In recent years,the global incidence of hepatitis E virus(HEV)has been rising,leading to increased morbidity and mortality associated with hepatitis.Cas13,a CRISPR effector,shows promise as an antiviral agent against single-stranded RNA viruses.Cas13d,a type Ⅵ-D effector,exhibits higher efficiency in suppressing RNA viruses compared to other type Ⅵ variants.

Global evolutionary dynamics of influenza A virus (IAV) are fundamentally driven by the extent of virus diversity generated, transmitted, and shaped in individual hosts. How vaccination affects the degree of IAV genetic diversity that can be transmitted and expanded in pigs is unknown. To evaluate the effect of vaccination on the transmission of genetically distinct IAV variants and their diversity after transmission in pigs, we examined the whole genome of IAV recovered from the nasal cavities of pigs vaccinated with differentinfluenza immunization regimens after being infected simultaneously by H1N1 and H3N2 IAVs using a seeder pig model. We found that the seeder pigs harbored more diversified virus populations than the contact pigs. Among contact pigs, H3N2 and H1N1 viruses recovered from pigs vaccinated with a single dose of an unmatched modified live vaccine generally accumulated more extensive genetic mutations than non-vaccinated pigs. Furthermore, the non-sterilizing immunity elicited by the single-dose-modified live vaccine may have exerted positive selection on H1 antigenic regions as we detected significantly higher nonsynonymous but lower synonymous evolutionary rates in H1 antigenic regions than non-antigenic regions. In addition, we observed that the vaccinated pigs shared significantly less proportion of H3N2 variants with seeder pigs than unvaccinated pigs. These results indicated that vaccination might reduce the impact of transmitted influenza variants on the overall diversity of IAV populations harbored in recipient pigs and that within-host genetic selection of IAV is more likely to occur in pigs vaccinated with improperly matched vaccines. IMPORTANCE Understanding how vaccination shapes the diversity of influenza variants that transmit and propagate among pigs is essential for designing effective IAV surveillance and control programs. Current knowledge about the transmission of IAV variants has primarily been explored in humans during natural infection. However, how immunity elicited by improperly matched vaccines affects the degree of IAV genetic diversity that can be transmitted and expanded in pigs at the whole-genome level is unknown. We analyzed IAV sequences from samples collected daily from experimentally infected pigs vaccinated with various protocols in a field-represented IAV co-infection model. We found that vaccine-induced non-sterilizing immunity might promote genetic variation on the IAV genome and drive positive selection at antigenic sites during infection. In addition, a smaller proportion of H3N2 viral variants were shared between seeder pigs and vaccinated pigs, suggesting the influence of vaccination on shaping the virus genomic diversity in recipient pigs during the transmission events.

Pigs are recognized as amplifying hosts for influenza A virus (IAV) reassortant viruses. Understanding the extent of IAV reassortment occurring at the individual pig level in naturally infected pigs and how reassortment impacts virus diversity, persistence, and replicative fitness is essential for countering IAV threats to humans and animals. Here, 244 IAV plaques were isolated from 24 commercial pigs, resulting in 26 distinct genome segment constellations. About 33% (8/24) of pigs were infected with two or more different genotypes, with two pigs harboring two or more different subtypes. Our results indicate that few pigs in a population harbor significantly more genotypes than other pigs and generate most of the diversity, including the emergence of reassortants. However, detecting distinct genotypes during surveillance was dynamic, with most of the genotypes subsiding over time. All the IAV genotypes could replicate in various swine and human-sourced respiratory epithelial cells, and we observed that distinct reassortant genotypes recovered from a single pig could exhibit different growth abilities, especially in human cells. Overall, we demonstrated that multiple distinct IAV genotypes with distinct antigenic profiles and varying growth abilities on swine and human respiratory tracts can be shed simultaneously from a single pig, which contributes to the dynamic nature of IAV prevalence. The striking magnitude of IAV reassortment at the single pig level revealed in this study highlights the need to strengthen surveillance efforts and plans to eliminate IAV from swine farms because pigs have a high potential to produce diverse and potentially zoonotic influenza reassortant viruses. IMPORTANCE Pigs play a crucial role in driving influenza A virus (IAV) diversification and evolution by reassorting the viruses originating from different hosts. Despite IAV reassortment and diversity being well documented in pig populations at different scales (e.g., farm, region, country), limited field research has explored the extent of reassortment happening at the single pig level and how that contributes to the overall genetic and biological variation observed in populations. We provide initial information on levels of reassortment happening at the single pig level in naturally infected pigs, and that particular pigs can shed a plethora of distinct genotypes, with certain genotypes having distinct replicative fitness on swine and human respiratory tracts, which preserves the potential for IAV long-term evolution and facilitates the emergence of zoonotic/pandemic-capable reassortants.

The development of epigenetic maps, such as the ENCODE project in humans, provides resources for gene regulation studies and a reference for research of disease-related regulatory elements. However,epigenetic information, such as a bird-specific chromatin accessibility atlas, is currently lacking for the thousands of bird species currently described.The major genomic difference between birds and mammals is their shorter introns and intergenic distances, which seriously hinders the use of humans and mice as a reference for studying the function of important regulatory regions in birds. In this study, using chicken as a model bird species, we systematically compiled a chicken chromatin accessibility atlas using 53 Assay of Transposase Accessible Chromatin sequencing（ATAC-seq）samples across 11 tissues. An average of 50 ?796open chromatin regions were identified per sample,cumulatively accounting for 20.36% of the chicken genome. Tissue specificity was largely reflected by differences in intergenic and intronic peaks, with specific functional regulation achieved by two mechanisms: recruitment of several sequence-specific transcription factors and direct regulation of adjacent functional genes. By integrating data from genome-wide association studies, our results suggest that chicken body weight is driven by different regulatory variants active in growth-relevant tissues. We propose CAB39L（active in the duodenum）, RCBTB1（muscle and liver）, and novel long non-coding RNA ENSGALG00000053256（bone） as candidate genes regulating chicken body weight. Overall, this study demonstrates the value of epigenetic data in fine-mapping functional variants and provides a compendium of resources for further research on the epigenetics and evolution of birds and mammals.

Interspecies transmission of influenza A virus (IAV) between pigs and people represents a threat to both animal and public health. To better understand the risks of influenza transmission at the human-animal interface, we evaluated 1) the rate of IAV detection in swine farmworkers before and after work during two human influenza seasons, 2) assessed risk factors associated with IAV detection in farmworkers and 3) characterized the genetic sequences of IAV detected in both workers and pigs. Of 58 workers providing nasal passage samples during 8-week periods during the 2017/18 and 2018/19 influenza seasons, 33 (57%) tested positive by rRT-PCR at least once. Sixteen (27%) workers tested positive before work and 24 (41%) after work. At the sample level, 58 of 1,785 nasal swabs (3.2%) tested rRT-PCR positive, of which 20 of 898 (2.2%) were collected prior to work and 38 of 887 (4.3%) after work. Although farmworkers were more likely to test positive at the end of the working day (OR = 1.98, 95% CI 1.14-3.41), there were no influenza-like illness (ILI) symptoms, or other risk indicators, associated with IAV detection before or after reporting to work. Direct whole-genome sequencing from samples obtained from worker nasal passages indicated evidence of infection of a worker with pandemic 2009 H1N1 of human-origin IAV (H1-pdm 1A 3.3.2) when reporting to work, and exposure of several workers to a swine-origin IAV (H1-alpha 1A 1.1) circulating in the pigs on the farm where they were employed. Our study provides evidence of 1) risk of IAV transmission between pigs and people, 2) pandemic H1N1 IAV infected workers reporting to work and 3) workers exposed to swine harbouring swine-origin IAV in their nasal passages temporarily. Overall, our results emphasize the need to implement surveillance and transmission preventive protocols at the pig/human interface.

Insulin-like growth factor I (IGF-1) is a key regulator of tissue growth and development in response to growth hormone stimulation. In the skeletal system, IGF-1 derived from osteoblasts and chondrocytes are essential for normal bone development; however, whether bone marrow (BM)-resident cells provide distinct sources of IGF-1 in the adult skeleton remains elusive. Here, we show that BM stromal cells (BMSCs) and megakaryocytes/platelets (MKs/PLTs) express the highest levels of IGF-1 in adult long bones. Deletion of Igf1 from BMSCs by Lepr-Cre leads to decreased bone formation, impaired bone regeneration, and increased BM adipogenesis. Importantly, reduction of BMSC-derived IGF-1 contributes to fasting-induced marrow fat accumulation. In contrast, deletion of Igf1 from MKs/PLTs by Pf4-Cre leads to reduced bone formation and regeneration without affecting BM adipogenesis. To our surprise, MKs/PLTs are also an important source of systemic IGF-1. Platelet-rich plasma (PRP) from Pf4-Cre; Igf1f/fmice showed compromised osteogenic potential both in vivo and in vitro, suggesting that MK/PLT-derived IGF-1 underlies the therapeutic effects of PRP. Taken together, this study identifies BMSCs and MKs/PLTs as two important sources of IGF-1 that coordinate to maintain and regenerate the adult skeleton, highlighting reciprocal regulation between the hematopoietic and skeletal systems.

Although vaccination is broadly used in North American swine breeding herds, managing swine influenza is challenging primarily due to the continuous evolution of influenza A virus (IAV) and the ability of the virus to transmit among vaccinated pigs. Studies that have simultaneously assessed the impact of vaccination on the emergence of IAV reassortment and genetic variation in pigs are limited. Here, we directly sequenced 28 bronchoalveolar lavage fluid (BALF) samples collected from vaccinated and unvaccinated pigs co-infected with H1N1 and H3N2 IAV strains, and characterized 202 individual viral plaques recovered from 13 BALF samples. We identified 54 reassortant viruses that were grouped in 17 single and 16 mixed genotypes. Notably, we found that prime-boost vaccinated pigs had less reassortant viruses than nonvaccinated pigs, likely due to a reduction in the number of days pigs were co-infected with both challenge viruses. However, direct sequencing from BALF samples revealed limited impact of vaccination on viral variant frequency, evolutionary rates, and nucleotide diversity in any IAV coding regions. Overall, our results highlight the value of IAV vaccination not only at limiting virus replication in pigs but also at protecting public health by restricting the generation of novel reassortants with zoonotic and/or pandemic potential.

Pandemic influenza, typically caused by the reassortment of human and avian influenza viruses, can result in severe or fatal infections in humans. Timely identification of potential pandemic viruses must be a priority in influenza virus surveillance. However, the range of host species responsible for the generation of novel pandemic influenza viruses remains unclear. In this study, we conducted serological surveys for avian and human influenza virus infections in farmed mink and determined the susceptibility of mink to prevailing avian and human virus subtypes. The results showed that farmed mink were commonly infected with human (H3N2 and H1N1/pdm) and avian (H7N9, H5N6, and H9N2) influenza A viruses. Correlational analysis indicated that transmission of human influenza viruses occurred from humans to mink, and that feed source was a probable route of avian influenza virus transmission to farmed mink. Animal experiments showed that mink were susceptible and permissive to circulating avian and human influenza viruses, and that human influenza viruses (H3N2 and H1N1/pdm), but not avian viruses, were capable of aerosol transmission among mink. These results indicate that farmed mink could be highly permissive "mixing vessels" for the reassortment of circulating human and avian influenza viruses. Therefore, to reduce the risk of emergence of novel pandemic viruses, feeding mink with raw poultry by-products should not be permitted, and epidemiological surveillance of influenza viruses in mink farms should be urgently implemented.

Emerging and endemic animal viral diseases continue to impose substantial impacts on animal and human health. Most current and past molecular surveillance studies of animal diseases investigated spatio-temporal and evolutionary dynamics of the viruses in a disjointed analytical framework, ignoring many uncertainties and made joint conclusions from both analytical approaches. Phylodynamic methods offer a uniquely integrated platform capable of inferring complex epidemiological and evolutionary processes from the phylogeny of viruses in populations using a single Bayesian statistical framework. In this study, we reviewed and outlined basic concepts and aspects of phylodynamic methods and attempted to summarize essential components of the methodology in one analytical pipeline to facilitate the proper use of the methods by animal health researchers. Also, we challenged the robustness of the posterior evolutionary parameters, inferred by the commonly used phylodynamic models, using hemagglutinin (HA) and polymerase basic 2 (PB2) segments of the currently circulating human-like H3 swine influenza (SI) viruses isolated in the United States and multiple priors. Subsequently, we compared similarities and differences between the posterior parameters inferred from sequence data using multiple phylodynamic models. Our suggested phylodynamic approach attempts to reduce the impact of its inherent limitations to offer less biased and biologically plausible inferences about the pathogen evolutionary characteristics to properly guide intervention activities. We also pinpointed requirements and challenges for integrating phylodynamic methods in routine animal disease surveillance activities.

Influenza A viruses evolve rapidly to escape host immunity. In swine, this viral evolution has resulted in the emergence of multiple H1 and H3 influenza A virus (IAV) lineages in the United States (US) pig populations. The heterologous prime-boost vaccination strategy is a promising way to deal with diverse IAV infection in multiple animal models. However, whether or not this vaccination strategy is applicable to US swine to impart immunity against infection from North American strains of IAV is still unknown. We performed a vaccination-challenge study to evaluate the protective efficacy of using multivalent inactivated vaccine and/or a live attenuated IAV vaccine (LAIV) in pigs following multiple prime-boost vaccination protocols against a simultaneous H1N1 and H3N2 IAV infection. Our data show that pigs in the heterologous prime-boost vaccination group had more favorable outcomes consistent with a better response against virus challenge than non-vaccinated pigs. Additionally, delivering a multivalent heterologous inactivated vaccine boost to pigs following a single LAIV administration was also beneficial. We concluded the heterologous prime boost vaccination strategy may potentiate responses to suboptimal immunogens and holds the potential applicability to control IAV in the North American swine industry. However, more studies are needed to validate the application of this vaccination approach under field conditions.

Pigs are considered as important hosts or "mixing vessels" for the generation of pandemic influenza viruses. Systematic surveillance of influenza viruses in pigs is essential for early warning and preparedness for the next potential pandemic. Here, we report on an influenza virus surveillance of pigs from 2011 to 2018 in China, and identify a recently emerged genotype 4 (G4) reassortant Eurasian avian-like (EA) H1N1 virus, which bears 2009 pandemic (pdm/09) and triple-reassortant (TR)-derived internal genes and has been predominant in swine populations since 2016. Similar to pdm/09 virus, G4 viruses bind to human-type receptors, produce much higher progeny virus in human airway epithelial cells, and show efficient infectivity and aerosol transmission in ferrets. Moreover, low antigenic cross-reactivity of human influenza vaccine strains with G4 reassortant EA H1N1 virus indicates that preexisting population immunity does not provide protection against G4 viruses. Further serological surveillance among occupational exposure population showed that 10.4% (35/338) of swine workers were positive for G4 EA H1N1 virus, especially for participants 18 y to 35 y old, who had 20.5% (9/44) seropositive rates, indicating that the predominant G4 EA H1N1 virus has acquired increased human infectivity. Such infectivity greatly enhances the opportunity for virus adaptation in humans and raises concerns for the possible generation of pandemic viruses.

Interferon-induced transmembrane protein 3 (IFITM3) as an antiviral factor can inhibit replication of several viruses including influenza virus. A single-nucleotide polymorphism rs12252-C of IFITM3 results in a truncated IFITM3 protein lacking its first 21 amino acids, which is much higher in the Han Chinese population and associated with severe illness in adults infected with pandemic influenza H1N1/09 virus. To investigate if IFITM3 or IFITM3 rs12252-C could affect the antibody response after influenza vaccination, we detected the haemagglutination inhibition (HI) of 171 healthy young adult volunteers (IFITM3 rs12252-C/C, C/T, T/T carriers) and in an IFITM3-deletion mouse model (Ifitm3(-/-)) after trivalent inactivated vaccine (TIV) immunization. Seroconversion rates for H1N1, H3N2 and B viruses in IFITM3 rs12252-C/C genotype carriers was lower compared with C/T and T/T donors. Significantly lower levels of specific antibodies to H1N1, H3N2 and B viruses and total IgG were observed in Ifitm3(-/-) mice. Correspondingly, the numbers of splenic germinal centre (GC) B cells, plasma cells, TIV-specific IgG(+) antibody secreting cells and T follicular helper cells in Ifitm3(-/-) mice were lower compared with wild type mice. However, the number of memory B cells was higher in Ifitm3(-/-) mice at day 7 after booster. The HI level of Ifitm3(-/-) mice remained lower than WT mice after third vaccination. Moreover, the transcriptional network regulating GC B cell and plasma cell differentiation was abnormal in Ifitm3(-/-) mice. Our results indicate that IFITM3 deletion attenuated the antibody response. The mechanism of influenza-IFITM3 interactions affecting the antibody response requires further investigation.

Domestic ducks are considered as the interface between wild aquatic birds and terrestrial poultry and play an important role in the transmission and evolution of avian influenza viruses (AIVs). However, the infectivity of H9N2 AIVs in different domestic duck species has not been systematically evaluated. Here we investigated the infectivity of various genotypes of chicken H9N2 AIVs in Pekin duck (Anas Platyrhynchos), Mallard duck (Anas Platyrhynchos) and Muscovy duck (Cairina Moschata) through intranasal inoculation. We found that Pekin ducks and Mallard ducks were generally resistant to chicken H9N2 virus infection, while Muscovy ducks were relatively susceptible to H9N2 AIVs. All the tested viruses were isolated from oropharynx, trachea and lung tissues of Muscovy ducks. Additionally, genotype 57 (G57) H9N2 AIVs, which was predominant in chickens since 2010, showed increased virus replication in this duck species, indicating an improved interspecies transmission ability of recent H9N2 viruses from chickens to ducks. Our results demonstrated the role of Muscovy ducks in the ecology of H9N2 AIVs. More attentions should be paid to this host during viral surveillances. Additionally, inactivated H9N2 vaccine may be unnecessarily used in Pekin and Mallard ducks.

Influenza H9N2 subtype viruses and their reassortants (such as H7N9) are posing increasing threats to birds and humans in China. During 2009-2013, multiple novel subtype viruses with H9N2 original genes emerged in China. Yet, the genetic evolution of H9N2 viruses in various host organisms in China has not been systematically investigated since 2009. In the present study, we performed large-scale sequence analysis of H9N2 viral genomes from public databases, representing the spectrum of viruses isolated from birds, mammals and humans in China from 1994 to 2013, and updated the clade classification for each segment. We identified 117 distinct genotypes in 730 H9N2 viruses. We analyzed the sequences of all eight segments in each virus and found three important time points: the years 2000, 2006 and 2010. In the periods divided by these years, genotypic diversity, geographic distribution and host range changed considerably. Genotypic diversity fluctuated greatly in 2000 and 2006. Since 2010, a single genotype became predominant in poultry throughout China, and the eastern coastal region became the newly identified epidemic center. Throughout their 20-year prevalence in China, H9N2 influenza viruses have emerged and adapted from aquatic birds to chickens. The minor avian species and wild birds exacerbated H9N2 genotypes by providing diversified genes, and chickens were the most prevalent vector in which the viruses evolved and expanded their prevalence. It is the necessity for surveillance and disease control on live-bird markets, poultry farms and wild-bird habitats in China.

Cases of human infection with a novel H7N9 avian influenza virus (AIV) were first reported in March 2013, which caused 115 deaths within a single year. Beyond that, other subtypes of H7 AIV were isolated from poultry in eastern China during the same period, including H7N7 and H7N2 AIV. In the present study, a subtype H3N2 AIV was isolated from ducks from Anhui Province, China. Sequence and phylogenetic analyses revealed that seven gene segments of this virus showed the highest sequence homology with that of the H7 subtype influenza virus, which is presumed to be the reassortants of the H3 and H7 subtypes AIV. The present study also reconfirmed that the reassortment between the H7 subtype and waterfowl-originating AIVs universally occurred in waterfowl. Animal inoculation tests showed that the virus has low pathogenicity in chickens; however, it could be replicated in the lungs of mice. The emergence of this H3N2 isolate emphasizes the importance of enhancing the surveillance of waterfowl-originating AIVs, the identification of novel reassortant strains, and characterization of their biological properties.

ABSTRACT Since May 2014, highly pathogenic avian influenza H5N6 virus has been reported to cause six severe human infections three of which were fatal. The biological properties of this subtype, in particular its relative pathogenicity and transmissibility in mammals, are not known. We characterized the virus receptor-binding affinity, pathogenicity, and transmissibility in mice and ferrets of four H5N6 isolates derived from waterfowl in China from 2013-2014. All four H5N6 viruses have acquired a binding affinity for human-like SAα2,6Gal-linked receptor to be able to attach to human tracheal epithelial and alveolar cells. The emergent H5N6 viruses, which share high sequence similarity with the human isolate A/Guangzhou/39715/2014 (H5N6), were fully infective and highly transmissible by direct contact in ferrets but showed less-severe pathogenicity than the parental H5N1 virus. The present results highlight the threat of emergent H5N6 viruses to poultry and human health and the need to closely track their continual adaptation in humans. IMPORTANCE Extended epizootics and panzootics of H5N1 viruses have led to the emergence of the novel 2.3.4.4 clade of H5 virus subtypes, including H5N2, H5N6, and H5N8 reassortants. Avian H5N6 viruses from this clade have caused three fatalities out of six severe human infections in China since the first case in 2014. However, the biological properties of this subtype, especially the pathogenicity and transmission in mammals, are not known. Here, we found that natural avian H5N6 viruses have acquired a high affinity for human-type virus receptor. Compared to the parental clade 2.3.4 H5N1 virus, emergent H5N6 isolates showed less severe pathogenicity in mice and ferrets but acquired efficient in-contact transmission in ferrets. These findings suggest that the threat of avian H5N6 viruses to humans should not be ignored.

Outbreaks of pandemic H1N1 2009 (pH1N1) in turkeys have been reported in several countries. Co-infection of pH1N1 and avian H9N2 influenza viruses in turkeys provide the opportunity for their reassortment, and novel reassortant viruses might further be transmitted to other avian species. However, virulence and transmission of those reassortant viruses in poultry remain unclear. In the present study, we generated 16 single-gene reassortant influenza viruses including eight reassortants on the pH1N1 background by individual replacement with a corresponding gene segment from H9N2 and eight reassortants on the H9N2 background replaced individually with corresponding gene from pH1N1, and characterized reassortants viruses in turkeys and chickens. We found that the pH1N1 virus dramatically increased its infectivity and transmissibility in turkeys and chickens after introducing any gene (except for PB2) from H9N2 virus, and H9N2 virus acquired single gene (except for HA) of pH1N1 almost did not influence its replication and transmission in turkeys and chickens. Additionally, 13 reassortant viruses transmitted from turkeys to chickens. Our results indicate that turkeys and chickens are susceptible to pH1N1-H9N2 reassortant viruses, and mixing breeding of different avian species would facilitate the transmission of these reassortant viruses.