Adelaide Milani

Antigenic Drift in H5N1 Avian Influenza Virus in Poultry Is Driven by Mutations in Major Antigenic Sites of the Hemagglutinin Molecule Analogous to Those for Human Influenza Virus

ABSTRACT H5N1 highly pathogenic avian influenza virus has been endemic in poultry in Egypt since 2008, notwithstanding the implementation of mass vaccination and culling of infected birds. Extensive circulation of the virus has resulted in a progressive genetic evolution and an antigenic drift. In poultry, the occurrence of antigenic drift in avian influenza viruses is less well documented and the mechanisms remain to be clarified. To test the hypothesis that H5N1 antigenic drift is driven by mechanisms similar to type A influenza viruses in humans, we generated reassortant viruses, by reverse genetics, that harbored molecular changes identified in genetically divergent viruses circulating in the vaccinated population. Parental and reassortant phenotype viruses were antigenically analyzed by hemagglutination inhibition (HI) test and microneutralization (MN) assay. The results of the study indicate that the antigenic drift of H5N1 in poultry is driven by multiple mutations primarily occurring in major antigenic sites at the receptor binding subdomain, similarly to what has been described for human influenza H1 and H3 subtype viruses.

Highly pathogenic avian influenza H5N1 subtype in mute swans in Italy.

SIR, – The mute swan (Cygnus olor) is a semi-sedentary, migratory species that is particularly common in Eurasia and Europe north of the 46° parallel. In Italy, the nesting population does not exceed 500 couples, but as an overwintering species it is relatively common. The overwintering birds migrating from eastern Europe generally prefer the wetlands in northern/central Italy. However, due to persistent low temperatures during the past winter, some individuals reached the coastal regions of southern Italy. The first cases of dead birds or of birds with suspect clinical signs were reported simultaneously from three southern regions, Calabria, Sicily and Puglia, to the Italian Reference Laboratory in the first week of February. The dead or sick birds were part of small flocks of 10 to 20 birds that included several young individuals. The general condition of the majority of the birds was poor, as is normally seen at the end of the winter season. Ill birds exhibited nervous signs such as somnolence, incoordination and ataxia. Other clinical signs observed were greenish diarrhoea and conjunctivitis. Gross pathology in dead or euthanased birds included hyperaemia and haemorrhage of the mucosal surface of the proventriculus and duodenum, airsacculitis, catarrhal rhinosinusitis and serohaemorrhagic tracheobronchitis. In total, the presence of the highly pathogenic H5N1 virus was confirmed in 16 swans all originating from the south of Italy. Although the birds did not have any identification tags, migration routes indicate that they most probably originated from the Black Sea. Phylogenetic analysis of the isolates from the Italian swans revealed a very high homology to other highly pathogenic avian influenza H5N1 isolates from wild birds in Asia and Europe (Table 1). Information is lacking on the incubation period, the duration and extent of shedding and other aspects of infection, which makes any forecast of the role of these birds in avian influenza epidemiology impossible. The greatest concern is that infection could have spread to resident wild bird populations, generating an endemic cycle. Increased surveillance in wild bird populations across Europe is crucial to the early detection of infection, which is a prerequisite to the prevention of infection spreading to rural and intensively reared poultry.

The combination of marker gene swapping and fluorescence-activated cell sorting improves the efficiency of recombinant modified vaccinia virus Ankara vaccine production for human use.

Modified vaccinia virus Ankara (MVA) is employed as a human vaccine vector for the high expression of heterologous genes and the lack of replication in mammalian cells. This study demonstrates that cells infected by recombinant viruses can be obtained by fluorescence-activated cell sorting. Recombinant viruses are generated by a swapping event between a red fluorescent protein gene in the acceptor virus and a plasmid cassette coding for both a green fluorescent marker and a transgene. To prevent the carry-over of parental virus, due to superinfection of the cells harbouring recombinant viruses, the sorting is performed on cells infected at low m.o.i. in the presence of a reversible inhibitor of viral particle release. Terminal dilution cloning is then used to isolate both green and marker-free recombinant viruses, which can be identified by whole-plate fluoroimaging. The differential visualization of all the viral types involved allows a stepwise monitoring of all recombinations and leads to a straightforward and efficient flow cytometry-based cell sorting purification protocol. As an example of the efficacy of this sorting procedure, the construction of rMVAs coding for the rat nuclear protein HMGB1 and H5N1 influenza A virus hemagglutinin is reported. The entire recombinant MVA production process is carried out in serum-free media employing primary chicken embryo fibroblasts (CEF), which are certified for the preparation of human vaccines. This rMVA production method is faster, simpler and more reliable than any other available procedure for obtaining safe vaccine stocks for human use.

The Quenching Effect of Flavonoids on 4-Methylumbelliferone, a Potential Pitfall in Fluorimetric Neuraminidase Inhibition Assays

Many assays aimed to test the inhibitory effects of synthetic molecules, and naturally occurring products on the neuraminidase activity exploit the hydrolysis of 2′-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (4-MUNANA). The amount of the released product, 4-methylumbelliferone (4-MU), is then measured fluorimetrically. The authors attempted an analysis of the inhibitory properties of 35 naturally occurring flavonoids on neuraminidase N3, where only 29 of them were sufficiently soluble in the assay medium. During the analysis, the authors noticed a strong quenching effect due to the test compounds on the fluorescence of 4-MU. The quenching constants for the flavonoids were determined according to the Stern-Volmer approach. The extent of fluorescence reduction due to quenching and the magnitude of the fluorescence reduction measured in the inhibition assays were comparable: for 11 of 29 compounds, the two values were found to be coincident within the experimental uncertainty. These data were statistically analyzed for correlation by calculating the pertinent Pearson correlation coefficient. Inhibition and quenching were found to be positively correlated (r = 0.71, p(uncorr) = 1.5 × 10−5), and the correlation was maintained for the whole set of tested compounds. Altogether, the collected data imply that all of the tested flavonoids could produce false-positive results in the neuraminidase inhibition assay using 4-MUNANA as a substrate.

Genetic Diversity of Highly Pathogenic Avian Influenza A(H5N8/H5N5) Viruses in Italy, 2016–17

In winter 2016–17, highly pathogenic avian influenza A(H5N8) and A(H5N5) viruses of clade 2.3.4.4 were identified in wild and domestic birds in Italy. We report the occurrence of multiple introductions and describe the identification in Europe of 2 novel genotypes, generated through multiple reassortment events.

In vitro study of the replication capacity of the RGNNV and the SJNNV betanodavirus genotypes and their natural reassortants in response to temperature

Betanodaviruses are the causative agents of viral nervous necrosis and affect a broad range of fish species worldwide. Their bi-segmented genome is composed of the RNA1 and the RNA2 molecules encoding the viral polymerase and the coat protein, respectively. In southern Europe the presence of the RGNNV and the SJNNV genotypes, and the RGNNV/SJNNV and RGNNV/SJNNV reassortants has been documented. Several studies have reported a correlation between water temperature and disease onset. To explore the replication efficiency of betanodaviruses with different genomes in relation to temperature and to understand the role of genetic reassortment on viral phenotype, RGNNV, SJNNV, RGNNV/SJNNV and RGNNV/SJNNV field isolates were fully sequenced, and growth curves generated in vitro at four different temperatures (15, 20, 25, 30 °C) were developed for each isolate. The data obtained, corroborated by statistical analysis, demonstrated that viral titres of diverse betanodavirus genotypes varied significantly in relation to the incubation temperature of the culture. In particular, at 30 °C betanodaviruses under investigation presented different phenotypes, and viruses containing the RNA1 of the RGNNV genotype showed the best replication efficiency. Laboratory results demonstrated that viruses clustering within the same genotype based on the polymerase gene, possess similar growth kinetics in response to temperature, thus highlighting the key role of RNA1 in controlling viral replication at different environmental conditions. The results generated might have practical implications for the inference of viral phenotype according to genetic features and may contribute to a better understanding of betanodavirus ecology.

Synthesis and in vitro study of novel neuraminidase inhibitors against avian influenza virus

Evidences of oseltamivir resistant influenza patients raised the need of novel neuraminidase inhibitors. In this study, five oseltamivir analogs PMC-31-PMC-36, synthesised according to the outcomes of a rational design analysis aimed to investigate the effects of substitution at the 5-amino and 4-amido groups of oseltamivir on its antiviral activity, were screened for their inhibition against neuraminidase N1 and N3. The enzymes used as models were from the avian influenza A H7N1 and H7N3 viruses. The neuraminidase inhibition assay was carried out by using recombinant species obtained from a baculovirus expression system and the fluorogenic substrate MUNANA. The assay was validated by using oseltamivir carboxylate as a reference inhibitor. Among the tested compounds, PMC-36 showed the highest inhibition on N1 with an IC(50) of 14.6±3.0nM (oseltamivir 25±4nM), while PMC-35 showed a significant inhibitory effect on N3 with an IC(50) of 0.1±0.03nM (oseltamivir 0.2±0.02nM). The analysis of the inhibitory properties of this panel of compounds allowed a preliminary assessment of a structure-activity relationship for the modification of the 4-amido and 5-amino groups of oseltamivir carboxylate. The substitution of the acetamido group in the oseltamivir structure with a 2-butenylamido moiety reduced the observed activity, while the introduction of a propenylamido group was well tolerated. Substitution of the free 5-amino group of oseltamivir carboxylate with an azide, decreased the activity against both N1 and N3. When these structural changes were both introduced, a dramatic reduction of activity was observed for both N1 and N3. The alkylation of the free 5-amino group in oseltamivir carboxylate introducing an isopropyl group seemed to increase the inhibitory effect for both N1 and N3 neuraminidases, displaying a more pronounced effect against N1.

The mouse model is suitable for the study of viral factors governing transmission and pathogenesis of highly pathogenic avian influenza (HPAI) viruses in mammals

Highly pathogenic avian influenza (HPAI) viruses of the H5 and H7 subtype pose a major public health threat due to their capacity to cross the species barrier and infect mammals, for example dogs, cats and humans. In the present study we tested the capacity of selected H7 and H5 HPAI viruses to infect and to be transmitted from infected BALB/c mice to contact sentinels. Previous experiments have shown that viruses belonging to both H5 and H7 subtypes replicate in the respiratory tract and central nervous system of experimentally infected mice. In this study we show that selected H7N1 and H5N1 HPAI viruses can be transmitted from mouse-to-mouse by direct contact, and that in experimentally infected animals they exhibit a different pattern of replication and transmission. Our results can be considered as a starting point for transmission experiments involving other influenza A viruses with α 2-3 receptor affinity in order to better understand the viral factors influencing transmissibility of these viruses in selected mammalian species.

The PDZ-Ligand and Src-Homology Type 3 Domains of Epidemic Avian Influenza Virus NS1 Protein Modulate Human Src Kinase Activity during Viral Infection

The Non-structural 1 (NS1) protein of avian influenza (AI) viruses is important for pathogenicity. Here, we identify a previously unrecognized tandem PDZ-ligand (TPL) domain in the extreme carboxy terminus of NS1 proteins from a subset of globally circulating AI viruses. By using protein arrays we have identified several human PDZ-cellular ligands of this novel domain, one of which is the RIL protein, a known regulator of the cellular tyrosine kinase Src. We found that the AI NS1 proteins bind and stimulate human Src tyrosine kinase, through their carboxy terminal Src homology type 3-binding (SHB) domain. The physical interaction between NS1 and Src and the ability of AI viruses to modulate the phosphorylation status of Src during the infection, were found to be influenced by the TPL arrangement. These results indicate the potential for novel host-pathogen interactions mediated by the TPL and SHB domains of AI NS1 protein.

Comparative Serological Assays for the Study of H5 and H7 Avian Influenza Viruses

The nature of influenza virus to randomly mutate and evolve into new types is an important challenge in the control of influenza infection. It is necessary to monitor virus evolution for a better understanding of the pandemic risk posed by certain variants as evidenced by the highly pathogenic avian influenza (HPAI) viruses. This has been clearly recognized in Egypt following the notification of the first HPAI H5N1 outbreak. The continuous circulation of the virus and the mass vaccination programme undertaken in poultry have resulted in a progressive genetic evolution and a significant antigenic drift near the major antigenic sites. In order to establish if vaccination is sufficient to provide significant intra- and interclade cross-protection, lentiviral pseudotypes derived from H5N1 HPAI viruses (A/Vietnam/1194/04, A/chicken/Egypt-1709-01/2007) and an antigenic drift variant (A/chicken/Egypt-1709-06-2008) were constructed and used in pseudotype-based neutralization assays (pp-NT). pp-NT data obtained was confirmed and correlated with HI and MN assays. A panel of pseudotypes belonging to influenza Groups 1 and 2, with a combination of reporter systems, was also employed for testing avian sera in order to support further application of pp-NT as an alternative valid assay that can improve avian vaccination efficacy testing, vaccine virus selection, and the reliability of reference sera.