(1) To develop animal, especially nonhuman primate, models that mimic human disease: We have established nonhuman primate (NHPs) models using Cynomolgus macaques for several influenza A viruses including the swine-origin H1N1 (SOIV) and a seasonal H1N1 virus (Safronetz 2011), a seasonal H3N2 virus (Kobayashi 2009, poster), and H2N2 pandemic and H2N3 swine virus (Ma, in preparation). Infections differ in virus replication, clinical symptoms, disease progression and pathology. However, they mimic human disease quite well with the above mentioned strains. Seasonal influenza viruses H1N1 &H3N2 lead to either asymptomatic or fairly mild disease in Cynomolgus macaques. Shortly after infection animals show reduced food intake and occasionally mild respiratory signs. Gross pathology is limited to a few areas in the lungs. Histopathological investigations of those areas reveal alveolar edema and fibrin, hyaline membrane formation and type II alveolar pneumocyte hyperplasia. Animals recover quickly form the infection and clear virus within the first few days. There is limited evidence for virus shedding from mucosal membranes of the upper respiratory tract (Brining 2010;Safronetz 2011). The pandemic H2N2 strain A/Singapore/1/57 (H2N2) leads to a moderate infection compared to the seasonal strains with moderate clinical symptoms and respiratory signs. Lung infiltrates, gross pathology and histopathology are in general slightly enhanced compared to the infections with seasonal viruses. Animals clear the infection with a delay and fully recover. There is limited evidence for virus shedding from mucosal membranes of the upper respiratory tract (Ma, in preparation). We observed different degrees of clinical severity, gross pathology, lung infiltration and histology in macaques when infected with three distinct SOIV isolates. A Mexican strain isolated from a moderately sick human A/Mexico/4108/2009 (H1N1) (M4108) behaved similarly to the pandemic H2N2 strain. A California isolate from a sick boy with moderate disease A/California/4/2009 (H1N1) (Ca04) was more severe and another Mexico strain from a cluster of severe cases A/Mexico/4487/2009 (H1N1) (M4487) was severe in clinical disease and pathology. No infection was lethal but the animals with the severe infection had not completely cleared virus at the end of the study (day 14). This clearly indicates that a variety of SOIV strains co-circulate with different pathogenic potential (Brining 2010;Safronetz 2011;Feldmann, in preparation). Infection with the swine H2N3 virus A/swine/MS/06 (H2N3) was also more severe and was similar to the disease caused by the California strain of SOIV. Thus, this newly described swine virus has potential to infect primates and could potentially also infect humans (Ma, in preparation). Furthermore, we have established the ferret model for influenza viruses here at RML. In a first round of experiments we have compared disease progression and virus replication of two pandemic H1N1 viruses, M4108 and M4487. In contrast to the differences we observed in the nonhuman primate model, both infections did not really differ in the ferret model with both viruses causing mild respiratory disease (Tsuda, in preparation;Tsuda 2011, poster) In conclusion, we have established nonhuman primate models for seasonal and pandemic influenza A viruses which mimic influenza-associated disease in humans. These models seem to be of particular use for studying severe influenza disease. We also have established the influenza ferret models which we intend to use as screening models for influenza viruses. These models will in the future support research on intervention strategies (vaccines and therapeutics). (2) To identify and characterize determinants of pathogenicity in animal models: To better understand the features that define highly lethal influenza viruses, we exploited a technology for the artificial generation and modification of influenza viruses (reverse genetics system). Using this technology, we have generated a consensus pandemic SOIV (H1N1) virus (parent virus). In order to study virulence of the different strains (isolates) and the mechanisms of pathogenesis we have generated selected recombinant viruses and characterized those mutants in vitro. For this we have compared the genome sequences of two early circulating SOIV strains with different pathogenicity in humans;M4108 and M4487. Sequence comparison revealed amino acid changes in 5 genomic segments, PB2, PA, HA, NP and M. The recombinant viruses were generated on the background of M4108 by replacing the corresponding genomic segment using the reverse genetics system. We have extensively characterized the recombinant viruses in tissue culture and found that the growth kinetics are similar (Tsuda 2011, poster). We are planing to investigate the in vivo characteristics of these recombinant viruses first in ferrets followed by selected recombinants in macaques. (3) To identify potential targets for intervention: The inherent virulence properties of the 1918 virus together with the emergence of pandemic SOIV and the continued pandemic potential posed by circulation of the highly virulent H5N1 virus indicated the need for evaluating antiviral options against infections with virulent influenza viruses. Therefore, the recently developed Cynomolgus macaque model for the 1918 influenza virus was used for a drug efficacy study. We demonstrated that oseltamivir phosphate is effective in preventing severe disease in Cynomolgus macaques caused by the 1918 influenza virus if given prophylactically. Efficacy was reduced in a treatment regime through emergence of oseltamivir-resistant mutants that lead to death of one of four animals. This emphasizes the importance of implementing combination therapy and vaccination strategies early in a pandemic such as the current one caused by the emerged swine-origin H1N1 influenza virus. The study is currently still under revision after initial journal submission;the revision included further animal experiments. We are planing to resubmit shortly (Feldmann, in revision). (4)To develop cross-protective vaccines and test their efficacy in the developed animal models: In collaboration with Dr. J. Roses laboratory we have generated and characterized VSV-based vaccines that express the HK/156 (clade 0) H5 HA from the first position of the VSV genome. These vectors induce broadly cross-neutralizing antibodies against homologous and heterologous H5N1 viruses of different clades in mice. The vaccines provide complete protection against morbidity and mortality after heterologous challenge with clade 0 and clade 1 strains in animals even 1 year after vaccination. Post-challenge pulmonary virus loads show that these vectors provide sterilizing immunity. Therefore, VSV-based avian influenza vaccines are potent, broadly cross-protective pandemic vaccine candidates (Schwartz 2010). In the next fiscal year we will continue our efforts to develop influenza vaccines. (5) Increased pathogenicity upon viral and bacterial co-infection in nonhuman primates: Severe influenza virus infections are often associated with bacterial co-infections. This seems also be the case with the current pandemic SOIV strains. In order to study a potentiating effect of influenza and bacterial co-infection we performed a co-infection study in cynomolgus macaques using a moderately severe pandemic H1N1 strain (Ca04) and Methicillin-resistant Staphylococcus aureus (MRSA). Animals infected with MRSA were either asymptomatic or developed mild clinical symptoms. Animals infected with Ca04 developed mild to moderate clinical symptoms. Upon co-infection at different time points there was no significant increase in clinical disease in any of the animals. Virology and histopathology are still ongoing.

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de Wit, Emmie; Feldmann, Friederike; Okumura, Atsushi et al. (2018) Prophylactic and therapeutic efficacy of mAb treatment against MERS-CoV in common marmosets. Antiviral Res 156:64-71
Malachowa, Natalia; Freedman, Brett; Sturdevant, Daniel E et al. (2018) Differential Ability of Pandemic and Seasonal H1N1 Influenza A Viruses To Alter the Function of Human Neutrophils. mSphere 3:
de Wit, Emmie; Siegers, Jurre Y; Cronin, Jacqueline M et al. (2018) 1918 H1N1 Influenza Virus Replicates and Induces Proinflammatory Cytokine Responses in Extrarespiratory Tissues of Ferrets. J Infect Dis 217:1237-1246
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Falzarano, Darryl; Kamissoko, Badian; de Wit, Emmie et al. (2017) Dromedary camels in northern Mali have high seropositivity to MERS-CoV. One Health 3:41-43
de Wit, Emmie; Feldmann, Friederike; Horne, Eva et al. (2017) Domestic Pig Unlikely Reservoir for MERS-CoV. Emerg Infect Dis 23:985-988
van Doremalen, Neeltje; Falzarano, Darryl; Ying, Tianlei et al. (2017) Efficacy of antibody-based therapies against Middle East respiratory syndrome coronavirus (MERS-CoV) in common marmosets. Antiviral Res 143:30-37
de Wilde, Adriaan H; Falzarano, Darryl; Zevenhoven-Dobbe, Jessika C et al. (2017) Alisporivir inhibits MERS- and SARS-coronavirus replication in cell culture, but not SARS-coronavirus infection in a mouse model. Virus Res 228:7-13
Prescott, Joseph; Feldmann, Heinz; Safronetz, David (2017) Amending Koch's postulates for viral disease: When ""growth in pure culture"" leads to a loss of virulence. Antiviral Res 137:1-5
Borisevich, Viktoriya; Lee, Benhur; Hickey, Andrew et al. (2016) Escape From Monoclonal Antibody Neutralization Affects Henipavirus Fitness In Vitro and In Vivo. J Infect Dis 213:448-55

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