BackgroundSwine herd immunization through modified live vaccination or live virus inoculation is a core strategy for controlling porcine reproductive and respiratory syndrome (PRRS), one of the most economically important endemic diseases in the United States that is caused by PRRSV-2. While antigenically homologous PRRSV-2 viruses are expected to elicit better immunological cross-protection, questions remain as to how genetic similarity translates to the degree of cross-protection elicited between the immunizing strain and subsequent challenge virus. For PRRSV-2, virus neutralization (VN) assays are commonly used to approximate humoral immune responses and to assess cross-variant vaccine efficacy; however, these assays are labor-intensive and difficult to scale for rapid immunization planning.MethodsHere, we developed a machine learning–based predictive model for PRRSV-2 cross-neutralization using viral genetic sequence data to address this gap. Virus neutralization assays were performed across a panel of viral isolates and anti-sera, representing a variety of contemporary and vaccine-like PRRSV-2 variants. Our internal dataset comprised 219 cross-neutralization pairs involving nine viral isolates and 25 antisera generated from animals inoculated with six isolates (immunizing viruses). Neutralization titers were classified as above versus below average using mean standardized log2 titers as the cutoff. Candidate predictors included residue-wise amino acid property differences, overall viral genetic distances, and protein structural comparison metrics. Eleven machine learning algorithms were trained and evaluated using internal and external test sets.ResultsThe best models, which utilized predictors from protein ectodomain features, effectively estimated whether a virus-antisera pair would exhibit above versus below average neutralization, achieving balanced accuracies of 87–92% on internal data, and 71–89% across three external VN datasets. Key predictors included amino acid properties in GP5 decoy epitope and hypervariable regions, residues within GP2–GP3 epitopes, and overall genetic distance.ConclusionThese models are publicly available through a web-based tool (https://stemma.shinyapps.io/PRRSLoom-NeutralizationPredictor/) for end-users to estimate the neutralization potential between genetically distinct PRRSV-2 viruses. Our integration of machine learning, in vitro experimental data, and webtool bridges experimental research with real-world application in veterinary decision-making.

Cuba is in a unique situation in which it has a large (220,000 managed colonies) and isolated honey bee population due to a 60+ year ban on the importation of bees. Despite this, the ectoparasitic mite Varroa destructor arrived in 1996, and with it came deformed wing virus (DWV). In 2018, an island-wide survey detected varroa and DWV in 91% of colonies. In this study, we conducted a full-virome analysis on some of these samples, along with additional samples collected in 2021. For the first time, we detected two variants of Lake Sinai Virus and confirmed the absence of the normally widespread black queen cell virus in Cuba. We also detected both DWV-A and DWV-B master variants, with DWV-B being the dominant variant. Interestingly, the DWV-B/A recombinant was also detected, indicating that despite Cuba’s isolated nature, the pattern of DWV evolution mirrors that found in the USA and Europe. However, this pattern is not found in neighboring Latin America, China, or Japan, where the DWV-A master variant continues to be dominant. How and why two distinct evolutionary DWV pathways have arisen remain a mystery.

The concept of viral quasispecies refers to a constantly mutating viral population occurring within hosts, which is essential for grasping the micro-evolutionary patterns of viruses. Despite its high error rate, long-read sequencing holds potential for advancing viral quasispecies research by resolving coverage limitations in next-generation sequencing. We introduce a refined workflow, QoALa, implemented in the longreadvqs R package. This workflow begins with nucleotide position-wise noise minimization of read alignments and sample size standardization, and extends to viral quasispecies comparison across related samples with integrated visualization capabilities. Benchmarking on simulated SARS-CoV-2 and HIV-1 datasets demonstrated that QoALa consistently outperformed existing error-correction methods in recovering quasispecies composition, particularly in preserving nucleotide diversity and hierarchical population structure. Real raw read samples from five studies of different viruses (HCV, HBV, HIV-1, SARS-CoV-2, and IAV), sequenced by major long-read platforms, were also used to evaluate these approaches. The comparative results provide novel insights into intra- and inter-host diversity dynamics in various scenarios and unveil rare haplotypes not reported in the original studies, underscoring the versatility and practicality of our methodology.

Bovine leukemia virus (BLV) is a highly prevalent retrovirus in US dairy cattle herds and has negatively affected dairy herds by reducing milk production, increasing culling rates, and contributing to economic losses. The objective of this study was to link pathogen genomic data with animal-level data to provide inference to BLV transmission pathways in dairy herds. Blood samples were collected from cattle of at least 8 mo of age during 5 different sampling periods in 2 different dairy herds. Following testing, we extracted, sequenced, and assembled the BLV proviral DNA using a prior validated and published protocol from positive samples. The assembled near-complete genomes were aligned and converted to phylogenetic trees, and single nucleotide variant (SNV) matrices were created. When compared with global BLV genomes published in GenBank, the BLV sequences from our 2 Minnesota study herds (herd A and F) showed additional clade diversity not shared with previously characterized global variants. The BLV genomes identified from the 2 study herds were organized in multiple clades, and herd A had more BLV genomic diversity than herd F. Of the 195 total BLV sequences from the herds, 51 were from cows at multiple time points, and from herds A and F, respectively 84.5% and 98.3%, of within-host pairs of cattle had ≤10 SNV differences between the pairs, and 82% and 98.4% of within-host pairs were in identical clades. These results indicated low variability of the BLV proviral DNA genome through time within individual infected cattle. Within the dam-daughter (DD) cattle pairs from herds A (n = 24) and F (n = 10), 62.5% and 60% had ≤10 SNV and 50% and 60% clustered in identical clades, respectively. In comparison, other between-cattle pairs in herds A and F showed 35.7% (n = 4,981) and 48.4% (n = 4,300) had ≤10 SNV and 11.2% and 50.6% in identical clades, respectively. These results are consistent with DD transmission in a subset of BLV-infected cattle. Pairs of cattle born at similar times in the herd had a similar SNV distribution to other cattle pairs within their herds, indicating minimal transmission early in life among similar age cohorts. Genomes from cattle with a high BLV proviral load (relative concentration of BLV proviral DNA to bovine DNA in copies/µL >2,000) were distributed across most clades, indicating the potential for local transmission for BLV from multiple clades rather than a single unusually transmissible lineage dominating the herd. In summary, this study shows the potential benefit of using near-complete genomes to identify high-resolution within-herd BLV differences to infer transmission.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses in the global swine industry, partly because current vaccination strategies offer limited protection against heterologous viral strains. Since modified medium-chain fatty acids (mMCFA) offer enhanced structural stability and antimicrobial properties compared with unmodified MCFA, we evaluated a proprietary mMCFA blend (D-Strike, Devenish Nutrition) against PRRSV in vitro and in vivo. In vitro efficacy was assessed using TCID50 assays on MARC-145 cells and a viability qPCR (V-qPCR). At 1:1000, 1:2000, and 1:5000 dilutions, the mMCFA inactivated >4 logs (99.99%) of the virus within 5 min of exposure. The results of V-qPCR were also compatible with those obtained by cell culture assays. For the in vivo study, 40 weaned male piglets were assigned to four dietary treatments: negative control (group A), PRRSV-challenge control (group B), 0.2% D-Strike (group C), and 0.4% D-Strike (group D). Pigs in groups B, C, and D were challenged intranasally with virulent PRRSV. The results demonstrated that dietary mMCFA reduced clinical signs, attenuated a febrile response, and decreased the severity of lung lesions. Some responses appeared more pronounced at the 0.4% inclusion level than at 0.2%, although a formal dose-response analysis was not statistically significant for most outcome measures. The sera from the 0.4% D-Strike group showed 65% lower PRRSV than the positive control, although this difference was not statistically significant. In addition, this group had 0% mortality as compared to 20% in the positive control group. Feed efficiency also improved in D-Strike-fed pigs. These findings indicate that mMCFA shows promise as a feed additive for mitigating PRRSV infection, although the in vivo antiviral effects require confirmation in larger studies.

Porcine reproductive and respiratory syndrome (PRRS) continues to be a major threat to U.S. swine industry, as a substantial number of herds become positive and can pose a risk to other nearby farms, especially in post weaning farms as multiple of them may be overseen by one worker. Personnel moving between farms without adequate biosecurity measures, may play a role in viral spread acting as a fomite. The ability to detect and distinguish between the free PRRS virus (PRRSV) genomic RNA vs its genome found in a viable virus form on frequently touched surfaces in growing pig farms was assess in this study. Ten PRRSV positive growing pig farms in the Midwestern U.S. were visited to collect 20 environmental surface samples and eight oral fluids from each one. Environmental samples were analyzed using standard RT-qPCR and viability RT-qPCR, while oral fluids were assessed using the VetMAX™ PRRSV EU & NA v3.0 kit. The virus' RNA was commonly detected on metal and plastic (non-porous) surfaces (e.g., pig pen top rail, mortality handling equipment's handle) from all farms, with 80 out of 200 environmental samples testing positive. Viable virus was detected in 48 samples across six farms, with non-porous materials testing positive more frequently. A generalized linear mixed effect model suggested a negative association (OR = 0.005; 95% CI 0.00, 6.82; p-value = 0.18) between the proportion of positive oral fluids and detecting viable virus from sampled surfaces. Agreement between the detection of RNA and viable PRRSV from surface samples using Cohen's kappa yielded perfect agreement (κ = 1.00) from doorknobs of different locations, to low agreement (κ = 0.29) in the floor of a specific area, among others. These results indicate the presence of viable virus on surfaces that are frequently touched by the farm's personnel. This study highlights the importance of biosecurity measures applied to the personnel and their potential role of environmental contamination and PRRSV dissemination. The use of viability RT-qPCR to detect viable PRRSV offers a practical tool in field settings to improve biosecurity protocols to reduce indirect transmission of PRRSV in swine production systems.

[This corrects the article DOI: 10.1371/journal.pone.0344714.].

For 65 years, the ectoparasitic mite Varroa destructor, along with the deformed wing virus (DWV) it vectors, has killed millions of Apis mellifera honey bee colonies globally. The coevolution of the 'bee-mite-virus' system in Hawaii over 17 years has been closely followed. Resistance to V. destructor evolved in the free-living honey bees on Oahu by detecting and removing a high proportion of mite-infested brood cells, as seen in other mite-resistant honey bees. On Big Island, miticides remain widely used, so on both islands, DWV prevalence decreased, but a more virulent recombinant form evolved. Thus, coevolution on Oahu has produced a new stable 'bee-mite-virus' state that has been exploited by beekeepers and provides a global solution to the V. destructor problem.

The objective of this study was to evaluate the time-course of incubation for the potential preventative mitigation of megaviruses using Termin-8 (a formaldehyde-based product) and Finio (non-formaldehyde solution) from Anitox. Emiliania huxleyi virus (EhV), an algal surrogate for African swine fever virus (ASFV), was treated with the recommended concentrations of Termin-8 (0.1% to 0.3%) and Finio (0.05% to 0.2%), and both viability qPCR (V-qPCR) and standard PCR (S-qPCR) were used to quantify EhV concentrations at 1 h, 5 h, 24 h and day 7 post-inoculation. Overall, Finio, and to a lesser extent Termin-8, at their highest treatment concentrations, showed the greatest log reduction of 4.5 and 2 log10 units, respectively, at 1 h post-inoculation. Although Termin-8 efficacy did not improve with time, due to its fixing of viral particles and rendering them non-infectious, treatment with Finio showed 100% viable viral inactivation (>5 log10 reduction units) at the lowest concentration after 7 days of exposure. Our results demonstrate that both Termin-8 and Finio can be used as effective chemical mitigants against megaviruses such as EhV and ASFV and can be used as effective preventive or mitigation strategies to prevent the transmission of ASFV by reducing particle viability in contaminated feed, although additional research is warranted.

The combination of Varroa destructor (Varroa) and the viruses it vectors is a major driver of honey bee ( Apis mellifera ) colony losses. Hygienic behavior and individual immunity enable bees to cope with some pests and parasites. Hygienic response of adult bees is driven by olfactory cues emanating from diseased brood. In this study, we tested the effect of viral inoculation on the chemical profile and immune response of pupae, independently of Varroa. We injected pupae with various doses of an enriched inocula of either a deformed wing virus (DWV) B-A recombinant or Israeli acute paralysis virus (IAPV) and followed their development and survival for five days. At the end of the five-day period, volatile profiles, viral loads, and expression of seven immune genes were assessed. Both IAPV and the DWV loads increased to equivalent high levels irrespective of the initial dose applied. Notably, greater rates of mortality (60% loss) were observed with the highest IAPV dose when compared to the lowest IAPV dose (15% loss). The DWV inoculum, caused limited mortality but nonetheless inhibited pupae development. The IAPV injected pupae showed evidence of a dose dependent DWV amplification from either a DWV variant in the original IAPV inoculum or from an endogenous source of DWV in the pupae themselves. IAPV-injected pupae had lower expression of immune genes than DWV-injected pupae, suggesting IAPV inhibits the pupae immune response. Overall, among the seven tested immune genes six were upregulated with only vago downregulated, suggesting inhibition of the RNAi pathway following infection. Chemical cues of mock and untreated pupae were similar, but notably different from the virus-injected pupae for both inocula. Our findings show that Varroa-independent virus inoculated pupae produce unique virus-specific chemical cues; the ultimate consequence of such a change might lead to virus specific bee behavioral responses. Authors summary This study investigated how two major honey bee viruses, Deformed Wing Virus (DWV) and Israeli Acute Paralysis Virus (IAPV), affect pupae independent of the Varroa mite, which typically spreads them. This study was conducted in the laboratory on incubated pupae isolated from the colony and injected with two viral preparations (inocula). We tested the impact after five-days incubation. The results show that both viruses reach high levels in pupae, though IAPV inoculum proved far more deadly (up to 60% mortality) and appeared to suppress the pupae’s immune response, possibly even amplifying co-occurring DWV. While less lethal, the DWV inoculum still inhibited normal development. Interestigly, the virus-infected pupae produced distinct, virus-specific chemical cues—unique volatile profiles different from healthy controls. Since adult honey bees use olfactory cues to detect and remove diseased brood (hygienic behavior), these findings suggest that the viruses themselves generate the “scent of sickness,” which could trigger colony-wide behavioral responses and offer a vital target for enhancing the bees’ natural defenses against these major drivers of colony loss. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. US-Israel Binational Agricultural Research and Development Fund, IS-5469-22 [1]: pending:yes

Background/Objectives: Porcine reproductive and respiratory syndrome virus (PRRSV) is classified into various lineages based on the phylogenetic variation of orf5, which encodes a major surface glycoprotein GP5 containing both neutralizing and non-neutralizing linear epitopes. Several positively selected sites have been identified on the GP5 ectodomain, indicating host immune pressure on these sites. This present study aimed to investigate the kinetics of antibody responses to GP5 and to map the epitope-specific response to the GP5 ectodomain from different PRRSV lineages after vaccination with commercially available modified live virus (MLV) vaccines. Methods: Post-weaning pigs were vaccinated with MLV vaccines derived from either lineage 1D (Prevacent PRRS®) or lineage 5 (Ingelvac PRRS®). Animals were challenged with a heterologous (lineage 1A) strain at 64 days post-vaccination (dpv). Blood samples were collected at various times post-vaccination and challenge. Kinetics of antibody response to different PRRSV antigens were monitored and virus neutralization against archetypal and contemporary strains belonging to lineage 5 and 1A were evaluated. In addition, antibody responses to peptides derived from the GP5 ectodomain of different viral lineages were assessed. Results: Our results showed that the GP5-specific antibody response observed between 18 and 35 dpv was delayed compared to responses to the viral nucleocapsid protein. The polyclonal antibody response in both vaccinated groups showed similar levels of binding to variant GP5 peptides from different sub-lineages. Notably, in both vaccinated groups, the antibody directed to a peptide representing the GP5 ectodomain of a lineage 1C strain (variant 1C.5) displayed a rise in titer at 64 dpv, which was further increased by the challenge with the lineage 1A strain. Less than 50% of animals developed heterologous neutralizing antibodies post-vaccination with both MLV vaccines. However, higher neutralization titers were observed in all vaccinated animal post-challenge. Conclusions: Together, these data provide insights into the antibody responses to the GP5 ectodomain in MLV-vaccinated swine herds.

Introduction Anitox has developed and markets Termin-8 (a formaldehyde-based product) and Finio (non-formaldehyde solution) for the control microbial contamination in feed. To date, both Finio and Termin-8 have not been tested for their potential antiviral activity against megaviruses, such African swine fever virus (ASFV) and its surrogate algal virus, Emiliania huxleyi virus (EhV). Given the limited access and great expense for routine chemical mitigation testing with ASFV, we focused on using EhV as a safe and effective surrogate to evaluate the antiviral activity of these Anitox products. The specific objective of the current study was to evaluate the time course of incubation from hours to days to mimic possible field relevant exposure times for the potential preventative mitigation of megaviruses using Termin-8 and Finio. Methods Emiliania huxleyi virus was treated with the Anitox recommended concentrations of Termin-8 (0.1% to 0.3% final) and Finio (0.05% to 0.2% final) in biological triplicate experiments. Both viability qPCR (V-qPCR) and standard PCR (S-qPCR) were conducted for EhV copies at 1 hr, 5 hrs, 24 hrs and day 7 post-inoculation. Results We observed that both Termin-8 and Finio, at their highest treatment concentrations, showed the greatest log reduction of 2 and 4.5 log10 units, respectively, at the earliest 1 hr post-inoculation time point. Although Termin-8 efficacy did not improve with time, treatment with Finio showed 100% viable viral inactivation (>5 log10 reduction units) at the lowest concentration after 7 days of exposure. In addition, Finio showed negligible viral DNA removal post-treatment as observed via S-qPCR, but use of Termin-8 resulted in a reduction of viral DNA (S-qPCR) equivalent to that observed using the V-qPCR assay. Discussion Our results demonstrate for the first time that both Termin-8 and Finio can be used as effective chemical mitigants against megaviruses such as EhV and ASFV. Moreover, the mechanism of chemical mitigation involves degradation of the virus particle itself. However, although Termin-8 efficacy appeared to be less than that of Finio, this is likely not the case because Termin-8 cross-links protein viral capsids and fixes the viral particle to make it non-infectious. With the threat of ASFV introduction into the North American swine industry and other non-infected regions around the world, the use of both Termin-8 and Finio as an effective preventive or mitigation strategy to prevent the transmission of ASFV by reducing particle viability in contaminated feed. Additional research is warranted with ASFV, in the presence of various types of feed ingredients and complete feeds and use bioassays to evaluate infectivity because results from V-qPCR revealed that intact viable particles remain post-treatment. ### Competing Interest Statement The authors have declared no competing interest.

Pathogen spillover events are of global concern as they have the potential to cause significant harm to the novel host species. The potential of viral spillover from the western honey bee (Apis mellifera) to other insects is well established. New variants should inevitably emerge following a host expansion, yet to our knowledge no study has shown this within this system. To investigate the outcome of viral spillover, we sequenced the RNA biased viromes of sympatric A. mellifera (n = 389) and common eastern bumblebee Bombus impatiens (n = 117) over three years. Distinct viromes occurred within each bee species throughout the study duration, with only one of the well-characterized honey bee viruses, sacbrood virus, consistently found in the bumblebee virome. Viruses shared by both bees shared over 98% nucleotide identity, and no bumblebee-specific strains of honey bee viruses occurred, as expected if spillover led to a true host expansion involving bumblebee-bumblebee transmission. Honey bee viruses, namely deformed wing virus, black queen cell virus, and sacbrood virus, which were present in the bumblebees did not show evidence of diversification among hosts, suggesting environmental exposure or dead-end spillover, rather than spillover host expansion.

Despite extensive use of vaccination, porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) continues to evolve, likely driven by escape from natural or vaccine-derived immunity. However, direct evidence of vaccine-induced evolutionary pressure remains limited. Here, we tracked the evolution of PRRSV-2 sublineage 1A strain IA/2014 (variant 1A-unclassified) genome from infection chains of sequentially infected pigs under different immune conditions. Weaned pigs were divided into three groups: a non-immunized control group and two groups vaccinated with different modified live virus (MLV) vaccines, namely Prevacent® PRRS MLV (variant 1D.2) and Ingelvac PRRS® MLV (variant 5A.1). Sixty-four days post-vaccination, the pigs were challenged with IA/2014 PRRSV-2. Virus infection chains (which used serum from pigs in batch n to infect batch n + 1) were maintained across six sequential batches of roughly seven pigs each, allowing for virus evolution to occur across the ~ 84 days of the infection chain. A total of 110 serum samples were successfully sequenced. Vaccinated groups exhibited over twice the genetic divergence from the original challenge virus (0.3%-0.4% mean nucleotide distance) compared to non-immunized group (0.15%). Variability was concentrated in ORF1a and ORF1b. Deep sequencing revealed more rapid shifts of viral quasispecies composition in vaccinated pigs, and more homogeneous viral populations over batches compared to non-immunized pigs. Selection pressure analyses indicated strong purifying selection in one vaccinated group, though without clear signals at known antigenic sites in all treatment groups. However, vaccinated pigs had significantly higher cycle threshold values (P<.001), indicating lower viral loads and suggesting potential fitness limitations for highly diverged viruses in immunized pigs. These findings demonstrate that MLV vaccination can exert substantial evolutionary pressure on PRRSV-2, driving genetic diversification and highlighting the need for continuous PRRS monitoring and adaptive control strategies.

Despite extensive use of vaccination, porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) continues to evolve, likely driven by escape from natural or vaccine-derived immunity. However, direct evidence of vaccine-induced evolutionary pressure remains limited. Here, we tracked the evolution of PRRSV-2 sub-lineage 1A strain IA/2014 (variant 1A-unclassified) genome from infection chains of sequentially infected pigs under different immune conditions. Weaned pigs were divided into three groups: a non-immunized control group and two groups vaccinated with different modified live vaccines (MLVs), namely Prevacent® PRRS MLV (variant 1D.2) and Ingelvac PRRS® MLV (variant 5A.1). Sixty-four days post-vaccination, the pigs were challenged with IA/2014 PRRSV-2. Virus infection chains (which used serum from pigs in batch n to infect batch n + 1) were maintained across six sequential batches of roughly seven pigs each, allowing for virus evolution to occur across the ~ 84 days of the infection chain. A total of 110 serum samples were successfully sequenced. Vaccinated groups exhibited over twice the genetic divergence from the original challenge virus (0.3–0.4% mean nucleotide distance) compared to non-immunized group (0.15%). Variability was concentrated in ORF1a and ORF1b. Deep sequencing revealed more rapid shifts of viral quasispecies composition in vaccinated pigs, and more homogeneous viral populations over batches compared to non-immunized pigs. Selection pressure analyses indicated strong purifying selection in one vaccinated group, though without clear signals at known antigenic sites in all treatment groups. However, vaccinated pigs had significantly higher Ct values (p<.001), indicating lower viral loads and suggesting potential fitness limitations for highly diverged viruses in immunized pigs. These findings demonstrate that MLV vaccination can exert substantial evolutionary pressure on PRRSV-2, driving genetic diversification and highlighting the need for continuous PRRS monitoring and adaptive control strategies.

Porcine reproductive and respiratory syndrome virus remains one of the most economically significant pathogens in swine production, with PRRSV-2 being the dominant variant in the United States. While lineage classification has traditionally relied on ORF5 sequencing, recent studies suggest that this single-gene approach may overlook key evolutionary events such as recombination. In this study, we performed whole-genome sequencing and phylogenetic analysis of seven PRRSV-2 isolates collected in the U.S. between 2006 and 2024. Using reference-guided assembly, lineage assignment, and recombination detection with RDP5 and SIMplot, we identified discordant phylogenetic placements between ORF5 and whole genomes in four of the seven isolates. These discordances were explained by multiple recombination events affecting different genomic regions, particularly ORF2–ORF7. In contrast, three isolates showed phylogenetic concordance and no strong evidence of recombination. Our findings demonstrate that recombination plays a significant role in shaping PRRSV-2 evolution and highlight the limitations of ORF5-based lineage classification. Whole-genome surveillance is therefore essential to accurately track viral diversity, detect recombinant strains, and inform control strategies. This work underscores the need for a broader adoption of full-genome analysis in routine PRRSV surveillance and research.

IntroductionAfrican swine fever virus (ASFV) is extremely stable in the environment, and previous laboratory experiments and simulations have also shown it to be highly stable in animal feed ingredients. However, ASFV cannot be studied in real world demonstrations because it is a highly contagious virus. African swine fever virus is a member of the nucleocytoplasmic large DNA viruses (NCLDVs), and similar to Emiliania huxleyi virus (EhV), which has a restricted host range limited to a species of marine algae called Emiliania huxleyi. This algal NCLDV has many similar morphological and physical characteristics to ASFV, thereby making it a safe surrogate for generating experimental results that are applicable to ASFV and representative of real-world conditions. MethodsWe inoculated whole soybeans with EhV strain 86 (EhV-86) at a concentration of 1.80 × 108 virus/mL, which were then processed at a pilot solvent extraction facility to produce soybean hulls and meal. After processing, samples were evaluated for virus presence and viability using a previously validated viability qPCR (V-qPCR) method.ResultsNo detection of EhV-86 occurred on environmental surfaces, air, and dust samples pre- or post-processing. Viable EhV-86 was detected in conditioned soybeans, dehulled soybeans, soybean hulls, soybean flakes, air-dried solvent extracted soybean flakes, post-desolventizer toaster soybean flakes, and soybean meal after reaching steady state during solvent extraction processing.DiscussionIt is important to note that 95% of viable virus was recovered (2.43 × 106 virus/g in replicate A and 2.61 × 106 virus/g in replicate B) in soybean meal, suggesting that longer retention times or application of chemical mitigants may be needed for more complete inactivation. The high concentration of viable viruses remaining on the soybean hulls after processing (1.98 × 107 virus/g in replicate A and 2.12 × 107 virus/g in replicate B) is a major concern for potential virus transmission in animal feed. These results demonstrate for the first time that ASFV-like NCLDVs can retain viability in soybean hulls, flakes, and meal during solvent extraction processing in a pilot facility and remain a hazard for virus transmission. Future risk assessments focused on the role of contaminated feed ingredients in transmission of viruses to swine farms must consider the ingredient composition of complete feeds delivered to farms and the initial concentration of viable viruses.

The globally distributed marine alga Emiliania huxleyi has cooling effect on the Earth’s climate. The population density of E. huxleyi is restricted by Nucleocytoviricota viruses, including E. huxleyi virus 201 (EhV-201). Despite the impact of E. huxleyi viruses on the climate, there is limited information about their structure and replication. Here, we show that the dsDNA genome inside the EhV-201 virion is protected by an inner membrane, capsid, and outer membrane. EhV-201 virions infect E. huxleyi by using fivefold vertices to bind to and fuse the virus’ inner membrane with the cell plasma membrane. Progeny virions assemble in the cytoplasm at the surface of endoplasmic reticulum–derived membrane segments. Genome packaging initiates synchronously with the capsid assembly and completes through an aperture in the forming capsid. The genome-filled capsids acquire an outer membrane by budding into intracellular vesicles. EhV-201 infection induces a loss of surface protective layers from E. huxleyi cells, which enables the continuous release of virions by exocytosis.

Deformed wing virus (DWV) was first detected in dead honey bees in 1982 but has been in honey bees for at least 300 years. Due to its high prevalence and virulence, they have been linked with the ongoing decline in honey bee populations worldwide. A rapid, simple, semi-automated, high-throughput, and cost-effective method of screening colonies for viruses would benefit bee research and the beekeeping industry. Here we describe a semi-automated approach that combines an RNA-grade liquid homogenizer followed by magnetic bead capture for total virus nucleic acid extraction. We compare it to the more commonly applied nucleic acid column-based purification method and use qPCR plus Oxford Nanopore Technologies sequencing to evaluate the accuracy of analytical results for both methods. Our results showed high reproducibility and accuracy for both approaches. The semi-automated method described here allows for faster screening of viral loads in units of 96 samples at a time. We developed this method to monitor viral loads in honey bee colonies, but it could be easily applied for any PCR or genomic-based screening assays.