Kerstin Tauscher

Avian Influenza H7N9/13 and H7N7/13: a Comparative Virulence Study in Chickens, Pigeons, and Ferrets

ABSTRACT Human influenza cases caused by a novel avian H7N9 virus in China emphasize the zoonotic potential of that subtype. We compared the infectivity and pathogenicity of the novel H7N9 virus with those of a recent European avian H7N7 strain in chickens, pigeons, and ferrets. Neither virus induced signs of disease despite substantial replication in inoculated chickens and rapid transmission to contact chickens. Evidence of the replication of both viruses in pigeons, albeit at lower levels of RNA excretion, was also detected. No clear-cut differences between the two H7 isolates emerged regarding replication and antibody development in avian hosts. In ferrets, in contrast, greater replication of the avian H7N9 virus than of the H7N7 strain was observed with significant differences in viral presence, e.g., in nasal wash, lung, and cerebellum samples. Importantly, both viruses showed the potential to spread to the mammal brain. We conclude that efficient asymptomatic viral replication and shedding, as shown in chickens, facilitate the spread of H7 viruses that may harbor zoonotic potential. Biosafety measures are required for the handling of poultry infected with avian influenza viruses of the H7 subtype, independently of their pathogenicity for gallinaceous poultry. IMPORTANCE This study is important to the field since it provides data about the behavior of the novel H7N9 avian influenza virus in chickens, pigeons, and ferrets in comparison with that of a recent low-pathogenicity H7N7 strain isolated from poultry. We clearly show that chickens, but not pigeons, are highly permissive hosts of both H7 viruses, allowing high-titer replication and virus shedding without any relevant clinical signs. In the ferret model, the potential of both viruses to infect mammals could be demonstrated, including infection of the brain. However, the replication efficiency of the H7N9 virus in ferrets was higher than that of the H7N7 strain. In conclusion, valuable data for the risk analysis of low-pathogenicity avian influenza viruses of the H7 subtype are provided that could also be used for the risk assessment of zoonotic potentials and necessary biosafety measures.

Effect of Q211 and K222PRNP Polymorphic Variants in the Susceptibility of Goats to Oral Infection With Goat Bovine Spongiform Encephalopathy

BACKGROUND The prion protein-encoding gene (PRNP) is one of the major determinants for scrapie occurrence in sheep and goats. However, its effect on bovine spongiform encephalopathy (BSE) transmission to goats is not clear. METHODS Goats harboring wild-type, R/Q211 or Q/K222 PRNP genotypes were orally inoculated with a goat-BSE isolate to assess their relative susceptibility to BSE infection. Goats were killed at different time points during the incubation period and after the onset of clinical signs, and their brains as well as several peripheral tissues were analyzed for the accumulation of pathological prion protein (PrP(Sc)) and prion infectivity by mouse bioassay. RESULTS R/Q211 goats displayed delayed clinical signs compared with wild-type goats. Deposits of PrP(Sc) were detected only in brain, whereas infectivity was present in peripheral tissues too. In contrast, none of the Q/K222 goats showed any evidence of clinical prion disease. No PrP(Sc) accumulation was observed in their brains or peripheral tissues, but very low infectivity was detected in some tissues very long after inoculation (44-45 months). CONCLUSIONS These results demonstrate that transmission of goat BSE is genotype dependent, and they highlight the pivotal protective effect of the K222 PRNP variant in the oral susceptibility of goats to BSE.

A Unique Multibasic Proteolytic Cleavage Site and Three Mutations in the HA2 Domain Confer High Virulence of H7N1 Avian Influenza Virus in Chickens

ABSTRACT In 1999, after circulation for a few months in poultry in Italy, low-pathogenic (LP) avian influenza (AI) H7N1 virus mutated into a highly pathogenic (HP) form by acquisition of a unique multibasic cleavage site (mCS), PEIPKGSRVRR*GLF (asterisk indicates the cleavage site), in the hemagglutinin (HA) and additional alterations with hitherto unknown biological function. To elucidate these virulence-determining alterations, recombinant H7N1 viruses carrying specific mutations in the HA of LPAI A/chicken/Italy/473/1999 virus (Lp) and HPAI A/chicken/Italy/445/1999 virus (Hp) were generated. Hp with a monobasic CS or carrying the HA of Lp induced only mild or no disease in chickens, thus resembling Lp. Conversely, Lp with the HA of Hp was as virulent and transmissible as Hp. While Lp with a multibasic cleavage site (Lp_CS445) was less virulent than Hp, full virulence was exhibited when HA2 was replaced by that of Hp. In HA2, three amino acid differences consistently detected between LP and HP H7N1 viruses were successively introduced into Lp_CS445. Q450L in the HA2 stem domain increased virulence and transmission but was detrimental to replication in cell culture, probably due to low-pH activation of HA. A436T and/or K536R restored viral replication in vitro and in vivo. Viruses possessing A436T and K536R were observed early in the HPAI outbreak but were later superseded by viruses carrying all three mutations. Together, besides the mCS, stepwise mutations in HA2 increased the fitness of the Italian H7N1 virus in vivo. The shift toward higher virulence in the field was most likely gradual with rapid optimization. IMPORTANCE In 1999, after 9 months of circulation of low-pathogenic (LP) avian influenza virus (AIV), a devastating highly pathogenic (HP) H7N1 AIV emerged in poultry, marking the largest epidemic of AIV reported in a Western country. The HPAIV possessed a unique multibasic cleavage site (mCS) complying with the minimum motif for HPAIV. The main finding in this report is the identification of three mutations in the HA2 domain that are required for replication and stability, as well as for virulence, transmission, and tropism of H7N1 in chickens. In addition to the mCS, Q450L was required for full virulence and transmissibility of the virus. Nonetheless, it was detrimental to virus replication and required A436T and/or K536R to restore replication, systemic spread, and stability. These results are important for better understanding of the evolution of highly pathogenic avian influenza viruses from low-pathogenic precursors.

On the situation of African swine fever and the biological characterization of recent virus isolates

Die Afrikanische Schweinepest (ASP) ist eine bei der Weltorganisation fur Tiergesundheit (OIE) anzeigepflichtige Tierseuche, die mit schweren, unspezifischen Symptomen und einer hohen Mortalitat einhergeht. Empfanglich fur das gleichnamige DNA-Virus (ASPV) sind ausschlieslich Haus- und Wildschweine sowie Zecken der Gattung Ornithodoros. Da es bislang keinen zugelassenen Impfstoff gibt, beruht die Bekampfung ausschlieslich auf strikten veterinarhygienischen Masnahmen. Wichtigster Bestandteil dieser Masnahmen ist eine fruhzeitige Entdeckung einer Infektion in betroffenen Schweinebestanden und eine schnelle und zuverlassige Labordiagnose. Infektionen mit aktuellen Virusisolaten aus Armenien, Sardinien, Litauen oder Kenia fuhren bei Haus- und Wildschweinen zu akuten Krankheitsverlaufen, deren Kardinalsymptom sehr hohes Fieber (> 41 °C) ist. Begleitet wird dies von diversen unspezifischen Symptomen wie Lethargie, Inappetenz, Diarrhoe, respiratorischen Symptomen sowie einer erhohten Blutungsneigung. In experimentellen Untersuchungen lag die Mortalitat bei 100 %. Bei den durchgefuhrten pathologischanatomischen Untersuchungen wurden insbesondere ebenholzfarbene gastrohepatische Lymphknoten, Lungenodeme, Petechien in der Nierenrinde und Gallenblasenwandodeme gefunden. Vor dem Hintergrund der aktuellen Seuchenlage mit einem endemischen Geschehen auf Sardinien, Ausbruchen in Russland sowie mehreren ostlichen EU-Mitgliedstaaten und dem damit verbundenen Risiko fur die Einschleppung der ASP in weitere Staaten der EU, unter anderem auch Deutschland, sollten bei unspezifischer Klinik und Pathologie in Hausschweinebestanden geeignete Proben, wie Serum, EDTA-Blut und Milzmaterial differenzialdiagnostisch auf ASP und klassische Schweinepest untersucht werden.

Immunolabelling of non-phosphorylated neurofilament indicates damage of spinal cord axons in TSE-infected goats

TRANSMISSIBLE spongiform encephalopathies (TSEs) including bovine spongiform encephalopathy (BSE) are devastating neurodegenerative disorders caused by conversion of the normal cellular prion protein (PrPC) into an abnormal isoform (PrPSc; Prusiner, 1982, Chesebro, 2003). Following the discovery of goat BSE cases in the UK and France (Eloit and others 2005, Jeffrey and others 2006, Spiropoulos and others 2011) small ruminants were considered to pose a BSE infection risk/source for cattle, and human beings in particular. In goats experimental TSE susceptibility strongly depends on polymorphisms in the prion protein gene ( PRNP ) (Aguilar-Calvo and others 2014, 2015, Lacroux and others 2014). In particular, goats with R/Q211 polymorphism (IQQ/IRQ; single-letter amino acid code) and Q/K222 polymorphism (IRK/IRQ) show delayed or even absent clinical signs compared with wild-type goats (IRQ/IRQ) (Aguilar-Calvo and others 2015). After oral infection of ruminants, the agent is shown to spread among others via the autonomous nervous system, ultimately resulting in manifested disease in the brain and spinal cord (van Keulen and others 2002, Hoffmann and others 2007, Kaatz and others 2012). So far however, little attention has been paid to the cellular and molecular mechanisms that facilitate the spread of prions within the nervous system as well as to the involvement of the spinal cord in the pathogenesis of TSEs, even though PrPSc has been detected in this part of the nervous tissue (Flechsig and others 2000, Fukuda and others 2012, Kaatz and others 2012). The aim of the present study was to elucidate potential changes in the immunoreactivity of various immunohistochemical markers, in particular in regions of the sympathetic infection route, which have been demonstrated to represent key locations for the spread of the BSE agent (Kaatz and others 2012), including the coeliac and …

Progressive glycosylation of the haemagglutinin of avian influenza H5N1 modulates virus replication, virulence and chicken-to-chicken transmission without significant impact on antigenic drift

Highly pathogenic H5N1 avian influenza virus (A/H5N1) devastated the poultry industry and continues to pose a pandemic threat. Studying the progressive genetic changes in A/H5N1 after long-term circulation in poultry may help us to better understand A/H5N1 biology in birds. A/H5N1 clade 2.2.1.1 antigenic drift viruses have been isolated from vaccinated commercial poultry in Egypt. They exhibit a peculiar stepwise accumulation of glycosylation sites (GS) in the haemagglutinin (HA) with viruses carrying, beyond the conserved 5 GS, additional GS at amino acid residues 72, 154, 236 and 273 resulting in 6, 7, 8 or 9 GS in the HA. Available information about the impact of glycosylation on virus fitness and pathobiology is mostly derived from mammalian models. Here, we generated recombinant viruses imitating the progressive acquisition of GS in HA and investigated their biological relevance in vitro and in vivo. Our in vitro results indicated that the accumulation of GS correlated with increased glycosylation, increased virus replication, neuraminidase activity, cell-to-cell spread and thermostability, however, strikingly, without significant impact on virus escape from neutralizing antibodies. In vivo, glycosylation modulated virus virulence, tissue tropism, replication and chicken-to-chicken transmission. Predominance in the field was towards viruses with hyperglycosylated HA. Together, progressive glycosylation of the HA may foster persistence of A/H5N1 by increasing replication, stability and bird-to-bird transmission without significant impact on antigenic drift.

Histopathological and Immunohistochemical Studies of Cowpox Virus Replication in a Three-Dimensional Skin Model

Human cowpox virus (CPXV) infections are rare, but can result in severe and sometimes fatal outcomes. The majority of recent cases were traced back to contacts with infected domestic cats or pet rats. The aim of the present study was to evaluate a three-dimensional (3D) skin model as a possible replacement for animal experiments. We monitored CPXV lesion formation, viral gene expression and cell cycle patterns after infection of 3D skin cultures with two CPXV strains of different pathogenic potential: a recent pet rat isolate (RatPox09) and the reference Brighton red strain. Infected 3D skin cultures exhibited histological alterations that were similar to those of mammal skin infections, but there were no differences in gene expression patterns and tissue damage between the two CPXV strains in the model system. In conclusion, 3D skin cultures reflect the development of pox lesions in the skin very well, but seem not to allow differentiation between more or less virulent virus strains, a distinction that is made possible by experimental infection in suitable animal models.

Characterization of Simbu serogroup virus infections in type I interferon receptor knock-out mice

In late 2011, Schmallenberg virus (SBV), a novel, arthropod-borne, teratogenic orthobunyavirus, emerged near the German/Dutch border and thereafter spread rapidly throughout the continent thereby causing great economic losses in European livestock. SBV mainly infects ruminants and closely related viruses such as Sabo virus (SABOV), Simbu virus (SIMBUV) and Sathuperi virus (SATV) have been isolated from their insect-vectors or putative ruminant hosts. However, information about their pathogenesis and in vivo studies with SABOV, SIMBUV, and SATV are scarce. As experimental infections of ruminants are comprehensive and time-consuming, an SBV small animal model was assessed regarding its suitability for studying Simbu viruses. Adult type I interferon deficient mice (IFNAR-/-) were subcutaneously infected with the Simbu serogroup members SABOV, SIMV and SATV, respectively, and compared to SBV-infected mice. All animals were clinically, virologically, serologically, and pathologically examined. The clinical signs were mainly characterised by the loss of body weight and by paralysis. In blood, and samples from the spleen and brain, high loads of viral genome were detected using newly developed real-time PCR assays. The most common histologic lesions included meningo-encephalomyelitis, perivascular cuffing of lymphocytes and macrophages, neuronal degeneration and gliosis. These lesions have also been described in foetuses after transplacental infection with SBV. In-situ hybridisation signals were widely distributed in multiple neurons of the brain and spinal cord in all examined, inoculated mice. In conclusion, IFNAR-/- mice are a suitable animal model for pathogenesis studies of a broad range of Simbu serogroup viruses since all the viruses examined displayed a common pattern of viral organ and tissue distribution in this mouse model.