Association of viruses with two protozoal pathogens of the honey bee

Undisturbed honey bee colonies near Rothamsted were commonly infected with a filamentous DNA virus (FV) which was usually associated with Nosema apis in infected adult individuals and occurred most frequently about June. No symptoms were caused by FV, which appeared less harmful than either black queen-ceil virus (BQCV) or bee virus Y (BVY), two isometric RNA viruses also associated with N. apis. Bee virus X, another isometric RNA virus distantly related to BVY, was not associated with N. apis, but prevailed in winter and was then associated significantly with Malpighamoeba mellificae in dead individuals, although it frequently multiplied alone. Results of laboratory experiments supported conclusions made from the field observations about these relationships and their pathology.     

Detection of chronic bee paralysis virus and acute bee paralysis virus in Uruguayan honeybees

Chronic bee paralysis virus (CBPV) causes a disease characterized by trembling, flightless, and crawling bees, while Acute bee paralysis virus (ABPV) is commonly detected in apparently healthy colonies, usually associated to Varroa destructor. Both viruses had been detected in most regions of the world, except in South America. In this work, we detected CBPV and ABPV in samples of Uruguayan honeybees by RT-PCR. The detection of both viruses in different provinces and the fact that most of the analyzed samples were infected, suggest that, they are widely spread in the region. This is the first record of the presence of CBPV and ABPV in Uruguay and South America.     

The prevalence of pathogens in honey bee (Apis mellifera) colonies infested with the parasitic mite Varroa jacobsoni

The prevalence of nine honey bee viruses in samples of dead adult bees from Apis mellifera colonies in the Netherlands and Germany infested with the parasitic mite Varroa jacobsoni was compared with virus incidence in uninfested colonies in Britain. In colonies with low mite populations the viruses present and their incidence during the year were similar to the results obtained from British colonies. However, in marked contrast with findings in Britain, acute paralysis virus (APV) was the primary cause of adult bee mortality in German honey bee colonies severely infested with V. jacobsoni. Dead brood from unsealed and sealed infested cells from German colonies with high mite populations also contained much APV. The evidence suggests that V. jacobsoni activates APV replication in adult bees by its feeding behaviour and transmits virus from adult honey bees to pupae. In addition, adult bees, in which APV is multiplying, transmit the virus to unsealed brood in the larval food.     

Detection of honey bee (Apis mellifera) viruses with an oligonucleotide microarray

In recent years, declines in honey bee (Apis mellifera L.) colonies have been observed to varying degrees worldwide with the worst losses in the USA being termed Colony Collapse Disorder (CCD). Pathogen load and the prevalence of honey bee viruses have been implicated in these losses and many diseased hives have multiple viruses present. We have designed and tested an oligonucleotide microarray which enables the simultaneous detection of nine honey bee viruses: Acute bee paralysis virus, Black queen cell virus, Chronic bee paralysis virus, Deformed wing virus, Kashmir bee virus, Sacbrood virus, Israel acute paralysis virus, Varroa destructor virus 1 and Slow paralysis virus. The microarray can be used to robustly diagnose nine viruses in one test.     

The Acute bee paralysis virus–Kashmir bee virus–Israeli acute paralysis virus complex

Acute bee paralysis virus (ABPV), Kashmir bee virus (KBV) and Israeli acute paralysis virus (IAPV) are part of a complex of closely related viruses from the Family Dicistroviridae. These viruses have a widespread prevalence in honey bee (Apis mellifera) colonies and a predominantly sub-clinical etiology that contrasts sharply with the extremely virulent pathology encountered at elevated titres, either artificially induced or encountered naturally. These viruses are frequently implicated in honey bee colony losses, especially when the colonies are infested with the parasitic mite Varroa destructor. Here we review the historical and recent literature of this virus complex, covering history and origins; the geographic, host and tissue distribution; pathology and transmission; genetics and variation; diagnostics, and discuss these within the context of the molecular and biological similarities and differences between the viruses. We also briefly discuss three recent developments relating specifically to IAPV, concerning its association with Colony Collapse Disorder, treatment of IAPV infection with siRNA and possible honey bee resistance to IAPV.     

Molecular and phylogenetic characterization of honey bee viruses,Nosema microsporidia,protozoan parasites,and parasitic mites in China

China has the largest number of managed honey bee colonies, which produce the highest quantity of honey and royal jelly in the world; however, the presence of honey bee pathogens and parasites has never been rigorously identified in Chinese apiaries. We thus conducted a molecular survey of honey bee RNA viruses, Nosema microsporidia, protozoan parasites, and tracheal mites associated with nonnative Apis mellifera ligustica and native Apis cerana cerana colonies in China. We found the presence of black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), and sacbrood virus (SBV), but not that of acute bee paralysis virus (ABPV) or Kashmir bee virus (KBV). DWV was the most prevalent in the tested samples. Phylogenies of Chinese viral isolates demonstrated that genetically heterogeneous populations of BQCV, CBPV, DWV, and A. cerana‐infecting SBV, and relatively homogenous populations of IAPV and A. meliifera‐infecting new strain of SBV with single origins, are spread in Chinese apiaries. Similar to previous observations in many countries, Nosema ceranae, but not Nosema apis, was prevalent in the tested samples. Crithidia mellificae, but not Apicystis bombi was found in five samples, including one A. c. cerana colony, demonstrating that C. mellificae is capable of infecting multiple honey bee species. Based on kinetoplast‐encoded cytochrome b sequences, the C. mellificae isolate from A. c. cerana represents a novel haplotype with 19 nucleotide differences from the Chinese and Japanese isolates from A. m. ligustica. This suggests that A. c. cerana is the native host for this specific haplotype. The tracheal mite, Acarapis woodi, was detected in one A. m. ligustica colony. Our results demonstrate that honey bee RNA viruses, N. ceranae, C. mellificae, and tracheal mites are present in Chinese apiaries, and some might be originated from native Asian honey bees.     

Survey of six bee viruses using RT-PCR in Northern Thailand

Six honey bee viruses were surveyed using RT-PCR in Northern Thailand where about 80% of Thai apiaries are located. Tested samples were found to be positive for deformed wing virus (DWV), acute bee paralysis virus (ABPV), sacbrood virus (SBV) and Kashmir bee virus (KBV). In the collected samples, neither chronic bee paralysis virus nor black queen cell virus nucleic acids could be detected. It was found that DWV was the most widespread and ABPV was the second most prevalent. Kashmir bee virus was found only in the Lampang province where high infestation of Varroa destructor mite occurred. Tropilaelaps, European foulbrood, and Chalkbrood diseases were found in some apiaries.     

The ectoparasitic mite Tropilaelaps mercedesae (Acari, Laelapidae) as a vector of honeybee viruses

The ectoparasitic mites Varroa destructor and Tropilaelaps mercedesae share life history traits and both infect honeybee colonies, Apis mellifera. Since V. destructor is a biological vector of several honeybee viruses, we here test whether T. mercedesae can also be infected and enable virus replication. In Kunming (China), workers and T. mercedesae mites were sampled from three A. mellifera colonies, where workers were exhibiting clinical symptoms of deformed wing virus (DWV). We analysed a pooled bee sample (15 workers) and 29 mites for the presence of Deformed wing virus (DWV), Black queen cell virus (BQCV), Sacbrood virus (SBV), Kashmir bee virus (KBV), Acute bee paralysis virus (ABPV), and Chronic bee paralysis virus (CBPV). Virus positive samples were analysed with a qPCR. Only DWV +RNA was found but with a high titre of up to 10⁸ equivalent virus copies per mite and 10⁶ per bee. Moreover, in all DWV positive mites (N= 12) and in the bee sample virus–RNA was also detected using RT-PCR and tagged RT-PCR, strongly suggesting virus replication. Our data show for the first time that T. mercedesae may be a biological vector of DWV, which would open a novel route of virus spread in A. mellifera. Received 6 June 2008; revised 14 August 2008; accepted 10 September 2008.     

The Honey Bee Pathosphere of Mongolia: European Viruses in Central Asia

Parasites and pathogens are apparent key factors for the detrimental health of managed European honey bee subspecies, Apis mellifera. Apicultural trade is arguably the main factor for the almost global distribution of most honey bee diseases, thereby increasing chances for multiple infestations/infections of regions, apiaries, colonies and even individual bees. This imposes difficulties to evaluate the effects of pathogens in isolation, thereby creating demand to survey remote areas. Here, we conducted the first comprehensive survey for 14 honey bee pathogens in Mongolia (N = 3 regions, N = 9 locations, N = 151 colonies), where honey bee colonies depend on humans to overwinter. In Mongolia, honey bees, Apis spp., are not native and colonies of European A. mellifera subspecies have been introduced ~60 years ago. Despite the high detection power and large sample size across Mongolian regions with beekeeping, the mite Acarapis woodi, the bacteria Melissococcus plutonius and Paenibacillus larvae, the microsporidian Nosema apis, Acute bee paralysis virus, Kashmir bee virus, Israeli acute paralysis virus and Lake Sinai virus strain 2 were not detected, suggesting that they are either very rare or absent. The mite Varroa destructor, Nosema ceranae and four viruses (Sacbrood virus, Black queen cell virus, Deformed wing virus (DWV) and Chronic bee paralysis virus) were found with different prevalence. Despite the positive correlation between the prevalence of V. destructor mites and DWV, some areas had only mites, but not DWV, which is most likely due to the exceptional isolation of apiaries (up to 600 km). Phylogenetic analyses of the detected viruses reveal their clustering and European origin, thereby supporting the role of trade for pathogen spread and the isolation of Mongolia from South-Asian countries. In conclusion, this survey reveals the distinctive honey bee pathosphere of Mongolia, which offers opportunities for exciting future research.     

北京地区六种蜜蜂病毒病的流行病学研究

【目的】对蜜蜂的6种病毒:以色列急性麻痹病毒(Israeli acute paralysis virus,IAPV)、残翅病毒(Deformed wing virus,DWV)、囊状幼虫病病毒(Sacbrood virus,SBV)、急性蜜蜂麻痹病毒(Acute bee paralysis virus,ABPV)、黑蜂王台病毒(Black queen cell virus,BQCV)、慢性麻痹病毒(Chronic bee paralysis virus,CBPV)在北京地区的流行情况进行调查,以期为该地区蜜蜂病毒病的防控提供一定的理论依据。【方法】应用多重RT-PCR法确定上述6种病毒在该地区的感染情况,并通过序列分析确定特异性。【结果】在所有检测样本中均未检测到急性麻痹病病毒和慢性麻痹病病毒,感染率最高的是以色列急性麻痹病毒,其次是残翅病毒。检测的样本普遍存在混合感染。【结论】以色列急性麻痹病毒、残翅病毒、囊状幼虫病病毒、黑蜂王台病毒4种病毒可能在北京地区广泛分布。     

Chronic bee paralysis: A disease and a virus like no other?

Chronic bee paralysis which was called Paralysis is a rather unusual disease caused by a rather unusual virus. In this review, we explore current knowledge of the disease and its etiological agent. Paralysis is the only common viral disease of adult bees whose symptoms include both behavioural and physiological modifications: trembling and hair loss. The disease often affects the strong colonies of an apiary and thousands of dead individuals are then observed in front of the hives. Two sets of symptoms have traditionally been described in the existing literature, but nowadays we can define a general syndrome.The morphology of the Chronic bee paralysis virus (CBPV) particles and the multipartite organisation of the RNA genome are exceptional, as most honey bee viruses are picorna-like viruses belonging to the Iflavirus and Cripavirus genera with symmetric particles and monopartite positive, single-strand RNA genomes. CBPV is currently classified as an RNA virus but is not included in any family or genus. Although it shares several characteristics with viruses in the Nodaviridae and Tombusviridae families, it differs from previously known viruses according to the various demarcation criteria defined by the International Committee on Taxonomy of Viruses (ICTV). Thus, it should be considered as the type species of a new group of positive-strand RNA viruses.The recent sequencing of the complete CBPV genome has opened the way for phylogenetic studies and development of new molecular tools able to detect variable isolates and to quantify genomic loads. This article considers the results of such recent detection tests but also previous studies including: (i) the distribution of CBPV infection within the bees and the hive, (ii) the way the virus spreads and its persistence in the colony environment, and (iii) geographical and seasonal distribution and impact of CBPV infections.     

Infestation of Japanese Native Honey Bees by Tracheal Mite and Virus from Non-native European Honey Bees in Japan

Invasion of alien species has been shown to cause detrimental effects on habitats of native species. Insect pollinators represent such examples; the introduction of commercial bumble bee species for crop pollination has resulted in competition for an ecological niche with native species, genetic disturbance caused by mating with native species, and pathogen spillover to native species. The European honey bee, Apis mellifera, was first introduced into Japan for apiculture in 1877, and queen bees have been imported from several countries for many years. However, its effects on Japanese native honey bee, Apis cerana japonica, have never been addressed. We thus conducted the survey of honey bee viruses and Acarapis mites using both A. mellifera and A. c. japonica colonies to examine their infestation in native and non-native honey bee species in Japan. Honey bee viruses, Deformed wing virus (DWV), Black queen cell virus (BQCV), Israeli acute paralysis virus (IAPV), and Sacbrood virus (SBV), were found in both A. mellifera and A. c. japonica colonies; however, the infection frequency of viruses in A. c. japonica was lower than that in A. mellifera colonies. Based on the phylogenies of DWV, BQCV, and SBV isolates from A. mellifera and A. c. japonica, DWV and BQCV may infect both honey bee species; meanwhile, SBV has a clear species barrier. For the first time in Japan, tracheal mite (Acarapis woodi) was specifically found in the dead honey bees from collapsing A. c. japonica colonies. This paper thus provides further evidence that tracheal-mite-infested honey bee colonies can die during cool winters with no other disease present. These results demonstrate the infestation of native honey bees by parasite and pathogens of non-native honey bees that are traded globally.     

Honeybee viruses in Uruguay

Mortality of honeybees is a serious problem that beekeepers have to face periodically in Uruguay and worldwide. The presence of RNA viruses, in addition to other pathogens may be one of its possible causes. In this work, we detected Chronic bee paralysis virus, Acute bee paralysis virus, Black queen cell virus, Sacbrood virus and Deformed wing virus in samples of Uruguayan honeybees with or without Varroa destructor and Nosema apis. The detection of viruses in different provinces, simultaneous co-infection of colonies by several viruses and the fact that 96% of the samples were infected with one or more virus, indicates they are widely spread in the region.     

Occurrence and prevalence of seven bee viruses in Apis mellifera and Apis cerana apiaries in China

Populations of Apis mellifera and Apis cerana in China were surveyed for seven bee viruses: acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Kashmir bee virus (KBV), sacbrood virus (SBV), and Isreal acute paralysis virus (IAPV). No KBV was detected from any samples of the two species. In A. mellifera, DWV was the most prevalent virus, but in A. cerana, SBV was the dominant. Simultaneous multiple infections of viruses were common in both species. This is the first report of detection of IAPV and CBPV in A. cerana.     

The incidence of virus diseases in the honey bee

Chronic bee-paralysis virus and sacbrood virus occur commonly in apparently normal honey-bee colonies in Britain. Most sick adult bees not affected by Nosema apis, Malpighamoeba mellificae or Acarapis woodi have chronic paralysis and most dead larvae not affected by micro-organisms have sacbrood. Both virus diseases are probably limited by hereditary factors, but unknown environmental factors also seem to influence disease. Paralysed bees from Australia, Canada, Eire, Germany and Mexico were found to be infected with chronic paralysis virus.     

Prevalence and distribution of six bee viruses in Korean Apis cerana populations

The prevalence and distribution of six bee viruses was investigated in 527 Apis cerana samples which were collected from five provinces in South Korea. The most prevalent virus, black queen cell virus (BQCV), was present in 75.11% of 446 adult bee samples, followed by sacbrood virus (SBV) in 30.71%. Deformed wing virus (DWV), Kashmir bee virus (KBV), and chronic bee paralysis virus (CBPV) were present at lower levels of 8.07%, 1.56%, and 0.44%, respectively. The most prevalent virus in 81 larvae samples was SBV, with an incidence of 60.49%, followed by BQCV in 48.14%, DWV in 6.17%, and KBV in 1.23% of samples. CBPV infection was not detected in larvae samples, and acute bee paralysis virus (ABPV) was not present in both larvae and adult bee. Simultaneous infections with up to four viruses were also identified. Of these, infections with SBV and BQCV were most frequent in 25.61% of samples. The distribution of these viruses varied considerably throughout the geographic regions investigated. The three provinces of Gyeongbuk, Jeonnam, and Chungbuk had the highest frequency of bee viruses.     

Lower Virus Infections in Varroa destructor-Infested and Uninfested Brood and Adult Honey Bees (Apis mellifera) of a Low Mite Population Growth Colony Compared to a High Mite Population Growth Colony

A comparison was made of the prevalence and relative quantification of deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), Kashmir bee virus (KBV), acute bee paralysis virus (ABPV) and sac brood virus (SBV) in brood and adult honey bees (Apis mellifera) from colonies selected for high (HMP) and low (LMP) Varroa destructor mite population growth. Two viruses, ABPV and SBV, were never detected. For adults without mite infestation, DWV, IAPV, BQCV and KBV were detected in the HMP colony; however, only BQCV was detected in the LMP colony but at similar levels as in the HMP colony. With mite infestation, the four viruses were detected in adults of the HMP colony but all at higher amounts than in the LMP colony. For brood without mite infestation, DWV and IAPV were detected in the HMP colony, but no viruses were detected in the LMP colony. With mite infestation of brood, the four viruses were detected in the HMP colony, but only DWV and IAPV were detected and at lower amounts in the LMP colony. An epidemiological explanation for these results is that pre-experiment differences in virus presence and levels existed between the HMP and LMP colonies. It is also possible that low V. destructor population growth in the LMP colony resulted in the bees being less exposed to the mite and thus less likely to have virus infections. LMP and HMP bees may have also differed in susceptibility to virus infection.     

Seasonal dynamics and co‐occurrence patterns of honey bee pathogens revealed by high‐throughput RT‐qPCR analysis

The health of the honey bee Apis mellifera is challenged by introduced parasites that interact with its inherent pathogens and cause elevated rates of colony losses. To elucidate co‐occurrence, population dynamics, and synergistic interactions of honey bee pathogens, we established an array of diagnostic assays for a high‐throughput qPCR platform. Assuming that interaction of pathogens requires co‐occurrence within the same individual, single worker bees were analyzed instead of collective samples. Eleven viruses, four parasites, and three pathogenic bacteria were quantified in more than one thousand single bees sampled from sixteen disease‐free apiaries in Southwest Germany. The most abundant viruses were black queen cell virus (84%), Lake Sinai virus 1 (42%), and deformed wing virus B (35%). Forager bees from asymptomatic colonies were infected with two different viruses in average, and simultaneous infection with four to six viruses was common (14%). Also, the intestinal parasites Nosema ceranae (96%) and Crithidia mellificae/Lotmaria passim (52%) occurred very frequently. These results indicate that low‐level infections in honey bees are more common than previously assumed. All viruses showed seasonal variation, while N. ceranae did not. The foulbrood bacteria Paenibacillus larvae and Melissococcus plutonius were regionally distributed. Spearman's correlations and multiple regression analysis indicated possible synergistic interactions between the common pathogens, particularly for black queen cell virus. Beyond its suitability for further studies on honeybees, this targeted approach may be, due to its precision, capacity, and flexibility, a viable alternative to more expensive, sequencing‐based approaches in nonmodel systems.