Evolutionary Analysis of Inter-Farm Transmission Dynamics in a Highly Pathogenic Avian Influenza Epidemic

Phylogenetic studies have largely contributed to better understand the emergence, spread and evolution of highly pathogenic avian influenza during epidemics, but sampling of genetic data has never been detailed enough to allow mapping of the spatiotemporal spread of avian influenza viruses during a single epidemic. Here, we present genetic data of H7N7 viruses produced from 72% of the poultry farms infected during the 2003 epidemic in the Netherlands. We use phylogenetic analyses to unravel the pathways of virus transmission between farms and between infected areas. In addition, we investigated the evolutionary processes shaping viral genetic diversity, and assess how they could have affected our phylogenetic analyses. Our results show that the H7N7 virus was characterized by a high level of genetic diversity driven mainly by a high neutral substitution rate, purifying selection and limited positive selection. We also identified potential reassortment in the three genes that we have tested, but they had only a limited effect on the resolution of the inter-farm transmission network. Clonal sequencing analyses performed on six farm samples showed that at least one farm sample presented very complex virus diversity and was probably at the origin of chronological anomalies in the transmission network. However, most virus sequences could be grouped within clearly defined and chronologically sound clusters of infection and some likely transmission events between farms located 0.8–13 Km apart were identified. In addition, three farms were found as most likely source of virus introduction in distantly located new areas. These long distance transmission events were likely facilitated by human-mediated transport, underlining the need for strict enforcement of biosafety measures during outbreaks. This study shows that in-depth genetic analysis of virus outbreaks at multiple scales can provide critical information on virus transmission dynamics and can be used to increase our capacity to efficiently control epidemics

Role of Position 627 of PB2 and the Multibasic Cleavage Site of the Hemagglutinin in the Virulence of H5N1 Avian Influenza Virus in Chickens and Ducks

Highly pathogenic H5N1 avian influenza viruses have caused major disease outbreaks in domestic and free-living birds with transmission to humans resulting in 59% mortality amongst 564 cases. The mutation of the amino acid at position 627 of the viral polymerase basic-2 protein (PB2) from glutamic acid (E) in avian isolates to lysine (K) in human isolates is frequently found, but it is not known if this change affects the fitness and pathogenicity of the virus in birds. We show here that horizontal transmission of A/Vietnam/1203/2004 H5N1 (VN/1203) virus in chickens and ducks was not affected by the change of K to E at PB2-627. All chickens died between 21 to 48 hours post infection (pi), while 70% of the ducks survived infection. Virus replication was detected in chickens within 12 hours pi and reached peak titers in spleen, lung and brain between 18 to 24 hours for both viruses. Viral antigen in chickens was predominantly in the endothelium, while in ducks it was present in multiple cell types, including neurons, myocardium, skeletal muscle and connective tissues. Virus replicated to a high titer in chicken thrombocytes and caused upregulation of TLR3 and several cell adhesion molecules, which may explain the rapid virus dissemination and location of viral antigen in endothelium. Virus replication in ducks reached peak values between 2 and 4 days pi in spleen, lung and brain tissues and in contrast to infection in chickens, thrombocytes were not involved. In addition, infection of chickens with low pathogenic VN/1203 caused neuropathology, with E at position PB2-627 causing significantly higher infection rates than K, indicating that it enhances virulence in chickens

Spherical nanocomposite particles prepared from mixed cellulose–chitosan solutions

Novel cellulose–chitosan nanocomposite particles with spherical shape were successfully prepared via mixing of aqueous biopolymer solutions in three different ways. Macroparticles with diameters in the millimeter range were produced by dripping cellulose dissolved in cold LiOH/urea into acidic chitosan solutions, inducing instant co-regeneration of the biopolymers. Two types of microspheres, chemically crosslinked and non-crosslinked, were prepared by first mixing cellulose and chitosan solutions obtained from freeze thawing in LiOH/KOH/urea. Thereafter epichlorohydrin was applied as crosslinking agent for one of the samples, followed by water-in-oil (W/O) emulsification, heat induced sol–gel transition, solvent exchange, washing and freeze-drying. Characterization by X-ray photoelectron spectroscopy, total elemental analysis, and Fourier transform infrared spectroscopy confirmed the prepared particles as being true cellulose–chitosan nanocomposites with different distribution of chitosan from the surface to the core of the particles depending on the preparation method. Field emission scanning electron microscopy and laser diffraction was performed to study the morphology and size distribution of the prepared particles. The morphology was found to vary due to different preparation routes, revealing a core shell structure for macroparticles prepared by dripping, and homogenous nanoporous structure for the microspheres. The non-crosslinked microparticles exhibited a somewhat denser structure than the crosslinked ones, which indicated that crosslinking restricts packing of the chains before and under regeneration. From the obtained volume-weighted size distributions it was found that the crosslinked microspheres had the highest median diameter. The results demonstrate that not only the mixing ratio and distribution of the two biopolymers, but also the morphology and nanocomposite particle diameters are tunable by choosing between the different routes of preparation.First Online: 05 August 2016</p