Rescuing Newcastle disease virus with tag for screening viral-host interacting proteins based on highly efficient reverse genetics.

The interaction between viral proteins and host proteins plays a crucial role in the process of virus infecting cells. Tags such as HA, His, and Flag do not interfere with the function of fusion proteins and are commonly used to study protein-protein interactions. Adding these tags to viral proteins will address the challenge of the lack of antibodies for screening host proteins that interact with viral proteins during infection. Obtaining viruses with tagged fusion proteins is crucial. This study established a new reverse genetic system with T7 promoter and three plasmids, which efficiently rescued Newcastle disease virus (NDV) regardless of its ability to replicate in cells. Subsequently, using this system, NDV containing a HA-tagged structural protein and NDV carrying a unique tag on each structural protein were successfully rescued. These tagged viruses replicated normally and exhibited genetic stability. Based on tag antibodies, every NDV structural protein was readily detected and showed correct subcellular localization in infected cells. After infecting cells with NDV carrying HA-tagged M protein, several proteins interacting with the M protein during the infection process were screened using HA tag antibodies. The establishment of this system laid the foundation for comprehensive exploration of the interaction between NDV proteins and host proteins.

Transcriptional kinetics of NADC34-like porcine reproductive and respiratory syndrome virus during cellular infection.

NADC34-like porcine reproductive and respiratory syndrome virus (PRRSV) employs complex strategies to synthesize subgenomic RNAs (sgRNAs); however, their plasticity and temporal dynamics remain largely unexplored. Using next-generation sequencing (NGS), we examined the high-resolution landscape of the PRRSV subgenome, highlighting considerable heterogeneity in temporal kinetics and transcriptional control and revealing extensive coordination between TRSL-dependent and TRSL-independent sgRNAs. In addition, a comprehensive re-annotation of transcription regulatory sequence (TRS) locations was conducted, clarifying that their usage involved canonical, alternative, and non-canonical splicing events for annotated genes. These insights emphasize that the coding of genetic material in PRRSV is far more intricate than previously anticipated. Collectively, the altered sgRNA phenotype offers distinctive insights into PRRSV transcription and gives additional impetus for mining the functional short- and long-range RNA-RNA interactome at active viral replication sites.

Canthin-6-one analogs block Newcastle disease virus proliferation via suppressing the Akt and ERK pathways.

Newcastle disease virus, a member of the Paramyxoviridae family, causes significant economic losses in poultry worldwide. To identify novel antiviral agents against NDV, 36 canthin-6-one analogs were evaluated in this study. Our data showed that 8 compounds exhibited excellent inhibitory effects on NDV replication with IC50 values in the range of 5.26 to 11.76 µM. Besides, these analogs inhibited multiple NDV strains with IC50 values within 12 µM and exerted antiviral activity against peste des petits ruminants virus (PPRV) and canine distemper virus (CDV). Among these analogs, 16 presented the strongest anti-NDV activity (IC50 = 5.26 µM) and minimum cytotoxicity (CC50 > 200 µM) in DF-1 cells. Furthermore, 16 displayed antiviral activity in different cell lines. Our results showed that 16 did not affect the viral adsorption while it can inhibit the entry of NDV by suppressing the Akt pathway. Further study found that 16-treatment inhibited the NDV-activated ERK pathway, thereby promoting the expression of interferon-related genes. Our findings reveal an antiviral mechanism of canthin-6-one analogs through inhibition of the Akt and ERK signaling pathways. These results point to the potential value of canthin-6-one analogs to serve as candidate antiviral agents for NDV.

Characterization of Pseudomonas aeruginosa bacteriophages and control hemorrhagic pneumonia on a mice model.

Pseudomonas aeruginosa is one of the most common pathogens causing hemorrhagic pneumonia in Chinese forest musk deer. Multidrug-resistant P. aeruginosa is frequently isolated from the lungs of affected musk deer in Shaanxi Province, China. With the increasing bacterial drug resistance, commonly used antibiotics have shown limited efficacy against drug-resistant P. aeruginosa. Therefore, phages have garnered attention as a promising alternative to antibiotics among researchers. In this study, phages vB_PaeP_YL1 and vB_PaeP_YL2 (respectively referred to as YL1 and YL2) were isolated from mixed sewage samples from a farm. YL1 and YL2 exhibit an icosahedral head and a non-contractile short tail, belonging to the Podoviridae family. Identification results demonstrate good tolerance to low temperatures and pH levels, with minimal variation in potency within 30 min of UV irradiation. The MOI for both YL1 and YL2 was 0.1, and their one-step growth curve latent periods were 10 min and 20 min, respectively. Moreover, both single phage and phage cocktail effectively inhibited the growth of the host bacteria in vitro, with the phage cocktail showing superior inhibitory effects compared to the single phage. YL1 and YL2 possess double-stranded DNA genomes, with YL1 having a genome size of 72,187 bp and a total G + C content of 55.02%, while YL2 has a genome size of 72,060 bp and a total G + C content of 54.98%. YL1 and YL2 are predicted to have 93 and 92 open reading frames (ORFs), respectively, and no ORFs related to drug resistance or lysogeny were found in both phages. Genome annotation and phylogenetic analysis revealed that YL1 is closely related to vB_PaeP_FBPa1 (ON857943), while YL2 is closely related to vB_PaeP_FBPa1 (ON857943) and Phage26 (NC041907). In a mouse model of hemorrhagic pneumonia, phage cocktail treatment showed better control of the disease and significantly reduced lung bacterial load compared to single phage treatment. Therefore, YL1 and YL2 have the potential for the prevention and treatment of multidrug-resistant P. aeruginosa infections.

Genomic analysis of Clostridium perfringens type D isolates from goat farms.

C. perfringens type D strains are the leading cause of enterotoxaemia in ruminants such as goats, sheep, and cattle. However, there has been no prior research on the genomic characteristics of C. perfringens type D strains from various regions in China. Here, we investigated the antibiotic resistance, genomic characteristics, and phylogenetic relationship of C. perfringens type D isolates recovered from goat farms in Shaanxi, Gansu, and Ningxia provinces. The antibiotic resistance test indicated that the isolates displayed high minimum inhibitory concentration (MIC) values to sulfafurazole, whereas the other antibiotics tested, such as penicillin, enrofloxacin, and florfenicol, worked well on them. Additionally, only tetracycline resistance genes [tetA(P) and tetB(P)] were identified from the isolates. A collective of 13 toxin genes, including etx and cpe were detected among the isolates. Sequence comparison revealed that the etx and cpe genes shared high sequence identities, and they could coexist on a pCW3-like plasmid, representing a potential risk to both animal breeding and public health. Phylogenetic analysis using core genome multi-locus sequence typing (cgMLST) and core genome single nucleotide polymorphisms (SNPs) revealed the close genetic relationship and potential regional/transregional transmission of the C. perfringens type D isolates in Shaanxi and Gansu provinces. Furthermore, pan-genomic analysis suggested the functional differences at the protein-coding gene level, although isolates from the same source shared a close genetic relationship. In conclusion, this study indicated the antibiotic resistance, virulence markers, potential transregional transmission, and genomic diversity of C. perfringens type D strains from various regions in China, which could provide references for the prevention of C. perfringens foodborne diseases and further research.

Lineage 1 PRRSVs infection induces hemorrhagic injury in intestines of piglets: Effects on complement and coagulation cascades.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly transmissible disease of significant concern in the pig industry. Previous studies have demonstrated that the XM-2020 strain (a lineage 1.8 PRRSV IA/2012/NADC30) can induce special hemorrhagic injury in the small intestines. However, the specific mechanism underlying this injurious effect remains incompletely understood. In this study, we examined the pathogenic properties of XM-2020 and YC-2020 strains (a lineage 1.5 PRRSV IA/2014/NADC34) in piglets. Animal pathogenic tests revealed that with either Lineage 1 PRRSVs strains XM-2020 or YC-2020 demonstrated pronounced intestinal hemorrhage and suppression of peripheral immunological organs, comparing to JXA1 infection. Transcriptome analysis of diseased small intestines unveiled that PRRSV infection stimulated oxidative and inflammatory reactions. Remarkably, we also observed activation of the complement system alongside a notable down-regulation of complement and coagulation cascade pathways in the Lineage 1 PRRSVs infection group. Based on these findings, we propose that the primary mechanism driving the hemorrhagic injury of the small intestine caused by Lineage 1 PRRSVs is the suppression of complement and coagulation cascades resulting from immunosuppression. This discovery deepens our understanding of the pathogenicity of PRRSV in the small intestine and provides promising ways out for the development of innovative strategies aimed at controlling PRRSV.

The Ca2+-dependent phosphatase calcineurin dephosphorylates TBK1 to suppress antiviral innate immunity.

Tumor necrosis factor receptor-associated factor family member-associated NF-κB activator-binding kinase 1 (TBK1) plays a key role in the induction of the type 1 interferon (IFN-I) response, which is an important component of innate antiviral defense. Viruses target calcium (Ca2+) signaling networks, which participate in the regulation of the viral life cycle, as well as mediate the host antiviral response. Although many studies have focused on the role of Ca2+ signaling in the regulation of IFN-I, the relationship between Ca2+ and TBK1 in different infection models requires further elucidation. Here, we examined the effects of the Newcastle disease virus (NDV)-induced increase in intracellular Ca2+ levels on the suppression of host antiviral responses. We demonstrated that intracellular Ca2+ increased significantly during NDV infection, leading to impaired IFN-I production and antiviral immunity through the activation of calcineurin (CaN). Depletion of Ca²+ was found to lead to a significant increase in virus-induced IFN-I production resulting in the inhibition of viral replication. Mechanistically, the accumulation of Ca2+ in response to viral infection increases the phosphatase activity of CaN, which in turn dephosphorylates and inactivates TBK1 in a Ca2+-dependent manner. Furthermore, the inhibition of CaN on viral replication was counteracted in TBK1 knockout cells. Together, our data demonstrate that NDV hijacks Ca2+ signaling networks to negatively regulate innate immunity via the CaN-TBK1 signaling axis. Thus, our findings not only identify the mechanism by which viruses exploit Ca2+ signaling to evade the host antiviral response but also, more importantly, highlight the potential role of Ca2+ homeostasis in the viral innate immune response.IMPORTANCEViral infections disrupt intracellular Ca2+ homeostasis, which affects the regulation of various host processes to create conditions that are conducive for their own proliferation, including the host immune response. The mechanism by which viruses trigger TBK1 activation and IFN-I induction through viral pathogen-associated molecular patterns has been well defined. However, the effects of virus-mediated Ca2+ imbalance on the IFN-I pathway requires further elucidation, especially with respect to TBK1 activation. Herein, we report that NDV infection causes an increase in intracellular free Ca2+ that leads to activation of the serine/threonine phosphatase CaN, which subsequently dephosphorylates TBK1 and negatively regulates IFN-I production. Furthermore, depletion of Ca2+ or inhibition of CaN activity exerts antiviral effects by promoting the production of IFN-I and inhibiting viral replication. Thus, our results reveal the potential role of Ca2+ in the innate immune response to viruses and provide a theoretical reference for the treatment of viral infectious diseases.

The W195 Residue of the Newcastle Disease Virus V Protein Is Critical for Multiple Aspects of Viral Self-Regulation through Interactions between V and Nucleoproteins.

The transcription and replication of the Newcastle disease virus (NDV) strictly rely on the viral ribonucleoprotein (RNP) complex, which is composed of viral NP, P, L and RNA. However, it is not known whether other viral non-RNP proteins participate in this process for viral self-regulation. In this study, we used a minigenome (MG) system to identify the regulatory role of the viral non-RNP proteins V, M, W, F and HN. Among them, V significantly reduced MG-encoded reporter activity compared with the other proteins and inhibited the synthesis of viral mRNA and cRNA. Further, V interacted with NP. A mutation in residue W195 of V diminished V-NP interaction and inhibited inclusion body (IB) formation in NP-P-L-cotransfected cells. Furthermore, a reverse-genetics system for the highly virulent strain F48E9 was established. The mutant rF48E9-VW195R increased viral replication and apparently enhanced IB formation. In vivo experiments demonstrated that rF48E9-VW195R decreased virulence and retarded time of death. Overall, the results indicate that the V-NP interaction of the W195 mutant V decreased, which regulated viral RNA synthesis, IB formation, viral replication and pathogenicity. This study provides insight into the self-regulation of non-RNP proteins in paramyxoviruses.

Effects of a novel Bacillus subtilis GXYX crude lipopeptide against Salmonella enterica serovar Typhimurium infection in mice.

The increased rate of antibiotic resistance strongly limits the resolution of Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. Therefore, new strategies to control bacterial infections are urgently needed. Bacillus subtilis (B. subtilis) and its metabolites are desirable antibacterial agents. Here, we aimed to evaluate the antibacterial activity of the novel B. subtilis strain GXYX (No: PRJNA940956) crude lipopeptide against S. Typhimurium. In vitro, GXYX crude lipopeptides affected S. Typhimurium biofilm formation and swimming and attenuated the adhesion and invasion abilities of S. Typhimurium toward BHK-21 cells; in addition, it inhibited the mRNA expression of the filA, filC, csgA, and csgB genes, which are related to the adhesion and invasion ability of S. Typhimurium. In vivo, pretreatment with GXYX crude lipopeptide via intragastric administration improved the survival rate by 30%, which was related to reductions in organ bacterial loads and clinical signs in mice. Intragastric administration of GXYX crude lipopeptide significantly downregulated the mRNA levels of TNF-α, IL-1ß, IL-12 and IL-6 in response to S. Typhimurium-induced inflammation compared with intraperitoneal injection. Moreover, it significantly improved the intestinal barrier-related gene (ZO-1, claudin-1, occludin-1) mRNA levels in intestinal tissue damaged by S. Typhimurium infection. In conclusion, GXYX crude lipopeptides were effective at reducing S. Typhimurium colonization, laying a foundation for the further development of novel antibacterial agents.

Bovine parainfluenza virus type 3 infections induce ER stress-mediated autophagy to facilitate virus replication.

Bovine Parainfluenza Virus Type 3 (BPIV3) serves as a crucial pathogen in cattle, adept at triggering severe respiratory symptoms. This investigation explores the intricate interplay of endoplasmic reticulum stress (ER stress), unfolded protein response (UPR), and autophagy upon BPIV3 infection. In this study, we initially confirm a substantial increase in glucose regulatory protein 78 (GRP78) expression, accompanied by noticeable morphological changes and significant expansion of the ER lumen observed through transmission electron microscopy upon BPIV3 infection. Our findings indicate that ER Stress is induced during BPIV3 infection in vitro. Subsequently, we illustrate that BPIV3 triggers ER Stress to facilitate viral replication through heightened autophagy through treatment with the ER stress inhibitor 4-phenylbutyrate (4-PBA) and utilizing small interfering RNA (siRNA) technology to knock down GRP78. Additionally, we observe that the activation of ER stress initiates the UPR via PERK and ATF6 pathways, with the IRE1 pathway not contributing to the regulation of ER stress-mediated autophagy. Moreover, intervention with the PERK inhibitor GSK2606414, ATF6 inhibitor Ceapin-A7, and siRNA technology successfully reverses BPIV3-induced autophagy. In summary, these findings propose that BPIV3 induces ER stress to enhance viral replication through increased autophagy, with the PERK and ATF6 pathways playing a significant role in ER stress-mediated autophagy.

Construction and characterization of a reverse genetics system of bovine parainfluenza virus type 3c as a tool for rapid screening of antivirals in vitro.

Bovine parainfluenza virus type 3 (BPIV3) is a key pathogen associated with bovine respiratory disease complex (BRDC). However, its specific pathogenesis mechanisms have not been fully elucidated. Reverse genetics provides a useful method for understanding the pathogenic mechanism of BPIV3. To ensure the functionality of the rescue platforms, we first constructed a minigenome (MG) system of BPIV3 utilizing a 5-plasmid system in this investigation. Then, a full-length infection clone of BPIV3 was obtained from the SX-2021 strain, and different methods were employed to identify the rescued virus. Additionally, we recovered a recombinant BPIV3 using the reverse genetics system that could express enhanced green fluorescence protein (eGFP). Through the growth curve assays, the replicate capability of rBPIV3-SX-EGFP was found to be similar to that of the parental virus. Subsequently, the rBPIV3-SX-EGFP was used to determine the antiviral activity of ribavirin. The results showed that ribavirin had an anti-BPIV3 effect in MDBK cells. In conclusion, the successful development of a reverse genetic system for the SX-2021 strain establishes a foundation for future studies on BPIV3, including investigations into its pathogenic mechanism, gene function, and antiviral screening properties.

Bovine parainfluenza type 3 virus induces incomplete autophagy to promote viral replication by activated beclin1 in vitro.

Bovine Parainfluenza virus Type 3 (BPIV3) is one of the most important pathogens in cattle, capable of causing severe respiratory symptoms. Numerous studies have shown that autophagy plays a diverse role in the infection process of various pathogens. The influence of autophagy machinery on BPIV3 infection has not yet been confirmed. In the present study, we initially demonstrated that the expression of LC3 was significantly increased and exhibited a notable increase in double or single-membrane vesicles under a transmission electron microscope during BPIV3 infection. These observations unequivocally establish the induction of steady-state autophagy in vitro consequent to BPIV3 infection. Furthermore, quantification of autophagic flux substantiates the induction of an incomplete autophagic process during BPIV3 infection. Additionally, through targeted interventions, we demonstrate the regulatory impact of pharmacological agents influencing autophagy and RNA interference targeting an autophagy-associated protein on viral replication. Intriguingly, our data revealed that BPIV3 infection enhanced the phosphorylation of rapamycin kinase (mTOR). This result demonstrated that mTOR does not operate as a counteractive regulator of BPIV3-induced autophagy. Instead, we discern an augmentation in the expression of Beclin1, a key autophagy initiator, which complexes with Vps34, constituting a Class III phosphatidylinositol 3-kinase. This phenomenon serves as a hallmark in the inaugural phase of autophagy initiation during BPIV3 infection. Collectively, these discernments underscore that BPIV3 infection actively stimulates autophagy, thereby enhancing viral replication through the activation of Beclin1, independently of the mTOR signaling pathway. This nuanced comprehension significantly contributes to unraveling the intricate molecular mechanisms governing BPIV3-induced autophagy.

Porcine Epidemic Diarrhea Virus: Etiology, Epidemiology, Antigenicity, and Control Strategies in China.

Porcine epidemic diarrhea virus (PEDV) is a porcine enteric coronavirus, which is one of the main causative agents of porcine epidemic diarrhea (PED), with 100% morbidity and 80-100% mortality in neonatal piglets. Since 2010, large-scale PED caused by highly pathogenic variants of PEDV has occurred successively in China and other countries in the world, posing a great threat to the global pig industry. It has been demonstrated in many investigations that the classic attenuated vaccine strain, PEDV CV777, is insufficient to fully protect against the PEDV variants. Moreover, the maternally derived antibodies elicited by inactivated vaccines also cannot completely protect piglets from infection. In addition, feedback feeding poses a risk of periodic PEDV recurrence in pig farms, making it challenging to successfully limit the spread of PEDV in China. This review focuses on the etiology, epidemiology, antigenicity, and control strategies of PEDV in China and provides information for the formulation of effective control measures.

Erratum: MiR-375 Has Contrasting Effects on Newcastle Disease Virus Growth Depending on the Target Gene : Erratum.

[This corrects the article DOI: 10.7150/ijbs.25106.].

Characterization of Lung Microbiomes in Pneumonic Hu Sheep Using Culture Technique and 16S rRNA Gene Sequencing.

Hu sheep, a locally bred species in China known for its high productivity, is currently suffering from pneumonia. Here, we combine high-throughput 16SrRNA gene sequencing and bacterial culturing to examine the bacterial community in pneumonic Hu Sheep lungs (p < 0.05). The results showed that the abundance and diversity of lung bacteria in healthy sheep were significantly higher than those in pneumonia sheep (p = 0.139), while there was no significant difference between moderate and severe pneumonia. Furthermore, the composition of the lung microbiota community underwent significant alterations between different levels of pneumonia severity. The application of LEfSe analysis revealed a notable enrichment of Mannheimiae within the lungs of sheep afflicted with moderate pneumonia (p < 0.01), surpassing the levels observed in their healthy counterparts. Additionally, Fusobacterium emerged as the prevailing bacterial group within the lungs of sheep suffering from severe pneumonia. Integrating the results of bacterial isolation and identification, we conclusively determined that Mannheimia haemolytica was the primary pathogenic bacterium within the lungs of sheep afflicted with moderate pneumonia. Furthermore, the exacerbation of pneumonia may be attributed to the synergistic interplay between Fusobacterium spp. and other bacterial species. Our results provide new insights for guiding preventive and therapeutic measures for pneumonia of different severities in sheep.

Antigenic variation in hemagglutinin-neuraminidase of Newcastle disease virus isolated from Tibet, China.

Vaccines are widely used to prevent Newcastle disease virus (NDV). Under the pressure of immunization, NDVs with mutations among epitopes of F and HN protein were isolated, which indicates that the efficiency of vaccine may decrease in terms of preventing emerged NDV. However, the lack of evidences to support whether these mutations contribute to antigenic mutation and immune escape in NDV leading to the controversy that the matched vaccine is more effective than the mismatched vaccine. In this study, a genotype VII velogenic NDV strain (C22) was isolated from a vaccinated farm in Tibet, China. We found that this strain was close to NDV from east China, but it had a specific mutation (K138R) in one epitope (131DYIGGIGKE139) of HN protein. This mutation might change the interaction between amino acids in stalk-head link region of HN protein and then induce the specific antibody to worse recognize the C22 strain, but it did not alter viral virulence and growth ability. Then, the C22 strain was attenuated via modification of the F protein cleavage site to generate a matched vaccine. Comparing to a mismatched vaccine (LaSota), this matched vaccine showed advantages in inhibiting viral shedding and tissue damage. However, both vaccines induced chicken to generate similar level of neutralizing antibodies against C22, C22mut (R138K) and LaSota. These results suggest that the epitope mutation is insufficient to help NDV escaping neutralizing antibodies of vaccinated chicken, supporting that the merits of NDV matched vaccine are not totally related to humoral immunity.

Genome characteristics of the optrA-positive Clostridium perfringens strain QHY-2 carrying a novel plasmid type.

Clostridium perfringens is a bacterial species of importance to both public and animal health. The gene optrA is the first gene that confers resistance to the tedizolid, a last-resort antimicrobial agent in human medicine. Herein, we whole-genome sequenced and analyzed one optrA-positive C. perfringens strain QHY-2 from Tibetan sheep in Qinghai province and identified one optrA plasmid pQHY-2. The plasmid shared similar structure with the optrA-positive plasmids p2C45 and p21-D-5b previously identified in C. perfringens, demonstrating the potential horizontal transmission of the optrA plasmids among C. perfringens strains. Annotation of the optrA-positive plasmids showed optrA and erm(A) located on a segment flanked by IS element IS1216E, and fexA, optrA, and erm(A) located on a segment flanked by IS element ISVlu1, which revealed the possible dissemination mechanism. Additionally, a Tn6218-like transposon carrying aac(6')-aph(2″) and erm(B) was also detected on pQHY-2, demonstrating the transposition of Tn6218 and spread of antibiotic resistance among Clostridium bacteria. Molecular analysis indicated the optrA-positive plasmids belonged to a plasmid type distinct from the pCW3-like plasmids, pCP13-like plasmids, or pIP404-like plasmids. Further structure analysis showed they might be formed by inserting segments into plasmid pCPCPI53k-r1_1, which coexist with two pCW3-like plasmids and one pCP13-like plasmid in C. perfringens strain CPI 53k-r1 isolated from a healthy human in Finland. IMPORTANCE Antimicrobial resistance is now a global concern posing threats to food safety and public health. The pCW3-like plasmids can encode several main toxin genes and three antibiotic resistance genes (ARGs), including tetA(P), tetB(P), and erm(B), which used to be considered as the main carrier of ARGs in Clostridium perfringens. In this study, we found the optrA plasmids, which belonged to a novel plasmid type, could also harbor many other ARGs, indicating this type of plasmid might be the potential repository of ARGs in C. perfringens. Additionally, this type of plasmid could coexist with the pCW3-like plasmids and pCP13-like plasmids that encoded toxin genes associated with gastrointestinal diseases, which showed the potential threat to public health.

Genetic and pathogenic characterization of an NADC-34-like isolate of porcine reproductive and respiratory syndrome virus.

In this study, an NADC34-like strain of porcine reproductive and respiratory syndrome virus (PRRSV), YC-2020, was isolated from a pig farm in Yuncheng, Shanxi Province, China. Phylogenetic and molecular evolutionary analysis showed that the genome sequence of YC-2020 was very similar to those of NADC34-like PRRSV strains in the ORF2-7 region. However, it was more closely related to NADC30-like PRRSV and highly pathogenic (HP) PRRSV in the NSP2 and NSP3-9 coding regions, respectively, suggesting that recombination had occurred between viruses belonging to lineages 1 and 8. Piglets infected with YC-2020 exhibited mild clinical signs, but they had severe histopathological lesions in their lungs. These findings reveal novel genetic and pathogenic features of this isolate.

Newcastle disease virus forms inclusion bodies with features of liquid-liquid phase separation.

Formation of inclusion bodies (IBs) is a hallmark of infections with negative-strand RNA viruses. Although the Newcastle disease virus (NDV) IBs had been observed in the 1950s, the characteristics of NDV IBs remained largely unknown. Here, we show that NDV infection triggers the formation of IBs that contain newly synthesized viral RNA. The structures of NDV IBs, observed by electron microscopy, were not membrane-bound. Fluorescence recovery after photobleaching a region of NDV IBs occurred rapidly, and IBs were dissolved by 1,6-hexanediol treatment, demonstrating they exhibited properties consistent with liquid-liquid phase separation (LLPS). We find the nucleoprotein (NP) and phosphoprotein (P) are sufficient to generate IB-like puncta, with the N arm domain and N core region of NP and the C terminus of P playing important roles in this process. In summary, our findings suggest that NDV forms IBs containing viral RNA, and provide insights into the formation of NDV IBs.

Probiotics and vitamins modulate the cecal microbiota of laying hens submitted to induced molting.

Induced molting enables laying hens to relax, restore energy and prolong the laying hen cycle, resolving problems such as poor egg quality and minimizing economic losses caused by rising global feeding costs. However, traditional molting methods may disrupt gut microflora and promote potential pathogens infections. This study used a customized additive with a mixture of probiotics and vitamins to induce molting and examine the cecal microbiota post molting. A total of two hundred 377 day-of-ISA Brown laying hens were randomly assigned to four groups: non-molt with basal diet (C), 12-day feeding restriction (FR) in earlier-molting (B), feed again to 27.12% egg production in middle-molting (A) and reach second peak of egg production over 81.36% in post-molting (D). Sequencing 16S rRNA to analyze cecal microbial composition revealed that there is no significant change in bacterial community abundance post-molting. In contrast to group C, the number of potentially harmful bacteria such as E. coli and Enterococcus was not found to increase in groups B, A, or D. This additive keeps cecal microbiota diversity and community richness steady. In cecal contents, hens in group B had lower Lactobacillus, Lachnospiraceae and Prevotellaceae (vsC, A, and D), no significant differences were found between post-molting and the non-molting. Furthermore, cecal microbiota and other chemicals (antibodies, hormones, and enzymes, etc.) strongly affect immunological function and health. Most biochemical indicators are significantly positively correlated with Prevotellaceae, Ruminococcaceae and Subdoligranulum, while negatively with Phascolarctobacterium and Desulfovibrio. In conclusion, the additive of probiotics and vitamins improved the cecal microbiota composition, no increase in the associated pathogenic microbial community due to traditional molting methods, and enhances hepatic lipid metabolism and adaptive immunological function, supporting their application and induced molting technology in the poultry breeding industry.