**1. Introduction**

Among the factors that threaten the health and wellbeing of honeybees, a noteworthy variety of pathogens such as bacteria, viruses, fungi and parasites are mentioned. In recent decades, honeybee viruses have been studied for their potential impact on beekeeping productions, acquiring more and more importance in the research world. Viruses in honeybees were first described in 1913 [1] when an American researcher attributed to a virus the "sac" appearance showed by some diseased larvae, although the causative agent (Sacbrood virus) was not characterized until 1964 [2]. To date, at least 22 viruses that can infect honeybees have been described. Most investigated hives are found to be infected by at least one virus, but often multiple viruses are detected in one hive [3–5].

In addition to causing high economic losses, viruses negatively affect the morphology, physiology, and behavior of honeybees and, although individuals do not always show clinical signs, they are frequently associated with weakening and colony collapse [6]. Depending on the different pathways of infection and on the health status of the colonies, viruses can cause symptomatic infections, i.e., overt or clinical, and asymptomatic infections, i.e., covert or subclinical [7]. Symptomatic infections are characterized by clinical signs and by high levels of viral particle production, to which the insect either succumbs or survives according to the status of the immune system.

These symptomatic infections can be further divided into acute and chronic: the acute involve the active replication of the virus, with a high titer of viral particles in a short time and cause rapid death of the host with evident clinical signs. In extreme cases, when the

**Citation:** Power, K.; Martano, M.; Altamura, G.; Piscopo, N.; Maiolino, P. Histopathological Features of Symptomatic and Asymptomatic Honeybees Naturally Infected by Deformed Wing Virus. *Pathogens* **2021**, *10*, 874. https://doi.org/ 10.3390/pathogens10070874

Academic Editor: Giovanni Cilia

Received: 28 May 2021 Accepted: 8 July 2021 Published: 10 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

production of high viral titers occurs during a short time, sudden death can occur without previous clinical signs (hyperacute infections) [8]. Chronic infections, on the other hand, imply a slow but constant production of viral particles during the life of the host, or during the duration of the infected life stage, with subsequent appearance of clinical signs. On the contrary, asymptomatic infections are characterized by persistence of the virus beyond life stage, vertical transmission and the absence of obvious symptoms, although there could still be a hidden cost for the host [8–10]. Asymptomatic infections can be latent and persistent [6]. In the first case the viral genome may be present as an extrachromosomal episome or may be integrated into the host genome with incomplete replication or no replication at all. In the second case, there is a constant but low production of viral particles in the host cells, and either the infected cell survives, or the limited number of dead cells is counterbalanced by the production of new cells. Persistent infections, therefore, represent a balance between host and persistent viral replication, where despite the infection, the host does not die. Moreover, asymptomatic infections can become symptomatic when the host homeostasis is unbalanced by stressors such as other pathologies, food deficiencies, and other environmental factors [9].

Deformed wing virus (DWV) is positive single-stranded RNA virus belonging to the genus Iflavirus, family Iflaviridae of the order Picornavirales [11] and is the most prevalent virus in honeybees, with a minimum average of 55% of apiaries infected across 32 countries [12]. The virus was first isolated in the 1982 in the UK by Bill Baley and Brenda Ball from dead Japanese honeybees showing particular deformity of wings [13]. Soon after, honeybees from the UK, Belize and South Africa died showing DWV symptoms. Ten years after, in the UK the virus was found in Varroa destructor-infested colonies, and it was then found in every location where *V. destructor* was well established [12]. Due to the link with *V. destructor* and following the huge spread of it around the world between 1970 and 1980, DWV altered its epidemiology and has currently a global distribution [14]. Except for Australia, Uganda and the Canadian island of Newfoundland, where the *V. destructor* mite has not been found, the presence of this particular virus has been reported in Africa, Asia, Europe, North America and South America [15]. DWV appears in three master variants DWV-A, DWV-B and DWV-C, plus numerous recombinations, often more virulent than the masters [16]. DWV-A was the first variant to be detected and it is closely associated to colony collapse [17]; however, DWV-B, previously termed Varroa Destructor virus-1, was found to be equally or more virulent than DWV-A when injected in high viral loads [18]. DWV-C was first described in U.K. honeybee samples from 2007 and linked in combination with DWV-A to the death of overwintering colonies [19].

Recent studies have shown that DWV is present in more than 64 species of insects and highly prevalent not only in honeybees, but also in more than 29 arthropod species associated with honeybee hives [12,20,21]. Within insects, DWV was found in bumblebee species *Bombus terrestris* and *Bombus pascuorum*, wasp species *Vespula vulgaris* and *Vespa crabro* and *Lasius* spp. ants [12,22,23], *besides A. mellifera*. DWV is a major pathogen of honeybees and its prevalence, strongly connected with the ectoparasite *V. destructor*, strongly increases honeybee colony mortality [24]. DWV is a low pathogenic virus that is capable of infecting all stages of development of honeybees, from eggs to adults, although it shows a higher replication in pupae [25,26]. It takes its name from the characteristic symptom that manifests itself in newly hatched honeybees with deformed or underdeveloped wings; these honeybees, unable to fly, can die shortly after emerging from the cell. Initially, the deformity of the wings had been attributed to the action of the *V. destructor* mite, as the symptom was more evident in conjunction with a strong infestation by the parasite [27]. Subsequently, DWV was identified as the etiological agent of wing deformity, emphasizing the association between the viral titers and the symptom [25,28]. However, although DWV is one of the few honeybee viruses to have its own characteristic clinical manifestation, it is known also to be present in apparently healthy colonies [10].

Although the pathology and virulence of DWV remain linked to horizontal vectored transmission by *V. destructor*, the presence of DWV has been demonstrated also in the absence of *V. destructor* [29]. Varroa-mediated virus transmission from adult honeybees to developing pupae is responsible for the display of the symptoms [24,30] such as early pupal death, deformed wings, shortened and swollen abdomen and discoloration of the cuticle in adult bees, and learning deficiencies [14]. Symptomatic DWV infection occurs primarily during autumn and in highly mite-infested colonies, where it constitutes predictive marker for winter colony losses [30,31]. According to the epidemiological model proposed by Chen et al. [9] two distinct moments of viral presence and infection can be recognized: in healthy and viable colonies, the virus remains latent / persistent without determining evident symptoms. Vice versa, in weak "stressed" colonies, the virus can abandon the state of latency, considerably replicate, increasing its virulence and causing the death of single individuals and depopulation of the colony. Among the main stressors identified in DWV infection, temperature decline could increase severity of viral infection in newly emerged honeybees (probably explaining the high levels of winter losses), while pesticides and poor nutrition could trigger the honeybee immune system making them more susceptible to viral infection, leading to colony collapse [32,33]. However, the main trigger for DWV symptomatic infection remains the uncontrolled Varroa infestation.

There is no doubt that the impact of viral diseases, especially DWV, in apiaries is a global threat to beekeeping and it is associated to honeybee colony loss [34]. Possible treatments against viral infections in honeybees are not known and legally recognized to date. Currently, a suitable treatment against Varroa is the best approach to fight DWV, since, after treatment, there is a gradual reduction of viral titers in colonies [35,36].

A deep knowledge of the crucial aspects of the viral pathogenicity, is important for realizing an effective control program, therefore, the aim of this preliminary study was to analyze any anatomo-histopathological findings in symptomatic and asymptomatic honeybees collected from a hive infected from DWV to try to better understand the pathological events underlying the infection.
