Bacterial host resistance models in the evaluation of immunotoxicity

To assess potential immunomodulatory effects of a drug, pollutant, or natural product, an analysis of an exposed host's ability to resist challenge with a viable bacteria is one of the best gauges. Many factors govern whether a host exposed to a test agent and then infected becomes ill or dies at rates greater than infected control counterparts. Beyond the status of the host's immunocompetence, a bacterium's route of entry into the host and its inherent virulence are important variables determining how (and rate at which) an infection resolves. A pre-determination of endpoint(s) to be defined is critical during planning of resistance assays. If a study is to determine overall changes in immunocompetence due to exposure (regardless of regimen or dosage of test agent), then assessing shifts in morbidity/mortality at a defined lethal dose [LD(x)] value for the chosen route of infection would suffice. However, if a study is to define extent of immunomodulation in a particular body organ/cavity--or specific alterations in particular aspects of the humoral or cell-mediated immune responses--then careful selection of the pathogen, dose of the inoculum, means of infection of target site, and extent of the post-infection period to be examined, need to be made prior to host exposure to the test toxicant. This review will provide the Reader with background information about bacterial infections and how endpoint selection could be approached when designing resistance assays. An overview of protocols involved in the assays (e.g., bacterial preparation, host infection, post-infection endpoint analyses) and information about three bacteria that are among the most commonly employed in resistance assays is provided as well.     

Capsid processing requirements for abrogation of Fv1 and Ref1 restriction

Murine leukemia virus is restricted in mouse cells lines by a host factor known as Fv1 and in human cell lines by Ref1. Genetic evidence indicates that these restriction factors target the virus capsid (CA) protein. Restriction can be overcome by adding virus at a high multiplicity of infection, indicating that the restriction factors can be saturated. Cells preexposed to restricted virus will allow infection by a second virus which would normally be restricted. This phenomenon is known as abrogation; it provides us with a tool with which to study the interaction of virus with restriction factors. We tested the abilities of several Gag processing mutants to abrogate restriction. Our results show that CA must be cleaved from both p12 and nucleocapsid in order for the incoming virion to interact with the restriction factor. Endogenous expression of properly processed CA, however, failed to abrogate restriction. These results suggest that as well as being processed, CA must also be properly assembled in the form of a condensed viral core in order to interact with Fv1 and Ref1. This polymeric structure may contain restriction factor binding sites not present in monomeric CA.     

The effect of pneumonia induced in mice with Mycoplasma pulmonis on resistance to subsequent bacterial infection and the effect of a respiratory infection with Sendai virus on the resistance of mice to Mycoplasma pulmonis.

The effect of pneumonia induced by Mycoplasma pulmonis in mice on the resistance of the lung to additional bacterial infection was examined. The effect of pneumonia induced by Sendai virus on the resistance of mice to M. pulmonis was also investigated and compared with the effect of Sendai virus on resistance to Staphylococcus aureus. Sendai virus infection decreased subsequent resistance to M. pulmonis in proportion to the virus dose. Decreased resistance to subsequent S. aureus and M. pulmonis infection was greatest at about the same time after inoculation of virus and was related to virus-induced lesions. Besides affecting the resistance of mice to subsequent mycoplasma infection, Sendai virus could enhance an existing mycoplasma infection. Pneumonia induced by M. pulmonis did not decrease resistance to subsequent bacterial infection. The mechanism whereby Sendai virus decreases host resistance is therefore similar for bacteria and mycoplasmas, but pneumonia induced by mycoplasmas does not have the same effect.     

Vero Cell Proteomic Changes Induced by Zika Virus Infection

The re‐emergence and the recent spread of the Zika virus (ZIKV) has raised significant global concerns due to lack of information in patient diagnosis and management. Thus, in addition to gaining more basic information about ZIKV biology, appropriate interventions and management strategies are being sought to control ZIKV‐associated diseases and its spread. This study's objective is to identify host cell proteins that are significantly dysregulated during ZIKV infection. SOMAScan, a novel aptamer‐based assay, is used to simultaneously screen >1300 host proteins to detect ZIKV‐induced host protein dysregulation at multiple time points during infection. A total of 125 Vero cell host proteins, including cytokines such as CXCL11 and CCL5, interferon stimulated gene 15, and translation initiation factors EIF5A and EIF4G2, are significantly dysregulated after ZIKV infection. Bioinformatic analyses of 77 host proteins, that are significantly dysregulated ≥1.25‐fold, identify several activated biological processes, including the JAK/STAT, Tec kinase, and complement cascade pathways.     

Inferring the Source of Transmission with Phylogenetic Data

Identifying the source of transmission using pathogen genetic data is complicated by numerous biological, immunological, and behavioral factors. A large source of error arises when there is incomplete or sparse sampling of cases. Unsampled cases may act as either a common source of infection or as an intermediary in a transmission chain for hosts infected with genetically similar pathogens. It is difficult to quantify the probability of common source or intermediate transmission events, which has made it difficult to develop statistical tests to either confirm or deny putative transmission pairs with genetic data. We present a method to incorporate additional information about an infectious disease epidemic, such as incidence and prevalence of infection over time, to inform estimates of the probability that one sampled host is the direct source of infection of another host in a pathogen gene genealogy. These methods enable forensic applications, such as source-case attribution, for infectious disease epidemics with incomplete sampling, which is usually the case for high-morbidity community-acquired pathogens like HIV, Influenza and Dengue virus. These methods also enable epidemiological applications such as the identification of factors that increase the risk of transmission. We demonstrate these methods in the context of the HIV epidemic in Detroit, Michigan, and we evaluate the suitability of current sequence databases for forensic and epidemiological investigations. We find that currently available sequences collected for drug resistance testing of HIV are unlikely to be useful in most forensic investigations, but are useful for identifying transmission risk factors.     

Coxsackievirus B3-resistant mice become susceptible in Se/vitamin E deficiency

The severity of the heart damage caused by a coxsackievirus infection in mice is determined by several factors, including the genotype of the infecting virus as well as the genetic background of the infected host. Earlier work by us showed that the cardiovirulence of a given coxsackievirus genotype could be increased substantially by feeding the host a diet nutritionally deficient in either selenium or vitamin E. Here we report that host genetic background as a determinant of viral infection outcome is superseded by feeding the host a diet nutritionally deficient in both selenium and vitamin E. Mice of the C57Bl/6 strain, normally resistant to coxsackievirus B3-induced myocarditis, become susceptible when fed such a doubly deficient diet. Our results demonstrate the powerful influence of host nutritional status on the course of viral infection compared to other variables traditionally considered to play major roles in determining the extent of virally induced inflammatory heart disease.     

Impaired infectivity of ritonavir-resistant HIV is rescued by heat shock protein 90AB1

Certain ritonavir resistance mutations impair HIV infectivity through incomplete Gag processing by the mutant viral protease. Analysis of the mutant virus phenotype indicates that accumulation of capsid-spacer peptide 1 precursor protein in virus particles impairs HIV infectivity and that the protease mutant virus is arrested during the early postentry stage of HIV infection before proviral DNA synthesis. However, activation of the target cell can rescue this defect, implying that specific host factors expressed in activated cells can compensate for the defect in ritonavir-resistant HIV. This ability to rescue impaired HIV replication presented a unique opportunity to identify host factors involved in postentry HIV replication, and we designed a functional genetic screen so that expression of a given host factor extracted from activated T cells would lead directly to its discovery by rescuing mutant virus replication in nonactivated T cells. We identified the cellular heat shock protein 90 kDa α (cytosolic), class B member 1 (HSP90AB1) as a host factor that can rescue impaired replication of ritonavir-resistant HIV. Moreover, we show that pharmacologic inhibition of HSP90AB1 with 17-(allylamino)-17-demethoxygeldanamycin (tanespimycin) has potent in vitro anti-HIV activity and that ritonavir-resistant HIV is hypersensitive to the drug. These results suggest a possible role for HSP90AB1 in postentry HIV replication and may provide an attractive target for therapeutic intervention.     

Putative hepatitis C virus cell receptors

Virus entry into a host cell comprises the first step of the viral life cycle. Blockage of this process can stop or prevent the rise of the infection. Development of compounds exhibiting directed blocking activity requires information about host cell and viral molecules, which are involved into reciprocal recognition resulting in the virus entry into the cell. This review is devoted to the problems of the identification of plasma membrane molecules, involved in binding of hepatitis C virus and its subsequent transfer inside the cells. The putative role of these molecules as hepatitis C virus receptors and co-receptors in the beginning and development of the infection is discussed.     

Host factors against plant viruses

Plant virus genome replication and movement is dependent on host resources and factors. However, plants respond to virus infection through several mechanisms, such as autophagy, ubiquitination, mRNA decay and gene silencing, that target viral components. Viral factors work in synchrony with pro-viral host factors during the infection cycle and are targeted by antiviral responses. Accordingly, establishment of virus infection is genetically determined by the availability of the pro-viral factors necessary for genome replication and movement, and by the balance between plant defence and viral suppression of defence responses. Sequential requirement of pro-viral factors and the antagonistic activity of antiviral factors suggest a two-step model to explain plant–virus interactions. At each step of the infection process, host factors with antiviral activity have been identified. Here we review our current understanding of host factors with antiviral activity against plant viruses.     

Genome-Wide RNAi Screen Identifies Novel Host Proteins Required for Alphavirus Entry

The enveloped alphaviruses include important and emerging human pathogens such as Chikungunya virus and Eastern equine encephalitis virus. Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, relatively little is known about the host factors involved in alphavirus infection. We used a genome-wide siRNA screen to identify host factors that promote or inhibit alphavirus infection in human cells. Fuzzy homologue (FUZ), a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein TSPAN9 was critical for the efficient fusion of low pH-triggered virus with the endosome membrane. FUZ and TSPAN9 were broadly required for infection by the alphaviruses Sindbis virus, Semliki Forest virus, and Chikungunya virus, but were not required by the structurally-related flavivirus Dengue virus. Our results highlight the unanticipated functions of FUZ and TSPAN9 in distinct steps of alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy.     

Targeting host cofactors to inhibit viral infection

The majority of FDA-approved drugs indicated for the treatment of viral infections are inhibitors of viral proteins, of which the emergence of resistant strains is a major concern. This issue is exacerbated as most developed antiviral therapies are indicated for the treatment of viruses with error-prone replication. These problems may be addressed by the development of drugs that modulate the function of host factors involved in various aspects of a viral life cycle. Targeting host factors uncouples the mutation of a druggable protein gene from the replication and survival selection pressure exerted on a virus. Currently, a host-targeting antiviral (HTA), maraviroc, is approved for the treatment of human immunodeficiency virus (HIV) infection. In addition, several HTAs indicated for the treatment of hepatitis C virus (HCV) or HIV infection are at various stages of clinical evaluation. Targeting host factors is an attractive complement to therapies directly targeting a viral protein because of the expected higher genetic barrier for resistance and an overall increase in the diversity of treatment options. We examine how the integrated roles of emerging host cofactor screening approaches and drug development strategies may advance current treatment options.     

An integrated transcriptomic and meta-analysis of hepatoma cells reveals factors that influence susceptibility to HCV infection

Hepatitis C virus (HCV) is a global problem. To better understand HCV infection researchers employ in vitro HCV cell-culture (HCVcc) systems that use Huh-7 derived hepatoma cells that are particularly permissive to HCV infection. A variety of hyper-permissive cells have been subcloned for this purpose. In addition, subclones of Huh-7 which have evolved resistance to HCV are available. However, the mechanisms of susceptibility or resistance to infection among these cells have not been fully determined. In order to elucidate mechanisms by which hepatoma cells are susceptible or resistant to HCV infection we performed genome-wide expression analyses of six Huh-7 derived cell cultures that have different levels of permissiveness to infection. A great number of genes, representing a wide spectrum of functions are differentially expressed between cells. To focus our investigation, we identify host proteins from HCV replicase complexes, perform gene expression analysis of three HCV infected cells and conduct a detailed analysis of differentially expressed host factors by integrating a variety of data sources. Our results demonstrate that changes relating to susceptibility to HCV infection in hepatoma cells are linked to the innate immune response, secreted signal peptides and host factors that have a role in virus entry and replication. This work identifies both known and novel host factors that may influence HCV infection. Our findings build upon current knowledge of the complex interplay between HCV and the host cell, which could aid development of new antiviral strategies.     

A genome-wide CRISPR/Cas9 gene knockout screen identifies immunoglobulin superfamily DCC subclass member 4 as a key host factor that promotes influenza virus endocytosis

Influenza virus infection is dependent on host cellular factors, and identification of these factors and their underlying mechanisms can provide important information for the development of strategies to inhibit viral infection. Here, we used a highly pathogenic H5N1 influenza virus to perform a genome-wide CRISPR/Cas9 gene knockout screen in human lung epithelial cells (A549 cells), and found that knockout of transmembrane protein immunoglobulin superfamily DCC subclass member 4 (IGDCC4) significantly reduced the replication of the virus in A549 cells. Further studies showed that IGDCC4 interacted with the viral hemagglutinin protein and facilitated virus internalization into host cells. Animal infection studies showed that replication of H5N1 virus in the nasal turbinates, lungs, and kidneys of IGDCC4-knockout mice was significantly lower than that in the corresponding organs of wild-type mice. Half of the IGDCC4-knockout mice survived a lethal H5N1 virus challenge, whereas all of the wild-type mice died within 11 days of infection. Our study identifies a novel host factor that promotes influenza virus infection by facilitating internalization and provides insights that will support the development of antiviral therapies.     

Plant susceptibility genes as a source for potyvirus resistance

Virus infection depends on the resources provided by the host plant. A number of host proteins that enable potyvirus infection have been identified. The genes encoding them are called susceptibility genes (S-genes). Loss-of-susceptibility type of resistance is based on S-gene modifications leading to incompatible host–virus interactions. An increasing number of examples show that this is a viable method for resistance breeding. While the recent advancements in genome editing and sequencing have remarkably reduced the technical limitations, we still need to tackle many biological challenges to be able to utilise S-genes for durable and broad range potyvirus resistance to their full extent. Many lessons on functional redundancy between gene family members and durability of the resistance have been learned by studying the naturally occurring recessive resistance based on the interplay between eukaryotic initiation factors eIF4E and eIFiso4E and viral protein genome-linked (VPg). Nevertheless, the outcomes of the S-gene modifications on resistance or any other characteristic of the host plant cannot be predicted. In addition to the genetic background of the host, also the properties of the viral factors affect the efficiency of the resistance and the emergence of resistance-breaking mutations. Many potyviral protein–protein interactions occur in multiprotein complexes. This suggests that the susceptibility factors may interact with viral proteins as a part of multifaceted protein–protein interaction networks. Rather than reviewing exhaustively the S-genes involved in potyvirus infection, my intention here is to discuss in the light of selected examples, the prospects and challenges of the use of potyviral S-genes in resistance breeding.     

Effect of delta 9-tetrahydrocannabinol on herpes simplex virus type 2 vaginal infection in the guinea pig

The present investigation was undertaken to determine whether delta 9-tetrahydrocannabinol (delta 9-THC) decreases host resistance to herpes simplex virus type 2 vaginal infection in the guinea pig. The guinea pig was selected as the host since it has been shown to express a spectrum of primary herpes genitalis which is similar to that in humans. Animals were administered delta 9-THC or vehicle intraperitoneally on Days 1-4, 8-11, and 15-18. Herpes simplex virus was introduced intravaginally on Day 2. Host resistance to virus infection was assessed by comparing frequency and severity of lesions, virus shedding, and animal mortalities. Virus-infected animals treated with drug at doses of 4 and 10 mg/kg exhibited significantly greater severity of genital disease during the 30-day period of study when compared to virus-inoculated vehicle controls. A direct relationship was noted between dose of delta 9-THC and cumulative mortalities on Day 14 following primary infection. These results indicate that delta 9-THC decreases host resistance to herpes simplex virus type 2 vaginal infection in the guinea pig.     

Is flavivirus resistance interferon type I-independent?

Interferon type I comprises a group of major virus-inducible host antiviral factors that control infection with a great number of human and animal viruses. They are ubiquitously expressed cytokines that interfere with virus replication within different cell types by activating a number of host genes and several parallel antiviral pathways. Two major intracellular actors of IFN-I-induced antiviral states are ribonucleic acid-dependent protein kinase and 2'-5'-oligoadenylate synthetases/RNase L, both being induced by IFN-I and activated by viral double stranded ribonucleic acid. In addition, Mx proteins and ribonucleic acid-specific adenosine deaminase have also been implicated in IFN-I-induced antiviral responses to some RNA viruses. Viruses, in turn, have evolved different strategies to escape a control imposed by IFN-I and by IFN-I-induced antiviral factors. The fatal outcome of virus infection as well as the efficiency of IFN-I-based antiviral therapies in its prevention, are determined by complex interactions between viral virulence factors and cellular antiviral IFN-I inducible factors. In the light of these facts and current knowledge on IFN-I involvement in flavivirus infection, I discuss a possible role of IFN-I signalling in resistance to flavivirus infection in a model of congenic mouse strains that express different levels of susceptibility/resistance to common flaviviruses. Specifically, this review emphasizes importance of fully operative 2'-5'-oligoadenylate synthetases/RNase L pathway for the IFN-I-induced stimulation of flavivirus resistance conferred by Flv.