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Although extensive data exist on avian influenza in wild birds in North America, limited information is available from elsewhere, including Europe. Here, molecular diagnostic tools were employed for high-throughput surveillance of migratory birds, as an alternative to classical labor-intensive methods of virus isolation in eggs. This study included 36,809 samples from 323 bird species belonging to 18 orders, of which only 25 species of three orders were positive for influenza A virus. Information on species, locations, and timing is provided for all samples tested. Seven previously unknown host species for avian influenza virus were identified: barnacle goose, bean goose, brent goose, pink-footed goose, bewick's swan, common gull, and guillemot. Dabbling ducks were more frequently infected than other ducks and Anseriformes; this distinction was probably related to bird behavior rather than population sizes. Waders did not appear to play a role in the epidemiology of avian influenza in Europe, in contrast to the Americas. The high virus prevalence in ducks in Europe in spring as compared with North America could explain the differences in virus–host ecology between these continents. Most influenza A virus subtypes were detected in ducks, but H13 and H16 subtypes were detected primarily in gulls. Viruses of subtype H6 were more promiscuous in host range than other subtypes. Temporal and spatial variation in influenza virus prevalence in wild birds was observed, with influenza A virus prevalence varying by sampling location; this is probably related to migration patterns from northeast to southwest and a higher prevalence farther north along the flyways. We discuss the ecology and epidemiology of avian influenza A virus in wild birds in relation to host ecology and compare our results with published studies. These data are useful for designing new surveillance programs and are particularly relevant due to increased interest in avian influenza in wild birds., Author Summary Significant gaps in our knowledge of the ecology of avian influenza in wild migratory birds have become apparent during recent outbreaks of H5N1 highly pathogenic avian influenza, in particular in relation to the risk of virus spread by wild birds. An eight-year surveillance study, which included more than 36,000 wild birds tested for low pathogenic avian influenza, provides new information on host species, prevalence, and temporal and geographical variation of avian influenza in wild migratory birds in Europe. Dabbling ducks harbored nearly all known influenza virus subtypes, with the exception of H13 and H16, which were found primarily in gulls. In contrast to American studies, waders did not play a role in the epidemiology of avian influenza in Europe. This study provides important information on the ecology and epidemiology of avian influenza A virus and could assist in the design of new surveillance studies for high and low pathogenic avian influenza in wild birds.

The pathogenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) is likely mediated by disproportional immune responses and the ability of the virus to circumvent innate immunity. Using functional genomics, we analyzed early host responses to SARS-CoV infection in the lungs of adolescent cynomolgus macaques (Macaca fascicularis) that show lung pathology similar to that observed in human adults with SARS. Analysis of gene signatures revealed induction of a strong innate immune response characterized by the stimulation of various cytokine and chemokine genes, including interleukin (IL)-6, IL-8, and IP-10, which corresponds to the host response seen in acute respiratory distress syndrome. As opposed to many in vitro experiments, SARS-CoV induced a wide range of type I interferons (IFNs) and nuclear translocation of phosphorylated signal transducer and activator of transcription 1 in the lungs of macaques. Using immunohistochemistry, we revealed that these antiviral signaling pathways were differentially regulated in distinctive subsets of cells. Our studies emphasize that the induction of early IFN signaling may be critical to confer protection against SARS-CoV infection and highlight the strength of combining functional genomics with immunohistochemistry to further unravel the pathogenesis of SARS., Author Summary Severe acute respiratory syndrome coronavirus (SARS-CoV) infection causes a progressive atypical pneumonia. In typical cases, largely confined to adult and elderly individuals, acute respiratory distress syndrome develops, and admission to an intensive care unit is required. Although these complications can be fatal, most SARS patients recover, suggesting that protective immune responses are operational. In this study, we simultaneously examined virus replication and host–response gene expression profiles in macaque lungs during the acute phase of SARS to gain more insight into the early events that take place after SARS-CoV infection. We show that a strong host response is induced in the lungs of SARS-CoV–infected macaques, illustrated by the induction of several pathogenic cytokines and chemokines. Interestingly, antiviral pathways are activated as well, demonstrated by the presence of phosphorylated signal transducer and activator of transcription 1 (STAT1) transcription factors throughout the lung, but not in SARS-CoV–infected cells. A subset of cells was shown to produce interferon-β, a cytokine involved in the resistance to many viral infections and able to activate STAT1. Activation of this antiviral pathway upon SARS-CoV infection may be an important escape route of the host to withstand the devastating effects of SARS-CoV.

Identification of cellular receptors and characterization of viral tropism in animal models have vastly improved our understanding of morbillivirus pathogenesis. However, specific aspects of viral entry, dissemination and transmission remain difficult to recapitulate in animal models. Here, we used three virologically identical but phenotypically distinct recombinant (r) canine distemper viruses (CDV) expressing different fluorescent reporter proteins for in vivo competition and airborne transmission studies in ferrets (Mustela putorius furo). Six donor ferrets simultaneously received three rCDVs expressing green, red or blue fluorescent proteins via conjunctival (ocular, Oc), intra-nasal (IN) or intra-tracheal (IT) inoculation. Two days post-inoculation sentinel ferrets were placed in physically separated adjacent cages to assess airborne transmission. All donor ferrets developed lymphopenia, fever and lethargy, showed progressively increasing systemic viral loads and were euthanized 14 to 16 days post-inoculation. Systemic replication of virus inoculated via the Oc, IN and IT routes was detected in 2/6, 5/6 and 6/6 ferrets, respectively. In five donor ferrets the IT delivered virus dominated, although replication of two or three different viruses was detected in 5/6 animals. Single lymphocytes expressing multiple fluorescent proteins were abundant in peripheral blood and lymphoid tissues, demonstrating the occurrence of double and triple virus infections. Transmission occurred efficiently and all recipient ferrets showed evidence of infection between 18 and 22 days post-inoculation of the donor ferrets. In all cases, airborne transmission resulted in replication of a single-colored virus, which was the dominant virus in the donor ferret. This study demonstrates that morbilliviruses can use multiple entry routes in parallel, and co-infection of cells during viral dissemination in the host is common. Airborne transmission was efficient, although transmission of viruses expressing a single color suggested a bottleneck event. The identity of the transmitted virus was not determined by the site of inoculation but by the viral dominance during dissemination., Author summary Canine distemper virus (CDV) infection of ferrets is a tractable animal model for measles. Ferrets are highly susceptible to CDV, and inoculation with a low dose leads to lethal disease. We performed in vivo competition experiments to study virus entry, dissemination and transmission. Ferrets were simultaneously inoculated with CDV via the conjunctival, intra-nasal and intra-tracheal routes. The viruses were identical except for the fluorescent reporter protein encoded by the viral genome. By detecting cells expressing the different fluorescent reporter proteins at various sites in the host, we determined that CDV can enter the host in parallel at multiple sites. Virus spread in the ferret occurred via infected lymphocytes, which often turned out to be double- or triple-infected. Sentinel ferrets, placed in physically separated adjacent cages, became infected by airborne transmission. Transmission of the dominant single color despite replication of multicolor viruses in the upper respiratory tract suggested a bottleneck event.

A fatal human case of Duvenhage virus (DUVV) infection in a Dutch traveller who had returned from Kenya was reported in 2007. She exhibited classical symptoms of rabies encephalitis with distinct pathological findings. In the present study we describe the isolation and characterization of DUVV in vitro and its passage in BALB/c mice. The virus proved to be neuroinvasive in both juvenile and adult mice, resulting in about 50% lethality upon peripheral infection. Clinical signs in infected mice were those of classical rabies. However, the distribution of viral antigen expression in the brain differed from that of classical rabies virus infection and neither inclusion bodies nor neuronal necrosis were observed. This is the first study to describe the in vitro and in vivo isolation and characterization of DUVV., Author Summary Lyssaviruses have been known for centuries to cause lethal encephalitis in animals and humans, representing a serious public health problem especially in developing countries. Little is known about the way that lyssaviruses in general, and Duvenhage virus in particular cause disease. Studies of pathogenesis have been hampered by the fact that the virus has not yet been propagated and characterized extensively. In this paper, we describe the characterization of Duvenhage virus in vitro. Further, we characterized the virus in BALB/c mice. We compared Duvenhage virus with a wild type rabies virus (silver-haired bat rabies virus) and we found that while in vitro the differences of these two viruses were not significant, the in vivo characteristics of these two viruses differed significantly. Histological analyses of infected mouse brains suggest that differences in virulence may be associated with difference in tropism. Elucidating the differences in pathogenesis between different lyssaviruses might help us in the design of novel treatment protocols.

Measles remains a significant childhood disease, and is associated with a transient immune suppression. Paradoxically, measles virus (MV) infection also induces robust MV-specific immune responses. Current hypotheses for the mechanism underlying measles immune suppression focus on functional impairment of lymphocytes or antigen-presenting cells, caused by infection with or exposure to MV. We have generated stable recombinant MVs that express enhanced green fluorescent protein, and remain virulent in non-human primates. By performing a comprehensive study of virological, immunological, hematological and histopathological observations made in animals euthanized at different time points after MV infection, we developed a model explaining measles immune suppression which fits with the “measles paradox”. Here we show that MV preferentially infects CD45RA− memory T-lymphocytes and follicular B-lymphocytes, resulting in high infection levels in these populations. After the peak of viremia MV-infected lymphocytes were cleared within days, followed by immune activation and lymph node enlargement. During this period tuberculin-specific T-lymphocyte responses disappeared, whilst strong MV-specific T-lymphocyte responses emerged. Histopathological analysis of lymphoid tissues showed lymphocyte depletion in the B- and T-cell areas in the absence of apoptotic cells, paralleled by infiltration of T-lymphocytes into B-cell follicles and reappearance of proliferating cells. Our findings indicate an immune-mediated clearance of MV-infected CD45RA− memory T-lymphocytes and follicular B-lymphocytes, which causes temporary immunological amnesia. The rapid oligoclonal expansion of MV-specific lymphocytes and bystander cells masks this depletion, explaining the short duration of measles lymphopenia yet long duration of immune suppression., Author Summary Measles is associated with a transient immune suppression, resulting in increased susceptibility to opportunistic infections. Indeed, the main causes of measles mortality are secondary infections in the respiratory and digestive tract. Although measles is associated with lymphopenia, depletion of lymphocytes has often been dismissed as a cause of immune suppression. Lymphocyte counts rapidly return to normal after clearance of the virus, while immune suppression lasts several weeks to months. Many studies have focused on suppression of lymphocyte proliferation as an in vitro correlate of immune suppression. However, experimental infections of non-human primates show that in vivo lymphocyte proliferation is not impaired after measles. Instead, we hypothesize that massive expansion of MV-specific and bystander lymphocytes masks the fact that pre-existing memory lymphocytes have been depleted. We conclude that measles virus infection wipes out immunological memory, leaving individuals susceptible to opportunistic infectious agents that would normally be controlled by the immune system.

Only two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that influenza virus becomes resistant to these antiviral drugs and spreads in the human population. The 2009 pandemic A/H1N1 influenza virus is naturally resistant to adamantanes. Recently a novel neuraminidase I223R mutation was identified in an A/H1N1 virus showing cross-resistance to the neuraminidase inhibitors oseltamivir, zanamivir and peramivir. However, the ability of this virus to cause disease and spread in the human population is unknown. Therefore, this clinical isolate (NL/2631-R223) was compared with a well-characterized reference virus (NL/602). In vitro experiments showed that NL/2631-I223R replicated as well as NL/602 in MDCK cells. In a ferret pathogenesis model, body weight loss was similar in animals inoculated with NL/2631-R223 or NL/602. In addition, pulmonary lesions were similar at day 4 post inoculation. However, at day 7 post inoculation, NL/2631-R223 caused milder pulmonary lesions and degree of alveolitis than NL/602. This indicated that the mutant virus was less pathogenic. Both NL/2631-R223 and a recombinant virus with a single I223R change (recNL/602-I223R), transmitted among ferrets by aerosols, despite observed attenuation of recNL/602-I223R in vitro. In conclusion, the I223R mutated virus isolate has comparable replicative ability and transmissibility, but lower pathogenicity than the reference virus based on these in vivo studies. This implies that the 2009 pandemic influenza A/H1N1 virus subtype with an isoleucine to arginine change at position 223 in the neuraminidase has the potential to spread in the human population. It is important to be vigilant for this mutation in influenza surveillance and to continue efforts to increase the arsenal of antiviral drugs to combat influenza., Author Summary Recently, a 2009 pandemic A/H1N1 influenza virus was isolated from an immune compromised patient, with antiviral resistance to the neuraminidase inhibitor class of drugs. This virus had an amino acid change in the viral neuraminidase enzyme; an isoleucine at position 223 was substituted for an arginine (I223R). Patients infected with a pandemic virus that is resistant to all neuraminidase inhibitors, would leave physicians without antiviral treatment options, since these viruses are naturally resistant to the other class of antivirals, the adamantanes. To date, it is unknown if this I223R mutant virus is affected in its ability to cause severe disease and to transmit to other humans. Therefore, we have addressed this question by comparing the I223R mutant virus with a wild type reference virus in a ferret pathogenicity and transmission model. We found that the I223R mutant virus was not severely affected in its pathogenicity, although fewer lung lesions and alveolitis scores were found for the I223R mutant virus. In addition, we demonstrated that this virus transmitted efficiently to naïve ferrets. Consequently, we conclude that this I223R mutant virus has the potential to cause disease and may spread among humans. Therefore, influenza surveillance for this resistance pattern is advised.

Measles virus (MV) is hypothesized to enter the host by infecting epithelial cells of the respiratory tract, followed by viremia mediated by infected monocytes. However, neither of these cell types express signaling lymphocyte activation molecule (CD150), which has been identified as the receptor for wild-type MV. We have infected rhesus and cynomolgus macaques with a recombinant MV strain expressing enhanced green fluorescent protein (EGFP); thus bringing together the optimal animal model for measles and a virus that can be detected with unprecedented sensitivity. Blood samples and broncho-alveolar lavages were collected every 3 d, and necropsies were performed upon euthanasia 9 or 15 d after infection. EGFP production by MV-infected cells was visualized macroscopically, in both living and sacrificed animals, and microscopically by confocal microscopy and FACS analysis. At the peak of viremia, EGFP fluorescence was detected in skin, respiratory and digestive tract, but most intensely in all lymphoid tissues. B- and T-lymphocytes expressing CD150 were the major target cells for MV infection. Highest percentages (up to 30%) of infected lymphocytes were detected in lymphoid tissues, and the virus preferentially targeted cells with a memory phenotype. Unexpectedly, circulating monocytes did not sustain productive MV infection. In peripheral tissues, large numbers of MV-infected CD11c+ MHC class-II+ myeloid dendritic cells were detected in conjunction with infected T-lymphocytes, suggesting transmission of MV between these cell types. Fluorescent imaging of MV infection in non-human primates demonstrated a crucial role for lymphocytes and dendritic cells in the pathogenesis of measles and measles-associated immunosuppression., Author Summary Measles remains one of the most important causes of childhood mortality in developing countries. The virus is highly infectious and is spread via the respiratory route. According to textbook descriptions, measles virus first infects respiratory epithelial cells, followed by viremia mediated by infected monocytes. However, this order of events is inconsistent with current knowledge about receptor usage by measles virus strains. In this paper we have revisited the pathogenesis of measles by infecting non-human primates with a recombinant measles virus expressing enhanced green fluorescent protein. An important advantage of this system is that infected cells become fluorescent and can be detected with high sensitivity in living animals as well as tissue samples. Strikingly, at the peak of virus replication all lymphoid tissues were strongly fluorescent, and up to 10% of T-lymphocytes and 30% of B-lymphocytes were infected. In peripheral tissues the virus predominantly infected lymphocytes and dendritic cells, although to a lesser extent respiratory epithelial cells were also infected. We hypothesize that measles virus, like human immunodeficiency virus, utilizes dendritic cells as a vehicle to establish infection of the lymphoid system and cause immunosuppression. This study reshapes our basic view of measles pathogenesis.

Wild birds, particularly duck species, are the main reservoir of influenza A virus (IAV) in nature. However, knowledge of IAV infection dynamics in the wild bird reservoir, and the development of immune responses, are essentially absent. Importantly, a detailed understanding of how subtype diversity is generated and maintained is lacking. To address this, 18,679 samples from 7728 Mallard ducks captured between 2002 and 2009 at a single stopover site in Sweden were screened for IAV infections, and the resulting 1081 virus isolates were analyzed for patterns of immunity. We found support for development of homosubtypic hemagglutinin (HA) immunity during the peak of IAV infections in the fall. Moreover, re-infections with the same HA subtype and related prevalent HA subtypes were uncommon, suggesting the development of natural homosubtypic and heterosubtypic immunity (p-value = 0.02). Heterosubtypic immunity followed phylogenetic relatedness of HA subtypes, both at the level of HA clades (p-value = 0.04) and the level of HA groups (p-value = 0.05). In contrast, infection patterns did not support specific immunity for neuraminidase (NA) subtypes. For the H1 and H3 Clades, heterosubtypic immunity showed a clear temporal pattern and we estimated within-clade immunity to last at least 30 days. The strength and duration of heterosubtypic immunity has important implications for transmission dynamics of IAV in the natural reservoir, where immune escape and disruptive selection may increase HA antigenic variation and explain IAV subtype diversity., Author Summary Influenza A viruses (IAV) infect a range of hosts, with the largest diversity being found in waterfowl, particularly dabbling ducks. In these hosts, IAV causes only mild disease, while viruses that infect other hosts, such as poultry, horses or humans, can cause fatal infections. In fact, all known pandemic flu viruses have contained gene segments that originated in the wild bird reservoir. We sampled a wild population of Mallards over eight seasons and characterized infection histories in 7728 birds. For hemagglutinin (HA) the subtype recoveries indicated that once a Mallard has been infected, re-infection with the same HA subtype is uncommon within the next month, clearly indicating homosubtypic immunity. Moreover, we found evidence for natural heterosubtypic immunity, where phylogenetically related HA subtypes at clade and group levels were less common in re-infections than expected. On the contrary no specific patterns of immunity was found for neuraminidase subtypes. IAVs exist in numerous antigenic subtypes that co-circulate. The strength of heterosubtypic immunity in natural infections provides evidence that HA subtypes compete over hosts and that immune escape may result in positive selection for HA antigenic variation in the virus, and thus explain IAV subtype diversity.

Immunocompromised individuals tend to suffer from influenza longer with more serious complications than otherwise healthy patients. Little is known about the impact of prolonged infection and the efficacy of antiviral therapy in these patients. Among all 189 influenza A virus infected immunocompromised patients admitted to ErasmusMC, 71 were hospitalized, since the start of the 2009 H1N1 pandemic. We identified 11 (15%) cases with prolonged 2009 pandemic virus replication (longer than 14 days), despite antiviral therapy. In 5 out of these 11 (45%) cases oseltamivir resistant H275Y viruses emerged. Given the inherent difficulties in studying antiviral efficacy in immunocompromised patients, we have infected immunocompromised ferrets with either wild-type, or oseltamivir-resistant (H275Y) 2009 pandemic virus. All ferrets showed prolonged virus shedding. In wild-type virus infected animals treated with oseltamivir, H275Y resistant variants emerged within a week after infection. Unexpectedly, oseltamivir therapy still proved to be partially protective in animals infected with resistant virus. Immunocompromised ferrets offer an attractive alternative to study efficacy of novel antiviral therapies., Author Summary Immunocompromised patients, such as transplant recipients on immune suppressive therapy, are a substantial and gradually expanding patient group. Upon influenza virus infection, these patients clear the virus less efficiently and are more likely to develop severe pneumonia than immunocompetent individuals. Existing antiviral strategies are far from satisfactory for this patient group, as they show limited effectiveness with frequent emergence of antiviral resistance. For ethical and practical reasons antiviral efficacy studies are hard to conduct in these patients. Therefore, we developed an immunocompromised ferret, mimicking an immune suppressive regimen used for solid organ transplant recipients. Upon infection with 2009 pandemic influenza A/H1N1 virus these animals, like immunocompromised patients, develop severe respiratory disease with prolonged virus excretion. Interestingly, all immunocompromised ferrets on oseltamivir therapy excreted oseltamivir resistant viruses (H275Y) within one week after start of treatment. Furthermore, high dose oseltamivir therapy still proved to be partially effective against these oseltamivir resistant viruses. These immunocompromised ferrets provide a useful tool in the development of novel antiviral approaches for immunocompromised patients suffering from influenza.

Wild bird movements and aggregations following spells of cold weather may have resulted in the spread of highly pathogenic avian influenza virus (HPAIV) H5N1 in Europe during the winter of 2005–2006. Waterbirds are constrained in winter to areas where bodies of water remain unfrozen in order to feed. On the one hand, waterbirds may choose to winter as close as possible to their breeding grounds in order to conserve energy for subsequent reproduction, and may be displaced by cold fronts. On the other hand, waterbirds may choose to winter in regions where adverse weather conditions are rare, and may be slowed by cold fronts upon their journey back to the breeding grounds, which typically starts before the end of winter. Waterbirds will thus tend to aggregate along cold fronts close to the 0°C isotherm during winter, creating conditions that favour HPAIV H5N1 transmission and spread. We determined that the occurrence of outbreaks of HPAIV H5N1 infection in waterbirds in Europe during the winter of 2005–2006 was associated with temperatures close to 0°C. The analysis suggests a significant spatial and temporal association of outbreaks caused by HPAIV H5N1 in wild birds with maximum surface air temperatures of 0°C–2°C on the day of the outbreaks and the two preceding days. At locations where waterbird census data have been collected since 1990, maximum mallard counts occurred when average and maximum surface air temperatures were 0°C and 3°C, respectively. Overall, the abundance of mallards (Anas platyrhynchos) and common pochards (Aythya ferina) was highest when surface air temperatures were lower than the mean temperatures of the region investigated. The analysis implies that waterbird movements associated with cold weather, and congregation of waterbirds along the 0°C isotherm likely contributed to the spread and geographical distribution of outbreaks of HPAIV H5N1 infection in wild birds in Europe during the winter of 2005–2006., Author Summary Highly pathogenic avian influenza virus of the H5N1 subtype emerged more than a decade ago in poultry in South-East Asia. In 2005, it spread outside Asia infecting both poultry and wild birds in the Middle East, Europe and Africa. Both trade of poultry and movements of wild birds were likely implicated in the spread of the infection; however, the ability of wild birds to carry the virus to novel geographical areas is still highly debated and remains obscure. In Europe, the virus mainly infected wild birds, and emergence coincided with a spell of cold weather, which is known to result in massive movements of wild waterbirds. In this paper, we demonstrate that movements of wild waterbirds associated with cold weather contributed to the spread and geographical distribution of outbreaks in Europe during the winter of 2005–2006. Higher density of wild waterbirds on bodies of water that remain unfrozen ahead of the freezing line likely favoured transmission of the virus and resulted in distinctive distribution of outbreaks at locations where surface air temperatures were 0°C–2°C. This has important implications for surveillance, which should target areas where temperatures are close to freezing in winter, especially in poultry-dense regions close to areas where waterfowl aggregate.