Josanne H. Verhagen

How a virus travels the world

Wild birds may spread the H5N8 virus In November and December 2014, highly pathogenic avian influenza (HPAI) viruses of the H5 subtype originating from China were detected in poultry and wild birds in various countries of Asia and Europe, and, for the first time, in North America. These incursions of newly emerging HPAI H5 viruses constitute a threat to animal and potentially human health and raise questions about the routes of transmission.

Juveniles and migrants as drivers for seasonal epizootics of avian influenza virus

Similar to other infectious diseases, the prevalence of low pathogenic avian influenza viruses (LPAIV) has been seen to exhibit marked seasonal variation. However, mechanisms driving this variation in wild birds have yet to be tested. We investigated the validity of three previously suggested drivers for the seasonal dynamics in LPAIV infections in wild birds: (i) host density, (ii) immunologically naïve young and (iii) increased susceptibility in migrants. To address these questions, we sampled a key LPAIV host species, the mallard Anas platyrhynchos, on a small spatial scale, comprehensively throughout a complete annual cycle, measuring both current and past infection (i.e. viral and seroprevalence, respectively). We demonstrate a minor peak in LPAIV prevalence in summer, a dominant peak in autumn, during which half of the sampled population was infected, and no infections in spring. Seroprevalence of antibodies to a conserved gene segment of avian influenza virus (AIV) peaked in winter and again in spring. The summer peak of LPAIV prevalence coincided with the entrance of unfledged naïve young in the population. Moreover, juveniles were more likely to be infected, shed higher quantities of virus and were less likely to have detectable antibodies to AIV than adult birds. The arrival of migratory birds, as identified by stable hydrogen isotope analysis, appeared to drive the autumn peak in LPAIV infection, with both temporal coincidence and higher infection prevalence in migrants. Remarkably, seroprevalence in migrants was substantially lower than viral prevalence throughout autumn migration, further indicating that each wave of migrants amplified local AIV circulation. Finally, while host abundance increased throughout autumn, it peaked in winter, showing no direct correspondence with either of the LPAIV infection peaks. At an epidemiologically relevant spatial scale, we provide strong evidence for the role of migratory birds as key drivers for seasonal epizootics of LPAIV, regardless of their role as vectors of these viruses. This study exemplifies the importance of understanding host demography and migratory behaviour when examining seasonal drivers of infection in wildlife populations.

Wild bird surveillance around outbreaks of highly pathogenic avian influenza A(H5N8) virus in the Netherlands, 2014, within the context of global flyways.

Highly pathogenic avian influenza (HPAI) A(H5N8) viruses that emerged in poultry in east Asia since 2010 spread to Europe and North America by late 2014. Despite detections in migrating birds, the role of free-living wild birds in the global dispersal of H5N8 virus is unclear. Here, wild bird sampling activities in response to the H5N8 virus outbreaks in poultry in the Netherlands are summarised along with a review on ring recoveries. HPAI H5N8 virus was detected exclusively in two samples from ducks of the Eurasian wigeon species, among 4,018 birds sampled within a three months period from mid-November 2014. The H5N8 viruses isolated from wild birds in the Netherlands were genetically closely related to and had the same gene constellation as H5N8 viruses detected elsewhere in Europe, in Asia and in North America, suggesting a common origin. Ring recoveries of migratory duck species from which H5N8 viruses have been isolated overall provide evidence for indirect migratory connections between East Asia and Western Europe and between East Asia and North America. This study is useful for better understanding the role of wild birds in the global epidemiology of H5N8 viruses. The need for sampling large numbers of wild birds for the detection of H5N8 virus and H5N8-virus-specific antibodies in a variety of species globally is highlighted, with specific emphasis in north-eastern Europe, Russia and northern China.

One health, multiple challenges : The inter-species transmission of influenza A virus

Influenza A viruses are amongst the most challenging viruses that threaten both human and animal health. Influenza A viruses are unique in many ways. Firstly, they are unique in the diversity of host species that they infect. This includes waterfowl (the original reservoir), terrestrial and aquatic poultry, swine, humans, horses, dog, cats, whales, seals and several other mammalian species. Secondly, they are unique in their capacity to evolve and adapt, following crossing the species barrier, in order to replicate and spread to other individuals within the new species. Finally, they are unique in the frequency of inter-species transmission events that occur. Indeed, the consequences of novel influenza virus strain in an immunologically naïve population can be devastating. The problems that influenza A viruses present for human and animal health are numerous. For example, influenza A viruses in humans represent a major economic and disease burden, whilst the poultry industry has suffered colossal damage due to repeated outbreaks of highly pathogenic avian influenza viruses. This review aims to provide a comprehensive overview of influenza A viruses by shedding light on interspecies virus transmission and summarising the current knowledge regarding how influenza viruses can adapt to a new host.

Avian Influenza Virus Surveillance in Wild Birds in Georgia: 2009-2011

The Caucasus, at the border of Europe and Asia, is important for migration and over-wintering of wild waterbirds. Three flyways, the Central Asian, East Africa-West Asia, and Mediterranean/Black Sea flyways, converge in the Caucasus region. Thus, the Caucasus region might act as a migratory bridge for influenza virus transmission when birds aggregate in high concentrations in the post-breeding, migrating and overwintering periods. Since August 2009, we have established a surveillance network for influenza viruses in wild birds, using five sample areas geographically spread throughout suitable habitats in both eastern and western Georgia. We took paired tracheal and cloacal swabs and fresh feces samples. We collected 8343 swabs from 76 species belonging to 17 families in 11 orders of birds, of which 84 were real-time RT-PCR positive for avian influenza virus (AIV). No highly pathogenic AIV (HPAIV) H5 or H7 viruses were detected. The overall AIV prevalence was 1.6%. We observed peak prevalence in large gulls during the autumn migration (5.3–9.8%), but peak prevalence in Black-headed Gulls in spring (4.2–13%). In ducks, we observed increased AIV prevalence during the autumn post-moult aggregations and migration stop-over period (6.3%) but at lower levels to those observed in other more northerly post-moult areas in Eurasia. We observed another prevalence peak in the overwintering period (0.14–5.9%). Serological and virological monitoring of a breeding colony of Armenian Gulls showed that adult birds were seropositive on arrival at the breeding colony, but juveniles remained serologically and virologically negative for AIV throughout their time on the breeding grounds, in contrast to gull AIV data from other geographic regions. We show that close phylogenetic relatives of viruses isolated in Georgia are sourced from a wide geographic area throughout Western and Central Eurasia, and from areas that are represented by multiple different flyways, likely linking different host sub-populations.

Avian influenza a(H10n7) virus–associated mass deaths among harbor seals

To the Editor: Avian influenza A viruses occasionally cross the species barrier; influenza A(H5N1) virus and the recently emerged influenza A(H7N9) virus are prime examples of bird-to-human transmission (1,2). In addition, avian influenza A viruses can cross to various other mammalian species, including pinnipeds (e.g., seals) (3,4). Recently, mass deaths have occurred among harbor seals (Phoca vitulina); hundreds of carcasses washed up the shores of Sweden (March 2014), Denmark (July 2014), and Germany (October 2014). Approximately 1,400 dead harbor seals were seen in the coastal waters of Schleswig-Holstein in Germany alone, where the population is ≈12,000 animals. We report the investigation of the deaths of 17 seals from different age groups that were found dead on the islands of Helgoland and Sylt, Germany, during the second week of October 2014. Complete necropsies were performed on the carcasses, which were in variable nutritional conditions, ranging from very poor to good. Necropsy results showed consistently poorly retracted lungs with severe congestion, occasional diffuse consolidation, and multifocal firm nodular areas of gray-yellow discoloration with varying numbers of metazoic parasites. Histologic examinations showed acute necrotizing bronchitis and adenitis of bronchial glands with sloughing of epithelial cells (Figure, panel A). Occasionally, mild interstitial pneumonia was found. Multifocal pyogranulomatous to necrotizing pneumonia was associated with parasite infestation. A few animals had suppurative to necrotizing or nonsuppurative rhinitis and tracheitis. Figure Histopathologic and phylogenetic analyses of necropsy samples from harbor seals infected with avian influenza A(H10N7) virus, Germany, 2014. A) Lung of harbor seal showing marked necrosis and sloughing of epithelial cells in bronchial glands (arrows); ... Because mass deaths among seals were caused by phocine distemper virus in the same area in 1988 and 2002, we tested lung and throat swab samples for morbillivirus using reverse transcription PCR (RT-PCR) and immunohistochemical analysis (5). In addition, real-time RT-PCR targeting the influenza A virus matrix gene was performed (6). No indications for the morbillivirus were detected by RT-PCR and immunohistochemistry; however, in lung lesions and throat swab samples of 11 animals, a positive signal was observed by the influenza A matrix gene real-time RT-PCR (cycle threshold values 15.0–33.9). Influenza A virus (A/harbor seal/Germany/1/2014) was subsequently isolated from lung and throat swab samples; the virus replicated to high titers in 11-day-old embryonated chicken eggs and on MDCK cells. By PCR using specific primers and subsequent Sanger sequencing of the hemagglutinin and neuraminidase genes, this virus was characterized as an influenza A virus of the H10N7 subtype, commonly found in migratory waterfowl (6). In addition, genetic analyses of all other gene segments indicated that the influenza virus A/harbor seal/Germany/1/2014 was most closely related to various influenza A viruses detected in wild birds. Specifically, the hemagglutinin and neuraminidase genes were genetically most closely related to subtype H10N7 viruses recently found in migratory ducks in Georgia, Egypt, and the Netherlands (Figure, panel C) (7). Genetic analyses were based on BLAST analyses using public databases available as of October 17, 2014 (http://www.ncbi.nlm.nih.gov, http://www.gisaid.com) and supplemented with H10 and N7 sequences from the international wild bird surveillance program of Erasmus Medical Center (Technical Appendix Table). A maximum-likelihood phylogenetic tree of the hemagglutinin gene was generated by using PhyML version 3.1 (8) with the general time reversible +I+Γ model of nucleotide substitution; a full heuristic search and subtree pruning and regrafting searches were performed. The tree was visualized by using Figtree version 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree). To further elucidate the role of influenza A(H10N7) virus in the pathogenesis of the disease causing deaths among the seals, we conducted immunohistochemical analysis on the lungs using an influenza A virus nucleoprotein-specific monoclonal antibody (9). Evaluation of the lung tissues of the dead seals showed viral antigen in cytoplasm and nuclei of epithelial cells of bronchi and bronchial glands of affected lung areas (Figure, panel B), which suggests that this virus played a major role in the deaths. Immunohistochemical analysis performed on various organs (including brain and olfactory bulb) indicated that viral antigen was restricted to the respiratory tract. Although avian influenza A virus infections previously have caused mass deaths in seals (3,4,10), subtype H10N7 has not been associated with such events. We can speculate that the ongoing deaths could eventually affect all harbor seal populations of northwestern Europe and have consequences for wildlife management and seal rehabilitation activities. In addition, preliminary analysis of the hemagglutinin sequence of the influenza A(H10N7) virus suggests the presence of molecular determinants that indicate mammalian adaptation. Various analyses are ongoing to answer questions about the route of transmission among seals and possible transmissibility to humans. Note added in proof: Zohari et al. also recently reported the involvement of avian influenza A(H10N7) virus in mass deaths of harbor seals in Sweden (Euro Surveill. 2014;19:pii: 20967). Technical Appendix: Details of hemagglutinin sequences shown in the Figure. Click here to view.(22K, pdf)

Avian influenza a virus in wild birds in highly urbanized areas

Avian influenza virus (AIV) surveillance studies in wild birds are usually conducted in rural areas and nature reserves. Less is known of avian influenza virus prevalence in wild birds located in densely populated urban areas, while these birds are more likely to be in close contact with humans. Influenza virus prevalence was investigated in 6059 wild birds sampled in cities in the Netherlands between 2006 and 2009, and compared with parallel AIV surveillance data from low urbanized areas in the Netherlands. Viral prevalence varied with the level of urbanization, with highest prevalence in low urbanized areas. Within cities virus was detected in 0.5% of birds, while seroprevalence exceeded 50%. Ring recoveries of urban wild birds sampled for virus detection demonstrated that most birds were sighted within the same city, while few were sighted in other cities or migrated up to 2659 km away from the sample location in the Netherlands. Here we show that urban birds were infected with AIVs and that urban birds were not separated completely from populations of long-distance migrants. The latter suggests that wild birds in cities may play a role in the introduction of AIVs into cities. Thus, urban bird populations should not be excluded as a human-animal interface for influenza viruses.

Epidemiology of Influenza A Virus among Black- headed Gulls, the Netherlands, 2006-2010

We sampled 7,511 black-headed gulls for influenza virus in the Netherlands during 2006–2010 and found that subtypes H13 and H16 caused annual epidemics in fledglings on colony sites. Our findings validate targeted surveillance of wild waterbirds and clarify underlying factors for influenza virus emergence in other species.

Migratory birds reinforce local circulation of avian influenza viruses

Migratory and resident hosts have been hypothesized to fulfil distinct roles in infectious disease dynamics. However, the contribution of resident and migratory hosts to wildlife infectious disease epidemiology, including that of low pathogenic avian influenza virus (LPAIV) in wild birds, has largely remained unstudied. During an autumn H3 LPAIV epizootic in free-living mallards (Anas platyrhynchos) — a partially migratory species — we identified resident and migratory host populations using stable hydrogen isotope analysis of flight feathers. We investigated the role of migratory and resident hosts separately in the introduction and maintenance of H3 LPAIV during the epizootic. To test this we analysed (i) H3 virus kinship, (ii) temporal patterns in H3 virus prevalence and shedding and (iii) H3-specific antibody prevalence in relation to host migratory strategy. We demonstrate that the H3 LPAIV strain causing the epizootic most likely originated from a single introduction, followed by local clonal expansion. The H3 LPAIV strain was genetically unrelated to H3 LPAIV detected both before and after the epizootic at the study site. During the LPAIV epizootic, migratory mallards were more often infected with H3 LPAIV than residents. Low titres of H3-specific antibodies were detected in only a few residents and migrants. Our results suggest that in this LPAIV epizootic, a single H3 virus was present in resident mallards prior to arrival of migratory mallards followed by a period of virus amplification, importantly associated with the influx of migratory mallards. Thus migrants are suggested to act as local amplifiers rather than the often suggested role as vectors importing novel strains from afar. Our study exemplifies that a multifaceted interdisciplinary approach offers promising opportunities to elucidate the role of migratory and resident hosts in infectious disease dynamics in wildlife.

Tissue tropism and pathology of natural influenza virus infection in black-headed gulls (Chroicocephalus ridibundus)

Black-headed gulls (Chroicocephalus ridibundus) are a suitable host species to study the epidemiology of low-pathogenic avian influenza virus (LPAIV) infection in wild waterbirds because they are a common colony-breeding species in which LPAIV infection is detected frequently, limited mainly to the H13 and H16 subtypes. However, the sites of virus replication and associated lesions are poorly understood. We therefore performed virological and pathological analyses on tissues of black-headed gulls naturally infected with LPAIV. We found that 24 of 111 black-headed gulls collected from breeding colonies were infected with LPAIV (10 birds with H16N3, one bird with H13N8, 13 birds undetermined), based on virus and viral genome detection in pharyngeal and cloacal swabs. Of these 24 gulls, 15 expressed virus antigen in their tissues. Virus antigen expression was limited to epithelial cells of intestine and cloacal bursa. No histological lesions were detected in association with virus antigen expression. Our findings show that LPAIV replication in the intestinal tract of black-headed gulls is mainly a superficial infection in absence of detectable lesions, as determined recently for natural LPAIV infection in free-living mallards (Anas platyrhynchos). These findings imply that LPAIV in black-headed gulls has adapted to minimal pathogenicity to its host and that potentially the primary transmission route is faecal–oral.