The NLRP3 inflammasome consists of NLRP3, ASC, and pro-caspase-1 and is an important arm of the innate immune response against influenza A virus (IAV) infection. Upon infection, the inflammasome is activated, resulting in the production of IL-1β and IL-18, which recruits other immune cells to the site of infection. It has been suggested that in the presence of stress molecules such as nigericin, the trans-Golgi network (TGN) disperses into small puncta-like structures where NLRP3 is recruited and activated. Here, we investigated whether IAV infection could lead to TGN dispersion, whether dispersed TGN (dTGN) is responsible for NLRP3 inflammasome activation, and which viral protein is involved in this process. We showed that the IAV causes dTGN formation, which serves as one of the mechanisms of NLRP3 inflammasome activation in response to IAV infection. Furthermore, we generated a series of mutant IAVs that carry mutations in the M2 protein. We demonstrated the M2 proton channel activity, specifically His37 and Trp41 are pivotal for the dispersion of TGN, NLRP3 conformational change, and IL-1β induction. The results revealed a novel mechanism behind the activation and regulation of the NLRP3 inflammasome in IAV infection.

Rotaviruses (RVs) are a major cause of diarrhea in young children worldwide. The currently available and licensed vaccines contain live attenuated RVs. Optimization of live attenuated RV vaccines or developing non-replicating RV (e.g., mRNA) vaccines is crucial for reducing the morbidity and mortality from RV infections. Herein, a nucleoside-modified mRNA vaccine encapsulated in lipid nanoparticles (LNP) and encoding the VP7 protein from the G1 type of RV was developed. The 5' untranslated region of an isolated human RV was utilized for the mRNA vaccine. After undergoing quality inspection, the VP7-mRNA vaccine was injected by subcutaneous or intramuscular routes into mice. Mice received three injections in 21 d intervals. IgG antibodies, neutralizing antibodies, cellular immunity, and gene expression from peripheral blood mononuclear cells were evaluated. Significant differences in levels of IgG antibodies were not observed in groups with adjuvant but were observed in groups without adjuvant. The vaccine without adjuvant induced the highest antibody titers after intramuscular injection. The vaccine elicited a potent antiviral immune response characterized by antiviral clusters of differentiation CD8+ T cells. VP7-mRNA induced interferon-γ secretion to mediate cellular immune responses. Chemokine-mediated signaling pathways and immune response were activated by VP7-mRNA vaccine injection. The mRNA LNP vaccine will require testing for protective efficacy, and it is an option for preventing rotavirus infection.

Inactivated vaccines based on cell culture are very useful in the prevention and control of many diseases. The most popular strategy for the production of inactivated vaccines is based on monkey-derived Vero cells, which results in high productivity of the virus but has a certain carcinogenic risk due to non-human DNA contamination. Since human diploid cells, such as MRC-5 cells, can produce a safer vaccine, efforts to develop a strategy for inactivated vaccine production using these cells have been investigated using MRC-5 cells. However, most viruses do not replicate efficiently in MRC-5 cells. In this study, we found that rabies virus (RABV) infection activated a robust interferon (IFN)-β response in MRC-5 cells but almost none in Vero cells, suggesting that the IFN response could be a key limiting factor for virus production. Treatment of the MRC-5 cells with IFN inhibitors increased RABV titers by 10-fold. Additionally, the RABV titer yield was improved five-fold when using IFN receptor 1 (IFNAR1) antibodies. As such, we established a stable IFNAR1-deficient MRC-5 cell line (MRC-5IFNAR1-), which increased RABV production by 6.5-fold compared to normal MRC-5 cells. Furthermore, in a pilot-scale production in 1500 square centimeter spinner flasks, utilization of the MRC-5IFNAR1- cell line or the addition of IFN inhibitors to MRC cells increased RABV production by 10-fold or four-fold, respectively. Thus, we successfully established a human diploid cell-based pilot scale virus production platform via inhibition of IFN response for rabies vaccines, which could also be used for other inactivated virus vaccine production.

An increasing number of studies have begun considering human endogenous retroviruses (HERVs) as potential pathogenic phenomena. Our previous research suggests that HERV-W Envelope (HERV-W ENV), a HERV-W family envelope protein, is elevated in schizophrenia patients and contributes to the pathophysiology of schizophrenia. The dopamine (DA) hypothesis is the cornerstone in research and clinical practice related to schizophrenia. Here, we found that the concentration of DA and the expression of DA receptor D2 (DRD2) were significantly higher in schizophrenia patients than in healthy individuals. Intriguingly, there was a positive correlation between HERV-W ENV and DA concentration. Depth analyses showed that there was a marked consistency between HERV-W ENV and DRD2 in schizophrenia. Studies in vitro indicated that HERV-W ENV could increase the DA concentration by regulating DA metabolism and induce the expression of DRD2. Co-IP assays and laser confocal scanning microscopy indicated cellular colocalization and a direct interaction between DRD2 and HERV-W ENV. Additionally, HERV-W ENV caused structural and functional abnormalities of DA neurons. Further studies showed that HERV-W ENV could trigger the PP2A/AKT1/GSK3 pathway via DRD2. A whole-cell patch-clamp analysis suggested that HERV-W ENV enhanced sodium influx through DRD2. In conclusion, we uncovered a relationship between HERV-W ENV and the dopaminergic system in the DA neurons. Considering that GNbAC1, a selective monoclonal antibody to the MSRV-specific epitope, has been promised as a therapy for treating type 1 diabetes and multiple sclerosis (MS) in clinical trials, understanding the precise function of HERV-W ENV in the dopaminergic system may provide new insights into the treatment of schizophrenia.

Rotavirus (RV) is the major causes of severe diarrhea in infants and young children under five years of age. There are no effective drugs for the treatment of rotavirus in addition to preventive live attenuated vaccine. Recent evidence demonstrates that microRNAs (miRNAs) can affect RNA virus replication. However, the antiviral effect of miRNAs during rotavirus replication are largely unknown. Here, we determined that miR-7 is upregulated during RV replication and that it targets the RV NSP5 (Nonstructural protein 5). Results suggested that miR-7 affected viroplasm formation and inhibited RV replication by down-regulating RV NSP5 expression. Up-regulation of miR-7 expression is a common regulation method of different G-type RV-infected host cells. Then, we further revealed the antiviral effect of miR-7 in diarrhea suckling mice model. MiR-7 is able to inhibit rotavirus replication in vitro and in vivo. These data provide that understanding the role of cellular miR-7 during rotaviral replication may help in the identification of novel therapeutic small RNA molecule drug for anti-rotavirus.

Highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H7N9 viruses pose a severe threat to public health through zoonotic infection, causing severe respiratory disease in humans. While HPAI H5N1 human infections have typically been reported in Asian countries, avian H7N9 human infections have been reported mainly in China. However, Canada reported a case of fatal human infection by the HPAI H5N1 virus in 2014, and two cases of human illness associated with avian H7N9 virus infection in 2015. While the genomes of the causative viruses A/Alberta/01/2014 (H5N1) (AB14 (H5N1)) and A/British Columbia/1/2015 (H7N9) (BC15 (H7N9)) are reported, the isolates had not been evaluated for their pathogenicity in animal models. In this study, we characterized the pathogenicity of AB14 (H5N1) and BC15 (H7N9) and found that both strain isolates are highly lethal in mice. AB14 (H5N1) caused systemic viral infection and erratic proinflammatory cytokine gene expression in different organs. In contrast, BC15 (H7N9) replicated efficiently only in the respiratory tract, and was a potent inducer for proinflammatory cytokine genes in the lungs. Our study provides experimental evidence to complement the specific human case reports and animal models for evaluating vaccine and antiviral candidates against potential influenza pandemics.

An emerging virus-like flower yellowing disease (FYD) of green Sichuan pepper (Zanthoxylum armatum v. novemfolius) has been recently reported. Four new RNA viruses were discovered in the FYD-affected plant by the virome analysis using high-throughput sequencing of transcriptome and small RNAs. The complete genomes were determined, and based on the sequence and phylogenetic analysis, they are considered to be new members of the genera Nepovirus (Secoviridae), Idaeovirus (unassigned), Enamovirus (Luteoviridae), and Nucleorhabdovirus (Rhabdoviridae), respectively. Therefore, the tentative names corresponding to these viruses are green Sichuan pepper-nepovirus (GSPNeV), -idaeovirus (GSPIV), -enamovirus (GSPEV), and -nucleorhabdovirus (GSPNuV). The viral population analysis showed that GSPNeV and GSPIV were dominant in the virome. The small RNA profiles of these viruses are in accordance with the typical virus-plant interaction model for Arabidopsis thaliana. Rapid and sensitive RT-PCR assays were developed for viral detection, and used to access the geographical distributions. The results revealed a correlation between GSPNeV and the FYD. The viruses pose potential threats to the normal production of green Sichuan pepper in the affected areas due to their natural transmission and wide spread in fields. Collectively, our results provide useful information regarding taxonomy, transmission and pathogenicity of the viruses as well as management of the FYD.

Nucleotide-binding domain and leucine-rich repeat-containing protein 3 (NLRP3) inflammasome plays a pivotal role in modulating lung inflammation in response to the influenza A virus infection. We previously showed that the swine influenza virus (SIV) infection induced NLRP3 inflammasome-mediated IL-1β production in primary porcine alveolar macrophages (PAMs), and we were interested in examining the upstream signaling events that are involved in this process. Here, we report that the SIV-infection led to dynamin-related protein 1 (DRP1) phosphorylation at serine 579 and mitochondrial fission in PAMs. IL-1β production was dependent on the reactive oxygen species (ROS) production, and DRP1 phosphorylation resulted in the upregulation of the NLRP3 inflammasome. Furthermore, the requirement of the kinase activity of receptor-interacting protein kinase 1 (RIPK1) for the IL-1β production and RIPK1-DRP1 association suggested that RIPK1 is an upstream kinase for DRP1 phosphorylation. Our results reveal a critical role of the RIPK1/DRP1 signaling axis, whose activation leads to mitochondrial fission and ROS release, in modulating porcine NLRP3 inflammasome-mediated IL-1β production in SIV-infected PAMs.

The constant crosstalk between the large avian reservoir of influenza A viruses (IAV) and its mammalian hosts drives viral evolution and facilitates their host switching. Direct adaptation of an avian strain to human or reassortment between avian-origin gene segments with that of human strains are the two mechanisms for the emergence of pandemic viruses. While it was suggested that the 1918 pandemic virus is of avian origin, reassortment of 1918 human isolates and avian influenza viruses led to the generation of 1957 and 1968 pandemic viruses. Interestingly, the avian PB1 segment, which encodes the catalytic subunit of IAV polymerase, is present in the 1957 and 1968 pandemic viruses. The biological consequence and molecular basis of such gene exchange remain less well understood. Using the 2009 pandemic H1N1 virus as a model, whose polymerase contains a human-origin PB1 subunit, we demonstrate that the acquisition of an avian PB1 markedly enhances viral RNA synthesis. This enhancement is also effective in the absence of PB2 adaptive mutations, which are key determinants of host switching. Mechanistically, the avian-origin PB1 does not appear to affect polymerase assembly but imparts the reassorted pandemic polymerase-augmented viral primary transcription and replication. Moreover, compared to the parental pandemic polymerase, the reassorted polymerase displays comparable complementary RNA (cRNA)-stabilizing activity but is specifically enhanced in progeny viral RNA (vRNA) synthesis from cRNA in a trans-activating manner. Overall, our results provide the first insight into the mechanism via which avian-origin PB1 enhances viral RNA synthesis of the 2009 pandemic virus polymerase.

Influenza A viruses (IAVs) can cause a highly contagious respiratory disease for many mammalian species. In pigs, IAVs cause high morbidity and low mortality disease in susceptible populations that can have significant financial and production impacts. They can also present opportunities for mutations and gene reassortment, producing influenza strains with pandemic potential. Therefore, it is very important to prevent and control influenza infection in pigs, and the chief way to do so is through vaccination. The subtypes of IAV most prevalent in swine across the world are H1N1, H1N2, and H3N2; however, genetic diversity of these viruses can vary greatly by region. We previously developed an elastase-dependent bivalent live attenuated vaccine using two Canadian swine influenza A virus (swIAV) isolates, A/Swine/Alberta/SD0191/2016 (H1N2) [SD191] and A/Swine/Saskatchewan/SD0069/2015 (H3N2) [SD69], which provided protection against homologous strains. In this study, we demonstrate that this vaccine extends protection in pigs to more current, drifted non-homologous H1N2 and H3N2 strains, A/Swine/MB/SD0467/2019 (H1N2) [SD467] and A/Swine/AB/SD0435/2019 (H3N2) [SD435]. The vaccine elicited a robust immune response in the serum and the lung and reduced viral replication as well as lung pathology associated with these strains. Therefore, this bivalent vaccine remains a strong candidate that would be beneficial to the swine influenza vaccine market in North America.

Genital herpes (GH) has become one of the most common sexually transmitted diseases worldwide, and it is spreading rapidly in developing countries. Approximately 90% of GH cases are caused by HSV-2. Therapeutic HSV-2 vaccines are intended for people already infected with HSV-2 with the goal of reducing clinical recurrences and recurrent virus shedding. In our previous work, we evaluated recombinant adenovirus-based vaccines, including rAd-gD2ΔUL25, rAd-ΔUL25, and rAd-gD2, for their potency as prophylactic vaccines. In this study, we evaluated these three vaccines as therapeutic vaccines against acute and recurrent diseases in intravaginal challenged guinea pigs. Compared with the control groups, the recombinant vaccine rAd-gD2ΔUL25 induced a higher titer of the binding antibody, and rAd-gD2 + rAd-ΔUL25 induced a higher titer of the neutralizing antibody. Both rAd-gD2ΔUL25 and rAd-gD2 + rAd-ΔUL25 vaccines significantly enhanced the survival rate by 50% compared to rAd-gD2 and reduced viral replication in the genital tract and recurrent genital skin disease. Our findings provide a new perspective for HSV-2 therapeutic vaccine research and provide a new technique to curtail the increasing spread of HSV-2.