Unique Amino Acid Substitutions Research Articles

Short Communication: Genetic diversity and identification of unique Omicron variant markers of SARS-CoV-2 in Indonesia

Abstract. Suardana IBK, Mahardika BK, Santosa AAANA, Mahardika GN. 2024. Short Communication: Genetic diversity and identification of unique Omicron variant markers of SARS-CoV-2 in Indonesia. Biodiversitas 25: 1781-1787. The rapid and global spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused enormous socioeconomic disruptions, such as local and global travel, country or city lockdown, and manymore. We tested the hypothesis that global introduction and local substitution markers that are unique to Indonesia due to the global travel restrictions could be identified. All complete and high-coverage genomes of all Omicron variant sequences submitted from Indonesia were downloaded from the Global Initiative on Sharing All Influenza Data (GISAID), in October 27, 2022, and aligned with the Wuhan-Hu-1 strain and randomly selected Nextstrain Omicron clades. Genetic diversity was calculated using Tajima’s neutrality test, and phylogeny was constructed using the neighbour-joining method in Mega 11. The overall whole genome diversity of the Omicron variant detected in Indonesia is 0.0008, with the three highest coding region diversities being Open Reading Frame 6 (ORF6), Envelope, and Spike protein. The Tajima D value of the whole genome is -2.669, while the three highest Tajima values are ORF6, Envelope and ORF10. The phylogeny shows that there are 168 sequences of Indonesian Omicron variants, which do not share branches with Nextstrain Omicron clades. Unique amino acid substitutions to Wuhan-Hu-1 of the Omicron variants detected in Indonesia occurred in some coding regions, mainly in ORF1AB and Spike. We encourage country-based analysis as a lesson for future pandemic events.

Isolation and pathogenicity comparison of two novel natural recombinant porcine reproductive and respiratory syndrome viruses with different recombination patterns in Southwest China.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses in the swine industry. Frequent mutations and recombinations account for PRRSV immune evasion and the emergence of novel strains. In this study, we isolated and characterized two novel PRRSV-2 strains from Southwest China exhibiting distinct recombination patterns. They were designated SCABTC-202305 and SCABTC-202309. Phylogenetic results indicated that SCABTC-202305 was classified as lineage 8, and SCABTC-202309 was classified as lineage 1.8. Amino acid mutation analysis identified unique amino acid substitutions and deletions in ORF5 and Nsp2 genes. The results of the recombination analysis revealed that SCABTC-202305 is a recombinant with JXA1 as the major parental strain and NADC30 as the minor parental strain. At the same time, SCABTC-202309 is identified as a recombinant with NADC30 as the major parental strain and JXA1 as the minor parental strain. In this study, we infected piglets with SCABTC-202305, SCABTC-202309, or mock inoculum (control) to study the pathogenicity of these isolates. Although both isolated strains were pathogenic, SCABTC-202305-infected piglets exhibited more severe clinical signs and higher mortality, viral load, and antibody response than SCABTC-202309-infected piglets. SCABTC-202305 also caused more extensive lung lesions based on histopathology. Our findings suggest that the divergent pathogenicity observed between the two novel PRRSV isolates may be attributed to variations in the genetic information encoded by specific genomic regions. Elucidating the genetic determinants governing PRRSV virulence and transmissibility will inform efforts to control this devastating swine pathogen.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) is one of the most critical pathogens impacting the global swine industry. Frequent mutations and recombinations have made the control of PRRSV increasingly difficult. Following the NADC30-like PRRSV pandemic, recombination events involving PRRSV strains have further increased. We isolated two novel field PRRSV recombinant strains, SCABTC-202305 and SCABTC-202309, exhibiting different recombination patterns and compared their pathogenicity in animal experiments. The isolates caused higher viral loads, persistent fever, marked weight loss, moderate respiratory clinical signs, and severe histopathologic lung lesions in piglets. Elucidating correlations between recombinant regions and pathogenicity in these isolates can inform epidemiologic tracking of emerging strains and investigations into viral adaptive mechanisms underlying PRRSV immunity evasion. Our findings underscore the importance of continued genomic surveillance to curb this economically damaging pathogen.

A New Genotype of Trichophyton quinckeanum with Point Mutations in Erg11A Encoding Sterol 14-α Demethylase Exhibits Increased Itraconazole Resistance.

Trichophyton quinckeanum, the causative agent of mouse favus, has been responsible for several infections of animal owners in recent years and showed an infection peak around 2020 in Jena, Thuringia. The isolated T. quinckeanum strains from Thuringia differ in some positions of the ITS region compared to strains from the IHEM collection as well as to Trichophyton schoenleinii. All T. quinckeanum strains of the new genotype show up to a 100-fold increased itraconazole resistance as measured by microplate laser nephelometry (MLN) assays. Analysis of genes involved in Trichophyton indotineae azole resistance, such as Erg1, which encodes squalene epoxidase, and Erg11B, one of two copies of the sterol 14-α demethylase gene, show a 100% identity between the two T. quinckeanum genotypes. In contrast, Erg11A fragments differ in 15-nucleotide positions between both T. quinckeanum genotypes, resulting in the unique amino acid substitution Ala256Ser in resistant strains. The new T. quinckeanum genotype may have evolved through interspecies mating. Mating type analysis showed a nearly 100% identity of the minus type MAT1-1-1 fragment for all T. quinckeanum isolates. The closely related Trichophyton schoenleinii belongs to the plus mating type and has 100% identical fragments of Erg1 and Erg11B. Erg11A protein sequences of T. schoenleinii and T. quinckeanum showed increased diversity.

Sequencing and Partial Molecular Characterization of BAB-TMP, the Babeș Strain of the Fixed Rabies Virus Adapted for Multiplication in Cell Lines.

The rabies virus is a major zoonosis that causes severe nervous disease in humans, leading to paralysis and death. The world's second anti-rabies center was established in 1888 by Victor Babeș, in Bucharest, where an eponymous strain of rabies was isolated and used to develop a method for immunization. The Babeș strain of the rabies virus was used for over 100 years in Romania to produce a rabies vaccine for human use, based on animal nerve tissue, thus having a proven history of prophylactic use. The present study aimed to sequence the whole genome of the Babeș strain and to explore its genetic relationships with other vaccine strains as well as to characterize its relevant molecular traits. After being adapted for multiplication in cell lines and designated BAB-TMP, 99% of the viral genome was sequenced. The overall organization of the genome is similar to that of other rabies vaccine strains. Phylogenetic analysis indicated that the BAB-TMP strain is closely related to the Russian RV-97 vaccine strain, and both seem to have a common ancestor. The nucleoprotein gene of the investigated genome was the most conserved, and the glycoprotein showed several unique amino acid substitutions within the major antigenic sites and linear epitopes.

The dominance of co-circulating SARS-CoV-2 variants in wastewater

SARS-CoV-2 remains one of the biggest global health problems, which has already reached our wastewater through fecal shedding by COVID-19 patients. While the development of vaccines has mitigated the threats of the COVID-19 pandemic, the evolutionary dynamics of SARS-CoV-2 in wastewater lack monitoring and understanding. In this study, SARS-CoV-2 variants in wastewater were identified by analyzing 8511 wastewater-derived genome sequences from 9 countries from March 2020 to May 2023. The dominance of co-circulating variants was observed, namely B.1 in 2020, Alpha and Delta in 2021, then superseded by Omicron lineages in 2022 with a three-times increase. Mutations were also profiled, revealing nearly 5031 unique amino acid substitutions occurring approximately 371,591 times, some of which were associated with enhanced viral transmission and fitness. This study provided the first long-term multi-country overview of the prevalence of co-circulating SARS-CoV-2 lineages and mutations in wastewater and showed its comparison with conventional epidemiological surveillance. The results highlight the ability of wastewater-based genome monitoring to supplement clinical surveillance efforts in rapidly detecting viruses up to the strain level to keep track of their potential transmission routes and evolutionary dynamics.

Immunogenicity and efficacy of vaccine boosters against SARS-CoV-2 Omicron subvariant BA.5 in male Syrian hamsters

The SARS-CoV-2 Omicron subvariant BA.5 rapidly spread worldwide and replaced BA.1/BA.2 in many countries, becoming globally dominant. BA.5 has unique amino acid substitutions in the spike protein that both mediate immune escape from neutralizing antibodies produced by immunizations and increase ACE2 receptor binding affinity. In a comprehensive, long-term (up to 9 months post primary vaccination), experimental vaccination study using male Syrian hamsters, we evaluate neutralizing antibody responses and efficacy against BA.5 challenge after primary vaccination with Ad26.COV2.S (Janssen) or BNT162b2 (Pfizer/BioNTech) followed by a homologous or heterologous booster with mRNA-1273 (Moderna) or NVX-CoV2373 (Novavax). Notably, one high or low dose of Ad26.COV2.S provides more durable immunity than two primary doses of BNT162b2, and the NVX-CoV2373 booster provides the strongest augmentation of immunity, reduction in BA.5 viral replication, and disease. Our data demonstrate the immunogenicity and efficacy of different prime/boost vaccine regimens against BA.5 infection in an immune-competent model and provide new insights regarding COVID-19 vaccine strategies.

Predominance of DS‐1‐like G8P[8] rotavirus reassortant strains in children hospitalized with acute gastroenteritis in Thailand, 2018–2020

AbstractRotavirus A (RVA) is an important cause of acute gastroenteritis (AGE) in children. This study aims to investigate the molecular epidemiology of RVA in children hospitalized with AGE in Chiang Rai, Thailand in 2018–2020 by reverse transcription polymerase chain reaction. Of 302 samples, RVA was detected in 11.6% (35 samples): 11.3% (19/168) in 2018–2019 and 11.9% (16/134) in 2019–2020. G8P[8] was the predominant genotype at 68.4% in 2018–2019 and 81.2% in 2019–2020. G1P[8] (15.8%), G2P[4] (5.3%), G3P[8] (10.5%) in 2018–2019, and G9P[8] (18.8%) in 2019–2020 were also detected. Whole‐genome analysis of G8P[8] revealed a DS‐1‐like genetic backbone: G8‐P[8]‐I2‐R2‐C2‐M2‐A2‐N2‐T2‐E2‐H2. Phylogenetically, the VP7 genes of G8P[8] clustered in a major lineage with previously published 51 DS‐1‐like G8P[8] reference strains, closely related to 13 G8P[8] strains from Thailand and China. These G8P[8] strains contained two unique amino acid substitutions (A125S and N147D) in the VP7 antigenic epitopes. In addition, the VP1 and NSP2 genes of G8P[8] clustered in lineages separated from the DS‐1‐like G8P[8] reference strains with a high genetic divergence but were closely related to G1P[8], G2P[4], G3P[8], or G9P[8]. Several amino acid differences were observed in the VP7 and VP8* antigenic epitopes of G8P[8] compared with RVA vaccine strains. Homology modeling confirmed that these different amino acid residues were located on the surface‐exposed area of the structure. Taken together, the genetic analysis clearly defines the Chiang Rai DS‐1‐like G8P[8] strains as a novel reassortant strain that might have evolved genetically through reassortment events and consequently received their VP1 and NSP2 genes from locally cocirculating‐RVA genotypes.

The rough-toothed dolphin genome provides new insights into the genetic mechanism of its rough teeth.

The rough-toothed dolphin (Steno bredanensis) is characterized by having teeth covered in finely wrinkled vertical ridges, which is a general manifestation of amelogenesis imperfecta. The rough surfaces are hypothesized to be an evolutionary morphological trait of feeding adaptation to increase the dolphin's grip on prey. Here, we assembled a rough-toothed dolphin genome and performed the comparative genomic analysis to reveal the genetic basis of the special enamel. Results showed that genes related to enamel development or dental diseases have undergone diversified adaptive changes that may shape the special enamel morphology of this dolphin species, including positive selection (CLDN19, PRKCE, SSUH2, and WDR72), rapid evolution (LAMB3), or unique amino acid substitutions (AMTN, ENAM, MMP20, and KLK4). Meanwhile, the historical demography of rough-toothed dolphin indicated several distinct population fluctuations associated with climate change. The genome-wide heterozygosity of this dolphin is in the middle of all published data for cetaceans. Although the population is considerable, there may be population or subspecies differentiation, and with the global warming and the increasing disturbance of human activities, we should pay more attention to protection in the future. Together, our study brings new insights into the genetic mechanisms that may have driven the evolution of the special enamel morphology in rough-toothed dolphins and provides the first results of genetic heterozygosity and population historical dynamics of this species, which have important guiding implications for the conservation of this dolphin species.

Meta-Analysis of Microarray Data and Their Utility in Dissecting the Mapped QTLs for Heat Acclimation in Rice.

In the current global warming scenario, it is imperative to develop crops with improved heat tolerance or acclimation, for which knowledge of major heat stress-tolerant genes or genomic regions is a prerequisite. Though several quantitative trait loci (QTLs) for heat tolerance have been mapped in rice, candidate genes from these QTLs have not been reported yet. The meta-analysis of microarray datasets for heat stress in rice can give us a better genomic resource for the dissection of QTLs and the identification of major candidate genes for heat stress tolerance. In the present study, a database, RiceMetaSys-H, comprising 4227 heat stress-responsive genes (HRGs), was created using seven publicly available microarray datasets. This included in-house-generated microarray datasets of Nagina 22 (N22) and IR64 subjected to 8 days of heat stress. The database has provisions for searching the HRGs through genotypes, growth stages, tissues, and physical intervals in the genome, as well as Locus IDs, which provide complete information on the HRGs with their annotations and fold changes, along with the experimental material used for the analysis. The up-regulation of genes involved in hormone biosynthesis and signalling, sugar metabolism, carbon fixation, and the ROS pathway were found to be the key mechanisms of enhanced heat tolerance. Integrating variant and expression analysis, the database was used for the dissection of the major effect of QTLs on chromosomes 4, 5, and 9 from the IR64/N22 mapping population. Out of the 18, 54, and 62 genes in these three QTLs, 5, 15, and 12 genes harboured non-synonymous substitutions. Fifty-seven interacting genes of the selected QTLs were identified by a network analysis of the HRGs in the QTL regions. Variant analysis revealed that the proportion of unique amino acid substitutions (between N22/IR64) in the QTL-specific genes was much higher than the common substitutions, i.e., 2.58:0.88 (2.93-fold), compared to the network genes at a 0.88:0.67 (1.313-fold) ratio. An expression analysis of these 89 genes showed 43 DEGs between IR64/N22. By integrating the expression profiles, allelic variations, and the database, four robust candidates (LOC_Os05g43870, LOC_Os09g27830, LOC_Os09g27650, andLOC_Os09g28000) for enhanced heat stress tolerance were identified. The database thus developed in rice can be used in breeding to combat high-temperature stress.

In SARS-CoV-2 delta variants, Spike-P681R and D950N promote membrane fusion, Spike-P681R enhances spike cleavage, but neither substitution affects pathogenicity in hamsters

The SARS-CoV-2 delta (B.1.617.2 lineage) variant was first identified at the end of 2020 and possessed two unique amino acid substitutions in its spike protein: S-P681R, at the S1/S2 cleavage site, and S-D950N, in the HR1 of the S2 subunit. However, the roles of these substitutions in virus phenotypes have not been fully characterized. We used reverse genetics to generate Wuhan-D614G viruses with these substitutions and delta viruses lacking these substitutions and explored how these changes affected their viral characteristics invitro and invivo. S-P681R enhanced spike cleavage and membrane fusion, whereas S-D950N slightly promoted membrane fusion. Although S-681R reduced the virus replicative ability especially in VeroE6 cells, neither substitution affected virus replication in Calu-3cells and hamsters. The pathogenicity of all recombinant viruses tested in hamsters was slightly but not significantly affected. Our observations suggest that the S-P681R and S-D950N substitutions alone do not increase virus pathogenicity, despite of their enhancement of spike cleavage or fusogenicity. A full list of funding bodies that contributed to this study can be found under Acknowledgments.

Identification, molecular characterization, and pathological features of orf virus in sheep and goats in Punjab province, Pakistan

Orf virus (ORFV) causes an acute, contagious, skin disease of sheep and goats which is economically important. The objectives of this study were to identify ORFV and to explore its pathological and phylogenetic profiles in 350 goats and 91 sheep of 14 districts of Punjab, Pakistan, from July 2020 to July 2021. Skin scrapings (total no. of samples = 441) of suspected animals were subjected to polymerase chain reactions, phylogenetic analysis, and pathological observations. The partial length of GIF/IL-2 gene (408bp) was successfully amplified in 58/441 samples. Phylogenetic analysis of GIF/IL2 gene showed that the study isolates belonged to ORFV-cluster I, together with the viruses reported in India and China. Pakistan ORFV isolates were shared 97.6-98.7% nucleotide and 97.6-100% amino acid identities with the reference strain (NC_005336). Moreover, Chinese ORFV-isolates were detected unique multiple amino acid substitutions (F11L, Q21H, D27N, I46V, N49S, N82D, D103N, S129G) with study isolates. Naturally infected animals were anorexic, emaciated, dull, and depressed. The macroscopic lesions included multifocal to coalescing, ulceration followed by proliferative papules, pustules, and crust formation on the epidermis of gums, lips, mouth commissure, muzzles, nose, and udder. Histopathological examination revealed hyperplasia, anastomosing rete ridges formation and degenerative changes, including spongiosis and vacuolation of epidermal cells. Keratinocytes exhibited eosinophilic intracytoplasmic inclusion bodies with pyknotic and karyorrhexis nuclei. This is the first report on molecular characterization of ORFV from Pakistan, with insight into its pathogenesis and comparative analysis of pathological alterations and genetic diversity between ORFV strains reported in different geographical areas.

A Single Amino Acid Substitution in Porcine Reproductive and Respiratory Syndrome Virus Glycoprotein 2 Significantly Impairs Its Infectivity in Macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a restricted tropism for macrophages and CD163 is a key receptor for infection. In this study, the PRRSV strain NCV1 was passaged on MARC-145 cells for 95 passages, and two plaque-clones (C1 and C2) were randomly selected for further analysis. The C1 virus nearly lost the ability to infect porcine alveolar macrophages (PAMs), as well as porcine kidney cells expressing porcine CD163 (PK15-pCD163), while the C2 virus replicates well in these two cell types. Pretreatment of MARC-145 cells with an anti-CD163 antibody nearly blocked C1 virus infection, indicating that the virus still required CD163 to infect cells. The C1 virus carried four unique amino acid substitutions: three in the nonstructural proteins and a K160I in GP2. The introduction of an I160K substitution in GP2 of the C1 virus restored its infectivity in PAMs and PK15-pCD163 cells, while the introduction of a K160I substitution in GP2 of the low-passaged, virulent PRRSV strain NCV13 significantly impaired its infectivity. Importantly, pigs inoculated with the rNCV13-K160I mutant exhibited lower viremia levels and lung lesions than those infected with the parental rNCV13. These results demonstrated that the K160 residue in GP2 is one of the key determinants of PRRSV tropism.

First detection and molecular characteristics of bopivirus from goats in China.

A metavirome analysis was performed and detected bopivirus in the diarrhoeal fecal samples of goats in China. A total of 136 fecal samples were collected from yeanlings between the dates of June 2021 and January 2022 in Sichuan province, China. Moreover, "Bopivirus B" strains were detected by a specific RT-PCR targeting the 3D gene of the virus. The results showed that the overall detection rate of "Bopivirus B" was 19.12% (26/136). Additionally, there was a higher detection rate (24.05%, 19/79) in the fecal samples collected from yeanlings with diarrhea compared to those from asymptomatic animals (12.28%, 7/57). In these samples, no other common diarrhea-causing pathogens were detected except for three enteric viruses, namely caprine enterovirus, caprine kobuvirus and caprine hunnivirus (with detection rates of 13.97, 13.97, and 8.82%, respectively). Subsequently, full-length VP4, VP2, VP3, and VP1 genes from "Bopivirus B"-positive samples were amplified, cloned, sequenced, and analyzed. The phylogenetic analysis performed on the VP1 genes revealed that the identified bopivirus belonged to genotype B1 (seven strains) and B2 (three strains) and presented a high genetic diversity. Furthermore, a complete genome sequence of a "Bopivirus B" strain (SWUN/B1/2022) was obtained using PCR from fecal sample of a diarrhoeal yeanling. The complete genome was 7,309 nucleotides in length with a standard picornavirus genome organization, and shares 93.10% and 91.10% nucleotide similarity with bopivirus B1 genotype strain ovine/TB14/2010-HUN and bopivirus B2 genotype strain goat/AGK16/2020-HUN, respectively. According to the species classification criteria put forward by the International Committee on Taxonomy of Viruses and VP1 genotype, the strain SWUN/B1/2022 belongs to the bopivirus B1. This strain has unique amino acid substitutions in the VP4, VP2, VP3, and VP1 genes. Moreover, genomic recombination analysis revealed that this strain may be a minor parental strain of bopivirus B1 ovine/TB14/2010-HUN. Evolutionary analysis based on the 2C and 3CD genes revealed that the new bopivirus B1 strain SWUN/B1/2022 presents a unique evolutionary pattern. This study provided evidence to suggest that "Bopivirus B" is circulating with substantial genetic diversity in goats in China at present, and the mixed infection of "Bopivirus B" with other enteric viruses should be considered to be a composite factor in the occurrence of viral diarrhea in goats.

Notch Missense Mutations in Drosophila Reveal Functions of Specific EGF-like Repeats in Notch Folding, Trafficking, and Signaling.

Notch signaling plays various roles in cell-fate specification through direct cell-cell interactions. Notch receptors are evolutionarily conserved transmembrane proteins with multiple epidermal growth factor (EGF)-like repeats. Drosophila Notch has 36 EGF-like repeats, and while some play a role in Notch signaling, the specific functions of most remain unclear. To investigate the role of each EGF-like repeat, we used 19 previously identified missense mutations of Notch with unique amino acid substitutions in various EGF-like repeats and a transmembrane domain; 17 of these were identified through a single genetic screen. We assessed these mutants' phenotypes in the nervous system and hindgut during embryogenesis, and found that 10 of the 19 Notch mutants had defects in both lateral inhibition and inductive Notch signaling, showing context dependency. Of these 10 mutants, six accumulated Notch in the endoplasmic reticulum (ER), and these six were located in EGF-like repeats 8-10 or 25. Mutations with cysteine substitutions were not always coupled with ER accumulation. This suggests that certain EGF-like repeats may be particularly susceptible to structural perturbation, resulting in a misfolded and inactive Notch product that accumulates in the ER. Thus, we propose that these EGF-like repeats may be integral to Notch folding.

Molecular surveillance of equine parvovirus-hepatitis from oral, nasal, vaginal, and semen specimens collected from horses living in Korea.

Equine parvovirus-hepatitis (EqPV-H) is one of the etiological agents of Theiler's disease, causing fulminant hepatitis; however, its transmission route and pathogenesis remain unclear. In the present study, we aimed to determine EqPV-H shedding in oral/nasal/vaginal swabs or semen samples from horses living in Korea using nested polymerase chain reaction. We then used the data obtained to investigate various risk factors associated with EqPV-H including viral shedding, hepatopathological changes, and genetic diversity. Our data revealed occurrence of EqPV-H shedding in these animals (oral: 3/102 [2.9%]; nasal: 3/102 [2.9%]; semen: 1/9 [11.1%]) and identified that both age and country of foaling were significantly associated with EqPV-H shedding (p<.05). In addition, we noted that one of the newly isolated strains clustered separately from the other strains in the phylogenetic tree, revealing unique nucleotide and amino acid substitutions. This is a field surveillance study providing evidence of natural and venereal shedding of EqPV-H and describing its presence in both oral/nasal fluids and semen. This epidemiological and clinical analysis may help specify the clinicopathological features of EqPV-H and facilitate the development of novel disease prevention strategies.

Long Term Norovirus Infection in a Patient with Severe Common Variable Immunodeficiency.

Norovirus is the most common cause of acute non-bacterial gastroenteritis. Immunocompromised patients can become chronically infected, with or without symptoms. In Europe, common variable immunodeficiency (CVID) is one of the most common inborn errors of immunity. A potentially severe complication is CVID-associated enteropathy, a disorder with similar histopathology to celiac disease. Studies suggest that chronic norovirus infection may be a contributor to CVID enteropathy, and that the antiviral drug ribavirin can be effective against norovirus. Here, a patient with CVID-like disease with combined B- and T-cell deficiency, had chronic norovirus infection and enteropathy. The patient was routinely administered subcutaneous and intravenous immunoglobulin replacement therapy (SCIg and IVIg). The patient was also administered ribavirin for ~7.5 months to clear the infection. Stool samples (collected 2013–2016) and archived paraffin embedded duodenal biopsies were screened for norovirus by qPCR, confirming a chronic infection. Norovirus genotyping was done in 25 stool samples. For evolutionary analysis, the capsid (VP1) and polymerase (RdRp) genes were sequenced in 10 and 12 stool samples, respectively, collected before, during, and after ribavirin treatment. Secretor phenotyping was done in saliva, and serum was analyzed for histo-blood group antigen (HBGA) blocking titers. The chronic norovirus strain formed a unique variant subcluster, with GII.4 Den Haag [P4] variant, circulating around 2009, as the most recent common ancestor. This corresponded to the documented debut of symptoms. The patient was a secretor and had HBGA blocking titers associated with protection in immunocompetent individuals. Several unique amino acid substitutions were detected in immunodominant epitopes of VP1. However, HBGA binding sites were conserved. Ribavirin failed in treating the infection and no clear association between ribavirin-levels and quantity of norovirus shedding was observed. In conclusion, long term infection with norovirus in a patient with severe CVID led to the evolution of a unique norovirus strain with amino acid substitutions in immunodominant epitopes, but conservation within HBGA binding pockets. Regularly administered SCIg, IVIg, and ~7.5-month ribavirin treatment failed to clear the infection.

Single-Nucleotide Polymorphisms in Bmy1 Intron III Alleles Conferring the Genotypic Variations in β-Amylase Activity under Drought Stress between Tibetan Wild and Cultivated Barley

β-amylase activity is related to the polymorphisms of Bmy1 intron III; however, no attention has been given to such relationships under environmental stresses such as drought. In this study, 73 cultivated barley genotypes and 52 Tibetan wild barley accessions were used to test the association between Bmy1 gene intron III polymorphisms and β-amylase activity under drought stress. Our results showed that three alleles, Bmy1.a, Bmy1.b, and Bmy1.c, existed in the examined barley genotypes. Tibetan wild barley had a higher proportion of Bmy1.b, whereas cultivated barley showed a higher proportion of Bmy1.a. Impressively, barley genotypes with Bmy1.b showed a significant increase in β-amylase activity under drought stress, compared with those with Bmy1.a or Bmy1.c, indicating that the Bmy1.b allele might provide more chances for developing barley cultivars with higher β-amylase activity under water stress than both the Bmy1.a and Bmy1.c alleles. Furthermore, the Tibetan wild barley XZ147, belonging to the Bmy1.b allele type, showed significantly higher β-amylase activity than Triumph under drought stress. This might be the result of the unique amino acid substitution M527 or the amino acid composition of R115, D165, A233, S347, and M527 of XZ147.

Evidence for an Independent Hydrogenosome-to-Mitosome Transition in the CL3 Lineage of Fornicates

Fornicata, a lineage of a broader and ancient anaerobic eukaryotic clade Metamonada, contains diverse taxa that are ideally suited for evolutionary studies addressing various fundamental biological questions, such as the evolutionary trajectory of mitochondrion-related organelles (MROs), the transition between free-living and endobiotic lifestyles, and the derivation of alternative genetic codes. To this end, we conducted detailed microscopic and transcriptome analyses in a poorly documented strain of an anaerobic free-living marine flagellate, PCS, in the so-called CL3 fornicate lineage. Fortuitously, we discovered that the original culture contained two morphologically similar and closely related CL3 representatives, which doubles the taxon representation within this lineage. We obtained a monoeukaryotic culture of one of them and formally describe it as a new member of the family Caviomonadidae, Euthynema mutabile gen. et sp. nov. In contrast to previously studied caviomonads, the endobiotic Caviomonas mobilis and Iotanema spirale, E. mutabile possesses an ultrastructurally discernible MRO. We sequenced and assembled the transcriptome of E. mutabile, and by sequence subtraction, obtained transcriptome data from the other CL3 clade representative present in the original PCS culture, denoted PCS-ghost. Transcriptome analyses showed that the reassignment of only one of the UAR stop codons to encode Gln previously reported from I. spirale does not extend to its free-living relatives and is likely due to a unique amino acid substitution in I. spirale’s eRF1 protein domain responsible for termination codon recognition. The backbone fornicate phylogeny was robustly resolved in a phylogenomic analysis, with the CL3 clade amongst the earliest branching lineages. Metabolic and MRO functional reconstructions of CL3 clade members revealed that all three, including I. spirale, encode homologs of key components of the mitochondrial protein import apparatus and the ISC pathway, indicating the presence of a MRO in all of them. In silico evidence indicates that the organelles of E. mutabile and PCS-ghost host ATP and H2 production, unlike the cryptic MRO of I. spirale. These data suggest that the CL3 clade has experienced a hydrogenosome-to-mitosome transition independent from that previously documented for the lineage leading to Giardia.

Insights into the Evolution of Non‐shivering Thermogenesis from UCP1 of the Naked Mole Rat (Heterocephalus glaber)

Recognized as the primary location of non‐shivering thermogenesis (NST) in eutherian mammals, brown adipose tissue (BAT) functions to accelerate rewarming in hibernators and defend body temperature among newborns and small‐bodied species enduring cold stress. Uncoupling protein 1 (UCP1) is highly expressed in BAT and underlies the molecular mechanism of NST. UCP1 resides in the inner mitochondrial membrane where, upon functional induction by free fatty acids, it catalyzes mitochondrial proton leak, collapsing the proton gradient that stores oxidation energy. The ensuing futile cycling of protons increases substrate oxidation and thus heat production. Central questions remain regarding the exact mechanism by which UCP1 facilitates proton leak, stemming from the lack of a crystal structure of the protein. It is also unknown whether UCP1 function has been shaped by evolution to meet differing thermoregulatory demands of various lineages. We adopt a comparative approach, targeting species with extreme ecophysiological characteristics expected to culminate in drastic differences in thermoregulatory demands and thus UCP1 function, to provide insights into protein structure‐function relationships. For instance, the naked mole rat (NMR; Heterocephalus glaber) is well‐suited to survive in hypoxic burrows that exhibit high year‐round temperatures (30‐34°C). Hinting at a reduced NST capacity, NMRs also display poikilothermy, failing to maintain constant body temperatures when artificially cold challenged. Moreover, NMR UCP1 displays several unique amino acid substitutions, one of which, H145Q (histidine to glutamine), occurs at the well‐known “histidine pair motif”. This motif, typically comprised of histidines at positions 145 and 147, has been hypothesized to be a critical final proton acceptor/donor site shuttling protons into the mitochondrial matrix. The exact mutation that occurs in the NMR, H145Q, was previously shown to diminish hamster UCP1‐mediated proton conductance in proteoliposomes by 90%, calling the functional performance of NMR UCP1 into question. We functionally tested wildtype NMR UCP1 as well as histidine pair mutant combinations at positions 145 and 147, not in the highly artificial system of proteoliposomes, which may promote artefactual protein behaviour, but instead in the more native mammalian environment of HEK293 cells. Using plate‐based respirometry techniques, we demonstrate that wildtype NMR UCP1 uncouples, thus NMRs retain this mechanism of NST. While we hypothesized that reversion of the H145Q mutation would increase UCP1‐mediated proton leak, surprisingly both variants function similarly when activated with free fatty acids and inhibited with guanosine diphosphate (GDP). Likewise, mutation of the histidine at position 147, the other half of the “histidine pair motif”, does not drastically alter UCP1 function. Overall, these data suggest that the “histidine pair motif” may not be crucial for protein function, contrary to previous claims. Comparative functional assessments of UCP1 variants provide a deeper understanding of structure‐function relationships of this enigmatic protein and the evolution of mammalian NST in response to ecophysiological constraints.

Investigating the potential binding partners of H2-O to further elucidate its role in MHCII antigen presentation

Abstract The production of neutralizing antibodies in response to viral pathogens requires CD4+ T cell help. For this to occur, pathogen-derived peptides must be loaded onto class II major histocompatibility complex (MHC II) molecules in endosomal/lysosomal compartments and trafficked to the surface of antigen presenting cells. The αβ heterodimeric nonclassical MHCII molecules, H2-M and H2-O, modulate peptide loading. While H2-M directly catalyzes MHCII peptide loading, H2-O is a negative regulator of MHCII antigen presentation. Current dogma supports that H2-O is an MHCII structural mimic that exerts control over peptide loading via direct competition with MHCII for H2-M-binding. Work done in our labs, however, suggests this current model is incomplete and suggests the involvement of other, yet undefined, factors. I/LnJ mice, which are resistant to retroviruses through the production of potent neutralizing antibodies, inherit an H2-Ob allele that encodes nonfunctional H2-O. I/LnJ H2-O binds to but does not inhibit H2-M, directly challenging current dogma. Compared with H2-Oβ from retrovirus-susceptible mice, I/LnJ H2-Oβ contains three unique amino acid (AA) substitutions in the conserved immunoglobulin domain which we have found to be critical for the H2-O inhibitory activity of H2-M. We hypothesize that altered H2-O function in I/LnJ mice is due to an inability to interact with essential binding partners. We recently generated B6J mice in which we have knocked-in these three AA substitutions. This novel tool will be exploited to discover potential binding partners of variant H2-Oβ and elucidate the mechanism by which H2-O activity is modulated.