Oxygen Vacancy-Enriched CoFe2O4 for Electrochemically Sensitive Detection of the Breast Cancer CD44 Biomarker.

Enhancing the selectivity of detection methods is essential to distinguish breast cancer biomarker cluster of differentiation 44 (CD44) from other species and reduce false-positive or false-negative results. Here, oxygen vacancy-enriched CoFe2O4 (CoFe2O4-x) was crafted, and its implementation as an electrochemical electrode for the detection of CD44 biomarkers has been scrutinized. This unique electrode material offers significant benefits and novel features that enhance the sensitivity and selectivity of the detection process. The oxygen vacancy density of CoFe2O4-x was tuned by adjusting the mass ratios of iron to cobalt precursors (iron-cobalt ratio) and changing annealing atmospheres. Electrochemical characterization reveals that, when the iron-cobalt ratio is 1:0.54 and the annealing atmosphere is nitrogen, the as-synthesized CoFe2O4-x electrode manifests the best electrochemical activity. The CoFe2O4-x electrode demonstrates high sensitivity (28.22 µA (ng mL)-1 cm-2), low detection limit (0.033 pg mL-1), and robust stability (for 11 days). Oxygen vacancies can not only enhance the conductivities of CoFe2O4 but also provide better adsorption of -NH2, which is beneficial for stability and electrochemical detection performance. The electrochemical detection signal can be amplified using CoFe2O4-x as a signal probe. Additionally, it is promising to know that the CoFe2O4-x electrode has shown good accuracy in real biological samples, including melanoma cell dilutions and breast cancer patient sera. The electrochemical detection results are comparable to ELISA results, which indicates that the CoFe2O4-x electrode can detect CD44 in complex biological samples. The utilization of CoFe2O4-x as the signal probe may expand the application of CoFe2O4-x in biosensing fields.

[Progress in the role of immune cells in the pathogenesis of bronchial asthma].

Bronchial asthma is a chronic airway inflammatory disease that involves various immune cells. As the main roles in asthma immune mechanism, T lymphocytes [T helper type 1(Th1) cells, Th2 cells, Th17 cells, regulatory T cells (Tregs), T follicular helper (Tfh) cells and cytotoxic T (Tc) cells], innate lymphoid cells (ILCs), B cells, granulocytes (mast cells, eosinophils, basophils, neutrophils), macrophages as well as dendritic cells (DC) are activated by allergens and secrete their own specific cytokines. They interact with each other in function and form a complex asthma-related immune cell interaction network system. Asthma-related immune cells participate in the pathogenesis of asthma by conducting multi-target and multi-link dynamic regulation of immune mechanism through the innate and acquired immunity, cellular and humoral immunity. It needs to be further studied that the immunosuppressive effects of Tregs, Bregs, macrophages and dendritic cells, which are expected to become important targets for the treatment of asthma and development of new drugs.

A polysaccharide from Alhagi honey protects the intestinal barrier and regulates the Nrf2/HO-1-TLR4/MAPK signaling pathway to treat alcoholic liver disease in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: According to the theory of traditional Chinese medicine, the main factors related to alcoholic liver disease (ALD) are qi stagnation and blood stasis of the five viscera. Previously, we showed that the bioactive components of Alhagi honey have various pharmacological effects in treating liver diseases, but the influence of Alhagi honey on ALD (and its mechanism of action) is not known. AIM OF THE STUDY: To determine the efficacy of the main active component of Alhagi honey, the polysaccharide AHPN80, in ALD and to explore the potential mechanism of action. MATERIALS AND METHODS: AHPN80 was isolated from dried Alhagi honey and identified by transmission electron microscopy, Fourier-transform infrared spectroscopy, and gas chromatography. Venous blood, liver tissue, and colon tissue were collected in a mouse model of alcohol-induced acute liver injury. Histology, staining (Oil Red O, Alcian Blue-Periodic Acid Schiff) and measurement of reactive oxygen species (ROS) levels were used to detect histopathologic and lipid-accumulation changes in the liver and colon. Lipopolysaccharide (LPS) levels and the content of proinflammatory cytokines in serum were measured by enzyme-linked immunosorbent assays. Commercial kits were employed to detect biochemistry parameters in serum and the liver. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining kit was used to identify hepatocyte apoptosis. Expression of tight junction-associated proteins in colon tissues and nuclear factor erythroid 2-related factor 2/heme oxygenase-1/toll-like receptor-4/mitogen-activated protein kinase (Nrf2/HO-1/TLR4/MAPK) pathway-related proteins in liver tissues and HepG2 cells were analyzed by immunofluorescence or western blotting. RESULTS: In a mouse model of alcohol-induced acute liver injury, AHPN80 therapy: significantly improved liver parameters (cytochrome P450 2E1, alcohol dehydrogenase, aldehyde dehydrogenase, superoxide dismutase, malondialdehyde, glutathione peroxidase, catalase, total cholesterol, triglycerides, alanine transaminase, aspartate transaminase); reduced serum levels of LPS, interleukin (IL)-1ß, IL-6, and tumor necrosis faction-α; increased levels of IL-10 and interferon-gamma. AHPN80 reduced ALD-induced lipid accumulation and ROS production, improved alcohol-induced inflammatory damage to hepatocytes, and inhibited hepatocyte apoptosis. Immunofluorescence staining and western blotting suggested that AHPN80 might eliminate hepatic oxidative stress by activating the Nrf2/HO-1 signaling pathway, repair the intestinal barrier, inhibit the LPS/TLR4/MAPK signaling pathway, and reduce liver inflammation. CONCLUSIONS: AHPN80 may activate the Nrf2/HO-1 pathway to eliminate oxidative stress, protect the intestinal barrier, and regulate the TLR4/MAPK pathway to treat ALD in mice. AHPN80 could be a functional food and natural medicine to prevent ALD and its complications.

Structural characterization of a polysaccharide from Alhagi honey and its protective effect against inflammatory bowel disease by modulating gut microbiota dysbiosis.

The Alhagi honey polysaccharide (AHP) exhibits notable anti-inflammatory, antioxidant, and immunomodulatory properties, positioning it as a promising candidate in traditional Chinese medicine. In this investigation, we successfully isolated and purified a neutral AHP, designated AHPN50-1a, subsequently elucidating its structural attributes. AHPN50-1a was found to have a molecular weight of 1.756 × 106 Da, featuring a structural motif characterized by a recurring (1→6)-α-GlcP linker. To comprehensively evaluate its therapeutic potential, we explored the protective effects of AHPN50-1 in a murine model of dextran sodium sulfate-induced colitis. Administration of AHPN50-1 at doses of 200 and 400 mg/kg/day resulted in improved food intake, increased body weight, and increased colon length in mice with acute colitis. Simultaneously, a reduction in the disease activity index and histological scores was observed. AHPN50-1 effectively mitigated colon tissue damage, down-regulated the expression levels of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α) in colon tissue, restored intestinal microbiota diversity, and concentrations of short-chain fatty acids (SCFAs) of gut microbiota metabolites, thus alleviating intestinal inflammation in mice. In summary, our findings underscore the promise of AHPN50-1 as a valuable nutritional or dietary supplement for the treatment and prevention of inflammatory bowel disease.

Spatiotemporal Associations and Molecular Evolution of Highly Pathogenic Avian Influenza A H7N9 Virus in China from 2017 to 2021.

Highly pathogenic (HP) H7N9 avian influenza virus (AIV) emerged in China in 2016. HP H7N9 AIV caused at least 33 human infections and has been circulating in poultry farms continuously since wave 5. The genetic divergence, geographic patterns, and hemagglutinin adaptive and parallel molecular evolution of HP H7N9 AIV in China since 2017 are still unclear. Here, 10 new strains of HP H7N9 AIVs from October 2019 to April 2021 were sequenced. We found that HP H7N9 was primarily circulating in Northern China, particularly in the provinces surrounding the Bohai Sea (Liaoning, Hebei, and Shandong) since wave 6. Of note, HP H7N9 AIV phylogenies exhibit a geographical structure compatible with high levels of local transmission after unidirectional rapid geographical expansion towards the north of China in 2017. In addition, we showed that two major subclades were continually expanding with the viral population size undergoing a sharp increase after 2018 with an obvious seasonal tendency. Notably, the hemagglutinin gene showed signs of parallel evolution and positive selection. Our research sheds light on the current epidemiology, evolution, and diversity of HP H7N9 AIV that can help prevent and control the spreading of HP H7N9 AIV.

Emergence and adaptive evolution of influenza D virus.

As a novel member of the Orthomyxoviridae, influenza D virus (IDV) was firstly isolated from swine. However, cattle were found to serve as its primary reservoir. The study of IDV emergence can shed light into the dynamics of zoonotic infections and interspecies transmission. Although there is an increasing number of strains and sequenced IDV strains, their origin, epidemiology and evolutionary dynamics remain unclear. In this study, we reconstruct the diversity and evolutionary dynamics of IDVs. Molecular detection of swine tissue samples shows that six IDV positive samples were identified in the Eastern China. Phylogenetic analyses suggest three major IDV lineages designated as D/Japan, D/OK and D/660 as well as intermediate lineages. IDVs show strong association with geographical location indicating a high level of local transmission, which suggests IDVs tend to establish a local lineage of in situ evolution. In addition, the D/OK lineage widely circulates in swine in Eastern China, and all of the Chinese virus isolates form a distinct sub-clade (D/China sub-lineage). Furthermore, we identified important amino acids in the HEF gene under positive selection that might affect its receptor binding cavity relevant for its broader cell tropism. The combined results highlight that more attention should be paid to the potential threat of IDV to livestock and farming in China.

Phylogenetic and pathogenicity analysis of a novel lineage of caprine parainfluenza virus type 3.

Caprine parainfluenza virus type 3 (CPIV3) was first identified in goats named JS2013 in China. In 2019, a sheep herd broke a disease with respiratory disease in Hebei province, China. In order to confirm the pathogen of the disease, the nasal swabs, stool swabs and blood samples were collected from the sheep. Virus isolation was performed on MDBK cells and identification was conducted by RT-PCR. The complete genome of the isolate was sequenced and phylogenetic analyzed. In order to evaluate the pathogenicity of the virus, five seronegative sheep were experimental infected with the virus suspension. The phylogenetic analyses based on the complete genome and the M gene indicated that the isolate strain was distinguished distinct from previously reported CPIV3 lineage of JS2013. The virus-inoculated sheep displayed the syndrome with depression, cough, and fever. Virus shedding were detected by RT-PCR from nasal swabs. All infected showed virus shedding during 2 - 21dpi and viremia could be detected in serum samples. Gross pathological assessment of sheep in infected group showed gross lesion in the lungs. Histopathological observation results indicated that lungs had mild to moderate interstitial pneumonia, with thickened alveolar walls, decreased alveolar space, and increased amounts of inflammatory cells infiltration. This is the first report of pathogenicity of the novel lineage of sheep-derived CPIV3. The results would be helpful for further studies on the prevention and control strategies for CPIV3 infections in goat and sheep.

High-throughput immunosensor chip coupled with a fluorescent DNA dendrimer for ultrasensitive detection of cardiac troponin T.

A novel fluorescence (FL) imaging platform was established for ultrasensitive and rapid detection of cardiac troponin T (cTnT), based on a high-throughput immunosensor chip and a DNA dendrimer capped with a large number of fluorescent dyes (FDD@Cy5). Through an enzyme-free and step-by-step strategy, FDD@Cy5 was self-assembled facilely. After the formation of a sandwich immunocomplex and biotin-streptavidin conjugation, FDD@Cy5 could be captured on the chip. FL signals emerged from Cy5 under external light and the enrichment of Cy5 on the dendrimer led to signal amplification. A FL image containing 90 spots could be collected instantaneously by laser confocal scanning microscopy and the brightness of all the spots corresponded to the concentrations of target cTnT. Under optimal conditions, the immunosensor chip coupled with FDD@Cy5 exhibited an excellent detection limit of 0.10 pg L-1, a wide linear range from 0.20 pg L-1 to 2.0 ng L-1, a sample consumption down to 3.0 µL and a maximum throughput of 45 tests per h. The proposed approach was also applied to cTnT quantitation in serum samples with acceptable accuracy, providing a new avenue for early diagnosis and the prognosis evaluation of acute myocardial infarction.

Analysis of the Codon Usage Pattern of HA and NA Genes of H7N9 Influenza A Virus.

Novel H7N9 influenza virus transmitted from birds to human and, since March 2013, it has caused five epidemic waves in China. Although the evolution of H7N9 viruses has been investigated, the evolutionary changes associated with codon usage are still unclear. Herein, the codon usage pattern of two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), was studied to understand the evolutionary changes in relation to host, epidemic wave, and pathogenicity. Both genes displayed a low codon usage bias, with HA higher than NA. The codon usage was driven by mutation pressure and natural selection, although the main contributing factor was natural selection. Additionally, the codon adaptation index (CAI) and deoptimization (RCDI) illustrated the strong adaptability of H7N9 to Gallus gallus. Similarity index (SiD) analysis showed that Homo sapiens posed a stronger selection pressure than Gallus gallus. Thus, we assume that this may be related to the gradual adaptability of the virus to human. In addition, the host strong selection pressure was validated based on CpG dinucleotide content. In conclusion, this study analyzed the usage of codons of two genes of H7N9 and expanded our understanding of H7N9 host specificity. This aids into the development of control measures against H7N9 influenza virus.

Epidemiology, genetic diversity and evolution of canine astrovirus.

Astroviruses (AstVs) are major causative agents of gastroenteritis in children and have been reported in many species. Canine astrovirus (CaAstV), as an enteric pathogen, has been widely detected worldwide, but little is known about their genetic diversity and evolution, partly owing to a lack of genomic data. Here, we sequenced 12 nearly full-length CaAstV genomes to address the gap in knowledge. We found 14 (13.2%) and 7 (3.35%) CaAstV-positive samples from pet dogs with and without diarrhoea, respectively. Co-infections were with co-infection with Torque teno canis virus (TTCaV) reported for the first time. Phylogenetic analysis of the ORF2 gene revealed four major lineages. In particular, lineage 4 might have evolved from a recombinant virus from lineage 2 and lineage 3. The strains sequenced here clustered with lineages 2, 3 and 4 in contrast with other Chinese strains identified previously that clustered with lineages 2 and 4. Amino acid sequence alignment within lineage revealed intralineage amino acid diversity and that the type of epidemic strains within lineages changes over time. Three amino acids substitutions located in predicted B-cell epitopes, which might be involved escape of host immunity. Moreover, frequent inter-clade ORF2 gene recombinants were identified. The identification of individual recombination events and a recombinant lineage indicated that recombination plays a crucial role in CaAstV genetic evolution and diversity by generating divergent viruses. Moreover, phylogenetic analysis of ORF1b, the most conserved gene of astrovirus, revealed a close relationship between CaAstV and California sea lion astroviruses. Overall, we report detailed information on the genetic evolution and diversity of CaAstV, which indicates that CaAstV may pose challenges for diagnostics and future control strategies.

One-step multiplex TaqMan probe-based method for real-time PCR detection of four canine diarrhea viruses.

Viral canine diarrhea has high morbidity and mortality and is prevalent worldwide, resulting in severe economic and spiritual losses to pet owners. However, diarrhea pathogens have similar clinical symptoms and are difficult to diagnose clinically. Thus, fast and accurate diagnostic methods are of great significance for prevention and accurate treatment. In this study, we developed a one-step multiplex TaqMan probe-based real-time PCR for the differential diagnosis of four viruses causing canine diarrhea including, CPV (Canine Parvovirus), CCoV (Canine Coronavirus), CAstV (Canine Astrovirus), and CaKoV (Canine Kobuviruses). The limit of detection was up to 102 copies/µL and performed well with high sensitivity and specificity. This assay was optimized and used to identify possible antagonistic relationships between viruses. From this, artificial pre-experiments were performed for mixed infections, and a total of 82 canine diarrhea field samples were collected from different animal hospitals in Zhejiang, China to assess the method. The virus prevalence was significantly higher than what previously reported based on RT-PCR (Reverse Transcription-Polymerase Chain Reaction). Taken together, these results suggest that the method can be used as a preferred tool for monitoring laboratory epidemics, timely prevention, and effective monitoring of disease progression.

COVID-19: Epidemiology, Evolution, and Cross-Disciplinary Perspectives.

The recent outbreak of COVID-19 in Wuhan turned into a public health emergency of international concern. With no antiviral drugs nor vaccines, and the presence of carriers without obvious symptoms, traditional public health intervention measures are significantly less effective. Here, we report the epidemiological and virological characteristics of the COVID-19 outbreak. Originated in bats, 2019-nCoV/ severe acute respiratory syndrome coronavirus (SARS-CoV)-2 likely experienced adaptive evolution in intermediate hosts before transfer to humans at a concentrated source of transmission. Similarities of receptor sequence binding to 2019-nCoV between humans and animals suggest a low species barrier for transmission of the virus to farm animals. We propose, based on the One Health model, that veterinarians and animal specialists should be involved in a cross-disciplinary collaboration in the fight against this epidemic.

Epidemiology and evolutionary analysis of Torque teno sus virus.

Single stranded (ss) DNA viruses are increasingly being discovered due to the ongoing development of modern technologies in exploring the virosphere. Characterized by high rates of recombination and nucleotide substitutions, it could be comparable to RNA virus ones. Torque teno sus virus (TTSuV) is a standard ssDNA virus with a high population diversity, whose evolution is still obscure, further, it is frequently found in co-infections with other viruses threatening the porcine industry and therefore share the same host and epidemiological context. Here, we implement and describe approach to integrate viral nucleotide sequence analysis, surveillance data, and a structural approach to examine the evolution of TTSuVs, we collected samples from pigs displaying respiratory signs in China and revealed a high prevalence of TTSuV1 and TTSuVk2, frequently as part of co-infections with porcine circoviruses (PCVs), especially in spleen and lung. In addition, thirty six strains sequenced were obtained to investigate their genetic diversity in China. The evolutionary history of TTSuVs were unveiled as following: At the nucleotide sequence level, TTSuVs ORF1 was confirmed to be a robust phylogenetic maker to study evolution comparably to full genomes. Additionally, extensive recombination discovered within TTSuVk2a (also 5 out of the 36 sequenced strains in this study revealed to be recombination). Then, pairwise distance, phylogenetic trees, and amino acid analysis confirmed TTSuVs species, and allowed to define circulating genotypes (TTSuV1a-1, 1a-2, 1b-1, 1b-2, 1b-3, and k2a-1, k2a-2, k2b). Selection analysis uncovered seven and six positive selected sites in TTSuV1 and TTSuVk2, respectively. At the protein structure level, mapping of sites onto the three-dimensional structure revealed that several positive selected sites locate into potential epitopes, which might related to the potential escaping from host immune response. Our result could assist future studies on swine ssDNA virus classification, surveillance and control.

Genomic Epidemiology, Evolution, and Transmission Dynamics of Porcine Deltacoronavirus.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown once again that coronavirus (CoV) in animals are potential sources for epidemics in humans. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen of swine with a worldwide distribution. Here, we implemented and described an approach to analyze the epidemiology of PDCoV following its emergence in the pig population. We performed an integrated analysis of full genome sequence data from 21 newly sequenced viruses, along with comprehensive epidemiological surveillance data collected globally over the last 15 years. We found four distinct phylogenetic lineages of PDCoV, which differ in their geographic circulation patterns. Interestingly, we identified more frequent intra- and interlineage recombination and higher virus genetic diversity in the Chinese lineages compared with the USA lineage where pigs are raised in different farming systems and ecological environments. Most recombination breakpoints are located in the ORF1ab gene rather than in genes encoding structural proteins. We also identified five amino acids under positive selection in the spike protein suggesting a role for adaptive evolution. According to structural mapping, three positively selected sites are located in the N-terminal domain of the S1 subunit, which is the most likely involved in binding to a carbohydrate receptor, whereas the other two are located in or near the fusion peptide of the S2 subunit and thus might affect membrane fusion. Finally, our phylogeographic investigations highlighted notable South-North transmission as well as frequent long-distance dispersal events in China that could implicate human-mediated transmission. Our findings provide new insights into the evolution and dispersal of PDCoV that contribute to our understanding of the critical factors involved in CoVs emergence.

Etiology and genetic evolution of canine coronavirus circulating in five provinces of China, during 2018-2019.

As the outbreaks of COVID-19 in worldwide, coronavirus has once again caught the attention of people. Canine coronavirus is widespread among dog population, and sometimes causes even fatal cases. Here, to characterize the prevalence and evolution of current circulating canine coronavirus (CCoV) strains in China, we collected 213 fecal samples from diarrheic pet dogs between 2018 and 2019. Of the 213 samples, we found 51 (23.94%) were positive for CCoV. Co-infection with canine parvovirus (CPV), canine astrovirus (CaAstV), canine kobuvirus (CaKV), Torque teno canis virus (TTCaV) were ubiquitous existed. Mixed infection of different CCoV subtypes exists extensively. Considering the limited sequences data in recent years, we sequenced 7 nearly complete genomes and 10 complete spike gene. Phylogenetic analysis of spike gene revealed a new subtype CCoV-II Variant and CCoV-IIa was the most prevalent subtype currently circulating. Moreover, we identified strain B906_ZJ_2019 shared 93.24% nucleotide identifies with previous strain A76, and both of them clustered with CCoV-II Variant, which were not well clustered with the known subtypes. Recombination analysis of B906_ZJ_2019 indicated that strain B906_ZJ_2019 may a recombinant variant between CCoV-I and CCoV-II, which is consistent with strain A76. Furthermore, amino acid variations widely existed among current CCoV-IIa strains circulating in China and the classic CCoV-IIa strains, in spite of the unknown functions. In a word, we report a useful information as to the etiology and evolution of canine coronavirus in China based on the available sequences, which is urgent for the devise of future effective disease prevention and control strategies.

Adaption and parallel evolution of human-isolated H5 avian influenza viruses.

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.

A Shift in Porcine Circovirus 3 (PCV-3) History Paradigm: Phylodynamic Analyses Reveal an Ancient Origin and Prolonged Undetected Circulation in the Worldwide Swine Population.

The identification of a new circovirus (Porcine circovirus 3, PCV-3) has raised a remarkable concern because of some analogies with Porcine circovirus 2 (PCV-2). Preliminary results suggest an extremely recent PCV-3 emergence and high mutation rate. Retrospective studies prove its circulation at least since the early 1990s, revealing that PCV-3 could have been infecting pigs for an even longer period. Therefore, a new evaluation, based on an updated collection of PCV-3 sequences spanning more than 20 years, is performed using a phylodynamic approach. The obtained results overrule the previous PCV-3 history concept, indicating an ancient origin. These evidences are associated with an evolutionary rate far lower (10-5-10-6 substitution/site/year) than the PCV-2 one. Accordingly, the action of selective pressures on PCV-3 open reading frames (ORFs) seems to be remarkably lower compared to those acting on PCV-2, suggesting either a reduced PCV-3 plasticity or a less efficient host-induced natural selection. A complex and not-directional viral flow network is evidenced through phylogeographic analysis, indicating a long lasting circulation rather than a recent emergence followed by spreading. Being recent emergence has been ruled out, efforts should be devoted to understand whether its recent discovery is simply due to improved detection capabilities or to the breaking of a previous equilibrium.

Host-range shift of H3N8 canine influenza virus: a phylodynamic analysis of its origin and adaptation from equine to canine host.

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.

Comprehensive codon usage analysis of porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is a newly identified coronavirus of pigs that was first reported in Hong Kong in 2012. Since then, many PDCoV isolates have been identified worldwide. In this study, we analyzed the codon usage pattern of the S gene using complete coding sequences and complete PDCoV genomes to gain a deeper understanding of their genetic relationships and evolutionary history. We found that during evolution three groups evolved with a relatively low codon usage bias (effective number of codons (ENC) of 52). The factors driving bias were complex. However, the primary element influencing the codon bias of PDCoVs was natural selection. Our results revealed that different natural environments may have a significant impact on the genetic characteristics of the strains. In the future, more epidemiological surveys are required to examine the factors that resulted in the emergence and outbreak of this virus.