Baculoviruses establish systemic infections within susceptible insect hosts, even though host tissues are surrounded by basal lamlnae, extracellular matrices that exclude particles smaller than these viruses. Using a recombinant Autgrapha californwca M nuclear polyhedrosis virus containing a lacZ reporter gene under the control of a constitutive promoter, we followed the progression of infection in Tichoplusia ni larvae. We discovered that infection of the larval insect tracheal system (and not hemocytes, as thought previously) provides the major conduit for this virus to pass through basal laminae and to spread throughout the host. Tracheal epidermal cells, the only known cellular components of the tracheal system, share a common lymph system. Locally these cells contact one another by interdigitating cytoplasmic extensions called epidermal feet. These two features of the tracheal system are likely to facilitate the rapid systemic spread of the virus. The findings reported here have major implications for the fields of insect pathology and biological control and usher in an important consideration regarding host-range factors.Nuclear polyhedrosis viruses (NPV) (family Baculoviridae) are enveloped double-stranded DNA viruses that infect only arthropod hosts, primarily lepidopteran insect larvae. NPVs are unusual among viruses because they produce two phenotypes, one that transmits infection between hosts and one that spreads infection within the host. The first phenotype consists of one or more enveloped virions, each containing one or more nucleocapsids sequestered within a crystalline matrix ofprotein. Such viral occlusions are called polyhedra. As with the spore stages of many bacteria and fungi, polyhedra resist desiccation and allow for temporal escape from inhospitable environments. After ingestion by susceptible larval insect hosts, polyhedra rapidly dissociate in the alkaline gut juices and release occlusion-derived virus (ODV). Even though ODV lack surface spikes or peplomers, they enter midgut columnar epithelial cells by membrane fusion (1-3). These infected epithelial cells produce the second phenotype, budded virus (BV), which buds through the basal cell membrane at sites containing gp64, a viral-encoded glycoprotein that forms spikes on the surface of BV. These gp64-containing structures mediate entry of BV into target cells by promoting fusion of the BV envelope and endocytic vesicular membranes (4-6).Early investigations of baculovirus infections focused on host pathology and the potential use of baculoviruses for biological control purposes. Accurate descriptions of the progression of pathogenesis in virus-infected insects, however, were hampered by the limits of the light and electron microscopy techniques commonly used, both with and without accompanying immunohistochemistry. Rare events are difficult to capture by using these techniques because they require meticulous serial sectioning and the three-dimensional reconstruction of hundreds, if not thousands, of sections for each insect....

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Per os infectivity factors PIF1 (Ac119) and PIF2 (Ac022), like P74, are essential for oral infection of lepidopteran larval hosts of Autographa californica M nucleopolyhedrovirus (AcMNPV). Here we show that Ac115 also is a PIF (PIF3) and that, unlike PIF1 and PIF2, it does not mediate specific binding of AcMNPV occlusion-derived virus (ODV) to midgut target cells. We used an improved in vivo fluorescence dequenching assay to compare binding, fusion, and competition among control AcMNPV ODV and the ODVs of AcMNPV PIF1, PIF2, and PIF3 deletion mutants. Our results showed that binding and fusion of PIF1 and PIF2 mutants, but not the PIF3 mutant, were both qualitatively and quantitatively different from those of control ODV. Unlike control and PIF3-deficient ODV, an excess of PIF1-or PIF2-deficient ODV failed to compete effectively with control ODV's binding to specific receptors on midgut epithelial cells. Moreover, the levels of PIF1-and PIF2-deficient ODV binding were depressed threefold compared to control levels. Binding, fusion, and competition by PIF3-deficient ODV, however, were all indistinguishable from those of control ODV. These results implicated PIF1 and PIF2 as ODV envelope attachment proteins that mediate specific binding to primary target cells within the midgut. In contrast, PIF3 mediates another unidentified, but critical, early event during primary infection.

P74, an envelope protein of the occlusion-derived virus (ODV) of Autographa californica M nucleopolyhedrovirus (AcMNPV), is critical for oral infection of Trichoplusia ni larvae. The role of P74 during primary infection, however, is unknown. Here we provide evidence that P74 facilitates binding of AcMNPV ODV to a specific receptor within the larval midgut epithelia of another host species, Heliothis virescens. We adapted a fluorescence dequenching assay to compare binding, fusion, and competition of wild-type AcMNPV ODV in vivo with itself and with the ODV of a p74-deficient AcMNPV mutant. We found that relative to wild-type ODV, binding and fusion of ODV deficient in P74 were both qualitatively and quantitatively different. Unlike wild-type ODV, an excess of P74-deficient ODV failed to compete effectively with wild-type ODV binding, and the overall binding level of the mutant ODV was one-third that of the wild type. These results implicated P74 as an ODV attachment protein that binds to a specific receptor on primary target cells within the midgut.

Autographa californica M nucleopolyhedrovirus (AcMNPV) can infect and kill a wide range of larval lepidopteran hosts, but the dosage required to achieve mortal infection varies greatly. Using a reporter gene construct, we identified key differences between AcMNPV pathogenesis in Heliothis virescens and Helicoverpa zea, a fully permissive and a semipermissive host, respectively. Even though there was more than a 1,000-fold difference in the susceptibilities of these two species to mortal infection, there was no significant difference in their susceptibilities to primary infections in the midgut or secondary infections in the tracheal epidermis. Foci of infection within the tracheal epidermis of H. zea, however, were melanized and encapsulated by 48 h after oral inoculation, a host response not observed in H. virescens. Further, H. zea hemocytes, unlike those of H. virescens, were highly resistant to AcMNPV infection; reporter gene expression was observed only rarely even though virus was taken up readily, and nucleocapsids were transported to the nucleus. Collectively, these results demonstrated that hemocytes-by removing virus from the hemolymph instead of amplifying it and by participating in the encapsulation of infection foci-together with the host's melanization response, formed the basis of H. zea's resistance to fatal infection by AcMNPV. Autographa californica M nucleopolyhedrovirus (AcMNPV) is the type species of the Nucleopolyhedrovirus genus in the familyBaculoviridae. AcMNPV only infects larval lepidopterans and causes fatal infections in at least 32 species, but susceptibility to mortal infection varies greatly among the hosts (1,17,28,29,30,31). Like that of most baculoviruses, the infection cycle of AcMNPV is mediated by two phenotypically different viral particles: the occlusion-derived virus (ODV) and the budded virus (BV). ODV particles are packaged with varying numbers (one through many) of nucleocapsids within an envelope (the M trait), and many ODV particles are embedded within a crystalline matrix of polyhedrin protein forming an occlusion.