Detection of the sacbrood virus in bee larvae and honey, determination of its virulence

Sacbrood virus (SBV) is a prevalent and serious disease affecting bees and their brood. The virus primarily targets larvae that are 2 to 3 days old, as well as adult bees. Under natural conditions, larvae become infected when they are fed by nurse bees, which themselves become infected while cleaning cells containing deceased larvae. The sacbrood virus damages the cells of the hypopharyngeal glands and the fatty body of both bees and larvae, often resulting in larval mortality and a decreased lifespan for adult bees. The clinical presentation of sac brood in bees is characterized by variegated brood, where the head ends of some larvae are visible, dead larvae appear in the shape of a 'boat', many empty cells. Additionally, dead bee larvae are observed in the form of sacs filled with liquid. However, these larvae can only be detected in cases of severe infection. The discovery of insect viruses in honey indicates that healthy bees may become infected with these viruses through nectar collected from honey-producing plants. According to bibliographic monitoring, honey within the Russian Federation has not yet been studied for the presence of viruses. Today, the issue of bee infection with the SBV remains a significant concern. A clear clinical picture of the disease typically emerges only in severe cases. However, the disease can go undetected even with a low level of damage to the bee colony caused by the sacbrood virus. Up to 6% of bee larvae may die due to this insidious disease. There may be a correlation between the concentration of SBV in the brood and honey, as a virulent pathogen can persist in honey. Consequently, the detection of SBV in honey could serve as a non-invasive method for the early diagnosis of the disease. The purpose of this study is to identify the sacbrood virus in honey and bee larvae and to assess its potential virulence by infecting mammalian cell cultures. A veterinary examination of bee colonies in two apiaries located in Moscow Oblast was conducted, revealing a clinical presentation in seven colonies affected by the sacbrood virus (see Table 2). Molecular genetic analyses were performed at the Syntol LLC laboratory in Moscow, Russia. Samples of honey and bee larvae were tested for the presence of the sacbrood virus using real-time reverse transcription PCR (qRT-PCR) with the Iso Test-SBV (Syntol LLC, Moscow, Russia), following the manufacturer's guidelines. Amplification products were detected through real-time fluorescence on an ANK-32 analyzer. The following dyes were utilized: carboxyfluorescein (FAM - negative control) and carboxy-X-rhodamine (ROX). The detection of the bee brood sac virus was conducted using the A4xL cell culture, which consists of pig kidney SPEV-TK cells and horse lymphocytes (an interspecific hybrid strain) at the Department of Cellular Biotechnology and Nutrient Media of RES, Moscow, Russia. Honey samples for infecting the cell cultures were prepared as follows: 0.5 g of honey was dissolved in 1 ml of physiological saline, then centrifuged in a T-24 centrifuge at 10,000 rpm for 10 minutes. The supernatant was transferred to a test tube, and the centrifugate was filtered through a 0.45 pm micro filter. As a result of the studies, it was revealed that the sacbrood virus was present in honey and bee larvae across all examined bee colonies (see Table 3). Experiments involving the infection of a cell culture with the SBV isolated from honey demonstrated that after 168 hours of cultivation on the A4xL cell culture, cell degeneration was observed. This included the disruption of the monolayer's integrity, destruction of the cell membrane, vacuolization of the cytoplasm, and pyknosis of the nuclei. In contrast, the control group exhibited 98% preservation of the cell monolayer. The sac-brood virus isolated from honey induces cytopathic effects (CPE) on mammalian cell cultures. It is possible to identify the SBV in honey from infected colonies using a hybrid culture of mammalian cells, A4xL. The CPE of the virus was noted on the seventh day, indicating its virulence and confirming the potential for infection of bee colonies through honey. Similar quantities of the virus were detected in both larvae and honey (see Fig. 1, A). A strong correlation was also observed between the SBV content in honey and that in the A4xL hybrid cell culture (see Fig. 1, B). We have demonstrated for the first time in vitro that honey from clinically affected colonies retains the virulent sacbrood virus for at least 10 days from the time of sampling. The analysis of honey using qRT-PCR to detect SBV, along with the assessment of the virus's virulence on A4xL cell culture, can serve as an additional diagnostic method for sacbrood disease in bees. The article contains 1 Figure, 3 Tables and 31 References. The Authors declare no conflict of interest.

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