Gudrun S. Freidl

Influenza at the animal-human interface: A review of the literature for virological evidence of human infection with swine or avian influenza viruses other than A(H5N1)

Factors that trigger human infection with animal influenza virus progressing into a pandemic are poorly understood. Within a project developing an evidence-based risk assessment framework for influenza viruses in animals, we conducted a review of the literature for evidence of human infection with animal influenza viruses by diagnostic methods used. The review covering Medline, Embase, SciSearch and CabAbstracts yielded 6,955 articles, of which we retained 89; for influenza A(H5N1) and A(H7N9), the official case counts of t he World Health Organization were used. An additional 30 studies were included by scanning the reference lists. Here, we present the findings for confirmed infections with virological evidence. We found reports of 1,419 naturally infected human cases, of which 648 were associated with avian influenza virus (AIV) A(H5N1), 375 with other AIV subtypes, and 396 with swine influenza virus (SIV). Human cases naturally infected with AIV spanned haemagglutinin subtypes H5, H6, H7, H9 and H10. SIV cases were associated with endemic SIV of H1 and H3 subtype descending from North American and Eurasian SIV lineages and various reassortants thereof. Direct exposure to birds or swine was the most likely source of infection for the cases with available information on exposure.

Genetic Adaptation of Influenza A Viruses in Domestic Animals and Their Potential Role in Interspecies Transmission: A Literature Review

In December 2011, the European Food Safety Authority awarded a Grant for the implementation of the FLURISK project. The main objective of FLURISK was the development of an epidemiological and virological evidence-based influenza risk assessment framework (IRAF) to assess influenza A virus strains circulating in the animal population according to their potential to cross the species barrier and cause infections in humans. With the purpose of gathering virological data to include in the IRAF, a literature review was conducted and key findings are presented here. Several adaptive traits have been identified in influenza viruses infecting domestic animals and a significance of these adaptations for the emergence of zoonotic influenza, such as shift in receptor preference and mutations in the replication proteins, has been hypothesized. Nonetheless, and despite several decades of research, a comprehensive understanding of the conditions that facilitate interspecies transmission is still lacking. This has been hampered by the intrinsic difficulties of the subject and the complexity of correlating environmental, viral and host factors. Finding the most suitable and feasible way of investigating these factors in laboratory settings represents another challenge. The majority of the studies identified through this review focus on only a subset of species, subtypes and genes, such as influenza in avian species and avian influenza viruses adapting to humans, especially in the context of highly pathogenic avian influenza H5N1. Further research applying a holistic approach and investigating the broader influenza genetic spectrum is urgently needed in the field of genetic adaptation of influenza A viruses.

Serological evidence of influenza A viruses in frugivorous bats from Africa.

Bats are likely natural hosts for a range of zoonotic viruses such as Marburg, Ebola, Rabies, as well as for various Corona- and Paramyxoviruses. In 2009/10, researchers discovered RNA of two novel influenza virus subtypes – H17N10 and H18N11 – in Central and South American fruit bats. The identification of bats as possible additional reservoir for influenza A viruses raises questions about the role of this mammalian taxon in influenza A virus ecology and possible public health relevance. As molecular testing can be limited by a short time window in which the virus is present, serological testing provides information about past infections and virus spread in populations after the virus has been cleared. This study aimed at screening available sera from 100 free-ranging, frugivorous bats (Eidolon helvum) sampled in 2009/10 in Ghana, for the presence of antibodies against the complete panel of influenza A haemagglutinin (HA) types ranging from H1 to H18 by means of a protein microarray platform. This technique enables simultaneous serological testing against multiple recombinant HA-types in 5μl of serum. Preliminary results indicate serological evidence against avian influenza subtype H9 in about 30% of the animals screened, with low-level cross-reactivity to phylogenetically closely related subtypes H8 and H12. To our knowledge, this is the first report of serological evidence of influenza A viruses other than H17 and H18 in bats. As avian influenza subtype H9 is associated with human infections, the implications of our findings from a public health context remain to be investigated.

Livestock-associated risk factors for pneumonia in an area of intensive animal farming in the Netherlands.

Previous research conducted in 2009 found a significant positive association between pneumonia in humans and living close to goat and poultry farms. However, as this result might have been affected by a large goat-related Q fever epidemic, the aim of the current study was to re-evaluate this association, now that the Q-fever epidemic had ended. In 2014/15, 2,494 adults (aged 20–72 years) living in a livestock-dense area in the Netherlands participated in a medical examination and completed a questionnaire on respiratory health, lifestyle and other items. We retrieved additional information for 2,426/2,494 (97%) participants from electronic medical records (EMR) from general practitioners. The outcome was self-reported, physician-diagnosed pneumonia or pneumonia recorded in the EMR in the previous three years. Livestock license data was used to determine exposure to livestock. We quantified associations between livestock exposures and pneumonia using odds ratios adjusted for participant characteristics and comorbidities (aOR). The three-year cumulative frequency of pneumonia was 186/2,426 (7.7%). Residents within 2,000m of a farm with at least 50 goats had an increased risk of pneumonia, which increased the closer they lived to the farm (2,000m aOR 1.9, 95% CI 1.4–2.6; 500m aOR 4.4, 95% CI 2.0–9.8). We found no significant associations between exposure to other farm animals and pneumonia. However, when conducting sensitivity analyses using pneumonia outcome based on EMR only, we found a weak but statistically significant association with presence of a poultry farm within 1,000m (aOR: 1.7, 95% CI 1.1–2.7). Living close to goat and poultry farms still constitute risk factors for pneumonia. Individuals with pneumonia were not more often seropositive for Coxiella burnetii, indicating that results are not explained by Q fever. We strongly recommend identification of pneumonia causes by the use of molecular diagnostics and investigating the role of non-infectious agents such as particulate matter or endotoxins.

Getting more out of less--a quantitative serological screening tool for simultaneous detection of multiple influenza A hemagglutinin-types in chickens.

Current avian influenza surveillance in poultry primarily targets subtypes of interest for the veterinary sector (H5, H7). However, as virological and serological evidence suggest, surveillance of additional subtypes is important for public health as well as for the poultry industry. Therefore, we developed a protein microarray enabling simultaneous identification of antibodies directed against different HA-types of influenza A viruses in chickens. The assay successfully discriminated negative from experimentally and naturally infected, seropositive chickens. Sensitivity and specificity depended on the cut-off level used but ranged from 84.4% to 100% and 100%, respectively, for a cut off level of ≥1∶40, showing minimal cross reactivity. As this testing platform is also validated for the use in humans, it constitutes a surveillance tool that can be applied in human-animal interface studies.

Changes in heterosubtypic antibody responses during the first year of the 2009 A(H1N1) influenza pandemic

Seropositivity to avian influenza (AI) via low-level antibody titers has been reported in the general population and poultry-exposed individuals, raising the question whether these findings reflect true infection with AI or cross-reactivity. Here we investigated serological profiles against human and avian influenza viruses in the general population using a protein microarray platform. We hypothesized that higher antibody diversity across recent H1 and H3 influenza viruses would be associated with heterosubtypic reactivity to older pandemic- and AI viruses. We found significant heterogeneity in antibody profiles. Increased antibody diversity to seasonal influenza viruses was associated with low-level heterosubtypic antibodies to H9 and H7, but not to H5 AI virus. Individuals exposed to the recent 2009 A(H1N1) pandemic showed higher heterosubtypic reactivity. We show that there is a complex interplay between prior exposures to seasonal and recent pandemic influenza viruses and the development of heterosubtypic antibody reactivity to animal influenza viruses.

Weighing serological evidence of human exposure to animal influenza viruses − A literature review

Assessing influenza A virus strains circulating in animals and their potential to cross the species barrier and cause human infections is important to improve human influenza surveillance and preparedness. We reviewed studies describing serological evidence of human exposure to animal influenza viruses. Comparing serological data is difficult due to a lack of standardisation in study designs and in laboratory methods used in published reports. Therefore, we designed a scoring system to assess and weigh specificity of obtained serology results in the selected articles. Many studies report reliable evidence of antibodies to swine influenza viruses among persons occupationally exposed to pigs. Most avian influenza studies target H5, H7 and H9 subtypes and most serological evidence of human exposure to avian influenza viruses is reported for these subtypes. Avian influenza studies receiving a low grade in this review often reported higher seroprevalences in humans compared with studies with a high grade. Official surveillance systems mainly focus on avian H5 and H7 viruses. Swine influenza viruses and avian subtypes other than H5 and H7 (emphasising H9) should be additionally included in official surveillance systems. Surveillance efforts should also be directed towards understudied geographical areas, such as Africa and South America.

Epidemiological Risk Factors for Animal Influenza A Viruses Overcoming Species Barriers

AbstractDrivers and risk factors for Influenza A virus transmission across species barriers are poorly understood, despite the ever present threat to human and animal health potentially on a pandemic scale. Here we review the published evidence for epidemiological risk factors associated with influenza viruses transmitting between animal species and from animals to humans. A total of 39 papers were found with evidence of epidemiological risk factors for influenza virus transmission from animals to humans; 18 of which had some statistical measure associated with the transmission of a virus. Circumstantial or observational evidence of risk factors for transmission between animal species was found in 21 papers, including proximity to infected animals, ingestion of infected material and potential association with a species known to carry influenza virus. Only three publications were found which presented a statistical measure of an epidemiological risk factor for the transmission of influenza between animal species. This review has identified a significant gap in knowledge regarding epidemiological risk factors for the transmission of influenza viruses between animal species.

Pneumonia risk of people living close to goat and poultry farms - Taking GPS derived mobility patterns into account.

We previously observed an increased incidence of pneumonia in persons living near goat and poultry farms, using animal presence around the home to define exposure. However, it is unclear to what extent individual mobility and time spent outdoors close to home contributes to this increased risk. Therefore, the aim of the current study was to investigate the role of mobility patterns and time spent outdoors in the vicinity of goat or poultry farms in relation to pneumonia risk. In a rural Dutch cohort, 941 members logged their mobility using GPS trackers for 7 days. Pneumonia was diagnosed in 83 subjects (participants reported that pneumonia had been diagnosed by a medical doctor, or recorded in EMR from general practitioners, 2011-2014). We used logistic regression to evaluate pneumonia-risk by presence of goat farms within 500 and 1000 m around the home and around GPS-tracks (only non-motorised mobility), also we evaluated whether more time spent outdoors increased pneumonia-risks. We observed a clearly increased risk of pneumonia among people living in close proximity to goat farms, ORs increased with closer distances of homes to farms (500 m: 6.2 (95% CI 2.2-16.5) 1000 m: 2.5 (1.4-4.3)) The risk increased for individuals who spent more time outdoors close to home, but only if homes were close to goat farms (within 500 m and often outdoors: 12.7 (3.6-45.4) less often: 2.0 (0.3-9.2), no goat farms and often outdoors: 1.0 (0.6-1.6)). For poultry we found no increased risks. Pneumonia-risks increased when people lived near goat farms, especially when they spent more time outdoors, mobility does not seem to add to these risks.

Exploring novel sero-epidemiological tools—Effect of different storage conditions on longitudinal stability of microarray slides comprising influenza A-, measles- and Streptococcus pneumoniae antigens

In this study we evaluated the long-term stability of a microarray-based serological screening platform, containing antigens of influenza A, measles and Streptococcus pneumoniae, as part of a preparedness research program aiming to develop assays for syndromic disease detection. Spotted microarray slides were kept at four different storage regimes with varying temperature and humidity conditions. We showed that under the standard storage condition in a temperature-controlled (21°C) and desiccated environment (0% relative humidity), microarray slides remained stable for at least 22 months without loss of antigen quality, whereas the other three conditions (37°C, desiccated; Room temperature, non-desiccated; Frozen, desiccated) produced acceptable results for some antigens (influenza A, S.pneumoniae), but not for others (measles). We conclude that these arrays for multiplex antibody testing can be prepared and stored for prolonged periods of time, which aids laboratory-preparedness and facilitates sero-epidemiological studies.