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Avian-to-human transmission of highly pathogenic avian influenza viruses (HPAIV) and their subsequent adaptation to humans are of great concern to public health. Surveillance and early warning of AIVs with the potential to infect humans and pandemic potential is crucial. In this study, we determined whether adaptive evolution occurred in human-isolated H5 viruses. We evaluated all available genomes of H5N1 and H5N6 avian influenza A virus. Firstly, we systematically identified several new mutations in H5 AIV that might be associated with human adaptation using a combination of novel comparative phylogenetic methods and structural analysis. Some changes are the result of parallel evolution, further demonstrating their importance. In total, we identified 102 adaptive evolution sites in eight genes. Some residues had been previously identified, such as 227 in HA and 627 in PB2, while others have not been reported so far. Ten sites from four genes evolved in parallel but no obvious positive selection was detected. Our study suggests that during infection of humans, H5 viruses evolved to adapt to their new host environment and that the sites of adaptive/parallel evolution might play a role in crossing the species barrier and are the response to new selection pressure. The results provide insight to implement early detection systems for transitional stages in H5 AIV evolution before its potential adaptation for humans. Author summary line The prerequisite of surveillance and early warning of avian influenza viruses with the potential to infect humans depends on the identification of human-adaptation related mutations. In this study, we used a novel approach combining both phylogenetic and structural analysis to identify possible human-adaptation related mutations in H5 AIVs. Previous studies reported human-adaptation related mutations and some novel mutations exhibiting parallel evolution. Our result provides new insights into how AIVs adapt to humans by point mutations.

Background Pseudorabies virus (PRV) causes Aujeszky's disease in pigs and can be transmitted to other mammals, including humans. In the current study, we systematically studied the interspecies transmission and evolutionary history of PRV. Methods We performed comprehensive analysis on the phylodynamics, selection, and structural biology to summarize the phylogenetic and adaptive evolution of PRV based on all available full-length and major glycoprotein sequences. Results PRV can be divided into 2 main clades with frequent interclade and intraclade recombination. Clade 2.2 (variant PRV) is currently the most prevalent genotype worldwide, and most commonly involved in cross-species transmission events (including humans). We also found that the population size of clade 2.2 has increased since 2011, and the effective reproduction number was >1 from 2011 to 2016, indicating that PRV may be still circulating in swine herds and is still a risk in relation with cross-species transmission in China. Of note, we identified amino acid sites in some important glycoproteins gB, gC, gD, and gE that may be associated with PRV adaptation to new hosts and immune escape to vaccines. Conclusions Our study provides important genetic insight into the interspecies transmission and evolution of PRV within and between different hosts that warrant additional surveillance.

Feline calicivirus (FCV) is a highly prevalent pathogen of the domestic cat that causes acute infections of the oral and upper respiratory tract. The E region of the ORF2 protein is responsible for the induction of virus-neutralizing antibodies, thus it is important to understand the codon usage of this gene. Here, analysed 90 coding sequences of ORF2 and show that it undergoes a low codon usage bias. In addition, although mutational bias is one of the factors shaping the codon usage bias of this gene, natural selection plays a more significant role. Our results reveal part of the mechanisms driving FCV evolution, which will lay foundation for the further research of FCV., Highlights • The codon usage of the ORF2 gene of FCV was first analysed in this study. • The ORF2 gene of FCV shows a low codon usage bias.

Prior to the emergence of H3N8 canine influenza virus (CIV) and the latest avian-origin H3N2 CIV, there was no evidence of a circulating canine-specific influenza virus. Molecular and epidemiological evidence suggest that H3N8 CIV emerged from H3N8 equine influenza virus (EIV). This host-range shift of EIV from equine to canine hosts and its subsequent establishment as an enzootic CIV is unique because this host-range shift was from one mammalian host to another. To further understand this host-range shift, we conducted a comprehensive phylodynamic analysis using all the available whole-genome sequences of H3N8 CIV. We found that (1) the emergence of H3N8 CIV from H3N8 EIV occurred in approximately 2002; (2) this interspecies transmission was by a reassortant virus of the circulating Florida-1 clade H3N8 EIV; (3) once in the canine species, H3N8 CIV spread efficiently and remained an enzootic virus; (4) H3N8 CIV evolved and diverged into multiple clades or sublineages, with intra and inter-lineage reassortment. Our results provide a framework to understand the molecular basis of host-range shifts of influenza viruses and that dogs are potential “mixing vessels” for the establishment of novel influenza viruses.

Artificial intelligence (AI)-based methods have emerged as powerful tools to transform medical care. Although machine learning classifiers (MLCs) have already demonstrated strong performance in image-based diagnoses, analysis of diverse and massive electronic health record (EHR) data remains challenging. Here, we show that MLCs can query EHRs in a manner similar to the hypothetico-deductive reasoning used by physicians and unearth associations that previous statistical methods have not found. Our model applies an automated natural language processing system using deep learning techniques to extract clinically relevant information from EHRs. In total, 101.6 million data points from 1,362,559 pediatric patient visits presenting to a major referral center were analyzed to train and validate the framework. Our model demonstrates high diagnostic accuracy across multiple organ systems and is comparable to experienced pediatricians in diagnosing common childhood diseases. Our study provides a proof of concept for implementing an AI-based system as a means to aid physicians in tackling large amounts of data, augmenting diagnostic evaluations, and to provide clinical decision support in cases of diagnostic uncertainty or complexity. Although this impact may be most evident in areas where healthcare providers are in relative shortage, the benefits of such an AI system are likely to be universal.

The H3N2 canine influenza virus (CIV) originated from an avian species. Since its emergence, it has circulated in multiple states and has caused pandemics among dog populations; however, no comprehensive studies have explored the causes driving these ongoing cases. The study of the codon usage patterns of viruses can reveal the genetic changes required for the viruses to adapt to new hosts and the external environment. Here we performed a thorough genetic, evolutionary, and codon usage analysis. We identified three evolutionary H3N2 CIV clades from a timescaled phylogenetic tree, namely, Origin, China, and Korea/USA, by principal component analysis (PCA). Additionally, we found a low codon usage bias and that mutation pressure, natural selection, and dinucleotide abundance shape the codon usage bias of H3N2 CIVs, with natural selection being more crucial than the others. Moreover, the human codon adaptation index was similar to that of dogs (the natural host) and cats. In addition, the H3N2 CIV similarity index values were higher than those of the avian influenza virus (AIV), suggesting viral adaptation to the host. Therefore, H3N2 CIVs may pose a potential risk to public health in the future, and further epidemiologic, evolutionary, and pathogenetic studies are required.

Since its emergence, rabies virus (RABV) has been a major worldwide concern especially in developing countries. The nucleoprotein (N) of RABV is highly conserved and key for genetic typing, thus a better understanding of the N gene evolutionary trajectory can assist the development of control measures. We found that the N gene of RABV has a low codon usage bias with a mean effective number of codons (ENC) value of 56.33 influenced by both mutation pressure and natural selection. However, neutrality analysis indicated that natural selection dominates over mutation pressure. Additionally, we found that dinucleotide bias partly contributed to RABV codon usage bias. On the other hand, based on the clades of phylogenetic tree, we found that the evolutionary rate of the Africa 2 clade was the highest with a mean value of 3.75 × 10− 3 substitutions per site per year. Above all, our results regarding N gene of RABV codon usage will serve future RABV evolution research.

Rabies virus (RABV), the causative agent of rabies, is highly neurovirulent for warm-blooded animals with a mortality rate of up to 100%. The RABV matrix protein (M) is required for virus particle assembly and budding. However, little is known about antigenic differences in the M protein. In this study, five monoclonal antibodies (mAbs), designated 3B9, 4A1, 2B11, 2C1, and 4B11, against the RABV M protein were generated using a recombinant M protein. All five mAbs reacted with the CVS-11 strain but showed no reactivity against the HEP-Flury strain in indirect immunofluorescence and western blotting. The epitope targeted by these mAbs was further identified by peptide scanning using GST-fused peptides. The 25PPYDDD30 peptide was defined as the minimal linear epitope. Alignment of amino acid sequences and phylogenetic analysis of different RABV strains indicated that the variable epitope 25PPDGDD30 is only present in the HEP-Flury and variant Flury strains of clade III, while the other strains resembling ERA and SRVA9 within the clade had another variable epitope, 25PLDDDD30. A Y27D mutation within the epitope was found among the rest of the RABV strains distributed in different clades. However, a single D28G mutation eliminated the reactivity of these five mAbs. In addition, the mAbs were able to recognize wildtype RABV strain in indirect immunofluorescence and western blotting and detect RABV-infected brain tissue using immunohistochemistry. The newly established mAbs and identified epitope may facilitate future investigations in the structure and function of the M protein and the development of diagnostic methods for the detection of different RABV strains worldwide. Most importantly, the epitope recognized by the mAbs against M protein might serve as a novel target for the development of a vaccine targeting RABV virulent strains.

Objective Multiple myeloma (MM) is a heterogeneous disorder, encompassing several related entities that share the common characteristic of being composed of monoclonal plasma cells (PCs). MicroRNAs (miRNAs) are small noncoding RNAs that control the expression of many target messenger RNAs involved in normal cell functions. Two serum microRNAs, miRNA-720 and miRNA-1246, were found to have potential as diagnostic biomarkers in myeloma. Therefore, we investigated a possible correlation of peripheral blood (PB) miRNA expression with diagnosis and prognosis. Methods We measured the expression of PB miRNA-720 and miRNA-1246 in 60 newly diagnosed MM patients by quantitative real-time PCR analyses. And analysed the relationship about the expression levels of miRNAs with other clinical features. Results The expression levels of PB miRNAs are significantly higher in myeloma patients compared to controls and do not change with age, gender, disease stage, hemoglobin, bone marrow PC percentage, β 2 microglobulin, serum albumin, calcium serum, serum creatinine, and myeloma protein, and independent of the deletion of chromosome 13, suggesting that the expression levels of PB miRNA-720 and miRNA-1246 can be used as a diagnostic test for myeloma. We first discovered that increased expression of PB miRNA-720 and miRNA-1246 were associated with shorter progression-free survival, indicating poor prognosis. Conclusion Our study demonstrated that PB miRNA-720 and miRNA-1246 might act as a promoting factor in the development of MM and could be a diagnostic factor, therapeutic effect evaluator, and prognostic indicator in the prognosis of MM. The miRNAs have a significant value of appreciation of individual patients' behavior during the chemotherapy and evaluation the therapeutic strategies.

Porcine circovirus 3 (PCV3) is a novel virus associated with acute PDNS (porcine dermatitis and nephropathy syndrome)-like clinical signs identified by metagenomic sequencing from swine. Its high occurrence may pose a potential threat to the swine industry worldwide. The processes resulting in the emergence and spread of PCV3 remain poorly understood. Herein, the possible origin, genotypes, and evolutionary dynamics of PCV3 based on available genomic sequences are determined. The closest ancestor of PCV3 is found to be within the clade 1 bat CVs. Using different phylogenetic methods, two major genotypes are identified, PCV3a and PCV3b. It is found that the effective population size of PCV3 increased rapidly during late 2013 to early 2014 and this is associated with the diversification of PCV3a and PCV3b. A relatively high effective reproductive number (Re) value and higher evolutionary rate were found compared to other single-stranded DNA viruses, and positive selection on codons 122 and 320 (24 of ORF2) is identified. It is hypothesized that this, together with the prediction of a potential change of an antigenic epitope at position 320, might have allowed PCV3 to escape from the host immune response. Overall, this study has important implications for understanding the ongoing PCV3 cases worldwide and will guide future efforts to develop effective preventive and control measures.