Targeted phage hunting to specific Klebsiella pneumoniae clinical isolates is an efficient antibiotic resistance and infection control strategy.

Klebsiella pneumoniae is one of the most threatening multi-drug-resistant pathogens today, with phage therapy being a promising alternative for personalized treatments. However, the intrinsic capsule diversity in Klebsiella spp. poses a substantial barrier to the phage host range, complicating the development of broad-spectrum phage-based treatments. Here, we have isolated and genomically characterized phages capable of infecting each of the acquired 77 reference serotypes of Klebsiella spp., including capsular types widespread among high-risk K. pneumoniae clones causing nosocomial infections. We demonstrated the possibility of isolating phages for all capsular types in the collection, revealing high capsular specificity among taxonomically related phages, in contrast to a few phages that exhibited broad-spectrum infection capabilities. To decipher the determinants of the specificity of these phages, we focused on their receptor-binding proteins, with particular attention to depolymerases. We also explored the possibility of designing a broad-spectrum phage cocktail based on phages isolated in reference capsular-type strains and determining the ability to lyse relevant clinical isolates. A combination of 12 phages capable of infecting 55% of the reference Klebsiella spp. serotypes was tested on a panel of carbapenem-resistant K. pneumoniae clinical isolates. Thirty-one percent of isolates were susceptible to the phage cocktail. However, our results suggest that in a highly variable encapsulated bacterial host, phage hunting must be directed to the specific Klebsiella isolates. This work is a step forward in the understanding of the complexity of phage-host interactions and highlights the importance of implementing precise and phage-specific strategies to treat K. pneumoniae infections worldwide.IMPORTANCEThe emergence of resistant bacteria is a serious global health problem. In the absence of effective treatments, phages are a personalized and effective therapeutic alternative. However, little is still known about phage-host interactions, which are key to implementing effective strategies. Here, we focus on the study of Klebsiella pneumoniae, a highly pathogenic encapsulated bacterium. The complexity and variability of the capsule, where in most cases phage receptors are found, make it difficult for phage-based treatments. Here, we isolated a large collection of Klebsiella phages against all the reference strains and in a cohort of clinical isolates. Our results suggest that clinical isolates represent a challenge, especially high-risk clones. Thus, we propose targeted phage hunting as an effective strategy to implement phage-derived therapies. Our results are a step forward for new phage-based strategies to control K. pneumoniae infections, highlighting the importance of understanding phage-host interactions to design personalized treatments against Klebsiella spp.

Genetic diversity and cross-species transmissibility of bat-associated picornaviruses from Spain.

BACKGROUND: Emerging zoonotic diseases arise from cross-species transmission events between wild or domesticated animals and humans, with bats being one of the major reservoirs of zoonotic viruses. Viral metagenomics has led to the discovery of many viruses, but efforts have mainly been focused on some areas of the world and on certain viral families. METHODS: We set out to describe full-length genomes of new picorna-like viruses by collecting feces from hundreds of bats captured in different regions of Spain. Viral sequences were obtained by high-throughput Illumina sequencing and analyzed phylogenetically to classify them in the context of known viruses. Linear discriminant analysis (LDA) was performed to infer likely hosts based on genome composition. RESULTS: We found five complete or nearly complete genomes belonging to the family Picornaviridae, including a new species of the subfamily Ensavirinae. LDA suggested that these were true vertebrate viruses, rather than viruses from the bat diet. Some of these viruses were related to picornaviruses previously found in other bat species from distant geographical regions. We also found a calhevirus genome that most likely belongs to a proposed new family within the order Picornavirales, and for which genome composition analysis suggested a plant host. CONCLUSIONS: Our findings describe new picorna-like viral species and variants circulating in the Iberian Peninsula, illustrate the wide geographical distribution and interspecies transmissibility of picornaviruses, and suggest new hosts for calheviruses.

DepoScope: Accurate phage depolymerase annotation and domain delineation using large language models.

Bacteriophages (phages) are viruses that infect bacteria. Many of them produce specific enzymes called depolymerases to break down external polysaccharide structures. Accurate annotation and domain identification of these depolymerases are challenging due to their inherent sequence diversity. Hence, we present DepoScope, a machine learning tool that combines a fine-tuned ESM-2 model with a convolutional neural network to identify depolymerase sequences and their enzymatic domains precisely. To accomplish this, we curated a dataset from the INPHARED phage genome database, created a polysaccharide-degrading domain database, and applied sequential filters to construct a high-quality dataset, which is subsequently used to train DepoScope. Our work is the first approach that combines sequence-level predictions with amino-acid-level predictions for accurate depolymerase detection and functional domain identification. In that way, we believe that DepoScope can greatly enhance our understanding of phage-host interactions at the level of depolymerases.

Full-genome sequencing of dozens of new DNA viruses found in Spanish bat feces.

Bats are natural hosts of multiple viruses, many of which have clear zoonotic potential. The search for emerging viruses has been aided by the implementation of metagenomic tools, which have also enabled the detection of unprecedented viral diversity. Currently, this search is mainly focused on RNA viruses, which are largely over-represented in databases. To compensate for this research bias, we analyzed fecal samples from 189 Spanish bats belonging to 22 different species using viral metagenomics. This allowed us to identify 52 complete or near-complete viral genomes belonging to the families Adenoviridae, Circoviridae, Genomoviridae, Papillomaviridae, Parvoviridae, Polyomaviridae and Smacoviridae. Of these, 30 could constitute new species, doubling the number of viruses currently described in Europe. These findings open the door to a more thorough analysis of bat DNA viruses and their zoonotic potential. IMPORTANCE: Metagenomics has become a fundamental tool to characterize the global virosphere, allowing us not only to understand the existing viral diversity and its ecological implications but also to identify new and emerging viruses. RNA viruses have a higher zoonotic potential, but this risk is also present for some DNA virus families. In our study, we analyzed the DNA fraction of fecal samples from 22 Spanish bat species, identifying 52 complete or near-complete genomes of different viral families with zoonotic potential. This doubles the number of genomes currently described in Europe. Metagenomic data often produce partial genomes that can be difficult to analyze. Our work, however, has characterized a large number of complete genomes, thus facilitating their taxonomic classification and enabling different analyses to be carried out to evaluate their zoonotic potential. For example, recombination studies are relevant since this phenomenon could play a major role in cross-species transmission.

An efficient plasmid-based system for the recovery of recombinant vesicular stomatitis virus encoding foreign glycoproteins.

Viral glycoproteins mediate entry into host cells, thereby dictating host range and pathogenesis. In addition, they constitute the principal target of neutralizing antibody responses, making them important antigens in vaccine development. Recombinant vesicular stomatitis virus (VSV) encoding foreign glycoproteins can provide a convenient and safe surrogate system to interrogate the function, evolution, and antigenicity of viral glycoproteins from viruses that are difficult to manipulate or those requiring high biosafety level containment. However, the production of recombinant VSV can be technically challenging. In this work, we present an efficient and robust plasmid-based system for the production of recombinant VSV encoding foreign glycoproteins. We validate the system using glycoproteins from different viral families, including arenaviruses, coronaviruses, and hantaviruses, as well as highlight their utility for studying the effects of mutations on viral fitness. Overall, the methods described herein can facilitate the study of both native and recombinant VSV encoding foreign glycoproteins and can serve as the basis for the production of VSV-based vaccines.

Cell type-specific adaptation of the SARS-CoV-2 spike.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can infect various human tissues and cell types, principally via interaction with its cognate receptor angiotensin-converting enzyme-2 (ACE2). However, how the virus evolves in different cellular environments is poorly understood. Here, we used experimental evolution to study the adaptation of the SARS-CoV-2 spike to four human cell lines expressing different levels of key entry factors. After twenty passages of a spike-expressing recombinant vesicular stomatitis virus (VSV), cell-type-specific phenotypic changes were observed and sequencing allowed the identification of sixteen adaptive spike mutations. We used VSV pseudotyping to measure the entry efficiency, ACE2 affinity, spike processing, TMPRSS2 usage, and entry pathway usage of all the mutants, alone or in combination. The fusogenicity of the mutant spikes was assessed with a cell-cell fusion assay. Finally, mutant recombinant VSVs were used to measure the fitness advantage associated with selected mutations. We found that the effects of these mutations varied across cell types, both in terms of viral entry and replicative fitness. Interestingly, two spike mutations (L48S and A372T) that emerged in cells expressing low ACE2 levels increased receptor affinity, syncytia induction, and entry efficiency under low-ACE2 conditions. Our results demonstrate specific adaptation of the SARS-CoV-2 spike to different cell types and have implications for understanding SARS-CoV-2 tissue tropism and evolution.

Prediction of Klebsiella phage-host specificity at the strain level.

Phages are increasingly considered promising alternatives to target drug-resistant bacterial pathogens. However, their often-narrow host range can make it challenging to find matching phages against bacteria of interest. Current computational tools do not accurately predict interactions at the strain level in a way that is relevant and properly evaluated for practical use. We present PhageHostLearn, a machine learning system that predicts strain-level interactions between receptor-binding proteins and bacterial receptors for Klebsiella phage-bacteria pairs. We evaluate this system both in silico and in the laboratory, in the clinically relevant setting of finding matching phages against bacterial strains. PhageHostLearn reaches a cross-validated ROC AUC of up to 81.8% in silico and maintains this performance in laboratory validation. Our approach provides a framework for developing and evaluating phage-host prediction methods that are useful in practice, which we believe to be a meaningful contribution to the machine-learning-guided development of phage therapeutics and diagnostics.

Temperature impacts SARS-CoV-2 spike fusogenicity and evolution.

SARS-CoV-2 infects both the upper and lower respiratory tracts, which are characterized by different temperatures (33°C and 37°C, respectively). In addition, fever is a common COVID-19 symptom. SARS-CoV-2 has been shown to replicate more efficiently at low temperatures, but the effect of temperature on different viral proteins remains poorly understood. Here, we investigate how temperature affects the SARS-CoV-2 spike function and evolution. We first observed that increasing temperature from 33°C to 37°C or 39°C increased spike-mediated cell-cell fusion. We then experimentally evolved a recombinant vesicular stomatitis virus expressing the SARS-CoV-2 spike at these different temperatures. We found that spike-mediated cell-cell fusion was maintained during evolution at 39°C but was lost in a high proportion of viruses that evolved at 33°C or 37°C. Consistently, sequencing of the spikes evolved at 33°C or 37°C revealed the accumulation of mutations around the furin cleavage site, a region that determines cell-cell fusion, whereas this did not occur in spikes evolved at 39°C. Finally, using site-directed mutagenesis, we found that disruption of the furin cleavage site had a temperature-dependent effect on spike-induced cell-cell fusion and viral fitness. Our results suggest that variations in body temperature may affect the activity and diversification of the SARS-CoV-2 spike. IMPORTANCE: When it infects humans, SARS-CoV-2 is exposed to different temperatures (e.g., replication site and fever). Temperature has been shown to strongly impact SARS-CoV-2 replication, but how it affects the activity and evolution of the spike protein remains poorly understood. Here, we first show that high temperatures increase the SARS-CoV-2 spike fusogenicity. Then, we demonstrate that the evolution of the spike activity and variants depends on temperature. Finally, we show that the functional effect of specific spike mutations is temperature-dependent. Overall, our results suggest that temperature may be a factor influencing the activity and adaptation of the SARS-CoV-2 spike in vivo, which will help understanding viral tropism, pathogenesis, and evolution.

Cellular receptors for mammalian viruses.

The interaction of viral surface components with cellular receptors and other entry factors determines key features of viral infection such as host range, tropism and virulence. Despite intensive research, our understanding of these interactions remains limited. Here, we report a systematic analysis of published work on mammalian virus receptors and attachment factors. We build a dataset twice the size of those available to date and specify the role of each factor in virus entry. We identify cellular proteins that are preferentially used as virus receptors, which tend to be plasma membrane proteins with a high propensity to interact with other proteins. Using machine learning, we assign cell surface proteins a score that predicts their ability to function as virus receptors. Our results also reveal common patterns of receptor usage among viruses and suggest that enveloped viruses tend to use a broader repertoire of alternative receptors than non-enveloped viruses, a feature that might confer them with higher interspecies transmissibility.

Optimized Recovery of Viral DNA and RNA from Blood Plasma for Viral Metagenomics.

Metagenomics is vastly improving our ability to discover new viruses, as well as their possible associations with disease. However, metagenomics has also changed our understanding of viruses in general. This is because we can find viruses in healthy hosts in the absence of disease, which changes the perspective of viruses as mere pathogens and offers a new perspective in which viruses function as important components of ecosystems. In concrete, human blood metagenomics has revealed the presence of different types of viruses in apparently healthy subjects. These viruses are human anelloviruses and, to a lower extent, human pegiviruses. Viral metagenomics' major challenge is the correct isolation of the viral nucleic acids from a specific sample. For the protocol to be successful, all steps must be carefully chosen, in particular those that optimize the recovery of viral nucleic acids. Here, we present a procedure that allows the recovery of both DNA and RNA viruses from plasma samples.

Broad-range capsule-dependent lytic Sugarlandvirus against Klebsiella sp.

IMPORTANCE: The emergence of multi-drug resistant bacteria is a global health problem. Among them, Klebsiella pneumoniae is considered a high-priority pathogen, making it necessary to develop new therapeutic tools to reduce the bacterial burden in an effective and sustainable manner. Phages, bacterial viruses, are very promising tools. However, phages are highy specific, rendering large-scale therapeutics costly to implement. This is especially certain in Klebsiella, a capsular bacterium in which phages have been shown to be capsular type dependent, infecting one or a few capsular types through specific enzymes called depolymerases. In this study, we have isolated and characterized novel phages with lytic ability against bacteria from a wide variety of capsular types, representing the Klebsiella phages with the widest range of infection described. Remarkably, these broad-range phages showed capsule dependency, despite the absence of depolymerases in their genomes, implying that infectivity could be governed by alternative mechanisms yet to be uncovered.

Human Anelloviruses: Influence of Demographic Factors, Recombination, and Worldwide Diversity.

Anelloviruses represent the major and most diverse component of the healthy human virome, referred to as the anellome. In this study, we determined the anellome of 50 blood donors, forming two sex- and age-matched groups. Anelloviruses were detected in 86% of the donors. The number of detected anelloviruses increased with age and was approximately twice as high in men as in women. A total of 349 complete or nearly complete genomes were classified as belonging to torque teno virus (TTV), torque teno mini virus (TTMV), and torque teno midi virus (TTMDV) anellovirus genera (197, 88, and 64 sequences, respectively). Most donors had intergenus (69.8%) or intragenus (72.1%) coinfections. Despite the limited number of sequences, intradonor recombination analysis showed 6 intragenus recombination events in ORF1. As thousands of anellovirus sequences have been described recently, we finally analyzed the global diversity of human anelloviruses. Species richness and diversity were close to saturation in each anellovirus genus. Recombination was found to be the main factor promoting diversity, although its effect was significantly lower in TTV than in TTMV and TTMDV. Overall, our results suggest that differences in diversity between genera may be caused by variations in the relative contribution of recombination. IMPORTANCE Anelloviruses are the most common human infectious viruses and are considered essentially harmless. Compared to other human viruses, they are characterized by enormous diversity, and recombination is suggested to play an important role in their diversification and evolution. Here, by analyzing the composition of the plasma anellome of 50 blood donors, we find that recombination is also a determinant of viral evolution at the intradonor level. On a larger scale, analysis of anellovirus sequences currently available in databases shows that their diversity is close to saturation and differs among the three human anellovirus genera and that recombination is the main factor explaining this intergenus variability. Global characterization of anellovirus diversity could provide clues about possible associations between certain virus variants and pathologies, as well as facilitate the implementation of unbiased PCR-based detection protocols, which may be relevant for using anelloviruses as endogenous markers of immune status.

Inference of the Life Cycle of Environmental Phages from Genomic Signature Distances to Their Hosts.

The environmental impact of uncultured phages is shaped by their preferred life cycle (lytic or lysogenic). However, our ability to predict it is very limited. We aimed to discriminate between lytic and lysogenic phages by comparing the similarity of their genomic signatures to those of their hosts, reflecting their co-evolution. We tested two approaches: (1) similarities of tetramer relative frequencies, (2) alignment-free comparisons based on exact k = 14 oligonucleotide matches. First, we explored 5126 reference bacterial host strains and 284 associated phages and found an approximate threshold for distinguishing lysogenic and lytic phages using both oligonucleotide-based methods. The analysis of 6482 plasmids revealed the potential for horizontal gene transfer between different host genera and, in some cases, distant bacterial taxa. Subsequently, we experimentally analyzed combinations of 138 Klebsiella pneumoniae strains and their 41 phages and found that the phages with the largest number of interactions with these strains in the laboratory had the shortest genomic distances to K. pneumoniae. We then applied our methods to 24 single-cells from a hot spring biofilm containing 41 uncultured phage-host pairs, and the results were compatible with the lysogenic life cycle of phages detected in this environment. In conclusion, oligonucleotide-based genome analysis methods can be used for predictions of (1) life cycles of environmental phages, (2) phages with the broadest host range in culture collections, and (3) potential horizontal gene transfer by plasmids.

Genetic determinants of host tropism in Klebsiella phages.

Bacteriophages play key roles in bacterial ecology and evolution and are potential antimicrobials. However, the determinants of phage-host specificity remain elusive. Here, we isolate 46 phages to challenge 138 representative clinical isolates of Klebsiella pneumoniae, a widespread opportunistic pathogen. Spot tests show a narrow host range for most phages, with <2% of 6,319 phage-host combinations tested yielding detectable interactions. Bacterial capsule diversity is the main factor restricting phage host range. Consequently, phage-encoded depolymerases are key determinants of host tropism, and depolymerase sequence types are associated with the ability to infect specific capsular types across phage families. However, all phages with a broader host range found do not encode canonical depolymerases, suggesting alternative modes of entry. These findings expand our knowledge of the complex interactions between bacteria and their viruses and point out the feasibility of predicting the first steps of phage infection using bacterial and phage genome sequences.

Resistance of Omicron subvariants BA.2.75.2, BA.4.6, and BQ.1.1 to neutralizing antibodies.

Convergent evolution of SARS-CoV-2 Omicron BA.2, BA.4, and BA.5 lineages has led to the emergence of several new subvariants, including BA.2.75.2, BA.4.6. and BQ.1.1. The subvariant BQ.1.1 became predominant in many countries in December 2022. The subvariants carry an additional and often redundant set of mutations in the spike, likely responsible for increased transmissibility and immune evasion. Here, we established a viral amplification procedure to easily isolate Omicron strains. We examined their sensitivity to 6 therapeutic monoclonal antibodies (mAbs) and to 72 sera from Pfizer BNT162b2-vaccinated individuals, with or without BA.1/BA.2 or BA.5 breakthrough infection. Ronapreve (Casirivimab and Imdevimab) and Evusheld (Cilgavimab and Tixagevimab) lose antiviral efficacy against BA.2.75.2 and BQ.1.1, whereas Xevudy (Sotrovimab) remaine weakly active. BQ.1.1 is also resistant to Bebtelovimab. Neutralizing titers in triply vaccinated individuals are low to undetectable against BQ.1.1 and BA.2.75.2, 4 months after boosting. A BA.1/BA.2 breakthrough infection increases these titers, which remains about 18-fold lower against BA.2.75.2 and BQ.1.1, than against BA.1. Reciprocally, a BA.5 breakthrough infection increases more efficiently neutralization against BA.5 and BQ.1.1 than against BA.2.75.2. Thus, the evolution trajectory of novel Omicron subvariants facilitates their spread in immunized populations and raises concerns about the efficacy of most available mAbs.

Genetic complementation fosters evolvability in complex fitness landscapes.

The ability of natural selection to optimize traits depends on the topology of the genotype-fitness map (fitness landscape). Epistatic interactions produce rugged fitness landscapes, where adaptation is constrained by the presence of low-fitness intermediates. Here, we used simulations to explore how evolvability in rugged fitness landscapes is influenced by genetic complementation, a process whereby different sequence variants mutually compensate for their deleterious mutations. We designed our model inspired by viral populations, in which genetic variants are known to interact frequently through coinfection. Our simulations indicate that genetic complementation enables a more efficient exploration of rugged fitness landscapes. Although this benefit may be undermined by genetic parasites, its overall effect on evolvability remains positive in populations that exhibit strong relatedness between interacting sequences. Similar processes could operate in contexts other than viral coinfection, such as in the evolution of ploidy.

Cellular resistance to an oncolytic virus is driven by chronic activation of innate immunity.

The emergence of cellular resistances to oncolytic viruses is an underexplored process that could compromise the efficacy of cancer virotherapy. Here, we isolated and characterized B16 mouse melanoma cells that evolved resistance to an oncolytic vesicular stomatitis virus (VSV-D51). RNA-seq revealed that resistance was associated to broad changes in gene expression, which typically involved chronic upregulation of interferon-stimulated genes. Innate immunity activation was maintained in the absence of the virus or other infection signals, and conferred cross-resistance to wild-type VSV and the unrelated Sindbis virus. Furthermore, we identified differentially expressed genes with no obvious role in antiviral immunity, such as Mnda, Psmb8 and Btn2a2, suggesting novel functions for these genes. Transcriptomic changes associated to VSV resistance were similar among B16 clones and in some clones derived from the mouse colon carcinoma cell line CT26, suggesting that oncolytic virus resistance involves certain conserved mechanisms and is therefore a potentially predictable process.

Enveloped viruses show increased propensity to cross-species transmission and zoonosis.

The transmission of viruses between different host species is a major source of emerging diseases and is of particular concern in the case of zoonotic transmission from mammals to humans. Several zoonosis risk factors have been identified, but it is currently unclear which viral traits primarily determine this process as previous work has focused on a few hundred viruses that are not representative of actual viral diversity. Here, we investigate fundamental virological traits that influence cross-species transmissibility and zoonotic propensity by interrogating a database of over 12,000 mammalian virus-host associations. Our analysis reveals that enveloped viruses tend to infect more host species and are more likely to be zoonotic than nonenveloped viruses, while other viral traits such as genome composition, structure, size, or the viral replication compartment play a less obvious role. This contrasts with the previous notion that viral envelopes did not significantly impact or even reduce zoonotic risk and should help better prioritize outbreak prevention efforts. We suggest several mechanisms by which viral envelopes could promote cross-species transmissibility, including structural flexibility of receptor-binding proteins and evasion of viral entry barriers.

Resistance of Omicron subvariants BA.2.75.2, BA.4.6 and BQ.1.1 to neutralizing antibodies.

Convergent evolution of SARS-CoV-2 Omicron BA.2, BA.4 and BA.5 lineages has led to the emergence of several new subvariants, including BA.2.75.2, BA.4.6. and BQ.1.1. The subvariants BA.2.75.2 and BQ.1.1 are expected to become predominant in many countries in November 2022. They carry an additional and often redundant set of mutations in the spike, likely responsible for increased transmissibility and immune evasion. Here, we established a viral amplification procedure to easily isolate Omicron strains. We examined their sensitivity to 6 therapeutic monoclonal antibodies (mAbs) and to 72 sera from Pfizer BNT162b2-vaccinated individuals, with or without BA.1/BA.2 or BA.5 breakthrough infection. Ronapreve (Casirivimab and Imdevimab) and Evusheld (Cilgavimab and Tixagevimab) lost any antiviral efficacy against BA.2.75.2 and BQ.1.1, whereas Xevudy (Sotrovimab) remained weakly active. BQ.1.1 was also resistant to Bebtelovimab. Neutralizing titers in triply vaccinated individuals were low to undetectable against BQ.1.1 and BA.2.75.2, 4 months after boosting. A BA.1/BA.2 breakthrough infection increased these titers, which remained about 18-fold lower against BA.2.75.2 and BQ.1.1, than against BA.1. Reciprocally, a BA.5 breakthrough infection increased more efficiently neutralization against BA.5 and BQ.1.1 than against BA.2.75.2. Thus, the evolution trajectory of novel Omicron subvariants facilitated their spread in immunized populations and raises concerns about the efficacy of most currently available mAbs.

Spatially Segregated Transmission of Co-Occluded Baculoviruses Limits Virus-Virus Interactions Mediated by Cellular Coinfection during Primary Infection.

The occlusion bodies (OBs) of certain alphabaculoviruses are polyhedrin-rich structures that mediate the collective transmission of tens of viral particles to the same insect host. In addition, in multiple nucleopolyhedroviruses, occlusion-derived virions (ODVs) form nucleocapsid aggregates that are delivered to the same host cell. It has been suggested that, by favoring coinfection, this transmission mode promotes evolutionarily stable interactions between different baculovirus variants. To quantify the joint transmission of different variants, we obtained OBs from cells coinfected with two viral constructs, each encoding a different fluorescent reporter, and used them for inoculating Spodoptera exigua larvae. The microscopy analysis of midguts revealed that the two reporter genes were typically segregated into different infection foci, suggesting that ODVs show limited ability to promote the co-transmission of different virus variants to the same host cell. However, a polyhedrin-deficient mutant underwent inter-host transmission by exploiting the OBs of a fully functional virus and re-acquired the lost gene through recombination, demonstrating cellular coinfection. Our results suggest that viral spatial segregation during transmission and primary infection limits interactions between different baculovirus variants, but that these interactions still occur within the cells of infected insects later in infection.