Aa Changes Research Articles

Using IMGT unique numbering for IG allotypes and Fc-engineered variants of effector properties and half-life of therapeutic antibodies.

Therapeutic monoclonal antibodies (mAb) are usually of the IgG1, IgG2, and IgG4 classes, and their heavy chains may be modified by amino acid (aa) changes involved in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or half-life. Allotypes and Fc-engineered variants are classified using IMGT/HGNC gene nomenclature (e.g., Homo sapiens IGHG1). Allotype names follow the WHO/IMGT nomenclature. IMGT-engineered variant names use the IMGT nomenclature (e.g., Homsap G1v1), which comprises species and gene name (both abbreviated) followed by the letter v (for variant) and a number. Both allotypes and engineered variants are defined by their aa changes and positions, based on the IMGT unique numbering for C domain, identified in sequence motifs, referred to as IMGT topological motifs, as their limits and length are standardized and correspond to a structural feature (e.g., strand or loop). One hundred twenty-six variants are displayed with their type, IMGT numbering, Eu-IMGT positions, motifs before and after changes, and their property and function (effector and half-life). Three motifs characterize effector variants, CH2 1.6-3, 23-BC-41, and the FG loop, whereas three different motifs characterize half-life variants, two on CH2 13-AB-18 and 89-96 with H93, and one on CH3 the FG loop with H115.

Evolution and virulence of porcine epidemic diarrhea virus following in vitro and in vivo propagation

Practice of inoculating porcine epidemic diarrhea virus (PEDV) in piglets generating feedback material might influence the genetic evolution and attenuation of PEDV. The study was conducted to evaluate evolutionary rate and attenuation following serial in vitro and in vivo propagation. In the study, PED-JPFP0-PJ, Passage 0 (P0), was isolated from infected pigs and serially passaged in Vero cells for 5 consecutive times, P1-P5. P0, P2 and P5 were then subjected to orally inoculate 3-day-old piglets. At 24 h post inoculation, intestines of each passage (F1), were collected, and subsequently sub-passaged in piglets for 2 additional passages (F2-F3). Virus titration, PEDV genomic copies number, VH:CD ratios, and immunohistochemistry were evaluated. S and ORF3 genes were characterized. The results of the study demonstrated that virus titer and virulence were negatively correlated with increased passages, both in vitro and in vivo. Increased substitution rate was observed in higher passages. The evolutionary rate of S gene was higher than that of ORF3. Seven aa changes at positions 223, 291, 317, 607, 694, 1114 and 1199, with reduced N-linked glycan were observed in P5F3. In conclusion, serial passage of PEDV, both in vitro and in vivo, influence the genetic development and the attenuation of PEDV.

Persistence of SARS-CoV-2 infection and viral intra- and inter-host evolution in COVID-19 hospitalized patients.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) persistence in COVID-19 patients could play a key role in the emergence of variants of concern. The rapid intra-host evolution of SARS-CoV-2 may result in an increased transmissibility, immune and therapeutic escape which could be a direct consequence of COVID-19 epidemic currents. In this context, a longitudinal retrospective study on eight consecutive COVID-19 patients with persistent SARS-CoV-2 infection, from January 2022 to March 2023, was conducted. To characterize the intra- and inter-host viral evolution, whole genome sequencing and phylogenetic analysis were performed on nasopharyngeal samples collected at different time points. Phylogenetic reconstruction revealed an accelerated SARS-CoV-2 intra-host evolution and emergence of antigenically divergent variants. The Bayesian inference and principal coordinate analysis analysis showed a host-based genomic structuring among antigenically divergent variants, that might reflect the positive effect of containment practices, within the critical hospital area. All longitudinal antigenically divergent isolates shared a wide range of amino acidic (aa) changes, particularly in the Spike (S) glycoprotein, that increased viral transmissibility (K417N, S477N, N501Y and Q498R), enhanced infectivity (R346T, S373P, R408S, T478K, Q498R, Y505H, D614G, H655Y, N679K and P681H), caused host immune escape (S371L, S375F, T376A, K417N, and K444T/R) and displayed partial or complete resistance to treatments (G339D, R346K/T, S371F/L, S375F, T376A, D405N, N440K, G446S, N460K, E484A, F486V, Q493R, G496S and Q498R). These results suggest that multiple novel variants which emerge in the patient during persistent infection, might spread to another individual and continue to evolve. A pro-active genomic surveillance of persistent SARS-CoV-2 infected patients is recommended to identify genetically divergent lineages before their diffusion.

Geographical Expansion of Avian Metapneumovirus Subtype B: First Detection and Molecular Characterization of Avian Metapneumovirus Subtype B in US Poultry.

Avian metapneumovirus (aMPV), classified within the Pneumoviridae family, wreaks havoc on poultry health. It typically causes upper respiratory tract and reproductive tract infections, mainly in turkeys, chickens, and ducks. Four subtypes of AMPV (A, B, C, D) and two unclassified subtypes have been identified, of which subtypes A and B are widely distributed across the world. In January 2024, an outbreak of severe respiratory disease occurred on turkey and chicken farms across different states in the US. Metagenomics sequencing of selected tissue and swab samples confirmed the presence of aMPV subtype B. Subsequently, all samples were screened using an aMPV subtype A and B multiplex real-time RT-PCR kit. Of the 221 farms, 124 (56%) were found to be positive for aMPV-B. All samples were negative for subtype A. Six whole genomes were assembled, five from turkeys and one from chickens; all six assembled genomes showed 99.29 to 99.98% nucleotide identity, indicating a clonal expansion event for aMPV-B within the country. In addition, all six sequences showed 97.74 to 98.58% nucleotide identity with previously reported subtype B sequences, e.g., VCO3/60616, Hungary/657/4, and BR/1890/E1/19. In comparison to these two reference strains, the study sequences showed unique 49-62 amino acid changes across the genome, with maximum changes in glycoprotein (G). One unique AA change from T (Threonine) to I (Isoleucine) at position 153 in G protein was reported only in the chicken aMPV sequence, which differentiated it from turkey sequences. The twelve unique AA changes along with change in polarity of the G protein may indicate that these unique changes played a role in the adaptation of this virus in the US poultry. This is the first documented report of aMPV subtype B in US poultry, highlighting the need for further investigations into its genotypic characterization, pathogenesis, and evolutionary dynamics.

Genetic characterization of the parvovirus full-length VP2 gene in domestic cats in Brazil

Feline parvovirus (FPV) and canine parvovirus (CPV) are over 98% identical in their DNA sequences, and the new variants of CPV (2a/2b/2c) have gained the ability to infect and replicate in cats. The aim of this study was to determine the genetic diversity in the VP2 gene of parvovirus strains circulating in domestic cats in Brazil during a 10-year period (2008–2017). For parvovirus screening, specific PCR was performed, and 25 (34.7%) of 72 cats tested positive. The PCR-positive samples were further subjected to full-length VP2 sequencing (1755 bp), and eight sequences (36%) were characterized as FPV, seven (28%) as CPV-2a and (32%) nine (36%) as CPV-2b. One sequence (RJ1085/11) showing typical CPV amino acid (aa) at residues 80 R, 93 N, 103 A, 232 I, and 323 N could not be characterized at this time. The sequences in this study displayed aa changes previously described for FPV (A14T, A91S, I101T, N564S, and A568G) from cats and CPV-2a/2b (S297N and Y324L) from dogs. However, the Y324L mutation has not yet been reported in any CPV-2a/2b strains from cats. Phylogenetic analysis supported the division of these sequences into two well-defined clades, clade 1 for FPV and clade 2 for CPV2a/2b. Unusually, the sequence RJ1085/11 was grouped separately. Two recombination breakpoints were detected by Bootscan and 3Seq methods implemented in the RDP4. This study is the first report of CPV-2a/2b in cats in Brazil. The detection of FPV strains with mutations characteristic of CPV indicates that Brazilian FPV strains have undergone genetic changes.

Avian influenza virus cross-infections as test case for pandemic preparedness: From epidemiological hazard models to sequence-based early viral warning systems.

Pandemic preparedness starts with an early warning system of viruses with a pandemic potential. Based on information collected in a multitude of surveys, hazard models were developed identifying influenza viruses presenting a pandemic threat. Scores are attributed for 10 viral traits by expert panels which identified avian influenza viruses (AIV) belonging to subtypes H7N9 and H5N1 as representing the greatest pandemic risk. In 2013, more than 100 human cases infected with AIV H7N9 were observed in China. Case fatality rate (CFR) was high (27%), but the human-to-human transmission rate was low and by serological evidence H7N9 did not spread widely. Nevertheless, until 2019 more than 1500 H7N9 patients were identified characterized by a high CFR of 39%. Serology demonstrated that mild infections with H7N9 were widespread. In 2003, more than 400 people experienced AIV H7N7 cross-infection causing mainly conjunctivitis during a large poultry epidemic in The Netherlands. Between 1996 and 2019, a total of 881 human infections with avian H5N1 viruses were documented showing a CFR of 52%. Outbreaks were centred on South East Asia and showed close associations with epizootics in poultry. Mutations predisposing to human cross-infections were identified in the haemagglutinin (HA) and the RNA polymerase subunit PB2 of human H7N9 isolates. Human H5N1 isolates showed mutations in the receptor binding domain of HA and transmission in mammals could be obtained by as few as four additional aa changes introduced experimentally. Researchers have defined viral point mutations in HA, PB2 and the nucleoprotein NP that allowed AIV to cross the species barrier to mammals with respect to receptor recognition, RNA replication and escape from innate immunity respectively. Based on this insight a sequence-based early warning system for AIV preadapted to human transmission could be envisioned. Mink farms and live poultry markets are prime targets for such sequencing efforts.

135 Gene Variants in Bola-Dmb, DECR1, Fasn and SREBF1 Associated with Conceptus Death on Day 16 of Pregnancy in Holstein Cows

Abstract Investigating the influences of single nucleotide polymorphisms (SNP) associated with conceptus death (CD) on day 16 of pregnancy could distinguish cows who have inferior value for fertility traits. We hypothesized that CD is associated with SNP genotypes that disrupt maternal recognition and lead to early pregnancy loss. RNA sequences from Holstein cow pregnancies (n = 15) with normal conceptuses (Norm) or CD were subjected to SNP discovery via Qiagen CLC Genomics Workbench. Selected SNP were based on previous fertility studies, differentially expressed genes within pregnancy development analyses and proximity to SNP associated with fertility traits in the Cattle QTL Database. A second cohort of Holstein cows (n = 500) was used to conduct a validation and genotype to phenotype analysis of candidate SNP (cSNP) via DNA from blood samples and farm records. PLINK software was used to remove cows (n = 34) missing more than >20% cSNP and remove cSNP with monomorphic alleles, minor allele frequency (MAF) < 10% and not in Hardy-Weinberg Equilibrium (>1e-15). The GLM-one way ANOVA was used for all statistical models in SAS. Statistically significant models (P <0.05) were further analyzed for LSMEANS, Bonferroni adjustment of P-values, additive allele effect and predicted effect of amino acid (aa) change on protein function via SIFT analyses tool in the Ensembl variant table (scoring 0-1.0; < 0.05 being significant). Previously, we identified 69 cSNP on the RNA sequences of Norm or CD pregnancies from the initial group of Holstein cows and in proximity to >1 SNP associated with fertility traits from the Cattle QTL database. Quality control measures in PLINK reduced the number of Holstein cows in the second cohort to 466 and identified 4 cSNP associated with non-binary reproductive traits. Herein, we report an additional 4 cSNP discovered and validated with binary reproductive traits. The cSNP within genes were associated with the following roles: BOLA-DMB (antigen loading in the immune system), DECR1 (lipid metabolism), FASN (reconstitution of body reserves during pregnancy) and SREBF1 (synthesis of fatty acids). Cows with T allele, in BOLA-DMB and SREBF1, were less likely (P < 0.05) to become pregnant <150 days in milk (DIM). For DECR1 and FASN, cows were less likely (P < 0.05) to become pregnant at 1st artificial insemination with C or A allele, respectively. Only the cSNP in SREBF1 had both an additive allele effect and a predicted effect of aa change on protein function. When cows had C allele, in the SREBF1 cSNP, there was 6% greater (P < 0.01) probability of becoming pregnant <150 DIM. While SIFT revealed the cSNP in SREBF1 have a predicted significant effect with a score of 0.03 for the aa change of proline/leucine. The reported cSNP, specifically those influenced by CD, add valuable information for the development of improved genetic tools for the dairy industry. USDA-NIFA#2019-07133.

High-protein diet with excess leucine prevents inactivity-induced insulin resistance in women

Background and aimsMuscle inactivity leads to muscle atrophy and insulin resistance. The branched-chain amino acid (BCAA) leucine interacts with the insulin signaling pathway to modulate glucose metabolism. We have tested the ability of a high-protein BCAA-enriched diet to prevent insulin resistance during long-term bed rest (BR). MethodsStable isotopes were infused to determine glucose and protein kinetics in the postabsorptive state and during a hyperinsulinemic-euglycemic clamp in combination with amino acid infusion (Clamp+AA) before and at the end of 60 days of BR in two groups of healthy, young women receiving eucaloric diets containing 1 g of protein/kg per day (n=8) or 1.45 g of protein/kg per day enriched with 0.15 g/kg per day of BCAAs (leucine/valine/isoleucine=2/1/1) (n=8). Body composition was determined by Dual X-ray Absorptiometry. ResultsBR decreased lean body mass by 7.6±0.3% and 7.2±0.8% in the groups receiving conventional or high protein-BCAA diets, respectively. Fat mass was unchanged in both groups.At the end of BR, percent changes of insulin-mediated glucose uptake significantly (p=0.01) decreased in the conventional diet group from 155±23% to 84±10% while did not change significantly in the high protein-BCAA diet group from 126±20% to 141±27% (BR effect, p=0.32; BR/diet interaction, p=0.01; Repeated Measures ANCOVA). In contrast, there were no BR/diet interactions on proteolysis and protein synthesis Clamp+AA changes in the conventional diet and the high protein-BCAA diet groups. ConclusionA high protein-BCAA enriched diet prevented inactivity-induced insulin resistance in healthy women.

Dengue virus serotype 2 genotype III evolution during the 2019 outbreak in Mato Grosso, Midwestern Brazil.

DENV-2 was the main responsible for a 70% increase in dengue incidence in Brazil during 2019. That year, our metagenomic study by Illumina NextSeq on serum samples from acute febrile patients (n=92) with suspected arbovirus infection, sampled in 22 cities of the state of Mato Grosso (MT), in the middle west of Brazil, revealed eight complete genomes and two near-complete sequences of DENV-2 genotype III, one Human parvovirus B19 genotype I (5,391nt) and one Coxsackievirus A6 lineage D (4,514nt). These DENV-2 sequences share the aminoacidic identities of BR4 lineage on E protein domains I, II and III, and were included in a clade with sequences of the same lineage circulating in the southeast of Brazil in the same year. Nevertheless, 11/34 non-synonymous mutations are unique to three strains inthis study, distributed in the E (n=6), NS3 (n=2) and NS5 (n=3) proteins. Other 14 aa changes on C (n=1), E (n=3), NS1 (n=2), NS2A (n=1) and NS5 (n=7) were first reported in a genotype III lineage, having been already reported only in other DENV-2 genotypes. All 10 sequences have mutations in the NS5 protein (14 different aa changes). Nine E protein aa changes found in two sequences, six of which are unique, are in the ectodomain; where the E:M272T change is on the hinge of the E protein at domain II, in a region critical for the anchoring to the host cell receptor. The NS5:G81R mutation, in the methyltransferase domain, was found in one strain of this study. Altogether, these data points to an important evolution of DENV-2 genotype III lineage BR4 during this outbreak in 2019 in MT. Genomic surveillance is essential to detect virus etiology and evolution, possibly related to immune evasion and viral fitness changes leading to future novel outbreaks.

Rescue of a Live-Attenuated Porcine Epidemic Diarrhea Virus HSGP Strain Using a Virulent Strain and a Partially Attenuated Strain.

In South Korea in 2013, the G1-based vaccine failed to prevent an outbreak of G2b-type porcine epidemic diarrhea virus (PEDV), which is more pathogenic than the traditional G1-type strain, thereby allowing the virus to spread. In 2017 and 2018, field samples were cultured sequentially on Vero cells to isolate HS (virulent) and SGP-M1 (partially attenuated) strains, respectively, of the G2b type. The HS strain harbors a single amino acid (aa) change and two aa deletions in the N-terminal domain of S1 (55I56G57E→55K56Δ57Δ). The SGP-M1 strain harbors a seven aa deletion in the C-terminal domain of S2 (1380~1386ΔFEKVHVQ). By co-infecting various animal cells with these two strains (HS and SGP-M1), we succeeded in cloning strain HSGP, which harbors the mutations present in the two original viruses. The CPE pattern of the HSGP strain was different from that of the HS and SGP-M1 strains, with higher viral titers. Studies in piglets showed attenuated pathogenicity of the HSGP strain, with no clinical symptoms or viral shedding, and histopathologic lesions similar to those in negative controls. These findings confirm that deletion of specific sequences from the S gene attenuates the pathogenicity of PEDV. In addition, HSGP strains created by combining two different strains have the potential for use as novel attenuated live vaccine candidates.

Viral sequence analysis of chronic hepatitis B patients treated with the capsid assembly modulator JNJ-56136379 in the JADE phase 2a study

Background & aimsIn the monotherapy arms of the phase 2 JADE study (ClinicalTrials.gov Identifier: NCT03361956) evaluating the safety and efficacy of JNJ-56136379 (capsid assembly modulator-class E) with/without nucleos(t)ide analogue (NA), viral breakthroughs (VBT) were observed, leading to JNJ-56136379 monotherapy discontinuation. We present the viral sequencing analysis of JNJ-56136379±NA-treated hepatitis B virus (HBV)-infected patients. MethodsThe HBV full genome was sequenced using next generation sequencing. Baseline amino acid (aa) polymorphisms were defined as changes versus the universal HBV reference sequence (sequence read frequency >15%). Emerging mutations were defined as aa changes versus baseline sequence (frequency <1% at baseline and ≥15% post-baseline). Results6/28 JNJ-56136379 75 mg monotherapy arm patients experienced VBT; all 6 had emerging JNJ-56136379-resistant variants T33N (n = 5; fold change [FC] = 85) or F23Y (n = 1; FC = 5.2). 1/32 JNJ-56136379 250 mg arm patients (genotype-E) had <1 log10 IU/mL decline in HBV DNA at Week 4, experienced VBT at Week 8, and carried the I105T baseline polymorphism (FC = 7.9), but had no emerging variants. Eight additional monotherapy-treated patients had shallow second phases of their HBV DNA profile and emerging T33N (n = 7) or F23Y (n = 1) variants. NA initiation (switch [75 mg arm]; add-on [250 mg arm]) in all monotherapy patients with VBT resulted in HBV DNA decline in all patients. No VBT was observed during JNJ-56136379+NA combination therapy. ConclusionsJNJ-56136379 monotherapy resulted in VBT and was associated with the selection of JNJ-56136379-resistant variants. Efficacy of NA treatment (de novo combination or rescue therapy for VBT) was not impacted, confirming the lack of cross-resistance between these drug classes. Clinical trial numberNCT03361956.

Prevalence of HIV-1 Natural Polymorphisms and Integrase-Resistance-Associated Mutations in African Children.

Integrase inhibitors (INIs) are a potent option for HIV treatment. Limited data exist on INI resistance in West Africa, particularly in children living with HIV/AIDS. We determined the prevalence of integrase gene polymorphisms and the frequency of naturally occurring amino acid (aa) substitutions at positions associated with INI resistance. Dried blood spot (DBS) samples were obtained from one hundred and seven (107) HIV-1-infected children aged less than 15 years old in two West African countries, Benin and Mali. All children were naïve to INI treatment, 56 were naïve to anti-retroviral therapy (ART), and 51 had received ART. Genetic sequencing of HIV integrase was successful in 75 samples. The aa changes at integrase positions associated with INI resistance were examined according to the Stanford HIV Genotypic Resistance database. The median ages were 2.6 and 10 years for ART-naïve and -treated children, respectively. The most common subtypes observed were CRF02_AG (74.7%) followed by CRF06_cpx (20%). No major INI-resistance mutations at positions 66, 92, 121, 143, 147, 148, 155, and 263 were detected. The most prevalent INI accessory resistance mutations were: L74I/M (14/75, 18.6%) followed by E157Q (8/75, 10.6%), G163E/N/T/Q (5/75, 6.6%), Q95A/H/P (2/75, 2.6%), and T97A (4/75, 5.3%). Other substitutions observed were M50I/L/P, H51E/P/S/Q, I72V, T112V, V201I, and T206S. Polymorphisms at positions which may influence the genetic barrier and/or drive the selection of specific INI-resistance pathways were detected. However, no transmitted drug resistance (TDR) to INI was detected among samples of INI-naïve patients. These findings support the use of this treatment class for children with HIV-1, particularly in West Africa.

Genotyping and In Silico Analysis of Delmarva (DMV/1639) Infectious Bronchitis Virus (IBV) Spike 1 (S1) Glycoprotein

Genetic diversity and evolution of infectious bronchitis virus (IBV) are mainly impacted by mutations in the spike 1 (S1) gene. This study focused on whole genome sequencing of an IBV isolate (IBV/Ck/Can/2558004), which represents strains highly prevalent in Canadian commercial poultry, especially concerning features related to its S1 gene and protein sequences. Based on the phylogeny of the S1 gene, IBV/Ck/Can/2558004 belongs to the GI-17 lineage. According to S1 gene and protein pairwise alignment, IBV/Ck/Can/2558004 had 99.44–99.63% and 98.88–99.25% nucleotide (nt) and deduced amino acid (aa) identities, respectively, with five Canadian Delmarva (DMV/1639) IBVs isolated in 2019, and it also shared 96.63–97.69% and 94.78–97.20% nt and aa similarities with US DMV/1639 IBVs isolated in 2011 and 2019, respectively. Further homology analysis of aa sequences showed the existence of some aa substitutions in the hypervariable regions (HVRs) of the S1 protein of IBV/Ck/Can/2558004 compared to US DMV/1639 isolates; most of these variant aa residues have been subjected to positive selection pressure. Predictive analysis of potential N-glycosylation and phosphorylation motifs showed either loss or acquisition in the S1 glycoprotein of IBV/Ck/Can/2558004 compared to S1 of US DMV/1639 IBV. Furthermore, bioinformatic analysis showed some of the aa changes within the S1 protein of IBV/Ck/Can/2558004 have been predicted to impact the function and structure of the S1 protein, potentially leading to a lower binding affinity of the S1 protein to its relevant ligand (sialic acid). In conclusion, these findings revealed that the DMV/1639 IBV isolates are under continuous evolution among Canadian poultry.

Genetic Diversity and Low Therapeutic Impact of Variant-Specific Markers in HIV-1 Pol Proteins.

The emergence and spread of new HIV-1 variants pose a challenge for the effectiveness of antiretrovirals (ARV) targeting Pol proteins. During viral evolution, non-synonymous mutations have fixed along the viral genome, leading to amino acid (aa) changes that can be variant-specific (V-markers). Those V-markers fixed in positions associated with drug resistance mutations (DRM), or R-markers, can impact drug susceptibility and resistance pathways. All available HIV-1 Pol sequences from ARV-naïve subjects were downloaded from the United States Los Alamos HIV Sequence Database, selecting 59,733 protease (PR), 6,437 retrotranscriptase (RT), and 6,059 integrase (IN) complete sequences ascribed to the four HIV-1 groups and group M subtypes and circulating recombinant forms (CRFs). Using a bioinformatics tool developed in our laboratory (EpiMolBio), we inferred the consensus sequences for each Pol protein and HIV-1 variant to analyze the aa conservation in Pol. We analyzed the Wu–Kabat protein variability coefficient (WK) in PR, RT, and IN group M to study the susceptibility of each site to evolutionary replacements. We identified as V-markers the variant-specific aa changes present in >75% of the sequences in variants with >5 available sequences, considering R-markers those V-markers that corresponded to DRM according to the IAS-USA2019 and Stanford-Database 9.0. The mean aa conservation of HIV-1 and group M consensus was 82.60%/93.11% in PR, 88.81%/94.07% in RT, and 90.98%/96.02% in IN. The median group M WK was 10 in PR, 4 in RT, and 5 in IN. The residues involved in binding or catalytic sites showed a variability <0.5%. We identified 106 V-markers: 31 in PR, 28 in RT, and 47 in IN, present in 11, 12, and 13 variants, respectively. Among them, eight (7.5%) were R-markers, present in five variants, being minor DRM with little potential effect on ARV susceptibility. We present a thorough analysis of Pol variability among all HIV-1 variants circulating to date. The relatively high aa conservation observed in Pol proteins across HIV-1 variants highlights their critical role in the viral cycle. However, further studies are needed to understand the V-markers’ impact on the Pol proteins structure, viral cycle, or treatment strategies, and periodic variability surveillance studies are also required to understand PR, RT, and IN evolution.

Evolution of SARS-CoV-2 in Spain during the First Two Years of the Pandemic: Circulating Variants, Amino Acid Conservation, and Genetic Variability in Structural, Non-Structural, and Accessory Proteins.

Monitoring SARS-CoV-2’s genetic diversity and emerging mutations in this ongoing pandemic is crucial to understanding its evolution and ensuring the performance of COVID-19 diagnostic tests, vaccines, and therapies. Spain has been one of the main epicenters of COVID-19, reaching the highest number of cases and deaths per 100,000 population in Europe at the beginning of the pandemic. This study aims to investigate the epidemiology of SARS-CoV-2 in Spain and its 18 Autonomous Communities across the six epidemic waves established from February 2020 to January 2022. We report on the circulating SARS-CoV-2 variants in each epidemic wave and Spanish region and analyze the mutation frequency, amino acid (aa) conservation, and most frequent aa changes across each structural/non-structural/accessory viral protein among the Spanish sequences deposited in the GISAID database during the study period. The overall SARS-CoV-2 mutation frequency was 1.24 × 10−5. The aa conservation was >99% in the three types of protein, being non-structural the most conserved. Accessory proteins had more variable positions, while structural proteins presented more aa changes per sequence. Six main lineages spread successfully in Spain from 2020 to 2022. The presented data provide an insight into the SARS-CoV-2 circulation and genetic variability in Spain during the first two years of the pandemic.

HIV Transmembrane Glycoprotein Conserved Domains and Genetic Markers Across HIV-1 and HIV-2 Variants.

HIV envelope transmembrane glycoproteins gp41 (HIV-1) and gp36 (HIV-2) present high variability and play a key role in the HIV-host cell membrane's fusion, as a target for human broadly neutralizing antibodies (bnAbs) and drugs. Thus, a better knowledge of amino acid (aa) conservation across structural domains and HIV variants can help to identify conserved targets to direct new therapeutic and diagnostic strategies. All available gp41/gp36 nucleotide sequences were downloaded from Los Alamos National Laboratory (LANL) HIV Sequence Database, selecting 17,078 sequences ascribed to HIV-1 and HIV-2 variants with ≥3 sequences. After aligning and translating into aa with MEGAv6.0, an in-house bioinformatics program (EpiMolBio) was used to identify the most conserved aa and the aa changes that were specific for each variant (V-markers) vs. HXB2/BEN (HIV-1/HIV-2) reference sequence. We analyzed the presence of specific aa changes among V-markers affecting infectivity, gp41 structure, function, or resistance to the enfuvirtide viral fusion inhibitor (T-20). We also inferred the consensus sequences per HIV variant, describing in each HIV-1 group (M, N, O, P) the conservation level along the complete gp41 per structural domain and locating in each binding site the anti-gp41 human Abs (bnAbs and non bnAbs) described in LANL. We found 38.3/59.7% highly conserved aa present in ≥90% of the 16,803/275 gp41/gp36 sequences ascribed to 105/3 HIV-1/HIV-2 variants, with 9/12.6% of them showing complete conservation across LANL sequences. The fusion peptide, its proximal region, the N-heptad repeat, and the membrane-proximal external region were the gp41 domains with ≥84% of conserved aa in the HIV-1 consensus sequence, the target of most Abs. No natural major resistance mutations to T-20 were observed. Our results show, for the first time, a complete conservation study of gp41/gp36 per variant in the largest panel of HIV variants analyzed to date, providing useful information for a more rational design of drugs, vaccines, and molecular detection tests targeting the HIV transmembrane glycoprotein.

Genetic diversity and epidemiological features of respiratory syncytial virus, Beijing, 2015–2019: A multicenter and all-age groups study

To investigate the genetic variability and the epidemiological features of respiratory syncytial virus (RSV) in Beijing during five consecutive seasons from 2015 to 2019. We collected 36,927 samples (ages ranged from 1 day to 101 years old) from cases with acute respiratory tract infections (ARTI) using the Respiratory Pathogens Surveillance System (RPSS) in Beijing, 2015-2019. G gene sequencing and phylogenetic analysis were performed to identify RSV genotypes, clusters, and amino acid (aa) changes. In total, 764 (2.1%, 764/36927) cases were RSV positive, 52.1% cases were children under 5 years old, and 25.8% were elderly ≥ 60 years old. We obtained 369 sequences of the G gene. ON1 and BA9 were the dominant genotypes in Beijing. Sub-lineage 4 of ON1, which contains four aa substitutions (T113I, N178G, H258Q, and H266L), emerged in 2017 and became the predominant variant in 2018-2019. Sub-lineage 4 of BA9, which contains two aa changes (A131T, T137I), emerged in 2017 and became the predominant variant in 2019. We also observed 10 rarely reported nucleotide deletions in the 3' end of the G gene from five sequences of the ON1 genotype. With the exception of children < 5 years old, RSV infection mainly occurred in the elderly ≥ 60 years old. Newly emerged sub-lineages have replaced existing sub-lineage over time and become predominant in Beijing. Continued surveillance of the genetic diversity of RSV is necessary.

Impact of Vaccination on Rotavirus Genotype Diversity: A Nearly Two-Decade-Long Epidemiological Study before and after Rotavirus Vaccine Introduction in Sicily, Italy.

Sicily was the first Italian region to introduce rotavirus (RV) vaccination with the monovalent G1P[8] vaccine Rotarix® in May 2012. In this study, the seasonal distribution and molecular characterization of RV strains detected over 19 years were compared to understand the effect of Rotarix® on the evolutionary dynamics of human RVs. A total of 7846 stool samples collected from children < 5 years of age, hospitalized with acute gastroenteritis, were tested for RV detection and genotyping. Since 2013, vaccine coverage has progressively increased, while the RV prevalence decreased from 36.1% to 13.3% with a loss of seasonality. The local distribution of RV genotypes changed over the time possibly due to vaccine introduction, with a drastic reduction in G1P[8] strains replaced by common and novel emerging RV strains, such as equine-like G3P[8] in the 2018–2019 season. Comparison of VP7 and VP4 amino acid (aa) sequences with the cognate genes of Rotarix® and RotaTeq® vaccine strains showed specific aa changes in the antigenic epitopes of VP7 and of the VP8* portion of VP4 of the Italian RV strains. Molecular epidemiological surveillance data are required to monitor the emergence of novel RV strains and ascertain if these strains may affect the efficacy of RV vaccines.

Generation of Mild Recombinants of Papaya Ringspot Virus to Minimize the Problem of Strain-Specific Cross-Protection.

Papaya ringspot virus (PRSV) causes severe damage to papaya (Carica papaya L.) and is the primary limiting factor for papaya production worldwide. A nitrous acid-induced mild strain, PRSV HA 5-1, derived from Hawaii strain HA, has been applied to control PRSV by cross-protection for decades. However, the problem of strain-specific protection hampers its application in Taiwan and other geographic regions outside Hawaii. Here, sequence comparison of the genomic sequence of HA 5-1 with that of HA revealed 69 nucleotide changes, resulting in 31 aa changes, of which 16 aa are structurally different. The multiple mutations of HA 5-1 are considered to result from nitrous acid induction because 86% of nucleotide changes are transition mutations. The stable HA 5-1 was used as a backbone to generate recombinants carrying individual 3' fragments of Vietnam severe strain TG5, including NIa, NIb, and CP3' regions, individually or in combination. Our results indicated that the best heterologous fragment for the recombinant is the region of CP3', with which symptom attenuation of the recombinant is like that of HA 5-1. This mild recombinant HA51/TG5-CP3' retained high levels of protection against the homologous HA in papaya plants and significantly increased the protection against the heterologous TG-5. Similarly, HA 5-1 recombinants carrying individual CP3' fragments from Thailand SMK, Taiwan YK, and Vietnam ST2 severe strains also significantly increase protection against the corresponding heterologous strains in papaya plants. Thus, our recombinant approach for mild strain generation is a fast and effective way to minimize the problem of strain-specific protection.

In Silico Study to Predict the Structural and Functional Consequences of SNPs on Biomarkers of Ovarian Cancer (OC) and BPA Exposure-Associated OC

Background: Recently, we have shown that seven genes, namely GBP5, IRS2, KRT4, LINCOO707, MRPL55, RRS1 and SLC4A11, have prognostic power for the overall survival in ovarian cancer (OC). Methods: We present an analysis on the association of these genes with any phenotypes and mutations indicative of involvement in female cancers and predict the structural and functional consequences of those SNPS using in silico tools. Results: These seven genes present with 976 SNPs/mutations that are associated with human cancers, out of which 284 related to female cancers. We have then analysed the mutation impact on amino acid polarity, charge and water affinity, leading to the identification of 30 mutations in gynaecological cancers where amino acid (aa) changes lead to opposite polarity, charges and water affinity. Out of these 30 mutations identified, only a missense mutation (i.e., R831C/R804C in uterine corpus endometrial carcinomas, UCEC) was suggestive of structural damage on the SLC4A11 protein. Conclusions: We demonstrate that the R831C/R804C mutation is deleterious and the predicted ΔΔG values suggest that the mutation reduces the stability of the protein. Future in vitro studies should provide further insight into the role of this transporter protein in UCEC.