Nematode-resistance loci in Upland cotton genomes are associated with structural differences.

Reniform and root-knot nematode are two of the most destructive pests of conventional upland cotton, Gossypium hirsutum, L. and continue to be a major threat to cotton fiber production in semi-arid regions of the southern United States and Central America. Fortunately, naturally occurring tolerance to these nematodes has been identified in the Pima cotton species (G. barbadense) and several upland cotton varieties (G. hirsutum), which has led to a robust breeding program that has successfully introgressed and stacked these independent resistant traits into several upland cotton lineages with superior agronomic traits, e.g. BAR 32-30 and BARBREN-713. This work identifies the genomic variations of these nematode tolerant accessions by comparing their respective genomes to the susceptible, high-quality fiber producing parental line of this lineage: Phytogen 355 (PSC355). We discover several large genomic differences within marker regions that harbor putative resistance genes as well as expression mechanisms shared by the two resistant lines, with respect to the susceptible PSC355 parental line. This work emphasizes the utility of whole genome comparisons as a means of elucidating large and small nuclear differences by lineage and phenotype.  .

Evaluation of Cotton Fleahopper (Pseudatomoscelis seriatus (Reuter)) Feeding on Mpp51Aa2-Traited Cotton Utilizing Electrical Penetration Graph (EPG) Waveforms.

Prior to the recent implementation of the Mpp51Aa2 pesticidal protein (ThryvOn), transgenic cotton cultivars have historically offered no control of the cotton fleahopper (Pseudatomocelis seriatus (Reuter)). To evaluate the feeding behavior of cotton fleahoppers on ThryvOn cotton, electropenetrography (EPG) using a Giga-8 DC instrument was used to monitor the probing activity of fourth- and fifth-instar cotton fleahopper nymphs on both ThryvOn and non-ThryvOn cotton squares. Nymphs were individually placed on an excised cotton square for 8 h of EPG recording, after which resulting waveforms were classified as non-probing, cell rupturing, or ingestion. Although there were significantly more cell rupturing events per insect on ThryvOn (mean ± SEM, 14.8 ± 1.7) than on non-ThryvOn squares (mean ± SEM, 10.3 ± 1.6), there was no difference attributable to ThryvOn in the average number of ingestion events per insect. However, the average duration of ingestion events was significantly shorter on squares with ThryvOn (mean ± SEM, 509 ± 148 s) than on squares without (mean ± SEM, 914 ± 135 s). This suggests that cotton fleahoppers continued to probe despite their inability to sustain ingestion. These results provide conclusive evidence that the Mpp51Aa2 pesticidal protein affects the feeding behavior of cotton fleahopper nymphs.

Fungal seed treatments of cotton affect boll weevil development.

BACKGROUND: Nonpathogenic fungi associated with plants can enhance plant defenses against stress factors, including herbivory. This study assessed whether cotton plants grown from seeds treated with different fungi affected boll weevil, Anthonomus grandis grandis Boheman, development and reproduction along with plant tolerance. We used whole plants grown from seeds treated with different fungi (Chaetomium globosum TAMU520 and TAMU559, Phialemonium inflatum TAMU490, and Beauveria bassiana) versus non-treated controls to test insect growth, reproduction, and plant tolerance assays in a greenhouse. RESULTS: Regarding boll weevil reproduction, fewer larvae hatched and fewer adults emerged from fungal-treated plants. In addition, the developmental time from oviposition to adult emergence was delayed in the plants treated with all fungi. For plant tolerance, B. bassiana-treated plants attacked by boll weevils shed fewer squares than nonfungal-treated plants. CONCLUSION: Fungal treatments can affect boll weevil performance and reproduction on cotton plants, with potentially negative effects on population growth. Collectively, these results support the potential for cottonseed treatments with fungi as a novel tool for boll weevil management in the field. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A New qPCR Assay for the Rapid Diagnosis of Anthonomus grandis Subspecies.

Rapid and accurate identification of Anthonomus grandis subspecies is crucial for effective management and eradication. Current diagnostic methods have limitations in terms of time to diagnosis (up to seven days) and can yield ambiguous results. Here, we present the validation of a custom TaqMan SNP Genotyping Assay for the rapid and accurate identification of A. grandis grandis (boll weevil) and A. g. thurberiae (thurberia weevil) subspecies. To validate the assay, we conducted three main experiments: (1) a sensitivity test to determine the DNA concentration range at which the assay performs, (2) a non-target specificity test to ensure no amplification in non-target weevils (false positives), and (3) an accuracy test comparing the results of the new assay to previously established methods. These experiments were carried out in parallel at three independent facilities to confirm the robustness of the assay to variations in equipment and personnel. We used DNA samples from various sources, including field-collected specimens, museum specimens, and previously isolated DNA. The assay demonstrated high sensitivity (PCR success with ≥0.05 ng/µL DNA template), specificity (0.02 false positive rate), and accuracy (97.7%) in diagnosing boll weevil and thurberia weevil subspecies. The entire workflow, including DNA extraction, assay preparation, PCR run time, and data analysis, can be completed within a single workday (7-9 h) by a single technician. The deployment of this assay as a diagnostic tool could benefit boll weevil management and eradication programs by enabling same-day diagnosis of trap-captured or intercepted weevil specimens. Furthermore, it offers a more reliable method for identifying unknown specimens, contributing to the overall effectiveness of boll weevil research and control efforts.

The Transcriptomic Response of the Boll Weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), following Exposure to the Organophosphate Insecticide Malathion.

Insecticide tolerance and resistance have evolved countless times in insect systems. Molecular drivers of resistance include mutations in the insecticide target site and/or gene duplication, and increased gene expression of detoxification enzymes. The boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), is a pest of commercial cotton and has developed resistance in the field to several insecticides; however, the current organophosphate insecticide, malathion, used by USA eradication programs remains effective despite its long-term use. Here, we present findings from an RNA-seq experiment documenting gene expression post-exposure to field-relevant concentrations of malathion, which was used to provide insight on the boll weevil's continued susceptibility to this insecticide. Additionally, we incorporated a large collection of boll weevil whole-genome resequencing data from nearly 200 individuals collected from three geographically distinct areas to determine SNP allele frequency of the malathion target site, as a proxy for directional selection in response to malathion exposure. No evidence was found in the gene expression data or SNP data consistent with a mechanism of enhanced tolerance or resistance adaptation to malathion in the boll weevil. Although this suggests continued effectiveness of malathion in the field, we identified important temporal and qualitative differences in gene expression between weevils exposed to two different concentrations of malathion. We also identified several tandem isoforms of the detoxifying esterase B1 and glutathione S-transferases, which are putatively associated with organophosphate resistance.

Genome assembly of two nematode-resistant cotton lines (Gossypium hirsutum L.).

Upland cotton (Gossypium hirsutum L.) is susceptible to damage by the root-knot and the reniform nematodes, causing yield losses greater than 4% annually in the United States. In addition, these nematodes are synergistic with seeding disease and root rot pathogens that exacerbate diseases and subsequent yield losses. Production practices to minimize nematode damage include crop rotation and nematicides, but these techniques need to be repeated and are expensive. The use of resistant cultivars is deemed the most effective and economical approach for managing nematodes in cotton. Here, we describe the genomes of two nematode-resistant lines of cotton, BARBREN-713 and BAR 32-30. These genomes may expedite the development of DNA markers that can be used to efficiently introduce nematode resistance into commercially valuable Upland lines.

The Identification of Boll Weevil, Anthonomus grandis grandis (Coleoptera: Curculionidae), Genes Involved in Pheromone Production and Pheromone Biosynthesis.

Eradication programs for the boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), rely almost exclusively on pheromone traps to indicate the need for insecticide applications. However, the effectiveness of traps in detecting weevil populations is reduced during certain times of the year, particularly when cotton is actively fruiting. Consequently, this could result in fields becoming heavily infested with weevils. It is widely speculated that the lack of weevil captures in traps during this period is largely due to the overwhelming amount of pheromone released by weevils in the field, which outcompete the pheromone released from traps. Thus, this work sought to identify genes involved in pheromone production so that new control methods that target these genes can be explored. We conducted an RNA-seq experiment that revealed 2479 differentially expressed genes between pheromone-producing and non-pheromone-producing boll weevils. Of those genes, 1234 were up-regulated, and 1515 were down-regulated, and most had gene annotations associated with pheromone production, development, or immunity. This work advances our understanding of boll weevil pheromone production and brings us one step closer to developing gene-level control strategies for this cotton pest.

Foliar herbivory increases sucrose concentration in bracteal extrafloral nectar of cotton.

Cultivated cotton, such as Gossypium hirsutum L., produces extrafloral (EF) nectar on leaves (foliar) and reproductive structures (bracteal) as an indirect anti-herbivore defense. In exchange for this carbohydrate-rich substance, predatory insects such as ants protect the plant against herbivorous insects. Some EF nectar-bearing plants respond to herbivory by increasing EF nectar production. For instance, herbivore-free G. hirsutum produces more bracteal than foliar EF nectar, but increases its foliar EF nectar production in response to herbivory. This study is the first to test for systemically induced changes to the carbohydrate composition of bracteal EF nectar in response to foliar herbivory on G. hirsutum. We found that foliar herbivory significantly increased the sucrose content of bracteal EF nectar while glucose and fructose remained unchanged. Sucrose content is known to influence ant foraging behavior and previous studies of an herbivore-induced increase to EF nectar caloric content found that it led to increased ant activity on the plant. As a follow-up to our finding, ant recruitment to mock EF nectar solutions that varied in sucrose content was tested in the field. The ants did not exhibit any preference for either solution, potentially because sucrose is a minor carbohydrate component in G. hirsutum EF nectar: total sugar content was not significantly affected by the increase in sucrose. Nonetheless, our findings raise new questions about cotton's inducible EF nectar responses to herbivory. Further research is needed to determine whether an herbivore-induced increase in sucrose content is typical of Gossypium spp., and whether it constitutes a corollary of systemic sucrose induction, or a potentially adaptive mechanism which enhances ant attraction to the plant.

Population genomics and phylogeography of the boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae), in the United States, northern Mexico, and Argentina.

The boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae), is an important pest of commercial cotton across the Americas. In the United States, eradication of this species is complicated by re-infestations of areas where eradication has been previously successful and by the existence of morphologically similar variants that can confound identification efforts. To date, no study has applied a high-throughput sequencing approach to better understand the population genetic structure of the boll weevil. Furthermore, only a single study has investigated genetic relationships between populations in North and South America. We used double digest restriction site-associated DNA sequencing (ddRADseq) to resolve the population genomic structure of the boll weevil in the southern United States, northern Mexico, and Argentina. Additionally, we assembled the first complete mitochondrial genome for this species and generated a preliminary whole genome assembly, both of which were used to improve the identification of informative loci. Downstream analyses revealed two main lineages-one consisting of populations found geographically west of the Sierra Madre Occidental mountain range and the second consisting of populations found to the east-were revealed, and both were sub-structured. Population geographic structure was consistent with the isolation by distance model, indicating that geogrpahic distance is likely a primary mechanism driving divergence in this species. Boll weevil populations from Argentina were found to be more closely related to the eastern lineage, suggesting a recent colonization of South America by the eastern lineage, but additional sampling across Mexico, Central America and South America is needed to further clarify their origin. Finally, we uncovered an instance of population turnover or replacement, highlighting the temporal instability of population structure.

Adsorbent-SERS Technique for Determination of Plant VOCs from Live Cotton Plants and Dried Teas.

We developed a novel substrate for the collection of volatile organic compounds (VOCs) emitted from either living or dried plant material to be analyzed by surface-enhanced Raman spectroscopy (SERS). We demonstrated that this substrate can be utilized to differentiate emissions from blends of three teas, and to differentiate emissions from healthy cotton plants versus caterpillar-infested cotton plants. The substrate we developed can adsorb VOCs in static headspace sampling environments, and VOCs naturally evaporated from three standards were successfully identified by our SERS substrate, showing its ability to differentiate three VOCs and to detect quantitative differences according to collection times. In addition, volatile profiles from plant materials that were either qualitatively different among three teas or quantitatively different in abundance between healthy and infested cotton plants were confirmed by collections on Super-Q resin for dynamic headspace and solid-phase microextraction for static headspace sampling, respectively, followed by gas chromatography to mass spectrometry. Our results indicate that both qualitative and quantitative differences can also be detected by our SERS substrate although we find that the detection of quantitative differences could be improved.

Genome-wide markers reveal temporal instability of local population genetic structure in the cotton fleahopper, Pseudatomoscelis seriatus (Hemiptera: Miridae).

BACKGROUND: The cotton fleahopper, Pseudatomoscelis seriatus (Reuter) (Hemiptera: Miridae), is a pest of upland cotton, Gossypium hirsutum L. (Malvales: Malvaceae), that attacks pre-floral buds (squares), leading to abscission and yield losses. In the Brazos Valley cotton production area of Texas (USA), P. seriatus exhibits a seasonal pattern of host use. In spring, eggs hatch from stems of the overwintering host, woolly croton, Croton capitatus Michx. (Malpighiales: Euphorbiaceae). During the growing season, individuals feed on a variety of host plants, including cotton. Adults return to woolly croton at season end to oviposit. We investigated if genetic differentiation exists between populations infesting cotton and those infesting alternative hosts, and whether woolly croton serves as a year-end site of admixture that could be suitable as a natural refuge for the purposes of insect resistance management. We combined high-throughput DNA sequencing with fine-scale spatio-temporal sampling to test (i) whether a population genomic approach would recover patterns of genetic variation consistent with earlier studies and (ii) if local genetic population structure was robust to seasonal changes in local habitat over time. RESULTS: We found high gene flow among populations of P. seriatus collected from different host plants in the Brazos Valley. We also identified temporal instability of the local population genetic structure, including the near complete loss of a genotypic group that had been previously abundant. CONCLUSION: We support the status of woolly croton as a natural refuge that promotes year-end gene flow between genotypes infesting cotton and those infesting alternative hosts. © 2019 Society of Chemical Industry.

Termination of Diapause in the Boll Weevil (Coleoptera: Curculionidae).

The boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), is a major pest of cotton (Gossypium spp. (Malvales: Malvaceae)) in Mexico, South America, and South Texas in the United States. The ability of the boll weevil to survive extended cotton-free periods has been key to its persistence as a pest despite intensive control efforts. However, the mechanism facilitating survival has been subject to debate. Whereas adult diapause has long been considered the principal survival mechanism, some authors have characterized the dormancy as a quiescence. We induced dormancy in the weevil and examined whether food type, enforced starvation, or induced flight influenced termination of the dormancy. Providing dormant adult weevils a diet favoring reproduction for 7-14 d prompted a modest termination response in female weevils and virtually no response in males. Some weevils starved ≥21 d resumed reproduction after exposure to a favorable diet, but most weevils remained dormant. Induced flight followed by exposure to a favorable diet prompted >50% of the weevils to terminate the dormancy. Patterns of feeding and oviposition were also useful in interpreting the termination response. These results indicate that the dormancy exhibited by the weevil is a diapause of variable intensity rather than a quiescence. A conceptual model recognizing population heterogeneity in diapause induction and intensity is consistent with reports of host-free survival and accommodates perceived differences in boll weevil ecology among temperate, subtropical, and tropical regions. This model provides a framework that will be valuable to research, management, and eradication efforts in the tropics and subtropics.

Diapause Response of the Boll Weevil (Coleoptera: Curculionidae) to Feeding Period Duration and Cotton Square Size.

Distribution of the boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), in the United States has been greatly reduced by eradication efforts. Still, it remains a key pest of cotton (Gossypium spp., [Malvales: Malvaceae]) in the New World, and has proven difficult to eliminate from Mexico and from southern Texas. In those regions, improved knowledge of boll weevil overwintering ecology may benefit efforts by eradication and management programs. Adult diapause in the boll weevil is well documented, but influences of the feeding period duration between adult eclosion and assessment of diapause remain unstudied. We examined diapause incidence and associated survival for weevils fed for 7, 14, or 21 d after adult eclosion. Diapause incidence of females was less influenced by feeding duration compared with males. For males, highest diapause incidence occurred after 14 d of feeding compared with 7 or 21 d. Host-free survival tended to be higher after 14 d of feeding compared with 7 or 21 d, although many weevils were long-lived (≥80 d) after each feeding period duration. Males exhibited higher survival compared with females, and survival was higher for weevils fed large flower buds (squares) compared with smaller squares. Survival was most influenced by temperature; longevity increased with decreasing temperature except at the lowest temperature (12.8°C). These results suggest an optimal feeding period for induction of diapause and maximized host-free longevity. These findings may permit improved timing of late-season insecticide treatments aimed at reducing overwintering populations, and thereby improve effectiveness of eradication and management programs.

Morphology, Diet, and Temperature-Dependent Host-Free Survival of the Boll Weevil, Anthonomus grandis (Coleoptera: Curculionidae).

The boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), is an important pest of cotton (Gossypium spp. (Malvales: Malvaceae)) in South America, Mexico, and southernmost Texas in the United States. A key factor in the persistence of the boll weevil is its ability to survive the noncotton season. Mechanisms facilitating this survival in subtropical and tropical areas are incompletely known, and our understanding has been further complicated by recent reports of overwintering on noncotton hosts. In addition, the nature and even existence of the adult dormancy, and validity of the criteria used to distinguish it, have been questioned. We manipulated the boll weevil diet to produce a range of diapause responses and observed the corresponding host-free survival patterns. The estimated proportion of diapause was associated with subsequent survival. In addition, different diets producing similar incidences of diapause resulted in substantially different host-free longevity, and the longest survival was observed for weevils fed bolls as adults. Survival patterns exhibited a negative temperature dependence except for the lowest observed temperature (12.8°C), where survival was reduced compared with that at 18.3°C. This suggests chronic chilling injury in response to extended exposure to low, above freezing temperatures. All but the highest temperature (29.4°C) facilitated survival >6 mo, and survival of >1 yr was observed at 18.3°C. The temperatures permitting the greatest survival are typical for many subtropical and tropical regions during the noncotton season, indicating that diapausing boll weevils are capable of surviving the noncotton season in these regions without the benefit of presumptive noncotton hosts.

Different processing of CAPA and pyrokinin precursors in the giant mealworm beetle Zophobas atratus (Tenebrionidae) and the boll weevil Anthonomus grandis grandis (Curculionidae).

Capa and pyrokinin (pk) genes in hexapods share a common evolutionary origin. Using transcriptomics and peptidomics, we analyzed products of these genes in two beetles, the giant mealworm beetle (Zophobas atratus; Tenebrionidae) and the boll weevil (Anthonomus grandis grandis; Curculionidae). Our data revealed that even within Coleoptera, which represents a very well-defined group of insects, highly different evolutionary developments occurred in the neuropeptidergic system. These differences, however, primarily affect the general structure of the precursors and differential processing of mature peptides and, to a lesser degree, the sequences of the active core motifs. With the differential processing of the CAPA-precursor in Z. atratus we found a perfect example of completely different products cleaved from a single neuropeptide precursor in different cells. The CAPA precursor in abdominal ganglia of this species yields primarily periviscerokinins (PVKs) whereas processing of the same precursor in neurosecretory cells of the subesophageal ganglion results in CAPA-tryptoPK and a novel CAPA-PK. Particularly important was the detection of that CAPA-PK which has never been observed in the CNS of insects before. The three different types of CAPA peptides (CAPA-tryptoPK, CAPA-PK, PVK) each represent potential ligands which activate different receptors. In contrast to the processing of the CAPA precursor from Z. atratus, no indications of a differential processing of the CAPA precursor were found in A. g. grandis. These data suggest that rapid evolutionary changes regarding the processing of CAPA precursors were still going on when the different beetle lineages diverged. The sequence of the single known PVK of A. g. grandis occupies a special position within the known PVKs of insects and might serve asa basis to develop lineage-specific peptidomimetics capable of disrupting physiological processes regulated by PVKs.

Neuropeptides of the cotton fleahopper, Pseudatomoscelis seriatus (Reuter).

The cotton fleahopper, Pseudatomoscelis seriatus (Reuter), is an economically important pest of cotton, and increasing concerns over resistance, detrimental effects on beneficial insects and safety issues associated with traditional insecticide applications have led to an interest in research on novel, alternative strategies for control. One such approach requires a more basic understanding of the neurohormonal system that regulates important physiological properties of the fleahopper; e.g. the expression of specific messenger molecules such as neuropeptides. Therefore we performed a peptidomic study of neural tissues from the fleahopper which led to the first identification of the sequences of native peptide hormones. These peptide hormones include the following neuropeptides: corazonin, short neuropeptide F (sNPF), myosuppressin, CAPA-pyrokinin and CAPA-PVK peptides. The CAPA-pyrokinin, sNPF, and CAPA-PVK peptides represent novel sequences. A comparison of fleahopper neuropeptides with those of related heteropteran species indicates that they are quite different. The sNPF of P. seriatus shows, among others, a novel substitution of Leu with Phe within the C-terminal region; a modification that sets it apart from the known sNPFs of not only other Heteroptera but of other arthropod species as well. The identity of the neuropeptides native to the fleahopper can aid in the potential development of biostable, bioavailable mimetic agonists and antagonists capable of disrupting the physiological functions that these neuropeptides regulate.

The operative findings in re-do microvascular decompression for recurrent trigeminal neuralgia.

BACKGROUND: Re-do microvascular decompression (MVD) of the trigeminal nerve occasionally becomes necessary following recurrence of symptoms after a previous MVD. The indications include failure or contraindication of less invasive treatment options such as medication or trigeminal nerve lesioning and when supported by vascular compression on brain MRI scans. PURPOSE: To report on our findings following re-do MVD in the National Hospital for Neurology and Neurosurgery, Queen Square London from July 1995 to July 2008. METHODS: Case note review of all patients who underwent re-do MVD for the above reasons in the senior author's series in the above institution and stated period. The parameters of study interest were pre-operative MRI features, operative findings, and complete pain-free period with regards to the first and re-do MVD. RESULTS: Data analysis showed that 6 underwent a re-do MVD. Neurovascular conflict was confirmed in 5 cases (83.3%) involving the superior cerebellar artery and anterior inferior cerebellar artery in 2 cases each (33.3%) and ectatic basilar artery in 1 case (16.6%). Arachnoid adhesions around the previous teflon felt were also found in 5 cases (83.3%) mostly in association with other findings. Neo-arachnoid cyst formation in the corresponding cerebello-pontine angle, was an unexpected finding in 2 cases (33.3%), though of uncertain significance. CONCLUSION: Redo MVD should be considered as a safe and effective treatment option in recurrent trigeminal neuralgia, which fails to respond to non-surgical means.

Evaluation of kill strips on boll weevil (Coleoptera: Curculionidae) mortality in pheromone traps and impact on weevil escape.

A field study examined the temporal patterns of boll weevil mortality provided by two commercially available kill strips, Hercon VaporTape II and Plato Insecticide Strip, and to evaluate the impacts of these devices on weevil escape from traps. Both types of kill strips produced similar levels of weevil mortality with the exception of the last two inspection intervals (30 and 46 h after continual exposure to kill strips). At these intervals, the Plato Strip produced significantly higher mortality than the Hercon strip; however, these differences were numerically small (10 and 6%, respectively). Both types of kill strips produced a high level of weevil mortality in traps (> 90%) after 46 h of exposure. On average, 5-8% of weevils escaped from traps whether a kill strip was present or absent. A strong temporal pattern of escape was observed, with > or = 90% of escape occurring within the first hour after weevils were introduced into traps. Because > or = 90% of escape occurred within the first hour weevils were in the traps and < 3% of weevils died during the first hour of exposure to kill strips in traps, use of kill strips in large-scale boll weevil management programs is not justified on the basis of reduced weevil escape.

Immunopathogenesis and immune modulation of Venezuelan equine encephalitis virus-induced disease in the mouse.

The course of Venezuelan equine encephalitis (VEE) disease in immunodeficient and immunologically normal mice was compared to define the role of the immune system in this disease process. Immunocompetent mice infected with VEE exhibited a biphasic illness characterized by an early self-limiting lymphoid phase and a fatal CNS phase. The lymphoid phase of the illness was characterized by extensive viral replication within spleen, thymus, Peyer's patches, and lymph nodes, was accompanied by a high-titered serum viremia, and resolved with the production of VEE-specific IgM class antibody at 72 h postinfection (p.i.). Immunocompetent animals survived an average of 6.8 +/- 1.2 days before succumbing to fulminant encephalitis. In contrast, SCID mice infected with VEE showed a persistent replication of virus throughout all organs tested beginning at 24 h p.i. VEE-infected SCID mice exhibited a severe spongiform encephalopathy with 100% mortality and an average survival time of 8.9 +/- 0.9 days. These studies indicated that the characteristic organ tropism of VEE in the mouse is due in large part to an early anti-viral state, the establishment of which is dependent upon the presence of an intact immune system. Finally, the CNS pathology in a VEE-infected mouse had a significant immunologic component. However, in contrast to other neurovirulent alphaviruses, VEE was directly cytopathic for the cells of the CNS, even in the absence of an immune response.

A single-site mutant and revertants arising in vivo define early steps in the pathogenesis of Venezuelan equine encephalitis virus.

The early stages of Venezuelan equine encephalitis virus (VEE) pathogenesis in the mouse model have been examined using a genetic approach. Disease progression of a molecularly cloned single-site mutant was compared with that of the parental virus to determine the step in the VEE pathogenetic sequence at which the mutant was blocked. Assuming that such a block constitutes a genetic screen, isolates from different tissues thought to be distal to the block in the VEE pathogenetic sequence were analyzed to determine the pathogenetic step at which revertants of the mutant were selected. Directed mutation and analysis of reversion in vivo provide two powerful genetic tools for the dissection of the wild-type VEE pathogenetic sequence. Virus from the parental virulent clone, V3000, first replicated in the draining lymph node after subcutaneous inoculation in the left rear footpad. Movement of a cloned avirulent mutant, V3010 (E2 76 Glu to Lys), to the draining lymph node was impaired, replication in the node was delayed, and spread beyond the draining lymph node was sporadic. Serum, contralateral lymph node, spleen, and brain isolates from V3010 inoculated animals were invariably revertant with respect to sequence at E2 76 and/or virulence in mice. Revertants isolated from serum and contralateral lymph node retained the V3010 E2 Lys 76 mutation but also contained a second-site mutation, Glu to Lys at E2 116. Modification of the V3010 clone by addition of the second-site mutation at E2 116 produced a virus that bypassed the V3010 block at the draining lymph node but that did not possess full wild-type capacity for replication in the central nervous system or for induction of mortality. A control construct containing only the E2 116 reverting mutation on the V3000 background was identical to V3000 in terms of early pathogenetic steps and virulence. Therefore, analysis of mutant replication and reversion in vivo suggested (1) that the earliest steps in VEE pathogenesis are transit to the draining lymph node and replication at that site, (2) that the mutation in V3010 impairs transit to the draining lymph node and blocks dissemination to other tissues, and (3) that reversion can overcome the block without restoring full virulence.