Early Transcriptional Changes in Rabies Virus-Infected Neurons and Their Impact on Neuronal Functions

Rabies is a zoonotic disease caused by rabies virus (RABV). As rabies advances, patients develop a variety of severe neurological symptoms that inevitably lead to coma and death. Unlike other neurotropic viruses that can induce symptoms of a similar range, RABV-infected post-mortem brains do not show significant signs of inflammation nor the structural damages on neurons. This suggests that the observed neurological symptoms possibly originate from dysfunctions of neurons. However, many aspects of neuronal dysfunctions in the context of RABV infection are only partially understood, and therefore require further investigation. In this study, we used differentiated neurons to characterize the RABV-induced transcriptomic changes at the early time-points of infection. We found that the genes modulated in response to the infection are particularly involved in cell cycle, gene expression, immune response, and neuronal function-associated processes. Comparing a wild-type RABV to a mutant virus harboring altered matrix proteins, we found that the RABV matrix protein plays an important role in the early down-regulation of host genes, of which a significant number is involved in neuronal functions. The kinetics of differentially expressed genes (DEGs) are also different between the wild type and mutant virus datasets. The number of modulated genes remained constant upon wild-type RABV infection up to 24 h post-infection, but dramatically increased in the mutant condition. This result suggests that the intact viral matrix protein is important to control the size of host gene modulation. We then examined the signaling pathways previously studied in relation to the innate immune responses against RABV, and found that these pathways contribute to the changes in neuronal function-associated processes. We further examined a set of regulated genes that could impact neuronal functions collectively, and demonstrated in calcium imaging that indeed the spontaneous activity of neurons is influenced by RABV infection. Overall, our findings suggest that neuronal function-associated genes are modulated by RABV early on, potentially through the viral matrix protein-interacting signaling molecules and their downstream pathways.

A double deletion prevents replication of the pestivirus bovine viral diarrhea virus in the placenta of pregnant heifers

In contrast to wild type bovine viral diarhea virus (BVDV) specific double deletion mutants are not able to establish persistent infection upon infection of a pregnant heifer. Our data shows that this finding results from a defect in transfer of the virus from the mother animal to the fetus. Pregnant heifers were inoculated with such a double deletion mutant or the parental wild type virus and slaughtered pairwise on days 6, 9, 10 and 13 post infection. Viral RNA was detected via qRT-PCR and RNAscope analyses in maternal tissues for both viruses from day 6 p.i. on. However, the double deletion mutant was not detected in placenta and was only found in samples from animals infected with the wild type virus. Similarly, high levels of wild type viral RNA were present in fetal tissues whereas the genome of the double deletion mutant was not detected supporting the hypothesis of a specific inhibition of mutant virus replication in the placenta. We compared the induction of gene expression upon infection of placenta derived cell lines with wild type and mutant virus via gene array analysis. Genes important for the innate immune response were strongly upregulated by the mutant virus compared to the wild type in caruncle epithelial cells that establish the cell layer on the maternal side at the maternal–fetal interface in the placenta. Also, trophoblasts which can be found on the fetal side of the interface showed significant induction of gene expression upon infection with the mutant virus although with lower complexity. Growth curves recorded in both cell lines revealed a general reduction of virus replication in caruncular epithelial cells compared to the trophoblasts. Compared to the wild type virus this effect was dramtic for the mutant virus that reached only a TCID50 of 1.0 at 72 hours post infection.

Modelling Mutation in Equine Infectious Anemia Virus Infection Suggests a Path to Viral Clearance with Repeated Vaccination

Equine Infectious Anemia Virus (EIAV) is a lentivirus similar to HIV that infects horses. Clinical and experimental studies demonstrating immune control of EIAV infection hold promise for efforts to produce an HIV vaccine. Antibody infusions have been shown to block both wild-type and mutant virus infection, but the mutant sometimes escapes. Using these data, we develop a mathematical model that describes the interactions between antibodies and both wild-type and mutant virus populations, in the context of continual virus mutation. The aim of this work is to determine whether repeated vaccinations through antibody infusions can reduce both the wild-type and mutant strains of the virus below one viral particle, and if so, to examine the vaccination period and number of infusions that ensure eradication. The antibody infusions are modelled using impulsive differential equations, a technique that offers insight into repeated vaccination by approximating the time-to-peak by an instantaneous change. We use impulsive theory to determine the maximal vaccination intervals that would be required to reduce the wild-type and mutant virus levels below one particle per horse. We show that seven boosts of the antibody vaccine are sufficient to eradicate both the wild-type and the mutant strains. In the case of a mutant virus infection that is given infusions of antibodies targeting wild-type virus (i.e., simulation of a heterologous infection), seven infusions were likewise sufficient to eradicate infection, based upon the data set. However, if the period between infusions was sufficiently increased, both the wild-type and mutant virus would eventually persist in the form of a periodic orbit. These results suggest a route forward to design antibody-based vaccine strategies to control viruses subject to mutant escape.

Characterization of BoHV-1 gG-/tk-/gE- Mutant in Differential Protein Expression, Virulence, and Immunity

Infectious bovine rhinotracheitis (IBR), caused by bovine alphaherpesvirus 1 (BoHV-1), is an important disease affecting cattle worldwide resulting in great economic losses. Marker vaccines are effective in controlling infectious diseases including IBR, because they allow the discrimination between the natural infection and the vaccination. Therefore, a triple gene deleted strain BoHV-1 gG-/tk-/gE- was developed and evaluated in vivo and in vitro as a marker vaccine. In cell culture, this triple mutant virus showed significantly slower growth kinetics and smaller plaques when compared to wild-type (wt) BoHV-1 and double mutant BoHV-1 gG-/tk- (p < 0.01). On proteomic level, it revealed downregulation of some virulence related proteins including thymidine kinase, glycoproteins G, E, I, and K when compared to the wt. In vitro, the triple mutant virus showed a significantly lower and shorter viral shedding period (p < 0.001) in calves compared to double mutant. Moreover, the immunized calves with triple mutant virus showed protection rates of 64.2% and 68.6% against wt BoHV-1 and wt BoHV-5 challenge, respectively, without reactivation of latency after dexamethasone injection. In conclusion, BoHV-1 gG-/tk-/gE- is a safer marker vaccine against IBR although its immunogenicity in calves was decreased when compared to double mutant virus.

Influenza a virus NS1 resembles a TRAF3-interacting motif to target the RNA sensing-TRAF3-type I IFN axis and impair antiviral innate immunity

Background Influenza A virus (IAV) evolves strategies to counteract the host antiviral defense for establishing infection. The influenza A virus (IAV) non-structural protein 1 (NS1) is a key viral factor shown to counteract type I IFN antiviral response mainly through targeting RIG-I signaling. Growing evidence suggests that viral RNA sensors RIG-I, TLR3, and TLR7 function to detect IAV RNA in different cell types to induce type I IFN antiviral response to IAV infection. Yet, it remains unclear if IAV NS1 can exploit a common mechanism to counteract these RNA sensing pathways to type I IFN production at once, then promoting viral propagation in the host. Methods Luciferase reporter assays were conducted to determine the effect of NS1 and its mutants on the RIG-I and TLR3 pathways to the activation of the IFN-β and NF-κB promoters. Coimmunoprecipitation and confocal microscopic analyses were used to the interaction and colocalization between NS1 and TRAF3. Ubiquitination assays were performed to study the effect of NS1 and its mutants on TRAF3 ubiquitination. A recombinant mutant virus carrying NS1 E152A/E153A mutations was generated by reverse genetics for biochemical, ex vivo, and in vivo analyses to explore the importance of NS1 E152/E153 residues in targeting the RNA sensing-TRAF3-type I IFN axis and IAV pathogenicity. Results Here we report that NS1 subverts the RIG-I, TLR3, and TLR7 pathways to type I IFN production through targeting TRAF3 E3 ubiquitin ligase. NS1 harbors a conserved FTEE motif (a.a. 150-153), in which the E152/E153 residues are critical for binding TRAF3 to block TRAF3 ubiquitination and type I IFN production by these RNA sensing pathways. A recombinant mutant virus carrying NS1 E152A/E153A mutations induces higher type I IFN production ex vivo and in vivo, and exhibits the attenuated phenotype in infected mice, indicating the importance of E152/E153 residues in IAV pathogenicity. Conclusions Together our work uncovers a novel mechanism of IAV NS1-mediated immune evasion to promote viral infection through targeting the RNA sensing-TRAF3-type I IFN axis.

Collaboration of a detrimental HLA-B*35:01 allele with HLA-A*24:02 in co-evolution of HIV-1 with T-cells leading to poorer clinical outcomes

Although mutant-specific T-cells are elicited in some individuals infected with HIV-1 mutant viruses, the detailed characteristics of these T-cells remain unknown. A recent study showed that the accumulation of strains expressing Nef135F, which were selected by HLA-A*24:02-restricted T-cells, was associated with poor outcomes in individuals with the detrimental HLA-B*35:01 allele, and that HLA-B*35:01-restricted NefYF9(Nef135-143)-specific T-cells failed to recognize target cells infected with Nef135F mutant viruses. Here we investigated HLA-B*35:01-restricted T-cells specific for the NefFF9 epitope incorporating the Nef135F mutation. Longitudinal TCR clonotype analysis demonstrated that 3 types of HLA-B*35:01-restricted T-cells (wild type-specific, mutant-specific, and cross-reactive) with different T-cell repertoires were elicited during the clinical course. HLA-B*35:01 + individuals possessing wild type-specific T-cells had a significantly lower pVL than those with mutant-specific and/or cross-reactive T-cells, even though the latter T-cells effectively recognized the mutant virus-infected cells. These results suggest that mutant-specific and cross-reactive T-cells could only partially suppress HIV-1 replication in vivo. Ex vivo analysis of the T-cells showed higher expression of PD-1 on cross-reactive T-cells and lower expression of CD160/2B4 on the mutant-specific T cells than other T-cells, implying that these inhibitory and stimulatory molecules are key to the reduced function of these T-cells. In the present study, we demonstrate that mutant-specific and cross-reactive T-cells do not contribute to suppression of HIV-1 replication in HIV-1-infected individuals, even though they have the capacity to recognize mutant virus-infected cells. Thus, the collaboration of HLA-A*24:02 with the detrimental allele HLA-B*35:01 resulted in the co-evolution of HIV-1 alongside virus-specific T-cells, leading to poorer clinical outcomes. Importance HIV-1 escape mutations are selected under pressure from HIV-1-specific CD8 + T-cells. Accumulation of these mutations in circulating viruses impairs control of HIV-1 by HIV-1-specific T-cells. Although it is known that HIV-1-specific T-cells recognizing mutant virus were elicited in some individuals infected with mutant virus, the role of these T-cells remains unclear. Accumulation of Phenylalanine at HIV-1 Nef135 (Nef135F), which is selected by HLA-A*24:02-restricted T-cells, led to poor clinical outcome in individuals carrying the detrimental HLA-B*35:01 allele. In the present study, we found that HLA-B*35:01-restricted mutant-specific and cross-reactive T-cells were elicited in HLA-B*35:01 + individuals infected with Nef135F mutant virus. These T-cells could not effectively suppress HIV-1 replication in vivo even though they could recognize mutant virus-infected cells in vitro . Mutant-specific and cross-reactive T-cells expressed lower levels of stimulatory molecules and higher levels of inhibitory molecules, respectively, suggesting a potential mechanism whereby these T-cells fail to suppress HIV-1 replication in HIV-1-infected individuals.

Mutations at the Alphavirus E2/E1 inter-dimer interface have host-specific phenotypes

ABSTRACTAlphaviruses are enveloped viruses that are transmitted by an arthropod vector to vertebrate hosts. Alphaviruses have glycoprotein spikes on their particle surface which are essential for viral entry. Each of the 80 spikes on the surface of an alphavirus particle consists of a trimer of E2-E1 heterodimers. Two types of interactions make up the spikes: (1) interactions between E2 and E1 of the same heterodimer called intra-dimer contacts, and (2) inter-dimer interactions between E2 of one heterodimer and E1 of the adjacent heterodimer (called E1’). We hypothesized that the inter-dimer interactions are essential for trimerization of the E2-E1 heterodimers into a functional spike. In this work, we made a mutant virus where we replaced six inter-dimeric residues in Sindbis virus (WT SINV) with those from Chikungunya virus (CHIKV); the mutant is called CPB. CPB grew slower and to lower levels than WT SINV in mammalian cells, but not mosquito cells. When CPB virus was purified from mammalian cells, particles showed reduced amounts of glycoproteins relative to capsid protein, and defects in particle morphology compared to mosquito cells. CPB transported glycoproteins to the plasma membrane in similar amounts to WT SINV in mammalian cells. Two revertants, E2-H333N and E1-S247L, restored particle assembly to different degrees. The viruses were visualized by cryo-EM. We determined that the spikes of CPB had a different conformation than WT SINV or the revertants. We conclude that the inter-dimer mutant, CPB, has host-dependent defects in spike trimerization and/or particle budding in mammalian cells.IMPORTANCEAlphaviruses, which can cause disease when spread to humans by mosquitoes, have been classified as an emerging pathogen with a global distribution. The spikes on the surface of the alphavirus particle are absolutely required for the virus to enter a new host cell and initiate an infection. Using a structure-guided approach, we made a mutant virus that alters spike assembly in mammalian cells but not mosquito cells. This is important because it identifies a region in the spike that could be a target for antiviral drug design.

Infection spread simulation technology in a mixed state of multi valiant viruses

ATLM was extended to simulate the spread of infection in a mixed state of mutant virus and conventional virus. It is applied to the 4th wave of infection spread in Tokyo, and (1) the 4th wave bottoms out near the end of the state of emergency, and the number of infected people increases again. (2) The rate of increase will be mainly by L452R virus, while the increase by N501Y virus will be suppressed. (3) It is anticipated that the infection will spread during the Olympic Games. (4) When mutant virus competes, the infection of highly infectious virus rises sharply while the infection by weakly infectious ones has converged. (5) It is effective as an infection control measure to find an infected person early and shorten the period from infection to quarantine by PCR test or antigen test as a measure other than vaccine.