Amidst increasing concern about antibiotic resistance resulting from the overuse of antibiotics, there is a growing interest in exploring alternative agents. One such agent is citric acid, an organic compound commonly used for various applications. Our research findings indicate that the inclusion of citric acid can have several beneficial effects on the tight junctions found in the mouse intestine. Firstly, the study suggests that citric acid may contribute to weight gain by stimulating the growth of intestinal epithelial cells (IE-6). Citric acid enhances the small intestinal villus–crypt ratio in mice, thereby promoting intestinal structural morphology. Additionally, citric acid has been found to increase the population of beneficial intestinal microorganisms, including Bifidobacterium and Lactobacillus. It also promotes the expression of important protein genes such as occludin, ZO-1, and claudin-1, which play crucial roles in maintaining the integrity of the tight junction barrier in the intestines. Furthermore, in infected IEC-6 cells with H9N2 avian influenza virus, citric acid augmented the expression of genes closely associated with the influenza virus infection. Moreover, it reduces the inflammatory response caused by the viral infection and thwarted influenza virus replication. These findings suggest that citric acid fortifies the intestinal tight junction barrier, inhibits the replication of influenza viruses targeting the intestinal tract, and boosts intestinal immune function.

Proportions of QX-like genotype infectious bronchitis virus (IBV) isolates have increased over time. Here, to better understand the epidemiology and pathogenicity of IBV in China and control the spread of infectious bronchitis (IB), we conducted sequence analyses and examined the pathogenicity of 5 field isolates from diseased flocks in 2017 and 2018. Sequence analyses revealed that all the 5 strains, as well as many recent field isolates from other researchers, belonged to the QX-like IBV genotype, which were distantly related to commercial vaccine strains. Viral pathogenicity experiments showed that the isolates caused high morbidity and severe ciliostasis in chickens, although they caused milder lethality. This provides further evidence that QX-like IBV emergence remains a major problem in the poultry industry, and information on IBV epidemiology and pathogenicity may help to control IB.

H9N2 is currently the main subtype of avian influenza in China. In order to use reverse genetics to rapid preparation of seed strains for vaccine production, and intend to prevent and control the H9N2 subtype epidemic strains of avian influenza virus (AIV). In this study, we successfully rescued 2 H9N2 recombinant viruses based on the representative viruses of Southeast China and confirmed by RT-PCR and sequencing. Genetic stability, pathogenicity, transmissibility, and antigenicity of 2 recombinant viruses were evaluated. Compared to the FZ1, the growth kinetics of H9N2(HA+NA)/PR8 showed no significant difference, H9N2(HA+NA+M+PB1)/PR8 was slightly lower. Our study also confirmed 2 recombinant viruses had good genetic stability after 10 passages but possessed lower pathogenicity than FZ1. Although both recombinant viruses led to seroconversion in all inoculated birds on 14 dpi, they complete loss of viral transmission of the virus to contact birds. In addition, birds were immunized via hypodermic route by inactivated vaccines of H9N2(HA+NA)/PR8, H9N2(HA+NA+M+PB1)/PR8 and wild-type virus with a single dose, and the results showed that the hemagglutination inhibition titers on 21 dpv were 10.5, 9.6, and 10.5 log2, respectively. And recombinant viruses both provided a certain protection against wild-type virus challenge. In conclusion, these data indicated that 2 recombinant viruses will be expected to be used as inactivated vaccines to controlling the spread of H9N2 subtype AIV even have potential application for attenuated viral vaccines, which provides a reference for the prevention and control of influenza virus pandemics.

Long non-coding RNAs (lncRNAs) are critical regulators of various biological processes, yet their roles in chicken host-virus interactions remain largely unknown. This study investigated the impact of influenza A virus (IAV) H9N2 infection on the expression of two lncRNAs, Lnc9101 and Lnc8475, and the regulatory roles of these lncRNAs in the innate immune response. The results demonstrated that the expression of both lncRNAs were significantly upregulated in H9N2-infected chickens and could be induced by viral genomic RNA, poly (I:C) or LPS. Disruption of Lnc9101 expression in DF-1 cells enhanced the IAV replication, while its overexpression suppressed it. Conversely, Lnc8475 had the opposite effect on the IAV replication compared with Lnc9101. Silencing Lnc8475 expression in DF-1 cells attenuated IAV replication, while its overexpression promoted viral replication. Furthermore, we found that both lncRNAs regulated the expression of type I and type III interferons (IFNs), thereby affecting multiple interferon-stimulating genes (ISGs) expression and STAT1 activation upon IAV infection. Moreover, Lnc9101 knockdown impaired the expression of TLR7 and MDA5 and reduced IRF7 phosphorylation, whereas Lnc8475 knockdown promoted the expression of TLR3 and TLR7. In summary, Lnc9101 inhibits IAV replication by positively regulating the innate immune response, whereas Lnc8475 promotes IAV replication by suppressing it. These findings suggest that LncRNA9101 and Lnc8475 play important roles in pattern recognition receptors (PRRs)-dependent innate immune signaling and antiviral responses, advancing a better understanding of lncRNA functioning in chickens.

Vaccine adjuvants, serving as non-specific immune enhancers, play a pivotal role in the immunoprevention and control of animal diseases. This study utilized prokaryotic expression systems to express and purify chicken-derived cIFITM1, cIFITM3, and cViperin, which were then formulated as adjuvants with H9N2 avian influenza virus antigens to create inactivated vaccines. These vaccines were administered to SPF chickens to investigate their immunopotentiating functions. Additionally, the proteins were assessed for their ability to act as standalone immune enhancers. The results demonstrated that cIFITM1, cIFITM3, and cViperin significantly elevated serum hemagglutination inhibition (HI) antibody titers. Notably, when used individually, these proteins markedly enhanced the antiviral capabilities of challenged chickens, leading to alleviated clinical symptoms, reduced tracheal virus replication, diminished virus shedding, and lessened histopathological damage, with cIFITM1 exhibiting the most pronounced effect. Furthermore, the protective efficacy of two H9N2 recombinant virus inactivated vaccines supplemented with cIFITM1 adjuvant was validated, achieving a 100 % vaccine protection efficiency. In conclusion, cIFITM1, cIFITM3, and cViperin as adjuvants for influenza vaccines effectively inhibit virus replication and shedding, highlighting their significant potential in influenza prevention and control.

Vaccination is one of the most effective methods for controlling animal infectious diseases, and the use of adjuvants plays crucial role in enhancing the immune efficacy of vaccines, particularly in inactivated and subunit vaccines. With the continuous advancement of research in animal immunology and immune mechanisms, our understanding of the functions of cells and cytokines in immune responses has become increasingly comprehensive, laying a solid foundation for the development of novel vaccines and adjuvants. Cytokines are a class of proteins secreted by the animal body that regulate innate and adaptive immune responses through interaction with specific receptors. To date, numerous studies have investigated the potential of using cytokines as adjuvants to enhance the efficacy of veterinary vaccines. This review focuses on cytokines as veterinary vaccine adjuvants, with special attention to the current research progress and mechanisms of cytokines such as interleukins, interferons, chemokines, and colony-stimulating factors. Additionally, examples of the application of cytokine-based adjuvants in combination with veterinary vaccines will be discussed to provide further insights and references for the development of cytokine-based veterinary adjuvants.