Comparing Survival of Israeli Acute Paralysis Virus Infection among Stocks of U.S. Honey Bees

Among numerous viruses that infect honey bees (Apis mellifera), Israeli acute paralysis virus (IAPV) can be linked to severe honey bee health problems. Breeding for virus resistance may improve honey bee health. To evaluate the potential for this approach, we compared the survival of IAPV infection among stocks from the U.S. We complemented the survival analysis with a survey of existing viruses in these stocks and assessing constitutive and induced expression of immune genes. Worker offspring from selected queens in a common apiary were inoculated with IAPV by topical applications after emergence to assess subsequent survival. Differences among stocks were small compared to variation within stocks, indicating the potential for improving honey bee survival of virus infections in all stocks. A positive relation between worker survival and virus load among stocks further suggested that honey bees may be able to adapt to better cope with viruses, while our molecular studies indicate that toll-6 may be related to survival differences among virus-infected worker bees. Together, these findings highlight the importance of viruses in queen breeding operations and provide a promising starting point for the quest to improve honey bee health by selectively breeding stock to be better able to survive virus infections.

Transcriptomic and epigenomic dynamics of honey bees in response to lethal viral infection

Honey bees (Apis mellifera L) suffer from many brood pathogens, including viruses. Despite considerable research, the molecular responses and dynamics of honey bee pupae to viral pathogens remain poorly understood. Israeli Acute Paralysis Virus (IAPV) is emerging as a model virus since its association with severe colony losses. Using worker pupae, we studied the transcriptomic and methylomic consequences of IAPV infection over three distinct time points after inoculation. Contrasts of gene expression and 5mC DNA methylation profiles between IAPV-infected and control individuals at these time points—corresponding to the pre-replicative (5 hr), replicative (20 hr), and terminal (48 hr) phase of infection—indicate that profound immune responses and distinct manipulation of host molecular processes accompany the lethal progression of this virus. We identify the temporal dynamics of the transcriptomic response to with more genes differentially expressed in the replicative and terminal phases than in the pre-replicative phase. However, the number of differentially methylated regions decreased dramatically from the pre-replicative to the replicative and terminal phase. Several cellular pathways experienced hyper- and hypo-methylation in the pre-replicative phase and later dramatically increased in gene expression at the terminal phase, including the MAPK, Jak-STAT, Hippo, mTOR, TGF-beta signaling pathways, ubiquitin mediated proteolysis, and spliceosome. These affected biological functions suggest that adaptive host responses to combat the virus are mixed with viral manipulations of the host to increase its own reproduction, all of which are involved in anti-viral immune response, cell growth, and proliferation. Comparative genomic analyses with other studies of viral infections of honey bees and fruit flies indicated that similar immune pathways are shared. Our results further suggest that dynamic DNA methylation responds to viral infections quickly, regulating subsequent gene activities. Our study provides new insights of molecular mechanisms involved in epigenetic that can serve as foundation for the long-term goal to develop anti-viral strategies for honey bees, the most important commercial pollinator.Author SummaryHoney bees, the most important managed pollinators, are experiencing unsustainable mortality. Israeli Acute Paralysis Virus (IAPV) causes economically important disease in honey bees, and it is emerging as a model system to study viral pathogen-host interactions in pollinators. The pupation stage is important for bee development but individuals are particularly vulnerable for parasitic mite infestations and viral infections. Currently, it is unclear how honey bee pupae respond to this virus. However, these responses, including gene expression and DNA methylomic changes, are critical to understand so that anti-viral genes can be identified and new anti-viral strategies be developed. Here, we use next-generation sequencing tools to reveal the dynamic changes of gene expression and DNA methylation as pupae succumb to IAPV infections after 5, 20, and 48 hours. We found that IAPV causes changes in regions of DNA methylation more at the beginning of infection than later. The activity of several common insect immune pathways are affected by the IAPV infections, as are some other fundamental biological processes. Expression of critical enzymes in DNA methylation are also induced by IAPV in a temporal manner. By comparing our results to other virus studies of honey bees and fruit flies, we identified common anti-viral immune responses. Thus, our study provides new insight on the genome responses of honey bees over the course of a fatal virus infection with theoretical and practical implications.

The Pathogen Profile of a Honey Bee Queen Does Not Reflect That of Her Workers

Throughout a honey bee queen’s lifetime, she is tended to by her worker daughters, who feed and groom her. Such interactions provide possible horizontal transmission routes for pathogens from the workers to the queen, and as such a queen’s pathogen profile may be representative of the workers within a colony. To explore this further, we investigated known honey bee pathogen co-occurrence, as well as pathogen transmission from workers to queens. Queens from 42 colonies were removed from their source hives and exchanged into a second, unrelated foster colony. Worker samples were taken from the source colony on the day of queen exchange and the queens were collected 24 days after introduction. All samples were screened for Nosema spp., Trypanosome spp., acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), Israeli acute paralysis virus (IAPV), Lake Sinai virus (LSV), and deformed wing virus master variants (DWV-A, B, and C) using RT-qPCR. The data show that LSV, Nosema, and DWV-B were the most abundant pathogens in colonies. All workers (n = 42) were LSV-positive, 88% were Nosema-positive, whilst pathogen loads were low (<1 × 106 genome equivalents per pooled worker sample). All queens (n = 39) were negative for both LSV and Nosema. We found no evidence of DWV transmission occurring from worker to queen when comparing queens to foster colonies, despite DWV being present in both queens and workers. Honey bee pathogen presence and diversity in queens cannot be revealed from screening workers, nor were pathogens successfully transmitted to the queen.

A Reverse Genetics System for the Israeli Acute Paralysis Virus and Chronic Bee Paralysis Virus

Honey bee viruses are associated with honey bee colony decline. Israeli acute paralysis virus (IAPV) is considered to have a strong impact on honey bee survival. Phylogenetic analysis of the viral genomes from several regions of the world showed that various IAPV lineages had substantial differences in virulence. Chronic bee paralysis virus (CBPV), another important honey bee virus, can induce two significantly different symptoms. However, the infection characteristics and pathogenesis of IAPV and CBPV have not been completely elucidated. Here, we constructed infectious clones of IAPV and CBPV using a universal vector to provide a basis for studying their replication and pathogenesis. Infectious IAPV and CBPV were rescued from molecular clones of IAPV and CBPV genomes, respectively, that induced typical paralysis symptoms. The replication levels and expression proteins of IAPV and CBPV in progeny virus production were confirmed by qPCR and Western blot. Our results will allow further dissection of the role of each gene in the context of viral infection while helping to study viral pathogenesis and develop antiviral drugs using reverse genetics systems.

Addendum: Amiri, E. et al. Israeli Acute Paralysis Virus: Honey Bee Queen–Worker Interaction and Potential Virus Transmission Pathways. Insects 2019, 10, 9

It has been brought to our attention that one note was missing in the Funding section of our published paper [...]

The Israeli Acute Paralysis Virus IRES captures host ribosomes by mimicking a ribosomal state with hybrid tRNAs

TheColonyCollapseDisorder or CCD is a multi-faceted syndrome decimating bee populations worldwide[1]. A group of viruses of the widely distributedDicistroviridaefamily have been identified as a causing agent of CCD[2]. This family of viruses employ non-coding RNA sequences, calledInternalRibosomalEntrySite (IRES), to precisely exploit the host machinery for protein production. Using single-particle cryo-electron microscopy (cryo-EM) we have characterized at high resolution how the IRES of the intergenic region of theIsraeliAcuteParalysisVirus (IAPV) captures and redirects translating ribosomes towards viral messengers. Through a series of six structures at nominal resolutions close to 3Å, we could reconstruct the trajectory of IAPV-IRES from an early small subunit recruitment to a final post-translocated state in the ribosome. An early commitment of IRES/ribosome complexes for global pre-translocation mimicry explains the high efficiency observed for this IRES. The presented structures will help guide on-going efforts directed towards fighting CCD through RNA-interference technology [3].

Israeli Acute Paralysis Virus: Honey Bee Queen–Worker Interaction and Potential Virus Transmission Pathways

Queen loss or failure is an important cause of honey bee colony loss. A functional queen is essential to a colony, and the queen is predicted to be well protected by worker bees and other mechanisms of social immunity. Nevertheless, several honey bee pathogens (including viruses) can infect queens. Here, we report a series of experiments to test how virus infection influences queen–worker interactions and the consequences for virus transmission. We used Israeli acute paralysis virus (IAPV) as an experimental pathogen because it is relevant to bee health but is not omnipresent. Queens were observed spending 50% of their time with healthy workers, 32% with infected workers, and 18% without interaction. However, the overall bias toward healthy workers was not statistically significant, and there was considerable individual to individual variability. We found that physical contact between infected workers and queens leads to high queen infection in some cases, suggesting that IAPV infections also spread through close bodily contact. Across experiments, queens exhibited lower IAPV titers than surrounding workers. Thus, our results indicate that honey bee queens are better protected by individual and social immunity, but this protection is insufficient to prevent IAPV infections completely.

Detection of Israeli Acute Paralysis Virus (IAPV) and Apis mellifera Filamentous Virus (AmFV) in Honey Bees in Mexico

The recent alarming loss of honey bee colonies around the world is believed to be related to the presence of viruses. The aim of this study was to detect two major viral diseases, Apis mellifera Filamentous virus (AmFV) and Israeli Acute Paralysis Virus (IAPV) using Reverse Transcription - Polymerase Chain Reaction RT-PCR, in honey bees in Mexico. Adult and larvae honey bee samples were collected from asymptomatic colonies of six major beekeeping regions in the state of Chihuahua, Mexico. Both viruses were detected in both developmental stages of honey bees, IAPV at a higher prevalence (23.5%) as compared to AmFV, only in 0.9% of samples. However, this is the first report on AmFV infection in Mexican apiaries. Further studies are required to understand the AmFV and IAPV impact on colony loss in Mexico and to develop strategies for enhancing the control of viral diseases.