Abundance and Diversity of Dung Beetles (Coleoptera: Scarabaeoidea) as Affected by Grazing Management in the Nebraska Sandhills Ecosystem.

Dung beetles (Coleoptera: Scarabaeoidea) serve a significant role in regulating ecosystem services on rangelands. However, the influence of grazing management on dung beetle communities remains largely unknown. The purpose of this study was to investigate dung beetle abundance and diversity throughout the grazing season in the Nebraska Sandhills Ecoregion. Grazing treatments included: continuous grazing (CONT), low-stocking rotational grazing (LSR), high-stocking rotational grazing (HSR), and no grazing (NG). The abundance and diversity of dung beetles were measured in the 2014 and 2015 grazing seasons using dung-baited pitfall traps. Dung beetle abundance for each grazing treatment was characterized through four indices: peak abundance, species richness, Simpson's diversity index, and Simpson's evenness. A total of 4,192 dung beetles were collected through both years of trapping in this study. Peak abundance and species richness were greater in grazed treatments when compared to NG in both years. Peak abundance in the HSR was 200% (2014) and 120% (2015) higher than in the LSR. Species richness in the HSR was 70% (2014) and 61% (2015) higher than in the LSR, and 89% (2014) and 133% (2015) higher than in CONT. Simpson's diversity index was lower in the NG and CONT treatments when compared to the LSR or HSR treatments for both years. We conclude that rotational grazing, regardless of stocking density, promoted dung beetle abundance and diversity within the Nebraska Sandhills Ecoregion.

Differential Defense Responses of Upland and Lowland Switchgrass Cultivars to a Cereal Aphid Pest.

Yellow sugarcane aphid (YSA) (Sipha flava, Forbes) is a damaging pest on many grasses. Switchgrass (Panicum virgatum L.), a perennial C4 grass, has been selected as a bioenergy feedstock because of its perceived resilience to abiotic and biotic stresses. Aphid infestation on switchgrass has the potential to reduce the yields and biomass quantity. Here, the global defense response of switchgrass cultivars Summer and Kanlow to YSA feeding was analyzed by RNA-seq and metabolite analysis at 5, 10, and 15 days after infestation. Genes upregulated by infestation were more common in both cultivars compared to downregulated genes. In total, a higher number of differentially expressed genes (DEGs) were found in the YSA susceptible cultivar (Summer), and fewer DEGs were observed in the YSA resistant cultivar (Kanlow). Interestingly, no downregulated genes were found in common between each time point or between the two switchgrass cultivars. Gene co-expression analysis revealed upregulated genes in Kanlow were associated with functions such as flavonoid, oxidation-response to chemical, or wax composition. Downregulated genes for the cultivar Summer were found in co-expression modules with gene functions related to plant defense mechanisms or cell wall composition. Global analysis of defense networks of the two cultivars uncovered differential mechanisms associated with resistance or susceptibility of switchgrass in response to YSA infestation. Several gene co-expression modules and transcription factors correlated with these differential defense responses. Overall, the YSA-resistant Kanlow plants have an enhanced defense even under aphid uninfested conditions.

Greenbug (Schizaphis graminum) herbivory significantly impacts protein and phosphorylation abundance in switchgrass (Panicum virgatum).

Switchgrass (Panicum virgatum L.) is an important crop for biofuel production but it also serves as host for greenbugs (Schizaphis graminum Rondani; GB). Although transcriptomic studies have been done to infer the molecular mechanisms of plant defense against GB, little is known about the effect of GB infestation on the switchgrass protein expression and phosphorylation regulation. The global response of the switchgrass cultivar Summer proteome and phosphoproteome was monitored by label-free proteomics shotgun in GB-infested and uninfested control plants at 10 days post infestation. Peptides matching a total of 3,594 proteins were identified and 429 were differentially expressed proteins in GB-infested plants relative to uninfested control plants. Among these, 291 and 138 were up and downregulated by GB infestation, respectively. Phosphoproteome analysis identified 310 differentially phosphorylated proteins (DP) from 350 phosphopeptides with a total of 399 phosphorylated sites. These phosphopeptides had more serine phosphorylated residues (79%), compared to threonine phosphorylated sites (21%). Overall, KEGG pathway analysis revealed that GB feeding led to the enriched accumulation of proteins important for biosynthesis of plant defense secondary metabolites and repressed the accumulation of proteins involved in photosynthesis. Interestingly, defense modulators such as terpene synthase, papain-like cysteine protease, serine carboxypeptidase, and lipoxygenase2 were upregulated at the proteome level, corroborating previously published transcriptomic data.

Aphid-Responsive Defense Networks in Hybrid Switchgrass.

Aphid herbivory elicits plant defense-related networks that are influenced by host genetics. Plants of the upland switchgrass (Panicum virgatum) cultivar Summer can be a suitable host for greenbug aphids (Schizaphis graminum; GB), and yellow sugarcane aphids (Sipha flava, YSA), whereas the lowland cultivar Kanlow exhibited multi-species resistance that curtails aphid reproduction. However, stabilized hybrids of Summer (♀) x Kanlow (♂) (SxK) with improved agronomics can be damaged by both aphids. Here, hormone and metabolite analyses, coupled with RNA-Seq analysis of plant transcriptomes, were utilized to delineate defense networks induced by aphid feeding in SxK switchgrass and pinpoint plant transcription factors (TFs), such as WRKYs that potentially regulate these responses. Abscisic acid (ABA) levels were significantly higher in GB infested plants at 5 and 10 days after infestation (DAI). ABA levels were highest at 15DAI in YSA infested plants. Jasmonic acid levels were significantly elevated under GB infestation, while salicylic acid levels were signifi40cantly elevated only at 15 DAI in YSA infested plants. Similarly, levels of several metabolites were altered in common or specifically to each aphid. YSA infestation induced a significant enrichment of flavonoids consistent with an upregulation of many genes associated with flavonoid biosynthesis at 15DAI. Gene co-expression modules that responded singly to either aphid or in common to both aphids were differentiated and linked to specific TFs. Together, these data provide important clues into the interplay of metabolism and transcriptional remodeling accompanying defense responses to aphid herbivory in hybrid switchgrass.

Geographic Distribution of Bacillus thuringiensis Cry1F Toxin Resistance in Western Bean Cutworm (Lepidoptera: Noctuidae) Populations in the United States.

The western bean cutworm (WBC), Striacosta albicosta (Lepidoptera: Noctuidae), can be a severe pest of transgenic corn in the western Plains and Great Lakes regions of North America, including on hybrids expressing the Bacillus thuringiensis (Bt) Cry1F toxin. The level and geographic distribution of Cry1F resistance are not completely known. Neonate S. albicosta from 10 locations between Nebraska and New York state were subjected to dose-response trypsin-activated native Cry1F toxin overlay bioassays. In 2017, the mean estimated lethal concentration causing 50% larval mortality (LC50) ranged from 15.1 to 18.4 µg Cry1F cm-2, and were not significantly different among locations. In 2018, LC50 estimates at Scottsbluff, NE (22.0 µg Cry1F cm-2) and Watertown, NY (21.7 µg Cry1F cm-2) were significantly higher when compared to locations in Michigan (15.8 µg Cry1F cm-2). Significantly lower 14-day larval weight among survivors was correlated with higher Cry1F dose. Results from this study indicate that S. albicosta survivorship on purified Bt Cry1F toxin shows a relatively even distribution across the native and range expansion areas where seasonal field infestations typically occur.

Phenology and Dispersal of the Wheat Stem Sawfly (Hymenoptera: Cephidae) Into Winter Wheat Fields in Nebraska.

Historically, the wheat stem sawfly, Cephus cinctus Norton was a pest in spring wheat-growing regions of the northern Great Plains. However, in the 1980s, it was found infesting winter wheat fields in Montana. Infestations were first detected in western Nebraska in the 1990s, and have since spread throughout the Nebraska Panhandle. Larval damage occurs from stem-mining, but stem girdling that results in lodged stems that are not harvested results in the greatest yield losses. The biology and phenology of the wheat stem sawfly are well described in the northern portion of its range, but they are lacking in Colorado, southeast Wyoming, and Nebraska. In this study, the phenology and dispersal of the wheat stem sawfly in Nebraska winter wheat fields is described using sweep net and larval sampling. During this 2-yr study, adult activity began on May 23 and ended on June 21. Adult sex ratios were 2.32 males per female in 2014 and 0.46 males per female in 2015. Both sexes demonstrated an edge effect within the wheat fields, with greater densities near the field edge. The edge effect was stronger for male wheat stem sawfly than females. Wheat stem sawfly larval density also had an edge effect, regardless of the density of female wheat stem sawfly present. This information will be useful for developing management plans for the wheat stem sawfly in Nebraska and neighboring regions.

Bifenthrin Baseline Susceptibility and Evaluation of Simulated Aerial Applications in Striacosta albicosta (Lepidoptera: Noctuidae).

Striacosta albicosta (Smith) is a maize pest that has recently expanded its geographical range into the eastern United States and southeastern Canada. Aerial application of pyrethroids, such as bifenthrin, has been a major practice adopted to manage this pest. Reports of field failure of pyrethroids have increased since 2013. Striacosta albicosta populations were collected in 2016 and 2017 from maize fields in Nebraska, Kansas, and Canada and screened with bifenthrin active ingredient in larval contact dose-response bioassays. Resistance ratios estimated were generally low in 2016 (1.04- to 1.32-fold) with the highest LC50 in North Platte, NE (66.10 ng/cm2) and lowest in Scottsbluff, NE (50.10 ng/cm2). In 2017, O'Neill, NE showed the highest LC50 (100.66 ng/cm2) and Delhi, Canada exhibited the lowest (6.33 ng/cm2), resulting in a resistance ratio variation of 6.02- to 15.90-fold. Implications of bifenthrin resistance levels were further investigated by aerial application simulations. Experiments were conducted with a spray chamber where representative S. albicosta populations were exposed to labeled rates of a commercial bifenthrin formulation. Experiments resulted in 100% mortality for all populations, instars, insecticide rates, and carrier volumes, suggesting that levels of resistance estimated for bifenthrin active ingredient did not seem to impact the efficacy of the correspondent commercial product under controlled conditions. Results obtained from this research indicate that control failures reported in Nebraska could be associated with factors other than insecticide resistance, such as issues with the application technique, environmental conditions during and/or after application, or the insect's natural behavior. Data generated will assist future S. albicosta resistance management programs.

Fall armyworm (Spodoptera frugiperda Smith) feeding elicits differential defense responses in upland and lowland switchgrass.

Switchgrass (Panicum virgatum L.) is a low input, high biomass perennial grass being developed for the bioenergy sector. Upland and lowland cultivars can differ in their responses to insect herbivory. Fall armyworm [FAW; Spodoptera frugiperda JE Smith (Lepidoptera: Noctuidae)] is a generalist pest of many plant species and can feed on switchgrass as well. Here, in two different trials, FAW larval mass were significantly reduced when fed on lowland cultivar Kanlow relative to larvae fed on upland cultivar Summer plants after 10 days. Hormone content of plants indicated elevated levels of the plant defense hormone jasmonic acid (JA) and its bioactive conjugate JA-Ile although significant differences were not observed. Conversely, the precursor to JA, 12-oxo-phytodienoic acid (OPDA) levels were significantly different between FAW fed Summer and Kanlow plants raising the possibility of differential signaling by OPDA in the two cultivars. Global transcriptome analysis revealed a stronger response in Kanlow plant relative to Summer plants. Among these changes were a preferential upregulation of several branches of terpenoid and phenylpropanoid biosynthesis in Kanlow plants suggesting that enhanced biosynthesis or accumulation of antifeedants could have negatively impacted FAW larval mass gain on Kanlow plants relative to Summer plants. A comparison of the switchgrass-FAW RNA-Seq dataset to those from maize-FAW and switchgrass-aphid interactions revealed that key components of plant responses to herbivory, including induction of JA biosynthesis, key transcription factors and JA-inducible genes were apparently conserved in switchgrass and maize. In addition, these data affirm earlier studies with FAW and aphids that the cultivar Kanlow can provide useful genetics for the breeding of switchgrass germplasm with improved insect resistance.

Divergent Switchgrass Cultivars Modify Cereal Aphid Transcriptomes.

Schizaphis graminum Rondani (Hemiptera: Aphididae) and Sipha flava Forbes (Hemiptera: Aphididae) are two common pests of bioenergy grasses. Despite the fact that they are both considered generalists, they differ in their ability to colonize Panicum virgatum cultivars. For example, S. flava colonizes both P. virgatum cv. Summer and P. virgatum cv. Kanlow whereas S. graminum can only colonize Summer. To study the molecular responses of these aphids to these two switchgrass cultivars, we generated de novo transcriptome assemblies and compared the expression profiles of aphids feeding on both cultivars to profiles associated with feeding on a highly susceptible sorghum host and a starvation treatment. Transcriptome assemblies yielded 8,428 and 8,866 high-quality unigenes for S. graminum and S. flava, respectively. Overall, S. graminum responded strongly to all three treatments after 12 h with an upregulation of unigenes coding for detoxification enzymes while major transcriptional changes were not observed in S. flava until 24 h. Additionally, while the two aphids responded to the switchgrass feeding treatment by downregulating unigenes linked to growth and development, their responses to Summer and Kanlow diverged significantly. Schizaphis graminum upregulated more unigenes coding for stress-responsive enzymes in the Summer treatment compared to S. flava; however, many of these unigenes were actually downregulated in the Kanlow treatment. In contrast, S. flava appeared capable of overcoming host defenses by upregulating a larger number of unigenes coding for detoxification enzymes in the Kanlow treatment. Overall, these findings are consistent with previous studies on the interactions of these two cereal aphids to divergent switchgrass hosts.

Comparing the Effects of Two Tillage Operations on Beneficial Epigeal Arthropod Communities and Their Associated Ecosystem Services in Sugar Beets.

Beneficial arthropods provide important ecosystem services in terms of arthropod pest and weed management, but these services can be adversely affected by farming practices such as tillage. This study investigated the impact of two tillage operations (zone tillage and moldboard plow) on the activity density of several beneficial, epigeal arthropod taxa, and postdispersal weed seed and prey removal in sugar beet agroecosystems. In addition, four omnivorous ground beetle species were selected for a weed-seed choice feeding assay, whereas a single species was selected for a weed-seed age preference assay. Ground beetles were the most commonly collected taxon (via pitfall sampling), with only a few dominant species. Tillage operation did not affect ground beetle activity density; however, spider, centipede, and rove beetle activity densities were higher in the reduced-tillage treatment. Live prey consumption was similar between tillage practices, with more prey consumed during nocturnal hours. More weed seeds were consumed in the reduced-tillage treatment, whereas weed-seed preference differed between the four weed species tested [Setaria pumila (Poir.) Roem. & Schult., Echinochloa crus-galli (L.), Kochia scoparia (L.), and Chenopodium album (L.)]. In the weed-seed choice feeding assay, significantly more broad-leaf weed seeds (C. album and K. scoparia) were consumed compared with grassy weed seeds (E. crus-galli and S. pumila). No preference for seed age was detected for E. crus-galli, but Harpalus pensylvanicus (De Geer) preferred old C. album seeds over fresh seeds. Zone tillage is compatible with ecosystem services, providing critical habitat within agricultural ecosystems needed to conserve beneficial, edaphic arthropods.

Transcriptional analysis of defense mechanisms in upland tetraploid switchgrass to greenbugs.

BACKGROUND: Aphid infestation of switchgrass (Panicum virgatum) has the potential to reduce yields and biomass quality. Although switchgrass-greenbug (Schizaphis graminum; GB) interactions have been studied at the whole plant level, little information is available on plant defense responses at the molecular level. RESULTS: The global transcriptomic response of switchgrass cv Summer to GB was monitored by RNA-Seq in infested and control (uninfested) plants harvested at 5, 10, and 15 days after infestation (DAI). Differentially expressed genes (DEGs) in infested plants were analyzed relative to control uninfested plants at each time point. DEGs in GB-infested plants induced by 5-DAI included an upregulation of reactive burst oxidases and several cell wall receptors. Expression changes in genes linked to redox metabolism, cell wall structure, and hormone biosynthesis were also observed by 5-DAI. At 10-DAI, network analysis indicated a massive upregulation of defense-associated genes, including NAC, WRKY, and MYB classes of transcription factors and potential ancillary signaling molecules such as leucine aminopeptidases. Molecular evidence for loss of chloroplastic functions was also detected at this time point. Supporting these molecular changes, chlorophyll content was significantly decreased, and ROS levels were elevated in infested plants 10-DAI. Total peroxidase and laccase activities were elevated in infested plants at 10-DAI relative to control uninfested plants. The net result appeared to be a broad scale defensive response that led to an apparent reduction in C and N assimilation and a potential redirection of nutrients away from GB and towards the production of defensive compounds, such as pipecolic acid, chlorogenic acid, and trehalose by 10-DAI. By 15-DAI, evidence of recovery in primary metabolism was noted based on transcript abundances for genes associated with carbon, nitrogen, and nutrient assimilation. CONCLUSIONS: Extensive remodeling of the plant transcriptome and the production of ROS and several defensive metabolites in an upland switchgrass cultivar were observed in response to GB feeding. The early loss and apparent recovery in primary metabolism by 15-DAI would suggest that these transcriptional changes in later stages of GB infestation could underlie the recovery response categorized for this switchgrass cultivar. These results can be exploited to develop switchgrass lines with more durable resistance to GB and potentially other aphids.

Landscape epidemiology of bean pod mottle comovirus: molecular evidence of heterogeneous sources.

Bean pod mottle virus (BPMV) RNAs are grouped into subgroups (sgI and sgII). A BPMV partial diploid reassortant (IA-Di1) from the perennial Desmodium illinoense contained both RNA1 subgroups and an RNA1 recombinant. The RNA2 of IA-Di1 was characteristic of sgII. Additionally, ten BPMV isolates from a soybean field adjacent to the locality of IA-Di1 shared >98.5% nucleotide identity with RNA1 sgII of IA-Di1. The data demonstrate the co-existence of two differing consensus BPMV RNA1 subgroups in adjacent habitats and illustrate variation in virus genetic structure that can occur in a contiguous plant community.

The development of an efficient multipurpose bean pod mottle virus viral vector set for foreign gene expression and RNA silencing.

Plant viral vectors are valuable tools for heterologous gene expression, and because of virus-induced gene silencing (VIGS), they also have important applications as reverse genetics tools for gene function studies. Viral vectors are especially useful for plants such as soybean (Glycine max) that are recalcitrant to transformation. Previously, two generations of bean pod mottle virus (BPMV; genus Comovirus) vectors have been developed for overexpressing and silencing genes in soybean. However, the design of the previous vectors imposes constraints that limit their utility. For example, VIGS target sequences must be expressed as fusion proteins in the same reading frame as the viral polyprotein. This requirement limits the design of VIGS target sequences to open reading frames. Furthermore, expression of multiple genes or simultaneous silencing of one gene and expression of another was not possible. To overcome these and other issues, a new BPMV-based vector system was developed to facilitate a variety of applications for gene function studies in soybean as well as in common bean (Phaseolus vulgaris). These vectors are designed for simultaneous expression of multiple foreign genes, insertion of noncoding/antisense sequences, and simultaneous expression and silencing. The simultaneous expression of green fluorescent protein and silencing of phytoene desaturase shows that marker gene-assisted silencing is feasible. These results demonstrate the utility of this BPMV vector set for a wide range of applications in soybean and common bean, and they have implications for improvement of other plant virus-based vector systems.

Evaluation of management strategies for bean leaf beetles (Coleoptera: Chrysomelidae) and Bean pod mottle virus (Comoviridae) in soybean.

Cerotoma trifurcata Förster (Coleoptera: Chrysomelidae) and Bean pod mottle virus (Comoviridae) (BPMV) both can reduce yield and seed quality of soybean, Glycine max (L.) Merr. Field experiments were conducted to evaluate the effects of systemic, seed-applied, and foliar-applied insecticides for the management of this pest complex at three locations in central, northeastern, and northwestern Iowa during 2002-2004. Seed-applied insecticide was evaluated according to a currently recommended management program for Iowa (i.e., insecticide applications that target emerging overwintered beetles, F0, and the first seasonal generation, F1 ). The experimental treatments included seed-applied (thiamethoxam, 0.3-0.5 g [AI] kg(-1)] or clothianidin, 47.32 ml [AI] kg(-1)) and foliar-applied (A-cyhalothrin, 16.83-28.05 g [AI] ha(-1)) or esfenvalerate (43.74-54.69 g [AI] ha(-1)) insecticides. Applications of the foliar insecticides were timed to target F0, F1 or both F0 and F1 populations of C. trifurcata. Our results confirm that insecticides timed at F0 and F1 populations of C. trifurcata can reduce vector populations throughout the growing season, provide limited reduction in virus incidence, and improve both yield and seed coat color. Furthermore, seed-applied insecticides may be the more reliable option for an F0-targeted insecticide if used within this management strategy. An F0-targeted insecticide by itself only gave a yield improvement in one out of eight location-years. However, by adding an F1-targeted insecticide, there was a yield gain of 1.42-1.67 quintal ha(-1), based on contrast comparisons at three location-years.

Is preventative, concurrent management of the soybean aphid (Hemiptera: Aphididae) and bean leaf beetle (Coleoptera: Chrysomelidae) possible?

In Iowa, the management of insect pests in soybean, Glycine max (L.) Merr., has been complicated by the arrival of the invasive species soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), and occasional outbreaks of bean leaf beetle, Cerotoma trifurcata (Förster) (Coleoptera: Chrysomelidae), populations leading to economic losses. Several insecticide programs designed to reduce abundance of the overwintered and first generation C. trifurcata and the incidence of bean pod mottle virus were evaluated over 3 yr (2004-2006) for their impacts on A. glycines populations, at three locations in Iowa (Floyd, Lucas, and Story counties). There was no significant overlap of either overwintered (early May) or the first (early July) generations of C. trifurcata with A. glycines, because aphids were first detected in June and they did not reach economically damaging levels until August, if at all. During this study, insecticides targeting the overwintered population or the first generation of C. trifurcata provided a limited impact on A. glycines populations compared with untreated controls, and they did not prevent economic populations from occurring. Furthermore, the highest populations of A. glycines were frequently observed when a low rate of lambda-cyhalothrin (178 ml/ha) was applied targeting the overwintered population of C. trifurcata. Soybean yields were not protected by any of the insecticide treatments. Our results indicate that the use of either early season foliar or seed-applied insecticides for C. trifurcata management is of limited value for A. glycines management.

No-choice preference of cerotoma trifurcata (coleoptera: chrysomelidae) to potential host plants of bean pod mottle virus (Comoviridae) in Iowa.

To better understand the naturally occurring host range of Bean pod mottle virus (family Comoviridae, genus Comovirus, BPMV) and its principal vector Cerotoma trifurcata (Förster) (Coleoptera: Chrysomelidae), 18 field-collected perennial plant species were tested for the presence of BPMV. By using no-choice assays, we determined the preference of these plants by bean leaf beetle, by measuring their level of herbivory relative to soybean, Glycine max (L.). New food hosts for adult bean leaf beetles include Lespedeza capitata (Michaux), Lotus corniculatus L., Trifolium alexandrinum L., Trifolium ambiguum Bieberstein, and Trifolium incarnatum L. Desmodium illinoense Gray is discovered as a new naturally occurring host for BPMV.

Protective immunity to SIV challenge elicited by vaccination of macaques with multigenic DNA vaccines producing virus-like particles.

We utilized SIV(mne) infection of Macaca fascicularis to assess the efficacy of DNA vaccination alone, and as a priming agent in combination with subunit protein boosts. All SIV(mne) structural and regulatory genes were expressed using the human cytomegalovirus Immediate Early-1 promoter in plasmids that directed the formation of virus-like particles in vitro. Macaques (n = 4) were immunized intradermally and intramuscularly four times over 36 weeks with 3 mg plasmid DNA. A second group (n = 4) received two DNA priming inoculations followed by two intramuscular boosts consisting of 250 microg recombinant Env gp160 and 250 microg recombinant Gag-Pol particles in MF-59 adjuvant. These regimens elicited modest cellular immunity prior to challenge. Humoral immune responses to Env gp160 were elicited and sustained by both vaccine protocols, and as expected antibody titers were higher in the protein subunit-boosted animals. Neutralizing antibodies prior to challenge were measurable in two of four subunit-boosted macaques. The two vaccine regimens elicited comparable helper T cell responses at the time of challenge. Vaccinees and mock-immunized controls (n = 4) were challenged intrarectally at week 38 with uncloned SIV(mne). Following challenge all macaques became infected, but both vaccine regimens resulted in reduced peak virus loads (p = 0.07) and significantly improved maintenance of peripheral CD4(+) T cell counts postchallenge (p = 0.007, DNA alone and p = 0.01, all vaccinees). There was no significant difference between the two vaccine groups in levels of plasma viremia or maintenance of CD4(+) T cell counts postchallenge.