The 2009 flu pandemic and the appearance of oseltamivir-resistant H1N1 influenza strains highlight the need for treatment alternatives. One such option is the creation of a protective physical barrier in the nasal cavity. In vitro tests demonstrated that iota-carrageenan is a potent inhibitor of influenza A virus infection, most importantly also of pandemic H1N1/2009 in vitro. Consequently, we tested a commercially available nasal spray containing iota-carrageenan in an influenza A mouse infection model. Treatment of mice infected with a lethal dose of influenza A PR8/34 H1N1 virus with iota-carrageenan starting up to 48 hours post infection resulted in a strong protection of mice similar to mice treated with oseltamivir. Since alternative treatment options for influenza are rare, we conclude that the nasal spray containing iota-carrageenan is an alternative to neuraminidase inhibitors and should be tested for prevention and treatment of influenza A in clinical trials in humans.

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Adult, healthy mute swans were experimentally infected with highly pathogenic avian influenza virus A/ Cygnus cygnus /Germany/R65/2006 subtype H5N1. Immunologically naive birds died, whereas animals with preexisting, naturally acquired avian influenza virus–specific antibodies became infected asymptomatically and shed virus. Adult mute swans are highly susceptible, excrete virus, and can be clinically protected by preexposure immunity.

The potential role of wild birds as carriers of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 is still a matter of debate. Consecutive or simultaneous infections with different subtypes of influenza viruses of low pathogenicity (LPAIV) are very common in wild duck populations. To better understand the epidemiology and pathogenesis of HPAIV H5N1 infections in natural ecosystems, we investigated the influence of prior infection of mallards with homo- (H5N2) and heterosubtypic (H4N6) LPAIV on exposure to HPAIV H5N1. In mallards with homosubtypic immunity induced by LPAIV infection, clinical disease was absent and shedding of HPAIV from respiratory and intestinal tracts was grossly reduced compared to the heterosubtypic and control groups (mean GEC/100 µl at 3 dpi: 3.0×102 vs. 2.3×104 vs. 8.7×104; p<0.05). Heterosubtypic immunity induced by an H4N6 infection mediated a similar but less pronounced effect. We conclude that the epidemiology of HPAIV H5N1 in mallards and probably other aquatic wild bird species is massively influenced by interfering immunity induced by prior homo- and heterosubtypic LPAIV infections.

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We assessed the prediction that access of the viral NS1 protein to cellular PDZ domain protein networks enhances the virulence of highly pathogenic avian influenza A viruses. The NS1 proteins of most avian influenza viruses bear the C-terminal ligand sequence Glu-Ser-Glu-Val (ESEV) for PDZ domains present in multiple host proteins, whereas no such motif is found in the NS1 homologues of seasonal human virus strains. Previous analysis showed that a C-terminal ESEV motif increases viral virulence when introduced into the NS1 protein of mouse-adapted H1N1 influenza virus. To examine the role of the PDZ domain ligand motif in avian influenza virus virulence, we generated three recombinants, derived from the prototypic H5N1 influenza A/Vietnam/1203/04 virus, expressing NS1 proteins that either have the C-terminal ESEV motif or the human influenza virus RSKV consensus or bear a natural truncation of this motif, respectively. Cell biological analyses showed strong control of NS1 nuclear migration in infected mammalian and avian cells, with only minor differences between the three variants. The ESEV sequence attenuated viral replication on cultured human, murine, and duck cells but not on chicken fibroblasts. However, all three viruses caused highly lethal infections in mice and chickens, with little difference in viral titers in organs, mean lethal dose, or intravenous pathogenicity index. These findings demonstrate that a PDZ domain ligand sequence in NS1 contributes little to the virulence of H5N1 viruses in these hosts, and they indicate that this motif modulates viral replication in a strain-and host-dependent manner.

BackgroundBy using animal sera as sentinels, natural TBEV foci could be identified and further analyses including investigations of ticks could be initiated. However, antibody response against TBEV-related flaviviruses might adversely affect the readout of such a monitoring. Therefore, the cross-reactivity of the applied TBEV serology test systems – enzyme linked immunosorbent assay (ELISA) and virus neutralization test (VNT) – as well as the longevity of TBEV antibody titres in sheep and goats were investigated in this study.ResultsCross-reactivity of the TBEV antibody test systems with defined antibody-positive samples against selected members of the Flaviviridae family (e.g. Louping ill virus, West Nile virus) was observed for Louping-ill-positive sera only. In contrast, the commercial West Nile virus (WNV) competitive ELISA showed a high level of cross-reactivity with TBEV-specific positive sera.To assess the longevity of TBEV antibody titres, sera from two sheep and two goats, which had been immunized four times with a commercially available TBEV vaccine, were tested routinely over 28 months. In three of the four animals, TBEV-specific antibody titres could be detected over the whole test period.In addition, sera from the years 2010 and 2011 were collected in flocks in different villages of Baden-Württemberg and Thuringia to allow re-examination two to four years after the initial analysis. Interestingly, in most cases the results of the former investigations were confirmed, which may be caused by steadily existing natural TBEV foci.ConclusionCross-reactivity must be taken into consideration, particularly for TBEV serology in regions with a prevalence of Louping ill virus and for serological testing of WNV by cross-reactive ELISAs. Furthermore, over-interpretation of single TBEV-positive serological results should be avoided, especially in areas without a TBEV history.

Highly pathogenic avian influenza (HPAI) is a striking disease in susceptible poultry, which leads to severe economic losses. Inactivated vaccines are the most widely used vaccines in avian influenza virus (AIV) vaccination programs. However, these vaccines interfere with the serological detection of wild-type AIV infections in immunized populations. The use of vaccines that allow differentiation between infected and vaccinated animals (DIVA strategy) would stop current stamping-out policies. Therefore, novel vaccination strategies are needed to allow improved protection of animals and humans against HPAI virus (HPAIV) infection. The presented study analyzed for the first time the immunogenic capacity of plant-expressed full-length hemagglutinin (rHA0) of HPAIV H5N1 in several vaccine formulations within the highly relevant host species chicken. We were able to express plant-expressed rHA0 at high levels and could show that, when administered with potent adjuvants, it is highly immunogenic and can fully protect chicken against lethal challenge infection. Real-time reverse transcription (RT)-PCR and serological tests demonstrated only marginally increased virus replication in animals vaccinated with plant-derived rHA0 compared to animals immunized with an inactivated reference vaccine. In addition, the use of plant-expressed rHA0 also allowed an easy serological differentiation of vaccinated from AIV-infected animals based on antibodies against the influenza virus NP protein.Highly pathogenic avian influenza (AI) (HPAI) is a striking disease in susceptible poultry, which leads to severe economic losses (21). Since 2003, the H5N1 HPAI epidemic has claimed over 220 million poultry and other birds either through direct mortality from infection or from preemptive culling (22). The implementation of vaccination of poultry as a tool for the reduction of the viral load in the environment and, thus, for decreasing the risk of transmission within poultry-and, as a consequence, to humans-is still a discussed topic. Inactivated vaccines are the most widely used vaccines in AI vaccination programs. They are particularly addressed to protect adult chickens, turkeys, and other birds in emergency situations, e.g., when ring vaccination is used in an area of an HPAI virus (HPAIV) outbreak or when prophylactic vaccination is used in a region where H5 or H7 AI virus (AIV) infections are endemic. However, these vaccines limited the serological detection of wild-type AIV infections in immunized populations, as wild-type infection could be detectable only through higher antibody titers to nonstructural proteins or if the neuraminidase subtype of the vaccine differed from the subtype of the introduced wild-type virus (28).The use of vaccines that fit in any case to the strategy of differentiating infected from vaccinated animals (DIVA) would make a strong case for turning away from current stamping-out policies in many countries. Vaccines that consist of only one major antigenic protein related to influenza A virus (e.g., hemagglutini...