Testing the anxiety reduction function of grooming interactions in wild Barbary macaques (Macaca sylvanus)

Together with its hygienic and social function, grooming is thought to reduce anxiety. However, empirical evidence on the anxiety-reduction function of grooming is scarce. We collected 10-minute focal data on the donor and recipient of grooming using the post-grooming / matchedcontrol (PG-MC) method. In these PGs and MCs sessions, we recorded the occurrence of selfdirected behaviours (i.e. scratching and self-grooming), which are behavioural indicators of anxiety. We found mixed evidence of the relationship between anxiety and grooming interactions. The link between grooming and anxiety may be more complex than originally thought

Multiplex evaluation of influenza neutralizing antibodies with potential applicability to in-field serological studies

The increased number of outbreaks of H5 and H7 LPAI and HPAI viruses in poultry has major public and animal health implications. The continuous rapid evolution of these subtypes and the emergence of new variants influence the ability to undertake effective surveillance. Retroviral pseudotypes bearing influenza haemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins represent a flexible platform for sensitive, readily standardized influenza serological assays. We describe a multiplex assay for the study of neutralizing antibodies that are directed against both influenza H5 and H7 HA. This assay permits the measurement of neutralizing antibody responses against two antigenically distinct HAs in the same serum/plasma sample thus increasing the amount and quality of serological data that can be acquired from valuable sera. Sera obtained from chickens vaccinated with a monovalent H5N2 vaccine, chickens vaccinated with a bivalent H7N1/H5N9 vaccine, or turkeys naturally infected with an H7N3 virus were evaluated in this assay and the results correlated strongly with data obtained by HI assay. We show that pseudotypes are highly stable under basic cold-chain storage conditions and following multiple rounds of freeze-thaw. We propose that this robust assay may have practical utility for in-field sero-surveillance and vaccine studies in resource-limited regions worldwide

The use of equine influenza pseudotypes for serological screening

Standard assays used for influenza serology present certain practical issues, such as inter-laboratory variability, complex protocols and the necessity for handling certain virus strains in high biological containment facilities. In an attempt to address this, avian and human influenza HA pseudotyped retroviruses have been successfully employed in antibody neutralization assays. In this study we generated an equine influenza pseudotyped lentivirus for serological screening. This was achieved by co-transfection of HEK293T cells with plasmids expressing the haemagglutinin (HA) protein of an H3N8 subtype equine influenza virus strain, HIV gag-pol and firefly luciferase reporter genes and harvesting virus from supernatant. In order to produce infective pseudotype particles it was necessary to additionally co-transfect a plasmid encoding the TMPRSS2 endoprotease to cleave the HA. High titre pseudotype virus (PV) was then used in PV antibody neutralization assays (PVNAs) to successfully distinguish between vaccinated and non-vaccinated equines. The sera were also screened by single radial haemolysis (SRH) assay. There was a 65% correlation between the results of the two assays, with the PVNA assay appearing slightly more sensitive. Future work will extend the testing of the PVNA with a larger number of serum samples to assess sensitivity/specificity, inter/intra-laboratory variability and to define a protective titre

The production and development of H7 Influenza virus pseudotypes for the study of humoral responses against avian viruses

In recent years, high pathogenicity avian influenza (HPAI) virus, H5N1, low pathogenicity avian influenza (LPAI) virus, H9N2, and both HPAI and LPAI H7 viruses have proved devastating for the affected economies reliant on poultry industry, and have posed serious public health concerns. These viruses have repeatedly caused zoonotic disease in humans, raising concerns of a potential influenza pandemic. Despite the focus on the HPAI H5N1 outbreak in 1997 some H7 strains have also shown to be occasionally adaptable to infecting humans. Therefore, applying knowledge of the H5 virus evolution and spread to the development of sensitive serological methods is likely to improve our ability to understand and respond to the emergence of other HPAI and LPAI viruses, present within the avian populations, with the potential to infect humans and other species. In the present study we describe the construction and production of lentiviral pseudotypes bearing envelope glycoproteins of LPAI and HPAI H7 avian influenza viruses, which have been responsible for several outbreaks in the past decade. The H7 pseudotypes were evaluated in pseudotype-based neutralization (pp-NT) assays in order to detect and quantify the presence of neutralizing antibodies in avian sera, which were confirmed H7 positive by inhibition of haemagglutination (HI) test. Overall, our results substantiate influenza virus pseudotype neutralization as a robust tool for influenza sero-surveillance

An optimised method for the production of MERS-CoV spike expressing viral pseudotypes

The production and use of pseudotyped viral particles is widely established for many viruses, and applications in the fields of serology and vaccine development are manifold. Viral pseudotypes have proven to be powerful tools to study the effects of viral evolution on serological outcomes, viral tropism and immunogenicity studies. Pseudotyped viruses are chimeric constructs in which the outer (surface) glycoprotein(s) of one virus is combined with the replication-defective viral “core” of another virus. Pseudotypes allow for accurate, sequence-directed, sensitive antibody neutralization assays and antiviral screening to be conducted within a low biosecurity facility and offer a safe and efficient alternative to wildtype virus use. The protocol outlined here represents a rapid and reliable method for the generation of high-titre pseudotype viral particles with the MERS-CoV spike protein on a lentiviral core, and is adapted from previously published protocols. This protocol is optimised for transfection in a 100mm petri dish with 7ml of supernatant harvested, however it can be readily scaled to different production volumes. This protocol has a number of advantages including. 1. Use of readily available reagents 2. Consistent, high virus titres 3. Rapid generation of novel glycoproteins for research into strain variatio

Un ruolo, un archetipo: studio della componente psicologica e motivazionale che differenzia il portiere dal gruppo/squadra

La tesi è incentrata sul difficile ruolo del portiere, sia in relazione alle caratteristiche tecniche che lo differenziano dalla squadra, sia per le sue abilità psicologiche e motivazionali che lo rendono unico rispetto agli altri nell' approccio alla gara. La tesi comprende anche un' indagine dove, tramite l'utilizzo di un questionario sociometrico creato e somministrato da me su un campione di 10 portieri, vado ad analizzare direttamente, le caratteristiche sopracitate. I risultati raccolti portano ad ipotizzare una correlazione tra i portieri e gli archetipi teorizzati dal filoso Jung

Grooming interactions and cooperation in wild Barbary macaques (Macaca sylvanus)

The study of cooperation has been crucial to research on the evolution of social living in human and animal societies. Grooming interactions have been used as model to investigate the exchange of services in animals. Using both established and novel methodologies, this thesis examines grooming interactions and cooperation in two populations of wild Barbary macaques living in the Middle Atlas Mountains of Morocco. It is important to have a comprehensive idea of the costs and benefits of grooming interactions, and of the effect of grooming interactions on the anxiety of the grooming partners. This thesis showed that, contrary to previous studies, anxiety increased after grooming interactions in both the donor and recipient. This highlights the need to further investigate the link between grooming and emotions. Individuals may also affect the grooming interactions of other group members. This thesis showed that individuals benefit from disrupting grooming interactions of group members by gaining grooming opportunities for themselves and by stopping the group members from grooming each other, although grooming disruptions may be risky. Monkeys may affect others’ grooming interactions to favour their own social and dominance positions. A key aspect of this thesis was also to assess whether grooming is reciprocated in the short-term and which type of reciprocity (i.e. direct, indirect and generalised) play a role in the exchanges of grooming. This study showed that direct but not indirect and generalised reciprocity play a role in the exchange of grooming. While there is a wide range of evidence that direct reciprocity plays a role in the exchange of services in animals, there is little evidence of indirect and generalised reciprocity. Additionally to exchanging grooming for grooming, animals also exchange grooming for other services such as tolerance around food resource and support during agonistic interactions. In this thesis, no evidence of short-term contingency between the exchange of grooming and food tolerance was found. The exchanges of services may be little affected by recent single events, and mechanisms involving an emotional mediation based on long-term social bonds between partners may play a more important role. The capacity to make effective choices among potential social partners is an important social skill, as choosing the best available partner improves the chances to establish successful cooperative interactions. This thesis highlighted, to some extents, the importance of factors such as tolerance and relationship quality between partners, in the performances of individuals and their choice of partners to solve a cooperative task. Tolerant relationships may have been a prerequisite for the evolution of cognitively complex cooperation. Testing a comprehensive framework of predictions, this thesis brings novel contributions to the understanding of grooming interactions and cooperation in wild Barbary macaque

The use of hyperimmune chicken reference sera is not appropriate for the validation of influenza pseudotype neutralization assays

The pseudotype particle neutralization test (pp-NT) is a next-generation serological assay employed for the sensitive study of influenza antibody responses against hemagglutinin (HA), including stalk-directed antibodies. However, a validation of this assay has yet to be performed, and this limits its use to primarily research laboratories. To identify possible serological standards to be used in optimization and validation of the pp-NT, we have evaluated the cross-reactivity of hyperimmune chicken reference antisera in this assay. Our findings show that the cross-reactivity detected by the pp-NT is only partly explained by phylogenetic relationships and protein homology between the HA subtypes analysed; further studies are necessary to understand the origin of the cross-reactivity detected, and reference standards with higher specificity should be evaluated or generated de novo for future use in pp-NT

Characterisation of SARS-CoV-2 Lentiviral Pseudotypes and Correlation between Pseudotype-Based Neutralisation Assays and Live Virus-Based Micro Neutralisation Assays

The recent outbreak of a novel Coronavirus (SARS-CoV-2) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against SARS-CoV-2 spike protein and to evaluate the serological immunity in humans. Since the accessibility of live virus microneutralisation (MN) assays with SARS-CoV-2 is limited and requires enhanced bio-containment, the approach based on "pseudotyping" can be considered a useful complement to other serological assays. After fully characterising lentiviral pseudotypes bearing the SARS-CoV-2 spike protein, we employed them in pseudotype-based neutralisation assays in order to profile the neutralising activity of human serum samples from an Italian sero-epidemiological study. The results obtained with pseudotype-based neutralisation assays mirrored those obtained when the same panel of sera was tested against the wild type virus, showing an evident convergence of the pseudotype-based neutralisation and MN results. The overall results lead to the conclusion that the pseudotype-based neutralisation assay is a valid alternative to using the wild-type strain, and although this system needs to be optimised and standardised, it can not only complement the classical serological methods, but also allows serological assessments to be made when other methods cannot be employed, especially in a human pandemic context