Effect of 1,3-1,6 β-Glucan on Natural and Experimental Deformed Wing Virus Infection in Newly Emerged Honeybees (Apis mellifera ligustica)

The Western Honeybee is a key pollinator for natural as well as agricultural ecosystems. In the last decade massive honeybee colony losses have been observed worldwide, the result of a complex syndrome triggered by multiple stress factors, with the RNA virus Deformed Wing Virus (DWV) and the mite Varroa destructor playing crucial roles. The mite supports replication of DWV to high titers, which exert an immunosuppressive action and correlate with the onset of the disease. The aim of this study was to investigate the effect of 1,3-1,6 β-glucan, a natural innate immune system modulator, on honeybee response to low-titer natural and high-titer experimental DWV infection. As the effects exerted by ß-glucans can be remarkably different, depending on the target organism and the dose administered, two parallel experiments were performed, where 1,3-1,6 ß-glucan at a concentration of 0.5% and 2% respectively, was added to the diet of three cohorts of newly emerged honeybees, which were sampled from a Varroa-free apiary and harboured a low endogenous DWV viral titer. Each cohort was subjected to one of the following experimental treatments: no injection, injection of a high-copy number DWV suspension into the haemocel (experimental DWV infection) or injection of PBS into the haemocoel (physical injury). Control bees fed a ß-glucan-free diet were subjected to the same treatments. Viral load, survival rate, haemocyte populations and phenoloxidase activity of each experimental group were measured and compared. The results indicated that oral administration of 0.5% ß-glucan to naturally infected honeybees was associated with a significantly decrease of the number of infected bees and viral load they carried, and with a significant increase of the survival rate, suggesting that this natural immune modulator molecule might contribute to increase honeybee resistance to viral infection

Emergence and pandemic spread of small ruminant lentiviruses

Small ruminant lentiviruses (SRLVs) cause chronic, persistent infections in populations of domestic sheep (Ovis aries) and goats (Capra hircus) worldwide. The vast majority of SRLV infections involve two genotypes (A and B) that spread in association with the emergence of global livestock trade. However, SRLVs have likely been present in Eurasian ruminant populations since at least the early Neolithic period. Here, we use phylogenetic and phylogeographic approaches to reconstruct the origin of pandemic SRLV strains and infer their historical pattern of global spread. We constructed an open computational resource (‘Lentivirus-GLUE’) via which an up-to-date database of published SRLV sequences, multiple sequence alignments (MSAs), and sequence-associated metadata can be maintained. We used data collated in Lentivirus-GLUE to perform a comprehensive phylogenetic investigation of global SRLV diversity. Phylogenies reconstructed from genome-length alignments reveal that the deep divisions in the SRLV phylogeny are consistent with an ancient split into Eastern (A-like) and Western (B-like) lineages as agricultural systems disseminated out of domestication centres during the Neolithic period. These findings are also consistent with historical and phylogeographic evidence linking the early 20th century emergence of SRLV-A to the international export of Central Asian Karakul sheep. Investigating the global diversity of SRLVs can help reveal how anthropogenic factors have impacted the ecology and evolution of livestock diseases. The open resources generated in our study can expedite these studies and can also serve more broadly to facilitate the use of genomic data in SRLV diagnostics and research

Generation of virus like particles for epizootic hemorrhagic disease virus

Epizootic hemorrhagic disease virus (EHDV) is a distinct species within the genus Orbivirus, within the family Reoviridae. The epizootic hemorrhagic disease virus genome comprises ten segments of linear, double stranded (ds) RNA, which are packaged within each virus particle. The EHDV virion has a three layered capsid-structure, generated by four major viral proteins: VP2 and VP5 (outer capsid layer); VP7 (intermediate, core-surface layer) and VP3 (innermost, sub-core layer). Although EHDV infects cattle sporadically, several outbreaks have recently occurred in this species in five Mediterranean countries, indicating a potential threat to the European cattle industry. EHDV is transmitted by biting midges of the genus Culicoides, which can travel long distances through wind-born movements (particularly over water), increasing the potential for viral spread in new areas/countries. Expression systems to generate self-assembled virus like particles (VLPs) by simultaneous expression of the major capsid-proteins, have been established for several viruses (including bluetongue virus). This study has developed expression systems for production of EHDV VLPs, for use as non-infectious antigens in both vaccinology and serology studies, avoiding the risk of genetic reassortment between vaccine and field strains and facilitating large scale antigen production. Genes encoding the four major-capsid proteins of a field strain of EHDV-6, were isolated and cloned into transfer vectors, to generate two recombinant baculoviruses. The expression of these viral genes was assessed in insect cells by monitoring the presence of specific viral mRNAs and by western blotting. Electron microscopy studies confirmed the formation and purification of assembled VLPs

EFFETTO NUTRACEUTICO DEI BETA-­‐GLUCANI SU API INFETTE DA DEFORMED WING VIRUS (DWV).

Il Deformed Wing Virus (DWV) o virus delle api deformi è responsabile di una infezione spesso in forma subclinica che è molto diffusa nell'Apis mellifera. Il virus appartiene alla famiglia Picornavirales e, come gli altri membri della famiglia, è caratterizzato da un virione nudo di piccole dimensioni racchiudente un genoma a ssRNA (+). Il virus infetta le forme larvali durante il loro sviluppo e l’infezione si manifesta con la comparsa di api neo-sfarfallate con gravi deformazioni a carico delle ali e con ridotte dimensioni del corpo. La malattia può portare al collasso della colonia ed in genere le forme più gravi sono associate ad infestazioni di Varroa destructor, un acaro che agisce sia come parassita che come amplificatore e vettore biologico del virus. Le api, così come altri invertebrati, hanno sviluppato un'ampia varietà di meccanismi di difesa innata contro i vari agenti patogeni, seppur in assenza di un sistema immunitario adattativo. In molti dei meccanismi immunodifensivi degli invertebrati sono coinvolte proteine secrete da cellule dell'emolinfa dotate di attività opsonizzante, chemiotattica, battericida e perossidasica. Tali meccanismi possono essere modulati dalla presenza di varie molecole definite immunomodulatori. Tra queste i ß-glucani, polisaccaridi ramificati non amidacei costituiti da molecole di glucosio unite insieme mediante legami glicosidici β(1,3) e β(1,6), possono rappresentare una alternativa all'utilizzo di sostanze di sintesi chimica. I ß-glucani sono costituenti della parte solubile della fibra vegetale, sono presenti in molti cereali ma anche nelle pareti cellulari di vari agenti patogeni. Al fine di comprendere se i ß-glucani possano avere una efficace azione immunomodulante sulle api, la molecola è stata integrata nell’ alimentazione di gruppi sperimentali di api in un modello di infezione naturale e sperimentale con DWV. Gli esperimenti condotti hanno permesso di quantificare il tasso di mortalità, di stimare il numero e il grado di attivazione delle cellule deputate all’attività immunitaria, di quantizzare il virus presente nei vari distretti delle api (testa/addome) e di valutarne la replicazione. I risultati ottenuti hanno dimostrato che la somministrazione di ß-glucani per via orale in api infette da DWV è associata a un significativo aumento del tasso di sopravvivenza, ad un incremento del numero di emociti, e ad una riduzione della carica virale. Tali risultati suggeriscono che questa molecola possa contribuire ad incrementare la difesa delle api verso agenti patogeni

Infectivity of DWV associated to flower pollen: experimental evidence of a horizontal transmission route.

Deformed wing virus (DWV) is a honeybee pathogen whose presence is generally associated with infestation of the colony by the mite Varroa destructor, leading to the onset of infections responsible for the collapse of the bee colony. DWV contaminates bee products such as royal jelly, bee-bread and honey stored within the infected hive. Outside the hive, DWV has been found in pollen loads collected directly from infected as well as uninfected forager bees. It has been shown that the introduction of virus-contaminated pollen into a DWV-free hive results in the production of virus-contaminated food, whose role in the development of infected bees from virus-free eggs has been experimentally demonstrated. The aim of this study was twofold: (i) to ascertain the presence of DWV on pollen collected directly from flowers visited by honeybees and then quantify the viral load and (ii) determine whether the virus associated with pollen is infective. The results of our investigation provide evidence that DWV is present on pollen sampled directly from visited flowers and that, following injection in individuals belonging to the pollinator species Apis mellifera, it is able to establish an active infection, as indicated by the presence of replicating virus in the head of the injected bees. We also provide the first indication that the pollinator species Osmia cornuta is susceptible to DWV infection

A newly developed BVDV-1 RT-qPCR Taqman assay based on Italian isolates: evaluation as a diagnostic tool

A single-step TaqMan® RT-qPCR was developed for the detection of bovine viral diarrhea virus type 1 (BVDV- 1), an important pathogen of cattle worldwide. The assay was based on conserved 5’UTR sequences of Italian BVDV-1 isolates. In order to establish a diagnostic protocol which simplifies sample collection and processing, the assay was tested on a variety of biological specimens collected from persistently infected calves. The samples analyzed included PBMCs, plasma, dry blood, ear notch and hair bulb. Time and costs required for the analysis of each type of specimen were compared. The RT-qPCR, whose lower limit of detection was 100 copies of viral RNA (1 TCID50), correctly identified all PI animals, irrespective of the type of specimen. The highest copy numbers were obtained from the RNAs extracted from PBMCs, ear notches and hair bulbs. Hair bulb-supernatants directly used as a template allowed identification of all PI animals. In conclusion, based on time and cost evaluation, the most effective and efficient protocol was the one based on the direct analysis of hair bulb-supernatants, avoiding the RNA extraction step

EFFECT OF 1,3-1,6 Β-GLUCAN ON NATURAL AND EXPERIMENTAL DEFORMED WING VIRUS INFECTION IN NEWLY EMERGED HONEYBEES (APIS MELLIFERA LIGUSTICA) Atti del 71° Congresso SISVet:

The European honeybee (Apis mellifera ligustica L.) is the most managed pollinator species. Honeybees have recently suffered extensive losses mainly due to exposure to pesticides, malnutrition and pathogens, including bacteria, fungi, mites and viruses. Among honeybee’s pathogens, viruses play an important role in honeybee diseases (1). Deformed Wing Virus (DWV) is associated with honeybee colony losses. DWV can be transmitted vertically and persist in the bee colony as asymptomatic and symptomatic infections with clinical signs consisting of crumpled wings and a bloated abdomen. Symptomatic infections leading ultimately to the collapse of the colony are observed in apiaries infested by Varroa destructor which supports replication of DWV to high titers. During the first stage of the infection DWV is detected in the abdomen, at later stages presence of an actively replicating virus is also detectable in the head (2). The aim of this study was to investigate the effect of 1,3-1,6 β-glucan, a natural immune-modulator molecule, on honeybee response to both low-titer natural and high-titer experimental DWV infection. To date there are still few studies done to know the effect of ß-glucans on the immune response of honeybees. The investigation on their possible nutraceutical antiviral therapy may represent an innovative alternative to the use of synthetic chemical substances also for other diseases, with important benefits for the welfare of animals, humans and a positive impact on the environment. Two parallel experiments were performed; newly emerged honeybees, which were sampled from a Varroa-free apiary and harbored a low endogenous DWV viral titer where fed with 1,3-1,6 ß-glucan at a concentration of 0.5% and 2% respectively. Bees feed a 0.5% and 2% 1,3-1,6 ß-glucan diet were divided in three experimental cohorts and each of those was subjected to one of the following experimental treatments: no injection, injection into the haemocel of a high-copy number DWV suspension (experimental DWV infection) and PBS injection into the haemocel (physical injury). Control bees fed a ß-glucan-free diet were subjected to the same treatments. Twenty-five small cages with a maximum of 30 honeybees per cage were kept at 28°C for 13 days. Viral load was measured by qRT-PCR performed on RNA extracted from abdomen and head on honeybees. Insects were monitored daily and those found dead were collected to calculated the survival rate. Results indicated that administration of 0.5% ß-glucan to infected honeybees was associated with a significant reduction DWV viral load and with a significant increase of the survival rate suggesting that this natural immune modulator molecule might contribute to increase honeybee resistance to viral infection by restraining viral replication ultimately prolonging the honeybee lifespan