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Background: Since 2005, highly pathogenic avian influenza A H5N1 viruses (Goose/Guangdong/1/96-H5 lineage) have spread from Asia to Africa and Europe, infecting poultry, humans and wild birds. Subsequently, avian influenza surveillance has increased worldwide. Mongolia is a good location to study influenza viruses in wild birds within Asia because the country has very low densities of domestic poultry and supports large concentrations of migratory water birds. We conducted avian influenza surveillance in Mongolia over two time periods from 2009 to 2018, utilizing environmental fecal sampling.Methods: Fresh fecal samples were collected in areas where wild water birds, including orders Anseriformes and Charadriiformes, congregated. Hemagglutinin (HA) and neuraminidase (NA) subtypes of positive samples were identified through viral isolation or molecular assays, with pathogenicity determined by HA subtype or sequencing the HA cleavage site.Results: A total of 10,222 samples were collected and tested for avian influenza virus. Of these, 7,025 fecal samples were collected from 2009-2013, and 3,197 fecal samples were collected from 2016-2018 period. Testing revealed 175 (1.7%) positive samples for low pathogenicity influenza A, including 118 samples from 2009-2013 (1.7%) and 57 samples from 2016-2018 (1.8%). Over the period and locations of surveillance no influenza A viruses were detected in association with a wild bird mortality event. HA and NA subtyping of all positives identified eleven subtypes of HA (H1-H8; H10-H12) and nine subtypes of NA (N1-N9) in 29 different combinations. Within periods, viruses were detected more frequently during the fall season (August to October, 2.5%; 95% CI 2.1-2.9%; 146/5,750) than the early summer (April to July, 0.6%; 95% CI 0.4-0.9%; 29/4,472).Conclusion: Mongolia is an important location for wild birds and is positioned as a crossroad of multiple migratory bird flyways. Breeding birds from Mongolia and further north pass through the country to wintering areas as widespread as southern Africa, the Indian Subcontinent, Southeast Asia and Australasia. Our work demonstrates the feasibility of using an affordable environmental fecal sampling approach for avian influenza surveillance and contributes to understanding the prevalence and ecology of low pathogenicity avian influenza viruses in this important location, where birds from multiple flyways mix.

Background Human spillovers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to dogs and the emergence of a highly contagious avian-origin H3N2 canine influenza virus have raised concerns on the role of dogs in the spread of SARS-CoV-2 and their susceptibility to existing human and avian influenza viruses, which might result in further reassortment. Methods We systematically studied the replication kinetics of SARS-CoV-2, SARS-CoV, influenza A viruses of H1, H3, H5, H7, and H9 subtypes, and influenza B viruses of Yamagata-like and Victoria-like lineages in ex vivo canine nasal cavity, soft palate, trachea, and lung tissue explant cultures and examined ACE2 and sialic acid (SA) receptor distribution in these tissues. Results There was limited productive replication of SARS-CoV-2 in canine nasal cavity and SARS-CoV in canine nasal cavity, soft palate, and lung, with unexpectedly high ACE2 levels in canine nasal cavity and soft palate. Canine tissues were susceptible to a wide range of human and avian influenza viruses, which matched with the abundance of both human and avian SA receptors. Conclusions Existence of suitable receptors and tropism for the same tissue foster virus adaptation and reassortment. Continuous surveillance in dog populations should be conducted given the many chances for spillover during outbreaks.

Influenza can cause acute lung injury. Because immune responses often play a role, antivirals may not ensure a successful outcome. To identify pathogenic mechanisms and potential adjunctive therapeutic options, we compared the extent to which avian influenza A/H5N1 virus and seasonal influenza A/H1N1 virus impair alveolar fluid clearance and protein permeability in an in vitro model of acute lung injury, defined the role of virus-induced soluble mediators in these injury effects, and demonstrated that the effects are prevented or reduced by bone marrow-derived multipotent mesenchymal stromal cells. We verified the in vivo relevance of these findings in mice experimentally infected with influenza A/H5N1. We found that, in vitro, the alveolar epithelium's protein permeability and fluid clearance were dysregulated by soluble immune mediators released upon infection with avian (A/Hong Kong/483/97, H5N1) but not seasonal (A/Hong Kong/54/98, H1N1) influenza virus. The reduced alveolar fluid transport associated with down-regulation of sodium and chloride transporters was prevented or reduced by coculture with mesenchymal stromal cells. In vivo, treatment of aged H5N1-infected mice with mesenchymal stromal cells increased their likelihood of survival. We conclude that mesenchymal stromal cells significantly reduce the impairment of alveolar fluid clearance induced by A/H5N1 infection in vitro and prevent or reduce A/H5N1-associated acute lung injury in vivo. This potential adjunctive therapy for severe influenza-induced lung disease warrants rapid clinical investigation.

Objective This study assessed the transmission of low pathogenic avian influenza in live poultry market setting, using paired fecal and drinking water samples from a longitudinal surveillance program. The relative contribution of transmission via direct fecal-oral route versus drinking water will be determined. Introduction Live poultry markets (LPMs) continue to operate in many Asian countries. Low pathogenic avian influenza (LPAI) viruses are often endemic in the poultry, and LPM presents the opportunity for human-poultry interactions and potential human infections with avian influenza viruses. As a series of interventions to control avian influenza transmission in Hong Kong LPMs, local health authority implemented market rest days once every month since mid-2001, and an additional rest day every month since 2003, during which all unsold poultry were slaughtered and the stalls cleaned and disinfected. Rest days were found to effectively reduce avian influenza A(H9N2) isolation rate to baseline level for a few days following the rest days. However, H9N2 isolation rate was still observed to be increasing between the rest days, indicating the existence of efficient transmission in spite of rapid turnover of poultry. In LPMs, poultry are usually stored in cages where drinking water is shared among poultry. This is analogous to environmental contamination in the wild, but transmissibility may even be higher due to the dense environment. The use of drinking water for avian influenza surveillance in LPM setting was suggested to be more sensitive than fecal samples (1). However, the relative contribution of direct fecal-oral versus water transmission routes in the LPM setting was not yet understood. This study aimed to determine their role, which will have implications in the control of avian influenza transmission. Methods We analyzed 7,321 paired fecal and drinking water samples from a longitudinal surveillance programme during the period with 2 monthly rest days in the LPMs. Samples were collected from chicken cages and subsequently cultured. Positive isolates were subtyped by hemagglutination-inhibition tests and neuraminidase inhibition test. Data were aggregated by sampling occasion and days after the rest days. A compartmental transmission model which incorporated turnover and overnight stay of poultry, virus contamination and decay in drinking water was fitted to the data (Figure 1). A 12-hour trading day was assumed. Based on the parameterized model, we simulated the scenario that water transmission was prohibited to assess the role of transmission via drinking water. Results H9N2 isolation rates ranged from 0-25% for fecal samples and 0-56% for drinking water samples. A clear increasing trend can be seen over days after the rest days (Figure 2). The estimated parameter for water transmission is higher than the parameter for direct fecal-oral transmission. Simulation results show that transmission via drinking water plays a major role in the amplification of LPAI in the LPM setting (Figure 2). Conclusions Our study showed that drinking water has a major role in the transmission and amplification of LPAI H9N2 in LPMs, comparing to direct fecal-oral transmission route. Given the relatively low prevalence of H9N2 in chicken, direct transmission is governed by chance events, while chickens are consistently exposed to viruses in drinking water if contaminated. Drinking water could be targeted for intervention to control LPAI transmission in LPM. The use of drinking fountain or frequent disinfection of drinking water may be considered. Avian influenza viruses (e.g. H5N1) may differ in their pattern of virus shedding via oral versus fecal routes and thus extrapolation of these results to other viruses needs to be done with caution. However, H7N9 viruses are similar to H9N2 viruses by being shed primarily via the respiratory / oral route (2) and it is reasonable to assume that these conclusions would apply to H7N9 virus which is of major public health concern. However, our model could not differentiate the effect of indirect fecal-oral transmission through contamination of drinking water by droppings versus contamination through drinking.

daszak, peter/0000-0002-2046-5695; Kelly, Jeffrey/0000-0002-8255-7990; Bridge, Eli/0000-0003-3453-2008; /0000-0003-3213-6693 WOS: 000340312100029 PubMed: 25045322 Satellite-based tracking of migratory waterfowl is an important tool for understanding the potential role of wild birds in the long-distance transmission of highly pathogenic avian influenza. However, employing this technique on a continental scale is prohibitively expensive. This study explores the utility of stable isotope ratios in feathers in examining both the distances traveled by migratory birds and variation in migration behavior. We compared the satellite-derived movement data of 22 ducks from 8 species captured at wintering areas in Bangladesh, Turkey, and Hong Kong with deuterium ratios (delta D) in the feathers of these and other individuals captured at the same locations. We derived likely molting locations from the satellite tracking data and generated expected isotope ratios based on an interpolated map of delta D in rainwater. Although delta D was correlated with the distance between wintering and molting locations, surprisingly, measured delta D values were not correlated with either expected values or latitudes of molting sites. However, population-level parameters derived from the satellite-tracking data, such as mean distance between wintering and molting locations and variation in migration distance, were reflected by means and variation of the stable isotope values. Our findings call into question the relevance of the rainfall isotope map for Asia for linking feather isotopes to molting locations, and underscore the need for extensive ground truthing in the form of feather-based isoscapes. Nevertheless, stable isotopes from feathers could inform disease models by characterizing the degree to which regional breeding populations interact at common wintering locations. Feather isotopes also could aid in surveying wintering locations to determine where high-resolution tracking techniques (e.g. satellite tracking) could most effectively be employed. Moreover, intrinsic markers such as stable isotopes offer the only means of inferring movement information from birds that have died as a result of infection. In the absence of feather based-isoscapes, we recommend a combination of isotope analysis and satellite-tracking as the best means of generating aggregate movement data for informing disease models. (C) 2014 Elsevier Ltd. All rights reserved. NIH/NSF Ecology and Evolution of Infectious Diseases award from the Fogarty International Center of the National Institutes of Health [3R01-TW005869]; International Influenza Funds from the Office of the Secretary of the Department of Health and Human Services; ARRA U.S. Postdoctoral Scientist Administrative Supplement Isotope analyses and manuscript generation was supported by an NIH/NSF Ecology and Evolution of Infectious Diseases award from the Fogarty International Center of the National Institutes of Health (3R01-TW005869) with an ARRA U.S. Postdoctoral Scientist Administrative Supplement. Field work was supported by the United Nations Food and Agriculture Organization, the U. S. Geological Survey Western Ecological Research Center and Avian Influenza Program, and Erasmus University. The work was conducted in the context of Zoonotic Influenza Collaborative Network, led by the Fogarty International Center, and supported by International Influenza Funds from the Office of the Secretary of the Department of Health and Human Services. Research on animals was conducted with the approval the Animal Care and Use Committees of the USGS Western Ecological Center, the Patuxent Wildlife Research Center, The University of Maryland, Baltimore County (Protocol EE070200710), and the University of Oklahoma (Animal Use Statement R09-019). We thank the following people and organizations for their aid in organizing and executing work at the various field sites: C. Ozsemir, S. Inak, N. Yavuz, N. Tubbs, J. Epstein, J. Desmond, P. Sathyiaselvam, J. Allcock, K. Leung, M. Peiris, S. Luby, Y. Bans, M. Leven, A. Mikolon, and the Bangladesh Bird Club. We also thank Eben Paxton, Susan Jones, and two anonymous scientists for their helpful reviews. The use of trade names in this document is for descriptive purposes only and does not imply endorsement by the U.S. Government.

A novel H7N9 influenza A virus first detected in March 2013 has since caused more than 130 human infections in China, resulting in 40 deaths. Preliminary analyses suggest that the virus is a reassortant of H7, N9 and H9N2 avian influenza viruses, and carries some amino acids associated with mammalian receptor binding, raising concerns of a new pandemic. However, neither the source populations of the H7N9 outbreak lineage nor the conditions for its genesis are fully known. Using a combination of active surveillance, screening of virus archives, and evolutionary analyses, here we show that H7 viruses probably transferred from domestic duck to chicken populations in China on at least two independent occasions. We show that the H7 viruses subsequently reassorted with enzootic H9N2 viruses to generate the H7N9 outbreak lineage, and a related previously unrecognized H7N7 lineage. The H7N9 outbreak lineage has spread over a large geographic region and is prevalent in chickens at live poultry markets, which are thought to be the immediate source of human infections. Whether the H7N9 outbreak lineage has, or will, become enzootic in China and neighbouring regions requires further investigation. The discovery here of a related H7N7 influenza virus in chickens that has the ability to infect mammals experimentally, suggests that H7 viruses may pose threats beyond the current outbreak. The continuing prevalence of H7 viruses in poultry could lead to the generation of highly pathogenic variants and further sporadic human infections, with a continued risk of the virus acquiring human-to-human transmissibility., link_to_subscribed_fulltext

Using a pan-astrovirus reverse transcription-PCR assay, a great diversity of novel avastroviruses was detected from wild bird and poultry samples. Two groups of astroviruses detected from wild birds are genetically related or highly similar to previously known viruses in poultry. Most interestingly, a novel group of astroviruses was detected in wild aquatic birds. Our results also reveal that different groups of astroviruses might have difference host ranges. This study has expanded our understanding regarding avastrovirus ecology.

We detected a high prevalence (12.5%) of novel avian coronaviruses in aquatic wild birds. Phylogenetic analyses of these coronaviruses suggest that there is a diversity of gammacoronaviruses and deltacoronaviruses circulating in birds. Gammacoronaviruses were found predominantly in Anseriformes birds, whereas deltacoronaviruses could be detected in Ciconiiformes, Pelecaniformes, and Anseriformes birds in this study. We observed that there are frequent interspecies transmissions of gammacoronaviruses between duck species. In contrast, deltacoronaviruses may have more stringent host specificities. Our analysis of these avian viral and host mitochondrial DNA sequences also suggests that some, but not all, coronaviruses may have coevolved with birds from the same order. © 2011, American Society for Microbiology., link_to_OA_fulltext

Previous studies showed that the anterior cingulate cortex (ACC) is activated when individuals engage in attention and inhibitory control tasks. The present study examined whether ACC activity is associated with behavioral performance of the two tasks. Twenty normal and 20 children with autism spectrum disorders (ASDs) were subjected to neuropsychological assessments on attention and inhibitory control, as well as electroencephalography recording. Children with ASD performed significantly worse than normal children on attention tasks as shown in their poorer performance on the Digit Span test, the greater number of Omission Errors on both the Continuous Performance Test II and the Go/No-Go tasks. They also performed significantly worse than normal children on inhibitory control tasks as shown by the greater number of False Alarms on the Object Recognition and Hong Kong List Learning Test. Their ACC activities, as indicated by relative theta power, were found to be significantly lower than those of normal controls during performance of the Go/No-Go task. Depressed ACC activities were further found to be significantly associated with poorer performance in attention and inhibition. Clinical implications on the use of theta activities in the ACC as an indicator to monitor intervention progress in children with ASD were discussed.

Avian influenza A H5N1 remains unusual in its virulence for humans. While infection of humans remains inefficient, many of those with H5N1 disease have a rapidly progressing viral pneumonia leading to acute respiratory distress syndrome and death but its pathogenesis remains an enigma. Comparisons in the virology and pathogenesis of human seasonal influenza viruses (H3N2 and H1N1) and H5N1 in patients, animal models and in relevant primary human cell cultures remains instructive. While the direct effects of viral replication and differences in the tropism of the virus for cells in the lower respiratory tract clearly contribute to the pathogenesis, we focus here on the possible contribution of the host innate immune response in the pathogenesis of this disease.

Background: Avian influenza virus (AIV) surveillance in birds is important for public health. Faecal droppings from wild-birds are more readily available for such studies, but the inability to identify the species-origin of faecal samples limits their value. Objectives: To develop, optimise, and field-test a method to simultaneously detect AIV and identify the species-origin from faecal samples. Study design: Analytical sensitivity of the species-identification RT-PCR was assessed on serial dilutions of faecal droppings. Overall sensitivity of the methods for species-identification and AIV detection was assessed on 92 faecal and cloacal samples collected from wildlife, poultry markets, and experimentally H5N1-infected birds. Results: All 92 samples were correctly identified to 24 different species, with a detection limit of 2.8 μg of faecal material. All 20 specimens previously shown by virus culture to be positive for influenza virus were correctly identified by RT-PCR for influenza A using the same nucleic-acid extracts used for species-identification. Conclusion: We have optimised and evaluated a method for identifying the species of origin and detecting AIV from bird faecal droppings that can be applied to routine surveillance of influenza viruses in wild-birds. © 2009 Elsevier B.V. All rights reserved., link_to_OA_fulltext

Background Bali Province was affected by avian influenza H5N1 outbreaks in birds in October 2003. Despite ongoing circulation of the virus, no human infection had been identified by December 2005. Objectives To assess behavioral patterns associated with poultry rearing in Bali, and to identify potential risk factors for H5N1 infection in humans and in household chickens, ducks and pigs. Methods A behavioral, virological and seroepidemiologic survey in 38 villages and three live bird markets was completed in December 2005. A multi-stage cluster design was used to select 291 households with 841 participants from all nine districts in Bali. Specimens were collected from participants as well as a maximum of three pigs, chickens and ducks from each household. Eighty-seven market vendors participated, where specimens were collected from participants as well as chickens and ducks. Results Twenty out of the 38 villages sampled had H5N1 outbreaks. Despite exposure to H5N1 outbreaks, none of the participants from villages or markets were seropositive for H5N1. None of the pigs tested were positive for H5N1. Virus isolation rate in ducks and chicken in markets was higher than in households. Transport of poultry in or out of villages was a risk factor for outbreaks in household chickens and ducks. Conclusions The study highlighted that the market chain and associated behaviors may play a role in maintaining the virus in household flocks. The study adds evidence that transmission of H5N1 to humans remains a rare event despite high level handling of both healthy and sick birds.

Realizable one-step RT-PCR assays specific for influenza PB2, PB1 and PA segments are described in this report. The designs of the consensus primers were based on more than five thousands polymerase genes derived from avian or mammalian viral strains. All the viral RNA tested in this study could be consistently amplified by the assays. The reaction products were specific and could be used for direct DNA sequencing. These assays might be useful tools to study the sequences of these genes.

The pathogenesis of human influenza H5N1 virus infection remains poorly understood and controversial. Cytokine dysregulation in human infection has been hypothesized to contribute to disease severity. We developed in vitro cultures of mouse bone marrow derived macrophages (BMDMΦ) from C57BL/6N mouse to compare influenza A (H5N1 and H1N1) virus replication and pro-inflammatory cytokine and chemokine responses. While both H1N1 and H5N1 viruses infected the mouse bone marrow derived macrophages, only the H1N1 virus had showed evidence of productive viral replication from the infected cells. In comparison with human seasonal influenza H1N1 (A/HK/54/98) and mouse adapted influenza H1N1 (A/WSN/33) viruses, the highly pathogenic influenza H5N1 virus (A/HK/483/97) was a more potent inducer of the chemokine, CXCL 10 (IP-10), while there was not a clear differential TNF-α protein expression pattern. Although human influenza viruses rarely cause infection in mice without prior adaption, the use of in vitro cell cultures of primary mouse cells is of interest, especially given the availability of gene-defective (knock-out) mice for specific genes., published_or_final_version

There were errors in the Funding section. The correct funding information is as follows: Support for this project was provided by the National Institute of Allergy and Infectious Disease, National Institutes of Health, Department of Health and Human Services (Contracts HHSN266200700007C, HHSN266200700009C, and HHSN266200700005C), the World Bank, and the Food and Agriculture Organization of the United Nations. This work was made possible through support provided by the Office of Health, Infectious Disease and Nutrition, Bureau for Global Health, U.S. Agency for International Development and Wildlife Conservation Society, under the terms of Leader Award No.LAG-A-00-99-00047-00, Cooperative Agreement: GHS-A-00-06-00005. The opinions expressed herein are those of the author(s) and do not necessarily reflect the views of the U.S. Agency for International Development or Wildlife Conservation Society. DOJ was supported by generous funding from the Dunemere private foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The protective efficacy of a subunit avian influenza virus H5 vaccine based on recombinant baculovirus expressed H5 haemagglutinin antigen and an inactivated H5N2 avian influenza vaccine combined with a marker antigen (tetanus toxoid) was compared with commercially available inactivated H5N2 avian influenza vaccine in young ducks. Antibody responses, morbidity, mortality, and virus shedding were evaluated after challenge with a Vietnamese clade 1 H5N1 HPAI virus [A/VN/1203/04 (H5N1)] that was known to cause a high mortality rate in ducks. All three vaccines, administered with water-in-oil adjuvant, provided significant protection and dramatically reduced the duration and titer of virus shedding in the vaccinated challenged ducks compared with unvaccinated controls. The H5 subunit vaccine was shown to provide equivalent protection to the other two vaccines despite the H5 antibody responses in subunit vaccinated ducks being significantly lower prior to challenge. Ducks vaccinated with the H5N2 marker vaccine consistently produced antitetanus toxoid antibody. The two novel vaccines have attributes that would enhance H5N1 avian influenza surveillance and control by vaccination in small scale and village poultry systems.

The mechanism for the pathogenesis of H5N1 infection in humans remains unclear. This study reveals that cyclooxygenase-2 (COX-2) was strongly induced in H5N1-infected macrophages in vitro and in epithelial cells of lung tissue samples obtained during autopsy of patients who died of H5N1 disease. Novel findings demonstrated that COX-2, along with tumor necrosis factor alpha and other proinflammatory cytokines were hyperinduced in epithelial cells by secretory factors from H5N1-infected macrophages in vitro. This amplification of the proinflammatory response is rapid, and the effects elicited by the H5N1-triggered proinflammatory cascade are broader than those arising from direct viral infection. Furthermore, selective COX-2 inhibitors suppress the hyperinduction of cytokines in the proinflammatory cascade, indicating a regulatory role for COX-2 in the H5N1-hyperinduced host proinflammatory cascade. These data provide a basis for the possible development of novel therapeutic interventions for the treatment of H5N1 disease, as adjuncts to antiviral drugs.

Vaccination of chickens with a commercially available killed H5N2 vaccine was being evaluated as an additional tool to enhanced biosecurity measures and intensive surveillance for control of highly pathogenic avian influenza subtype H5N1 disease in Hong Kong in 2002. In December 2002 to January 2003, there were outbreaks of H5N1 disease in waterfowl in two recreational parks, wild water birds, several poultry markets and five chicken farms. In addition to quarantine, depopulation of the affected sheds and increased biosecurity, vaccination of the unaffected sheds and surrounding unvaccinated farms was undertaken on three farms. In at least two farms, infection spread to the recently vaccinated sheds with low rates of H5N1 mortality in sheds when the chickens were between 9 and 18 days post-vaccination. However, after 18 days post-vaccination no more deaths from H5N1 avian influenza occurred and intensive monitoring by virus culture on these farms showed no evidence of asymptomatic shedding of the virus. This provides evidence that H5 vaccine can interrupt virus transmission in a field setting.

Mongolia combines a near absence of domestic poultry, with an abundance of migratory waterbirds, to create an ideal location to study the epidemiology of highly pathogenic avian influenza virus (HPAIV) in a purely wild bird system. Here we present the findings of active and passive surveillance for HPAIV subtype H5N1 in Mongolia from 2005-2011, together with the results of five outbreak investigations. In total eight HPAIV outbreaks were confirmed in Mongolia during this period. Of these, one was detected during active surveillance employed by this project, three by active surveillance performed by Mongolian government agencies, and four through passive surveillance. A further three outbreaks were recorded in the neighbouring Tyva Republic of Russia on a lake that bisects the international border. No HPAIV was isolated (cultured) from 7,855 environmental fecal samples (primarily from ducks), or from 2,765 live, clinically healthy birds captured during active surveillance (primarily shelducks, geese and swans), while four HPAIVs were isolated from 141 clinically ill or dead birds located through active surveillance. Two low pathogenic avian influenza viruses (LPAIV) were cultured from ill or dead birds during active surveillance, while environmental feces and live healthy birds yielded 56 and 1 LPAIV respectively. All Mongolian outbreaks occurred in 2005 and 2006 (clade 2.2), or 2009 and 2010 (clade 2.3.2.1); all years in which spring HPAIV outbreaks were reported in Tibet and/or Qinghai provinces in China. The occurrence of outbreaks in areas deficient in domestic poultry is strong evidence that wild birds can carry HPAIV over at least moderate distances. However, failure to detect further outbreaks of clade 2.2 after June 2006, and clade 2.3.2.1 after June 2010 suggests that wild birds migrating to and from Mongolia may not be competent as indefinite reservoirs of HPAIV, or that HPAIV did not reach susceptible populations during our study., published_or_final_version