehabilitation facilities could put valuable birds of prey and humans at risk and, therefore, this practice should be closely monitored.

Pathogenesis and transmissibility of highly (H7N1) and low (H7N9) pathogenic avian influenza virus infection in red-legged partridge (Alectoris rufa).

An experimental infection with highly pathogenic avian influenza virus (HPAIV) and low pathogenic avian influenza virus (LPAIV) was carried out in red-legged partridges (Alectoris rufa) in order to study clinical signs, gross and microscopic lesions, and viral distribution in tissues and viral shedding. Birds were infected with a HPAIV subtype H7N1 (A/Chicken/Italy/5093/1999) and a LPAIV subtype H7N9 (A/Anas crecca/Spain/1460/2008). Uninoculated birds were included as contacts in both groups. In HPAIV infected birds, the first clinical signs were observed at 3 dpi, and mortality started at 4 dpi, reaching 100% at 8 dpi. The presence of viral antigen in tissues and viral shedding were confirmed by immunohistochemistry and quantitative real time RT-PCR (qRRT-PCR), respectively, in all birds infected with HPAIV. However, neither clinical signs nor histopathological findings were observed in LPAIV infected partridges. In addition, only short-term viral shedding together with seroconversion was detected in some LPAIV inoculated animals. The present study demonstrates that the red-legged partridge is highly susceptible to the H7N1 HPAIV strain, causing severe disease, mortality and abundant viral shedding and thus contributing to the spread of a potential local outbreak of this virus. In contrast, our results concerning H7N9 LPAIV suggest that the red-legged partridge is not a reservoir species for this virus.

Detection of infectious rotaviruses by flow cytometry.

Human rotaviruses are considered the main cause of viral gastroenteritis in infants and young children throughout the world. Their transmission is through the fecal-oral route, mostly after ingestion of contaminated water and food. Since an extremely high number of virus particles are present in the feces during the acute gastroenteritis, methods based on electron microscopy, passive particle agglutination tests, or enzyme-linked immunosorbent assays are readily employed for clinical diagnosis. However, the sensitivity of these procedures is not high enough to detect the low number of viral particles sometimes present in the environment. In the case of environmental samples, amplification of viral nucleic acids by polymerase chain reaction assays coupled to reverse transcription (RT-PCR) has been increasingly applied to detect rotaviruses in water and shellfish samples. However, procedures based on molecular approaches have to face the drawback that they do not differentiate between infectious and noninfectious particles, which is of major relevance from the public health point of view. Virus propagation in cell culture prior to detection by immunological or molecular procedures accomplishes the dual purpose of increasing the amount of target material and incorporating an infectivity assay as well.Wild-type rotaviruses present difficulties in their in vitro replication, although some of them may be adapted to grow in several cell lines such as the monkey kidney cell line MA104 or the human intestinal cell line CaCo-2. More than a decade ago, an assay for the specific detection of infectious rotaviruses in environmental samples, involving an indirect immunofluorescence test (IIF) and optical microscopy (OM) counting of infected foci in infected MA-104 cell monolayers, was described. On the other hand, CaCo-2 cells have been successfully employed in our laboratory for infectivity assays of several fastidious enteric virus strains present in water samples.