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The number and variability of the neutralizing epitopes targeted by polyclonal antibodies in individuals who are SARS-CoV-2 convalescent and vaccinated are key determinants of neutralization breadth and the genetic barrier to viral escape 1 – 4 . Using HIV-1 pseudotypes and plasma selection experiments with vesicular stomatitis virus/SARS-CoV-2 chimaeras 5 , here we show that multiple neutralizing epitopes, within and outside the receptor-binding domain, are variably targeted by human polyclonal antibodies. Antibody targets coincide with spike sequences that are enriched for diversity in natural SARS-CoV-2 populations. By combining plasma-selected spike substitutions, we generated synthetic ‘polymutant’ spike protein pseudotypes that resisted polyclonal antibody neutralization to a similar degree as circulating variants of concern. By aggregating variant of concern-associated and antibody-selected spike substitutions into a single polymutant spike protein, we show that 20 naturally occurring mutations in the SARS-CoV-2 spike protein are sufficient to generate pseudotypes with near-complete resistance to the polyclonal neutralizing antibodies generated by individuals who are convalescent or recipients who received an mRNA vaccine. However, plasma from individuals who had been infected and subsequently received mRNA vaccination neutralized pseudotypes bearing this highly resistant SARS-CoV-2 polymutant spike, or diverse sarbecovirus spike proteins. Thus, optimally elicited human polyclonal antibodies against SARS-CoV-2 should be resilient to substantial future SARS-CoV-2 variation and may confer protection against potential future sarbecovirus pandemics. A complex range of mutations within the SARS-CoV-2 spike protein is needed to escape polyclonal plasma neutralizing antibodies, and plasma from individuals who were first infected then vaccinated display the greatest resilience to escape mutations.

Monoclonal antibodies targeting a variety of epitopes have been isolated from individuals previously infected with SARS-CoV-2, but the relative contributions of these different antibody classes to the polyclonal response remains unclear. Here we use a yeast-display system to map all mutations to the viral spike receptor-binding domain (RBD) that escape binding by representatives of three potently neutralizing classes of anti-RBD antibodies with high-resolution structures. We compare the antibody-escape maps to similar maps for convalescent polyclonal plasmas, including plasmas from individuals from whom some of the antibodies were isolated. While the binding of polyclonal plasma antibodies are affected by mutations across multiple RBD epitopes, the plasma-escape maps most resemble those of a single class of antibodies that target an epitope on the RBD that includes site E484. Therefore, although the human immune system can produce antibodies that target diverse RBD epitopes, in practice the polyclonal response to infection is skewed towards a single class of antibodies targeting an epitope that is already undergoing rapid evolution. Emerging SARS-CoV-2 mutants may escape neutralization by antibodies. Here, the authors use deep mutational scanning to identify mutations in the RBD that escape human monoclonal antibodies or convalescent plasmas.

Neutralizing antibodies elicited by prior infection or vaccination are likely to be key for future protection of individuals and populations against SARS-CoV-2. Moreover, passively administered antibodies are among the most promising therapeutic and prophylactic anti-SARS-CoV-2 agents. However, the degree to which SARS-CoV-2 will adapt to evade neutralizing antibodies is unclear. Using a recombinant chimeric VSV/SARS-CoV-2 reporter virus, we show that functional SARS-CoV-2 S protein variants with mutations in the receptor-binding domain (RBD) and N-terminal domain that confer resistance to monoclonal antibodies or convalescent plasma can be readily selected. Notably, SARS-CoV-2 S variants that resist commonly elicited neutralizing antibodies are now present at low frequencies in circulating SARS-CoV-2 populations. Finally, the emergence of antibody-resistant SARS-CoV-2 variants that might limit the therapeutic usefulness of monoclonal antibodies can be mitigated by the use of antibody combinations that target distinct neutralizing epitopes. The new coronavirus, SARS-CoV-2, which causes the disease COVID-19, has had a serious worldwide impact on human health. The virus was virtually unknown at the beginning of 2020. Since then, intense research efforts have resulted in sequencing the coronavirus genome, identifying the structures of its proteins, and creating a wide range of tools to search for effective vaccines and therapies. Antibodies, which are immune molecules produced by the body that target specific segments of viral proteins can neutralize virus particles and trigger the immune system to kill cells infected with the virus. Several technologies are currently under development to exploit antibodies, including vaccines, blood plasma from patients who were previously infected, manufactured antibodies and more. The spike proteins on the surface of SARS-CoV-2 are considered to be prime antibody targets as they are accessible and have an essential role in allowing the virus to attach to and infect host cells. Antibodies bind to spike proteins and some can block the virus’ ability to infect new cells. But some viruses, such as HIV and influenza, are able to mutate their equivalent of the spike protein to evade antibodies. It is unknown whether SARS-CoV-2 is able to efficiently evolve to evade antibodies in the same way. Weisblum, Schmidt et al. addressed this question using an artificial system that mimics natural infection in human populations. Human cells grown in the laboratory were infected with a hybrid virus created by modifying an innocuous animal virus to contain the SARS-CoV-2 spike protein, and treated with either manufactured antibodies or antibodies present in the blood of recovered COVID-19 patients. In this situation, only viruses that had mutated in a way that allowed them to escape the antibodies were able to survive. Several of the virus mutants that emerged had evolved spike proteins in which the segments targeted by the antibodies had changed, allowing these mutant viruses to remain undetected. An analysis of more than 50,000 real-life SARS-CoV-2 genomes isolated from patient samples further showed that most of these virus mutations were already circulating, albeit at very low levels in the infected human populations. These results show that SARS-CoV-2 can mutate its spike proteins to evade antibodies, and that these mutations are already present in some virus mutants circulating in the human population. This suggests that any vaccines that are deployed on a large scale should be designed to activate the strongest possible immune response against more than one target region on the spike protein. Additionally, antibody-based therapies that use two antibodies in combination should prevent the rise of viruses that are resistant to the antibodies and maintain the long-term effectiveness of vaccines and therapies.

Background A critical step in the HIV-1 replication cycle is the assembly of Gag proteins to form virions at the plasma membrane. Virion assembly and maturation are facilitated by the cellular polyanion inositol hexaphosphate (IP 6 ), which is proposed to stabilize both the immature Gag lattice and the mature capsid lattice by binding to rings of primary amines at the center of Gag or capsid protein (CA) hexamers. The amino acids comprising these rings are critical for proper virion formation and their substitution results in assembly deficits or impaired infectiousness. To better understand the nature of the deficits that accompany IP 6 binding deficiency, we passaged HIV-1 mutants that had substitutions in IP 6 coordinating residues to select for compensatory mutations. Results We found a mutation, a threonine to isoleucine substitution at position 371 (T371I) in Gag, that restored replication competence to an IP 6 -binding-deficient HIV-1 mutant. Notably, unlike wild-type HIV-1, the assembly and infectiousness of resulting virus was not impaired when IP 6 biosynthetic enzymes were genetically ablated. Surprisingly, we also found that the maturation inhibitor Bevirimat (BVM) could restore the assembly and replication of an IP 6 -binding deficient mutant. Moreover, using BVM-dependent mutants we were able to image BVM-induced assembly of individual HIV-1 particles assembly in living cells. Conclusions Overall these results suggest that IP 6 -Gag and Gag-Gag contacts are finely tuned to generate a Gag lattice of optimal stability, and that under certain conditions BVM can rescue IP 6 deficiency. Additionally, our work identifies an inducible virion assembly system that can be utilized to visualize HIV-1 assembly events using live cell microscopy.

These data identify a host pathway by which VPS29 and associated factors control the endosomal environment in a manner that influences susceptibility to viral infection. This pathway could serve as a pharmaceutical target for intervention in zoonotic viral diseases, including those caused by coronaviruses, influenza viruses, and filoviruses, all of which are pandemic threats. Emerging zoonotic viral pathogens threaten global health, and there is an urgent need to discover host and viral determinants influencing infection. We performed a loss-of-function genome-wide CRISPR screen in a human lung cell line using HCoV-OC43, a human betacoronavirus. One candidate gene, VPS29, a component of the retromer complex, was required for infection by HCoV-OC43, SARS-CoV-2, other endemic- and pandemic-threat coronaviruses, as well as ebolavirus. Notably, we observed a heightened requirement for VPS29 by the recently described Omicron variant of SARS-CoV-2 compared to the ancestral variant. However, VPS29 deficiency had no effect on certain other viruses that enter cells via endosomes and had an opposing, enhancing effect on influenza A virus infection. Deficiency in VPS29 or other retromer components caused changes in endosome morphology and acidity and attenuated the activity of endosomal proteases. These changes in endosome properties caused incoming coronavirus, but not influenza virus particles, to become entrapped therein. Overall, these data show how host regulation of endosome characteristics can influence cellular susceptibility to viral infection and identify a host pathway that could serve as a pharmaceutical target for intervention in zoonotic viral diseases. IMPORTANCE These data identify a host pathway by which VPS29 and associated factors control the endosomal environment in a manner that influences susceptibility to viral infection. This pathway could serve as a pharmaceutical target for intervention in zoonotic viral diseases, including those caused by coronaviruses, influenza viruses, and filoviruses, all of which are pandemic threats. Our findings show how host regulation of endosome characteristics can influence viral susceptibility in both a positive and negative manner.

The influence of environmental factors on germination and emergence of horseweed was examined in growth chamber experiments. Germination was highest (61%) under 24/20 C day/night temperature under light. Horseweed seed germination was observed under both light (13 h photoperiod) and complete darkness (24 h), but germination under continuous darkness was only 0 to 15% compared with 0 to 61% under light. All other experiments were conducted under 24/20 C and 13-h light conditions. Germination was 19 to 36% over a pH range from 4 to 10, with a trend toward higher germination under neutral-to-alkaline conditions. Horseweed germination was > 20% at < 40 mM NaCl concentration and lowest (4%) at 160 mM NaCl. These data suggest that even at high soil salinity conditions, horseweed can germinate. Germination of horseweed decreased from 25% to 2% as osmotic potential increased from 0 (distilled water) to −0.8 MPa, indicating that germination can still occur under moderate water stress conditions. Horseweed seedling emergence was at its maximum on the soil surface, and no seedlings emerged from seeds placed at a depth of 0.5 cm or higher. Nomenclature: Horseweed, Conyza canadensis (L.) Cronq. ERICA.

Greenhouse and laboratory studies were conducted to confirm and quantify glyphosate resistance, quantify pyrithiobac resistance, and investigate interaction between flumiclorac and glyphosate mixtures on control of Palmer amaranth from Mississippi. The GR50 (herbicide dose required to cause a 50% reduction in plant growth) values for two glyphosate-resistant biotypes, C1B1 and T4B1, and a glyphosate-susceptible (GS) biotype were 1.52, 1.3, and 0.09 kg ae ha−1 glyphosate, respectively. This indicated that the C1B1 and T4B1 biotypes were 17- and 14-fold resistant to glyphosate, respectively, compared with the GS biotype. The C1B1 and T4B1 biotypes were also resistant to pyrithiobac, an acetolactate synthase (ALS) inhibitor, with GR50 values of 0.06 and 0.07 kg ai ha−1, respectively. This indicated that the C1B1 and T4B1 biotypes were 7- and 8-fold, respectively, more resistant to pyrithiobac compared with the GS biotype, which had a GR50 value of 0.009 kg ha−1. Flumiclorac was antagonistic to glyphosate by reducing glyphosate translocation. The C1B1 and T4B1 absorbed less glyphosate 48 h after treatment (HAT) compared with the GS biotype. The majority of the translocated glyphosate accumulated in the shoot above the treated leaf (that contains the apical meristem) in the GS biotype and in the shoot below the treated leaf in the resistant biotypes, C1B1 and T4B1, by 48 HAT. The C1B1 biotype accumulated negligible shikimate levels, whereas the T4B1 and GS biotypes recorded elevated levels of shikimate. Metabolism of glyphosate to aminomethylphosphonic acid was not detected in either of the resistant biotypes or the susceptible GS biotype. The above results confirm multiple resistance to glyphosate and pyrithiobac in Palmer amaranth biotypes from Mississippi and indicate that resistance to glyphosate is partly due to reduced absorption and translocation of glyphosate. Nomenclature: Flumiclorac; glyphosate; pyrithiobac; Palmer amaranth, Amaranthus palmeri S. Wats. AMAPA.

Field, laboratory, and greenhouse experiments were conducted to determine the seed production potential and effect of environmental factors on germination, emergence, and survival of texasweed. Texasweed produced an average of 893 seed per plant, and 90% were viable. Seed exhibited dormancy, and prechilling did not release dormancy. Percent germination ranged from 56% for seed subjected to no prechilling to 1% for seed prechilled at 5 C for 140 d. Seed remained viable during extended prechilling conditions, with 80% of seed viable after 140 d of prechilling. Texasweed seed germinated over a range of 20 to 40 C, with optimum germination (54%) occurring with a fluctuating 40/30 C temperature regime. Seed germinated with fluctuating 12-h light/dark and constant dark conditions. Texasweed seed germinated over a broad range of pH, osmotic potential, and salt concentrations. Seed germination was 31 to 62% over a pH range from 4 to 10. Germination of texasweed ranged from 9 to 56% as osmotic potential decreased from − 0.8 MPa to 0 (distilled water). Germination was greater than 52% at less than 40 mM NaCl concentrations and lowest (27%) at 160 mM NaCl. Texasweed seedlings emerged from soil depths as deep as 7.5 cm (7% emergence), but emergence was > 67% for seed placed on the soil surface or at a 1-cm depth. Texasweed seed did not germinate under saturated or flooded conditions, but seed survived flooding and germinated (23 to 25%) after flood removal. Texasweed seedlings 2.5 to 15 cm tall were not affected by emersion in 10-cm-deep flood for up to 14 d. These results suggest that texasweed seed is capable of germinating and surviving in a variety of climatic and edaphic conditions, and that flooding is not a viable management option for emerged plants of texasweed. Nomenclature: Texasweed, Caperonia palustris (L.) St. Hil. CNPPA.

A threefold glyphosate tolerance was identified in two Italian ryegrass populations, T1 and T2, from Mississippi. Laboratory experiments were conducted to characterize the mechanism of glyphosate tolerance in these populations. The T1 population absorbed less 14C-glyphosate (43% of applied) compared to the susceptible (S) population (59% of applied) at 48 h after treatment (HAT). The T2 population absorbed 14C-glyphosate at levels (56% of applied at 48 HAT) that were similar to both T1 and S populations, but tended to be more comparable to the S population. The amount of 14C-glyphosate that remained in the treated leaf was significantly higher in both T1 (67% of absorbed) and T2 (65% of absorbed) populations compared to the S population (45% of absorbed) at 48 HAT. The amount of 14C-glyphosate that moved out of treated leaf to shoot and root was lower in both T1 (25% of absorbed in shoot and 9% of absorbed in root) and T2 (25% of absorbed in shoot and 11% of absorbed in root) populations compared to the S population (40% of absorbed in shoot and 16% of absorbed in root) at 48 HAT. There were no differences in epicuticular wax mass among the three populations. Treating a single leaf with glyphosate solution at the field use rate (0.84 kg ae ha−1) as 10 1-µl droplets killed the S plant but not the T1 and T2 plants (33 and 55% shoot fresh-weight reduction, respectively). Shikimic acid accumulated rapidly at higher levels in glyphosate-treated leaf segments of the S population compared to the T1 population up to 100 µM glyphosate. However, above 500 µM glyphosate, the levels of shikimate were similar in both the S and T1 populations. Furthermore, shikimic acid content was three- to sixfold more in whole plants of the S population treated with 0.22 kg ae ha−1 glyphosate compared to the T1 and T2 populations. No degradation of glyphosate to aminomethylphosphonic acid was detected among the tolerant and susceptible populations. These results indicate that tolerance to glyphosate in the T1 population is partly due to reduced absorption and translocation of glyphosate and in the T2 population it is partly due to reduced translocation of glyphosate.

Survival of horseweed in several glyphosate-tolerant cotton and soybean fields treated with glyphosate at recommended rates preplant and postemergence was observed in Mississippi and Tennessee in 2001 and 2002. Plants originating from seed collected from fields where horseweed escapes occurred in 2002 were grown in the greenhouse to the 5-leaf, 13- to 15-leaf, and 25- to 30-leaf growth stages and treated with the isopropylamine salt of glyphosate at 0, 0.025, 0.05, 0.1, 0.21, 0.42, 0.84, 1.68, 3.36, 6.72, and 13.44 kg ae/ha to determine if resistance to glyphosate existed in any biotype. All biotypes exhibited an 8- to 12-fold level of resistance to glyphosate when compared with a susceptible biotype. One resistant biotype from Mississippi was two- to fourfold more resistant than other resistant biotypes. Growth stage had little effect on level of glyphosate resistance. The glyphosate rate required to reduce biomass of glyphosate-resistant horseweed by 50% (GR50) increased from 0.14 to 2.2 kg/ha as plant size increased from the 5-leaf to 25- to 30-leaf growth stage. The GR50 rate for the susceptible biotype increased from 0.02 to 0.2 kg/ha glyphosate. These results demonstrate that the difficult-to-control biotypes were resistant to glyphosate, that resistant biotypes could survive glyphosate rates of up to 6.72 kg/ha, and that plant size affected both resistant and susceptible biotypes in a similar manner.