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.

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.

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.

Simultaneous Expression of Chicken Granulocyte Monocyte Colony-Stimulating Factor and the Hemagglutinin-Neuraminidase Epitope of the Virulent Newcastle Disease Virus Genotype VII C22 Strain in a Functional Synthetic Recombinant Adenovirus as a Genotype-Matched Vaccine with Potential Antiviral Activity.

When it comes to the prevention of clinical signs and mortality associated with infection of the Newcastle disease virus (NDV), vaccination has been very effective. However, recent evidence has proven that more highly virulent strains are emerging that bypass existing immune protection and pose a serious threat to the global poultry industry. Here, a novel rescued adenovirus 5-coexpressed chicken granulocyte monocyte colony-stimulating factor (ChGM-CSF) bio-adjuvant and C22-hemagglutinin-neuraminidase (HN) boosted chickens' immunological genetic resistance and thus improved the immunological effectiveness of the critical new-generation vaccine in vitro and in vivo. Accordingly, the hemagglutination inhibition (HI) titers (log2) of the recombinant adenovirus (rAdv)-ChGM-CSF-HN-immunized chickens had greater, more persistent, and longer-lasting NDV-specific antibodies than the La Sota and rAdv-HN-inoculated birds. Moreover, humoral and adaptive immunological conditions were shown to be in harmony after rAdv-ChGM-CSF-HN inoculation and uniformly enhanced the expression of alpha interferon (IFN-α), IFN-ß, IFN-γ, interleukin-1ß (IL-1ß), IL-2, IL-16, IL-18, and IL-22. Postchallenge, the control challenge (CC), wild-type adenovirus (wtAdv), and rAdv-ChGM-CSF groups developed unique NDV clinical manifestations, significant viral shedding, high tissue viral loads, gross and microscopic lesions, and 100% mortality within 7 days. The La Sota, rAdv-HN, and rAdv-ChGM-CSF-HN groups were healthy and had 100% survival rates. The rAdv-ChGM-CSF-HN group swiftly regulated and stopped viral shedding and had lower tissue viral loads than all groups at 5 days postchallenge (dpc). Thus, the antiviral activity of ChGM-CSF offered robust immune protection in the face of challenge and reduced viral replication convincingly. Our advance innovation concepts, combining ChGM-CSF with a field-circulating strain epitope, could lead to the development of a safe, genotype-matched, universal transgenic vaccine that could eradicate the disease globally, reducing poverty and food insecurity. IMPORTANCE We studied the biological characterization of the developed functional synthetic recombinant adenoviruses, which showed a high degree of safety, thermostability, and genetic stability for up to 20 passages. It was demonstrated through both in vitro and in vivo testing that the immunogenicity of the proposed vaccine, which uses the T2A peptide from the Thosea asigna virus capsid protein supported by glycine and serine, helps with efficiency to generate a multicistronic vector, enables expression of two functional proteins in rAdv-ChGM-CSF-HN, and is superior to that of comparable vaccines. Additionally, adenovirus can be used to produce vaccines matching the virulent field-circulating strain epitope. Because there is no preexisting human adenoviral immunity detected in animals, the potency of adenoviral vaccines looks promising. Also, it ensures that the living vector does not carry the resistance gene that codes for the kanamycin antibiotic. Accordingly, a human recombinant adenoviral vaccine that has undergone biological improvements is beneficial and important.

Chicken ISG12(2) attenuates Newcastle disease virus and enhances the efficiency of Newcastle disease vaccine via activating immune pathways.

Low virulence and strong immunogenicity are quite important for Newcastle disease virus (NDV) producing Newcastle disease (ND) living-attenuated vaccine. However, immunogenicity of NDV positively correlates to its virulence. Usually, the velogenic NDV induces stronger immune responses of poultry than the lentogenic strain, but virulent NDV poses a risk for chicken. In this study, we identified the chicken interferon (IFN)-stimulated gene 12-2 (ISG12(2)) not only attenuated NDV, but also increased immunogenicity of ND vaccine strain. First, we found that NDV infection or IFNs stimulation induced expression of chicken ISG12(2) that reinforced expression of IFNs. Over-expression or knock-down proved that chicken ISG12(2) inhibited NDV replication. Then, recombinant NDV LaSota strains (rLaSota/Fmut/ISG12(2) and rLaSota/ISG12(2)), expressing ISG12(2), were rescued. Pathogenicity tests showed that ISG12(2) expression attenuated NDV. RNA-seq or RT-qPCR demonstrated that, comparing to rLaSota/Fmut and rLaSota, rLaSota/Fmut/ISG12(2) and rLaSota/ISG12(2) induced hosts to produce cytokines enriching in innate and adaptive immune pathways in vitro and in vivo. Finally, we showed that rLaSota/ISG12(2) vaccination improved immune condition of chicken to quickly respond NDV infection and then enhance protection. These results suggest that chicken ISG12(2) is a potential novel molecular adjuvant to regulate immune responses, which decrease virulence and increase immunogenicity of NDV. The chicken ISG12(2) may contribute to the development of high efficient poultry vaccine.