Consensus insertion/deletions and amino acid variations of all coding and noncoding regions of the SARS-CoV-2 Omicron clades, including the XBB and BQ.1 lineages.

The currently dominant Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has swiftly diverged into clades. To predict the probable impact of these clades, the consensus insertions/deletions (indels) and amino acid substitutions of the whole genome of clades were compared with the original SARS-CoV-2 strain. The evolutionary history of representatives of clades and lineages was inferred using the maximum-likelihood method and tested using the bootstrap method. The indels and polymorphic amino acids were found to be either clade-specific or shared among clades. The 21K clade has unique indels and substitutions, which probably represent reverted indels/substitutions. Three variations that appear to be associated with SARS-CoV-2 attenuation in the Omicron clades included a deletion in the nucleocapsid gene, a deletion in the 3'untranslated region, and a truncation in open reading frame 8. Phylogenetic analysis showed that the Omicron clades and lineages form three separate clusters.

Whole-Genome Comparison of Representatives of All Variants of SARS-CoV-2, Including Subvariant BA.2 and the GKA Clade.

Since its discovery at the end of 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly evolved into many variants, including the subvariant BA.2 and the GKA clade. Genomic clarification is needed for better management of the current pandemic as well as the possible reemergence of novel variants. The sequence of the reference genome Wuhan-Hu-1 and approximately 20 representatives of each variant were downloaded from GenBank and GISAID. Two representatives with no track of in-definitive nucleotides were selected. The sequences were aligned using muscle. The location of insertion/deletion (indel) in the genome was mapped following the open reading frame (ORF) of Wuhan-Hu-1. The phylogeny of the spike protein coding region was constructed using the maximum likelihood method. Amino acid substitutions in all ORFs were analyzed separately. There are two indel sites in ORF1AB, eight in spike, and one each in ORF3A, matrix (MA), nucleoprotein (NP), and the 3'-untranslated regions (3'UTR). Some indel sites and residues/substitutions are not unique, and some are variant-specific. The phylogeny shows that Omicron, Deltacron, and BA2 are clustered together and separated from other variants with 100% bootstrap support. In conclusion, whole-genome comparison of representatives of all variants revealed indel patterns that are specific to SARS-CoV-2 variants or subvariants. Polymorphic amino acid comparison across all coding regions also showed amino acid residues shared by specific groups of variants. Finally, the higher transmissibility of BA.2 might be due at least in part to the 48 nucleotide deletions in the 3'UTR, while the seem-to-be extinction of GKA clade is due to the lack of genetic advantages as a consequence of amino acid substitutions in various genes.

Selecting Potential Neuronal Drug Leads from Conotoxins of Various Venomous Marine Cone Snails in Bali, Indonesia.

Many conotoxins, natural peptides of marine cone snails, have been identified to target neurons. Here, we provide data on pharmacological families of the conotoxins of 11 species of cone snails collected in Bali. The identified definitive pharmacological families possibly targeting neuronal tissues were α (alpha), ι (iota), κ (kappa), and ρ (rho). These classes shall target nicotinic acetylcholine receptors, voltage-gated Na channels, voltage-gated K channels, and α1-adrenoceptors, respectively. The VI/VII-O3 conotoxins might be prospected as an inhibitor of N-methyl-d-aspartate. Con-ikot-ikot could be applied as an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor blocker medicine. The definitive pharmacology classes of conotoxins as well as those yet to be elucidated need to be further established and verified.

Lack of Signal for the Impact of Conotoxin Gene Diversity on Speciation Rates in Cone Snails.

Understanding why some groups of organisms are more diverse than others is a central goal in macroevolution. Evolvability, or the intrinsic capacity of lineages for evolutionary change, is thought to influence disparities in species diversity across taxa. Over macroevolutionary time scales, clades that exhibit high evolvability are expected to have higher speciation rates. Cone snails (family: Conidae, $>$900 spp.) provide a unique opportunity to test this prediction because their toxin genes can be used to characterize differences in evolvability between clades. Cone snails are carnivorous, use prey-specific venom (conotoxins) to capture prey, and the genes that encode venom are known and diversify through gene duplication. Theory predicts that higher gene diversity confers a greater potential to generate novel phenotypes for specialization and adaptation. Therefore, if conotoxin gene diversity gives rise to varying levels of evolvability, conotoxin gene diversity should be coupled with macroevolutionary speciation rates. We applied exon capture techniques to recover phylogenetic markers and conotoxin loci across 314 species, the largest venom discovery effort in a single study. We paired a reconstructed timetree using 12 fossil calibrations with species-specific estimates of conotoxin gene diversity and used trait-dependent diversification methods to test the impact of evolvability on diversification patterns. Surprisingly, we did not detect any signal for the relationship between conotoxin gene diversity and speciation rates, suggesting that venom evolution may not be the rate-limiting factor controlling diversification dynamics in Conidae. Comparative analyses showed some signal for the impact of diet and larval dispersal strategy on diversification patterns, though detection of a signal depended on the dataset and the method. If our results remain true with increased taxonomic sampling in future studies, they suggest that the rapid evolution of conid venom may cause other factors to become more critical to diversification, such as ecological opportunity or traits that promote isolation among lineages.

Targeted Sequencing of Venom Genes from Cone Snail Genomes Improves Understanding of Conotoxin Molecular Evolution.

To expand our capacity to discover venom sequences from the genomes of venomous organisms, we applied targeted sequencing techniques to selectively recover venom gene superfamilies and nontoxin loci from the genomes of 32 cone snail species (family, Conidae), a diverse group of marine gastropods that capture their prey using a cocktail of neurotoxic peptides (conotoxins). We were able to successfully recover conotoxin gene superfamilies across all species with high confidence (> 100× coverage) and used these data to provide new insights into conotoxin evolution. First, we found that conotoxin gene superfamilies are composed of one to six exons and are typically short in length (mean = ∼85 bp). Second, we expanded our understanding of the following genetic features of conotoxin evolution: 1) positive selection, where exons coding the mature toxin region were often three times more divergent than their adjacent noncoding regions, 2) expression regulation, with comparisons to transcriptome data showing that cone snails only express a fraction of the genes available in their genome (24-63%), and 3) extensive gene turnover, where Conidae species varied from 120 to 859 conotoxin gene copies. Finally, using comparative phylogenetic methods, we found that while diet specificity did not predict patterns of conotoxin evolution, dietary breadth was positively correlated with total conotoxin gene diversity. Overall, the targeted sequencing technique demonstrated here has the potential to radically increase the pace at which venom gene families are sequenced and studied, reshaping our ability to understand the impact of genetic changes on ecologically relevant phenotypes and subsequent diversification.

Dietary breadth is positively correlated with venom complexity in cone snails.

BACKGROUND: Although diet is believed to be a major factor underlying the evolution of venom, few comparative studies examine both venom composition and diet across a radiation of venomous species. Cone snails within the family, Conidae, comprise more than 700 species of carnivorous marine snails that capture their prey by using a cocktail of venomous neurotoxins (conotoxins or conopeptides). Venom composition across species has been previously hypothesized to be shaped by (a) prey taxonomic class (i.e., worms, molluscs, or fish) and (b) dietary breadth. We tested these hypotheses under a comparative phylogenetic framework using ecological data from past studies in conjunction with venom duct transcriptomes sequenced from 12 phylogenetically disparate cone snail species, including 10 vermivores (worm-eating), one molluscivore, and one generalist. RESULTS: We discovered 2223 unique conotoxin precursor peptides that encoded 1864 unique mature toxins across all species, >90 % of which are new to this study. In addition, we identified two novel gene superfamilies and 16 novel cysteine frameworks. Each species exhibited unique venom profiles, with venom composition and expression patterns among species dominated by a restricted set of gene superfamilies and mature toxins. In contrast with the dominant paradigm for interpreting Conidae venom evolution, prey taxonomic class did not predict venom composition patterns among species. We also found a significant positive relationship between dietary breadth and measures of conotoxin complexity. CONCLUSIONS: The poor performance of prey taxonomic class in predicting venom components suggests that cone snails have either evolved species-specific expression patterns likely as a consequence of the rapid evolution of conotoxin genes, or that traditional means of categorizing prey type (i.e., worms, mollusc, or fish) and conotoxins (i.e., by gene superfamily) do not accurately encapsulate evolutionary dynamics between diet and venom composition. We also show that species with more generalized diets tend to have more complex venoms and utilize a greater number of venom genes for prey capture. Whether this increased gene diversity confers an increased capacity for evolutionary change remains to be tested. Overall, our results corroborate the key role of diet in influencing patterns of venom evolution in cone snails and other venomous radiations.

Molecular analysis of hemagglutinin-1 fragment of avian influenza H5N1 viruses isolated from chicken farms in Indonesia from 2008 to 2010.

Highly pathogenic avian influenza virus of subtype H5N1 (AIV-H5N1) has been circulating in Indonesia since 2003. To understand the genetic diversity of these viruses, and to predict vaccine efficacy, the hemaglutinin-1 (HA-1) fragment of viruses isolated from chicken farms in Indonesia from 2008 to 2010 was sequenced and analyzed. The effects of these molecular changes were investigated in challenge experiments and HI assays of homologous and heterologous strains. Molecular analysis showed that these AIV-H5N1 isolates had evolved into three distinct sub-lineages from an ancestor circulating since 2003. Although no significant positive selection of residues was detected, 12 negatively selected sites were identified (p<0.05). Moreover, four sites showed evidence of significant episodic diversifying selection. The findings indicated complete protectivity and high HI titers with homologous strains, compared with protectivity ranging from 40 to 100% and lower HI titers with heterologous strains resulting from polymorphisms at antigenic sites. Our findings provide valuable insight into the molecular evolution of AIV and have important implications for vaccine efficacy and future vaccination strategies.

An investigation of classical swine fever virus seroprevalence and risk factors in pigs in East Nusa Tenggara, eastern Indonesia.

Classical swine fever virus (CSFV) is a highly infectious disease of pigs. It has had significant impacts on East Nusa Tenggara, eastern Indonesia since its introduction in 1997. In spite of its importance to this region, little is known about its seroprevalence and distribution, and pig-level and farmer-level factors that may have an impact on the serological status of an individual pig. To address this knowledge deficit, a cross-sectional seroprevalence survey was conducted in 2010 involving 2160 pigs and 805 farmers from four islands in the region. Farmer questionnaires and pig record forms were used to collect data about the farmers and pigs surveyed. Blood was collected from each pig to determine its CSFV serological status. Apparent and true prevalence were calculated for each island, district, subdistrict, and village surveyed. CSFV serological status was used as an outcome variable in mixed effects logistic regression analyses. Overall true CSFV seroprevalence was estimated at 17.5% (lower CI 16.0%; upper CI 19.5%). Seroprevalence estimates varied widely across the islands, districts, subdistricts, and villages. Manggarai Barat, a district on the western end of Flores Island, contained pigs that were positive for antibody to CSFV. This result was unexpected, as no clinical cases had been reported in this area. Older pigs and pigs that had been vaccinated for CSFV were more likely to test positive for antibody to CSFV. The final multivariable model accounted for a large amount of variation in the data, however much of this variation was explained by the random effects with less than 2% of the variation explained by pig age and pig CSFV vaccination status. In this study we documented the seroprevalence of CSFV across four islands in East Nusa Tenggara, eastern Indonesia. We also identified risk factors for the presence of antibody to CSFV. Further investigation is needed to understand why clinical CSFV has not been reported on the western end of Flores Island, and to identify additional risk factors that explain CSFV serological status to inform disease control strategies.

Epidemiological and clinical features of human rabies cases in Bali 2008-2010.

BACKGROUND: Previously thought to be rabies free, Bali experienced an outbreak of animal and human rabies cases in November 2008. We describe the epidemiological and clinical data of human rabies cases occurring in the first two years of the outbreak. METHODS: We analysed the patient records of all rabies cases from the Sanglah General Hospital in Denpasar, and district hospitals in Buleleng and Tabanan. A conventional reverse transcriptase polymerase chain reaction was developed to detect the rabies virus genome in saliva, corneal swabs, and ante- and post-mortem cerebrospinal fluid (CSF). RESULTS: There were 104 human rabies cases in Bali during November 2008-November 2010. Patients' mean age was 36.6 years (range 3-84 years; SD 20.7), most were male (56.7%), and originated from rural districts. Almost all (92%) cases had a history of dog bite. Only 5.8% had their wounds treated and received an anti-rabies vaccine (ARV) after the bite incident. No patients received rabies immunoglobulin (RIG). The estimated time from dog bite to the onset of signs and symptoms was 110.4 days (range 12-720 days; SD 118.2). The mean length of medical care until death was 21.8 hours (range 1-220 hours; SD 32.6). Less than 50% of patients had prodromal symptoms. The most frequent prodromal symptom was pain or paraesthesia at the bite site (37.6%). The two most common central nervous system infection signs were agitation (89.2%) and confusion (83.3%). Signs of autonomic nervous system dysfunction included hydrophobia (93.1%), hypersalivation (88.2%), and dyspnea (74.4%). On admission, 22 of 102 patients (21.6%) showed paralytic manifestations, while the rest (78.4%) showed furious rabies manifestations. The case-fatality rate was 100%. The rabies virus genome was detected in 50 of 101 patients (49.5%) with the highest detection rate from post-mortem CSF samples. CONCLUSIONS: Rabies is a major public health problem in Bali. Human fatalities occur because of a lack of knowledge regarding rabies risk, the poor management of dog bites, and the limited availability of RIG. Increasing public awareness of dog bite management, increasing the availability of ARV and RIG, and implementing an island-wide dog vaccination campaign will help prevent human rabies cases.