Frontiers in Virology

Ensuring COVID-19 testing remains accurate and reliable is of critical importance as the SARS-CoV-2 virus continues to evolve. Currently, a number of Omicron variants are dominating infection across the globe in including BQ.1 and XBB. Both variants and their sublineages (BQ.1* and XBB*) contain a 28,311 C/U mutation inherited from the original Omicron variant (BA.1). This mutation overlaps with a commonly used fluorescent probe for N gene detection in many Emergency Use Authorization (EUA) assays, as this target was originally established by the U.S. Centers for Disease Control and Prevention (CDC) in their EUA test for COVID-19 (2019-nCoV_N1). This C to U mutation was previously shown to have no impact on CDC N1 target detection. The rise of Omicron sublineages has increased the likelihood of additional point mutations occurring within the same assay target. A subpopulation of BQ.1* has an additional 28,312 C/U mutation within the CDC 2019_nCoV_N1 fluorescent probe in addition to the 28,311 C/U mutation. The double mutation could adversely affect the ability of diagnostic assays to detect the virus in patient samples and therefore it is important to verify the impacts of this additional mutation. Using in vitro transcribed (IVT) N gene RNA representing the wildtype (GenBank/GISAID ID MN908947.3) and Omicron BQ.1.1 variant (BQ.1, GISAID ID EPI_ISL_ 15155651), we evaluated the performance of two different amplification protocols, both of which include the CDC 2019-nCoV_N1 primer-probe set. Both assays successfully detected the mutant N gene sequence efficiently even at 10 copies of input, although the double mutation caused a 0.5[~]1 Cq delay on average when compared to the wild-type sequence. These data suggest that circulating BQ.1* lineage viruses with this double mutation likely have minimal impact on diagnostic assays that use the 2019-nCoV-N1 primer-probe.

Successfully predicting the effects of amino acid substitutions on protein function and stability remains challenging. Recent efforts to improve computational models have included training and validation on high-throughput experimental datasets, such as those generated by deep mutational scanning (DMS) approaches. However, DMS signals typically conflate a substitutions effects on protein function with those on in vivo protein abundance; this limits the resolution of mechanistic insights that can be gleaned from DMS data. Distinguishing functional changes from abundance-related effects is particularly important for substitutions that exhibit intermediate outcomes (e.g., partial loss-of-function), which are difficult to predict. Here, we explored changes in in vivo abundance for substitutions at representative positions in the SARS-CoV-2 Main Protease (Mpro). For this study, we used previously published DMS results to identify "rheostat" positions, which are defined by having substitutions that sample a broad range of intermediate outcomes. We generated 10 substitutions at each of six positions and separately measured effects on function and abundance. Results revealed an [~]45-fold range of change for abundance, demonstrating that it can make significant contributions to DMS outcomes. Moreover, the six tested positions showed diverse substitution sensitivities for function and abundance. Some positions influenced only one parameter. Others exhibited rheostatic effects on both parameters, which to our knowledge, provides the first example of such behavior. Since effects on function and abundance may arise through different biophysical bases, these results underscore the need for datasets that independently measure these parameters in order to build predictors with enhanced mechanistic insights. ABSTRACT for broader audienceChanging one amino acid in a protein can affect its function, its abundance, or both. Understanding these separate effects will help scientists predict how protein changes alter biology, which is important for understanding pathogen evolution, improving personalized medicine, and bioengineering. This work reports a study to experimentally separate these effects for the main protease of SARS-CoV-2 and suggests strategies for building better prediction models for emerging variants of this key viral protein.

The geminivirus beet curly top Iran virus (BCTIV) is one of the main causal agents of the beet curly top disease in Iran and the newly established Becurtovirus genus type species. Although the biological features of known becurtoviruses are similar to those of curtoviruses, they only share a limited sequence identity, and no information is available on the function of their viral genes. In this work, we demonstrate that BCTIV V2, as the curtoviral V2, is also a strong local silencing suppressor in Nicotiana benthamiana and can delay the systemic silencing spreading, although it cannot block the cell-to-cell movement of the silencing signal to adjacent cells. BCTIV V2 shows the same subcellular localization as curtoviral V2, being detected in the nucleus and perinuclear region, and its ectopic expression from a PVX-derived vector also causes the induction of necrotic lesions in N. benthamiana like the ones produced during the HR, both at local and systemic levels. The results from the infection of N. benthamiana with a V2 BCTIV mutant showed that V2 is required for systemic infection but not for viral replication in a local infection. Considering all these results, we can conclude that BCTIV V2 is a functional homologue of curtoviral V2 and plays a crucial role in viral pathogenicity and systemic movement.

Identifying reservoir host species is crucial for understanding the risk of pathogen spillover from wildlife to people. Orthohantaviruses are zoonotic pathogens primarily carried by rodents that cause the diseases hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) in humans. Given their diversity and abundance, many orthohantaviruses are expected to be undiscovered, and several host relationships remain unclear, particularly in the Americas. Despite the increasing use of predictive models for understanding zoonotic reservoirs, explicit comparisons between different evidence types for demonstrating host associations, and relevance to model performance in applied settings, have not been previously made. Using multiple machine learning methods, we identified phylogenetic patterns in and predicted unidentified reservoir hosts of New World orthohantaviruses based on evidence of infection (RT-PCR data) and competence (live virus isolation data). Infection data were driven by phylogeny, unlike competence data, and boosted regression tree (BRT) models using competence data displayed higher accuracy and a narrower list of predicted reservoirs than those using infection data. Eight species were identified by both BRT models as likely orthohantavirus hosts, with a total of 98 species identified by our infection models and 14 species identified by our competence models. Hosts predicted by competence models are concentrated in the northeastern United States (particularly Myodes gapperi and Reithrodontomys megalotis) and northern South America (several members of tribe Oryzomyini) and should be key targets for empirical monitoring. More broadly, these results demonstrate the value of infection competence data for predictive models of zoonotic pathogen hosts, which can be applied across a range of settings and host-pathogen systems. Author SummaryHuman diseases with wildlife origins constitute a significant risk for human health. Orthohantaviruses are viruses found primarily in rodents that cause disease with high rates of mortality and other complications in humans. An important step in disease prevention is to identify which rodent species carry and transmit orthohantaviruses. By incorporating species relatedness and evidence of different levels of host capacity to be infected and transmit virus, we used predictive modeling to determine unidentified rodent hosts of orthohantaviruses. Models using host competence data outperformed models using host infection data, highlighting the importance of stronger data in model optimization. Our results highlighted roughly a dozen key target species to be monitored that are concentrated in two geographic regions--northeastern United States and northern South America. More broadly, the approaches used in this study can be applied to a variety of other host-pathogen systems that threaten public health.

Soybean mosaic virus (SMV) causes systemic infections in soybean plants, leading to chlorotic mosaic and producing significant yield losses. The virus is widely distributed in all soybean production areas in the world. In Argentina, three geographical isolates were identified: Marcos Juarez (MJ), Manfredi (M), and North Western Argentina (NOA), and another isolate named "Planta Vinosa" (PV), which causes severe necrosis symptoms in some cultivars. Here, the biological, molecular and physiological characterization of these isolates was performed for the first time. Three of the four isolates showed a low genetic divergence in the evaluated genes (P1, CI and CP). Although SMV-NOA and SMV-PV had high homology at the sequence level, they showed wide differences in pathogenicity, seed mottling and the ability of transmission by seeds or aphids, as well as in physiological effects. SMV-NOA caused early alterations (before symptom appearance, BS) in {Phi}PSII and MDA content in leaves with respect to the other isolates. After the appearance of macroscopic symptoms (late symptoms, LS), SMV-M caused a significant increase in the content of MDA, total soluble sugars, and starch with respect to the other isolates. Thus, early alterations of {Phi}PSII and soluble sugars might have an impact on late viral symptoms. Likewise, SMV-MJ developed more severe symptoms in the susceptible Davis cultivar than in DM 4800. Therefore, our results show differences in genome, biological properties and physiological effects among SMV isolates as well as different interactions of SMV-MJ with two soybean cultivars.

The necrosis syndrome of freesia, first described in 1970 in Northern Europe, is still jeopardizing freesia cultivation all over the world. Although several viruses have been listed as possible causal agents, the etiology of the disease is still not clear and is possibly linked to a combination of different factors. In this study, a high-throughput sequencing virome analysis was performed on total RNA extracts derived from symptomatic freesia leaves; a novel virus putatively belonging to the recently ratified Konkoviridae family in the Bunyaviricetes class has been identified and characterized, for which we propose the name of freesia konkovirus 1 (FreKV-1). This family, officially listing only one genus and two species, has been expanded by exploring publicly available metatranscriptomic datasets through the Serratus Project Database and reconstructing new viral entities; the phylogenetic position of the Konkoviridae family has been investigated and new genera belonging to the family have been proposed. Moreover, a further previously unknown virus, putatively belonging to the Yueviridae family was partially characterized and its phylogenetic position was discussed. Overall, the analysis increased our knowledge of the number of viral agents infecting freesia and possibly involved in freesia necrosis syndrome.

Seven members of the Coronaviridae family infect humans, but only three (SARS-CoV, SARS-CoV-2 and MERS-CoV) cause severe disease with a high case fatality rate. Using in silico analyses (machine learning techniques and comparative genomics), several features associated to coronavirus pathogenicity have been recently proposed, including a potential increase in the strength of a predicted novel nuclear export signal (NES) motif in the nucleocapsid protein. Here, we have used a well-established nuclear export assay to experimentally establish whether the recently proposed nucleocapsid NESs are capable of mediating nuclear export, and to evaluate if their activity correlates with coronavirus pathogenicity. The six NES motifs tested were functional in our assay, but displayed wide differences in export activity. Importantly, these differences in NES strength were not related to strain pathogenicity. Rather, we found that the NESs of the strains belonging to the genus Alphacoronavirus were markedly stronger than the NESs of the strains belonging to the genus Betacoronavirus. We conclude that, while some of the genomic features recently identified in silico could be crucial contributors to coronavirus pathogenicity, this does not appear to be the case of nucleocapsid NES activity, as it is more closely related to coronavirus genus than to pathogenic capacity.

In Cote dIvoire, banana (Musa sp.) ranks third among exportation products and represents 3% of the Gross Domestic Product with a national production up to 500000 tons in 2019. Banana is subject to numerous disease agents among which viruses cause significant losses. To figure out the impact of viruses in Ivorian industrial banana fields, surveys were conducted in the 7 main banana production departments. A total of 260 leaf fragments presenting viral symptoms were collected and analyzed. From the 65 leaf fragments used for biological indexing, 14 showed symptoms related to Cucumber mosaic virus (CMV). CMV presence was confirmed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using CMV polyclonal antibodies. CMV strains we isolated, appeared to be highly infectious and to produce various symptoms like mosaic, chlorosis, and necrotic spots on Cucumis sativus, Cucurbita pepo, and Nicotiana tabacum. Satellite RNAs (SatRNAs) associated with CMV isolates were also detected using reverse transcription polymerase chain reaction (RT-PCR) with a degenerate primer pair. CMVs coat protein as well as satRNAs was sequenced. Novel Ivorian coat proteins and satRNAs were compared to publicly available sequences. We noticed a single amino acid substitution (Serine to Leucine) at position 73 of the novel coat protein that allowed us to divide Ivorian CMV strains into two groups. Molecular and phylogenetic analysis suggested that Ivorian strains might be classified into CMV Subgroup IA. We also discovered that satellite RNA associated with Ivorian CMVs form a separate clade.

Borealpox virus (BRPV; formerly Alaskapox) is an orthopoxvirus that has caused seven reported human infections in Alaska since 2015, including a fatal case in 2023. The natural reservoir of BRPV is unknown, although previous investigations have raised the possibility of wild small mammals transmitting the virus to humans, either through direct contact or via domestic cats and dogs. To understand which species may be involved in the maintenance and/or spillover of BRPV in Alaska, we trapped and sampled wild small mammals (including voles, shrews, and squirrels) in 2021 and 2024 near reported human case locations in Fairbanks and the Kenai Peninsula, respectively. We found evidence of previous exposure to orthopoxviruses in five species (including the House Mouse, Mus musculus) and detected BRPV DNA as well as viable virus in Northern Red-backed Voles (Clethrionomys rutilus). Further, screening of tissues from historical museum specimens revealed BRPV DNA in C. rutilus specimens collected in Denali National Park and Preserve in 1998 and 1999, 17 years before the first reported human case of BRPV. Phylogenomic analysis of all human and animal BRPV isolates strongly supports the hypothesis of local human infections through multiple spillover events. These findings suggest C. rutilus as a possible reservoir species for BRPV and indicate that BRPV has been present in Alaskan wild small-mammal populations for at least 25 years. Our study highlights the potential of museum collections to elucidate the temporal, spatial, and host ranges of emerging pathogens. Further museum- and field-based sampling will clarify the true geographic range of BRPV, which is closely related to Old World orthopoxviruses and may be circulating beyond North America.

The complete sequence of Wineberry latent virus (WLV), a previously reported but uncharacterised Rubus-infecting virus with flexuous particles, has been determined. Analysis shows WLV to have 76% overall nucleotide sequence identity to the more recently discovered Blackberry virus E, an allexivirus belonging to the subgroup of these viruses that lack the 3 proximal cysteine-rich protein (CRP) gene present in Allium-infecting allexiviruses. An infectious cDNA clone of WLV was constructed and a mutation introduced into the P40 gene, which is an allexivirus-specific gene of unknown function. In addition, the infectious clone was modified to express the green fluorescent protein (GFP) as an N-terminal fusion to the WLV coat protein (CP). Using this GFP overcoat strategy it was possible to follow the multiplication and movement of the virus in infected Chenopodium quinoa and spinach leaves. Introduction of the frameshift mutation into the P40 gene of WLV reduced virus accumulation by 97%, and with the GFP overcoated WLV the P40 mutation almost entirely abolished GFP fluorescence in inoculated leaves suggesting that the WLV P40 protein is required for normal levels of virus multiplication. RepositoriesWLV complete sequences deposited at GenBank. Accession Nos. MZ944847.1, OQ877124.1

SARS-CoV-2 strains with both high transmissibility and potential to cause asymptomatic infection is expected to gain selective advantage over other circulating strains having either high transmissibility or ability to trigger asymptomatic infection. The D614G mutation in spike glycoprotein, the characteristic mutation A2a clade, has been associated with high transmissibility, whereas the A3 clade specific mutation L37F in NSP6 protein has been linked with asymptomatic infection. In this study, we performed a comprehensive mutational analysis of 3,77,129 SARS-CoV-2 genomes collected during January, 2020 to December, 2020 from all across the world for the presence of D614G and L37F mutations. Out of 3,77,129 SARS-CoV-2 strains analysed, 14, 598 (3.87%) were found to harbour both the D614G and L37F mutations. Majority of these double mutant SARS-CoV-2 strains were identified in Europe (11097) followed by North America (1915), Asia (980), Oceania (242), Africa (219), and South America (145). Geographical root surveillance revealed their first emergence during February-March in all the six continents. Temporal prevalence analysis from February, 2020 to December, 2020 showed a gradual upsurge in their frequencies worldwide, which strongly demonstrated the adaptive selection of these double mutants. Evolutionary analysis depicted that these double mutants emerged as a new clade in the dendrogram (named as A2a/3), and were sub-divided into four distinct clusters (Cluster I, II, III and IV) according to different sets of coexisting mutations. The frequency distribution pattern showed the global predominance of cluster III (41.42%), followed by cluster IV (23.31%), cluster II (21.02%) and cluster I (14.25%). Overall, our study highlighted the emergence of a unique phylogenetic clade encompassing the double-mutant SARS-CoV-2 strains which may provide a fitness advantage during course of virus evolution.

In recent several years, multiple predominant SARS-CoV-2 variants that spread worldwide were derived from genomic recombination between SARS-CoV-2 lineages with diversified genetic backgrounds. However, the current understanding about the effects of recombination on SARS-CoV-2 evolution and functional aspects is limited. In this study, to achieve one overview regarding the evolution of SARS-CoV-2 recombinant variants, phylogenetic analyses have been performed to evaluate the divergence of representative Omicron recombinant variants from the predicted original Omicron lineage as well as the phylogenetic distances among these variants. As one example of predominant recombinant variants, we also investigated the growth of XFG in Germany by means of virus genomic epidemiology approaches. The results of the evolutionary relationship analyses indicate that recombination between evolutionarily distant lineages or closely related lineages can both drive SARS-CoV-2 evolution, and has the potential of resulting in a novel predominant strain, indicating that recombination plays one important role in SARS-CoV-2 evolution. Furthermore, this study provided information about the growth of XFG in Germany, and revealed a clear relative growth advantage of XFG over co-circulating lineages, such as LP.8.1 and NB.1.8.1. The information acquired from these investigations underlines the need for continuous efforts to detect recombination events and track recombinant variants, which is important for evaluating the long-term effects of recombination on SARS-CoV-2 evolution and of significance for public health.

We identify a mutation in the N gene of SARS-CoV-2 that adversely affects annealing of a commonly used RT-PCR primer; epidemiologic evidence suggests the virus retains pathogenicity and competence for spread. This reinforces the importance of using multiple targets, preferably in at least 2 genes, for robust SARS-CoV-2 detection. Article Summary LineA SARS-CoV-2 variant that occurs worldwide and has spread in California significantly affects diagnostic sensitivity of an N gene assay, highlighting the need to employ multiple viral targets for detection.

COVID-19 pandemic in Brazil was characterized by the sequential circulation of the SARS-CoV-2 lineages B.1.1.33, and variants Zeta (P.2), Gamma (P.1/P.1.*), Delta (B.1.617.2/AY.*), and Omicron (BA.*). Our research aimed to compare the biological traits of these lineages and variants by analyzing aspects of viral replication including binding, entry, RNA replication, and viral protein production. We demonstrated that the replication capacity of these variants varies depending on the cell type, with Omicron BA.1 exhibiting the lowest replication in the human pulmonary cells. Additionally, the nucleocapsid proteoforms generated during infection exhibit distinct patterns across variants. Our findings suggest that factors beyond the initial stages of virus entry influence the efficiency of viral replication among different SARS-CoV-2 variants. Thus, our study underscores the significance of RNA replication and the role of nucleocapsid proteins in shaping the replicative characteristics of SARS-CoV-2 variants. Author summaryThe COVID-19 pandemic was characterized by the emergence of different viral variants that presents specific properties such as response to antibodies, pathogenicity and detection by diagnostic tests. The circulation of these variants presented a particular pattern depending on the global geographic regions. Despite the cessation of the pandemic, as officially declared by the World Health Organization in 2023, new viral variants continue to emerge while aspects of the virus-cell interaction that contribute to the replication of these variants have not yet been completely understood. In our study, we compared the biological characteristics of SARS-CoV-2 variants that circulated in Brazil during the pandemic, verifying aspects of entry, viral replication and production of viral RNA and proteins. Our results indicate that Omicron BA.1 variant has reduced replication and protein production in human lung cells. We also observed that the viral nucleocapsid protein presents proteoforms that vary according to the variant. These differences could help to explain the differences observed in viral replication in human pulmonary cells.

Several SARS-CoV-2 variants of concern have independently acquired some of the same Spike protein mutations - notably E484K, N501Y, S477N, and K417T - associated with increased viral transmission and/or reduced sensitivity to neutralization by antibodies. Repeated evolution of the same mutations, particularly in variants that are now rapidly spreading in various regions of the world, suggests a fitness advantage. Mutations at position P681 in Spike - possibly affecting viral transmission - have also evolved multiple times, including in two variants of concern. Here, we describe three variants circulating in New York State that have independently acquired a P681H mutation and the different trajectories they have taken. While one variant rose to high prevalence since later summer 2020 it appears to be in decline. The other two variants were more recently detected in New York and harbor additional Spike mutations that might be cause for continued monitoring. The latter two P681H variants have shown moderate increases in prevalence but ultimately all might be subject to the same fate as more competitive variants come to dominate the scene.

During the first nine months of the SARS-CoV-2 pandemic, Uruguay successfully kept it under control, even when our previous studies support a recurrent viral flux across the Uruguayan-Brazilian border that sourced several local outbreaks in Uruguay. However, towards the end of 2020, a remarkable exponential growth was observed and the TETRIS strategy was lost. Here, we aimed to understand the factors that fueled SARS-CoV-2 viral dynamics during the first epidemic wave in the country. We recovered 84 whole viral genomes from patients diagnosed between November, 2020 and February, 2021 in Rocha, a sentinel eastern Uruguayan department bordering Brazil. The lineage B.1.1.28 was the most prevalent in Rocha during November-December 2020, P.2 became the dominant one during January-February 2021, while the first P.1 sequences corresponds to February, 2021. The lineage replacement process agrees with that observed in several Brazilian states, including Rio Grande do Sul (RS). We observed a one to three month delay between the appearance of P.2 and P.1 in RS and their subsequent detection in Rocha. The phylogenetic analysis detected two B.1.1.28 and one P.2 main Uruguayan SARS-CoV-2 clades, introduced from the southern and southeastern Brazilian regions into Rocha between early November and mid December, 2020. One synonymous mutation distinguishes the sequences of the main B.1.1.28 clade in Rocha from those widely distributed in RS. The minor B.1.1.28 cluster, distinguished by several mutations, harbours non-synonymous changes in the Spike protein: Q675H and Q677H, so far not concurrently reported. The convergent appearance of S:Q677H in different viral lineages and its proximity to the S1/S2 cleavage site raise concerns about its functional relevance. The observed S:E484K-VOI P.2 partial replacement of previously circulating lineages in Rocha might have increased transmissibility as suggested by the significant decrease in Ct values. Our study emphasizes the impact of Brazilian SARS-CoV-2 epidemics in Uruguay and the need of reinforcing real-time genomic surveillance on specific Uruguayan border locations, as one of the key elements for achieving long-term COVID-19 epidemic control.

ABSTRACTPlant viral infections pose a significant threat to global crop productivity. Despite their profound impact on agriculture, plant viruses have been relatively understudied, primarily due to technological limitations associated with classical molecular methods. However, the advent of NGS RNA-seq analysis has revolutionized virus characterization in environmental settings, overcoming previous limitations and providing a powerful tool for studying plant viruses. In an RNA-seq experiment conducted on a diseased Colombian Cannabis sativa hemp plant, we identified a linear single-stranded RNA (ssRNA) genome belonging to Tobacco Necrosis Virus A (TNVA), a common cause of necrotic lesions in plants such as tobacco and tulipa. The affected Cannabis sativa hemp plant exhibited severe symptoms, including alterations in pigmentation, leaf morphology such as chlorosis, necrotic tissue formation, and surface wear on the leaves. The complete genome sequence of the Cannabis sativa TNVA was 3,656 nucleotides long, containing five putative ORFs, and was classified in the family Tombusviridae, genus Alphanecrovirus, and belonging to the Necro-like clade based on RdRp protein phylogenetic analysis. Our analysis revealed a well-conserved RdRp protein among the Alphanecroviruses, with 89% of the amino acid residues in the peptide being entirely conserved. In contrast, the coat protein exhibited significantly higher variability, with only 49.3% of the residues being 100% conserved. Regarding the viral genome expression of Cannabis sativa TNVA, we observed that the virus was highly abundant in the leaves of the diseased plant, ranking among the topmost abundant transcripts, occupying the percentile position of 3.06%. Overall, our study generated the first reference genome of TNVA virus in the tropical region and reported the first case of this virus infecting a Cannabis sativa plant.

Complexes of viruses inducing a syndrome in plants strongly hinder the identification of the causal agent of a disease. The Sida micrantha mosaic disease is associated with a complex of begomoviruses. For more than twenty years, two DNAs A (DNA A2 and DNA A3) belonging to this complex could neither be detected nor isolated from Sida micrantha Schr. plants, although one of them (DNA A2) now appears to be the major component of the complex. A random unintended Bemisia tabaci-mediated transmission of begomoviruses from several Sida species - including S. micrantha - to experimental Malva parviflora plants resulted in the serendipitous finding of these new DNAs A. Simultaneously, a number of other begomoviruses infecting Sida plants from several Latin American countries were transmitted to M. parviflora plants and the convergence of them resulted in natural pseudorecombinants. Pseudorecombination, however, took place exclusively between heterologous genomic components that shared identical binding sites for the replication-associated protein AC1. This case study constitutes an exceptional opportunity for the analysis of plant-virus- vector interactions in a quasi-natural environment. In addition to that, the methodology described here may be used to isolate and characterize different begomoviruses inducing a syndrome during mix infections.

Munger black raspberry (Rubus occidentalis) has been a preferred indicator for Rubus viruses. The working hypothesis behind the Munger elevated susceptibility to viruses is its ability to sustain elevated virus titers; however, no research has yet explored this concept. To address this, we utilized an infectious clone of blackberry chlorotic ringspot virus to study the differences in virus expression between two Rubus species: R. occidentalis ( Munger) and R. L. subgenus Rubus Watson (blackberry, Natchez). Our data demonstrate that virus accumulation in Munger is over 47,300 times higher compared to Natchez, enhancing our understanding of virus-host dynamics and providing valuable insights into why Munger is a preferred indicator species for virus detection in Rubus spp.

This study compares the collection dates and locations of the Omicron BA.1 lineage and other major SARS-CoV- 2 mutants registered in NCBI GenBank and provides a detailed analysis of the emergence patterns of pure reverse mutants, which contain only reverse mutations and no other mutations in the surface glycoprotein. The results indicate that Omicron BA.1.1 and its pure reverse mutants were widely distributed throughout the United States from the early days of their emergence, showing a statistically significant difference compared to other major variants, which spread from a small number of sources. The peak emergence of BA.1.1 and BA.1.1.18 pure reverse mutants occurred a few weeks before the peak of all collected samples, whereas the peak of pure reverse mutants in major BA.1 variants and BA.2 coincides with the overall sampling peak. Although the peaks of BA.1.x collections are not all synchronous, the peaks of pure reverse mutants in the BA.1 lineage completely overlap, with the number of such mutants declining abruptly after the peak. These regional and temporal anomalies in the Omicron BA.1 lineage, especially in the BA.1.1 lineage, are virtually impossible to explain by current theories of natural mutation and spread by human-to-human infection.