Modeling the Immune Response for Pathogenic and Nonpathogenic Orthohantavirus Infections in Human Lung Microvasculature Endothelial Cells.

Hantaviruses, genus Orthohantavirus, family Hantaviridae, order Bunyavirales, are negative-sense, single-stranded, tri-segmented RNA viruses that persistently infect rodents, shrews, and moles. Of these, only certain virus species harbored by rodents are pathogenic to humans. Infection begins with inhalation of virus particles into the lung and trafficking to the lung microvascular endothelial cells (LMVEC). The reason why certain rodent-borne hantavirus species are pathogenic has long been hypothesized to be related to their ability to downregulate and dysregulate the immune response as well as increase vascular permeability of infected endothelial cells. We set out to study the temporal dynamics of host immune response modulation in primary human LMVECs following infection by Prospect Hill (nonpathogenic), Andes (pathogenic), and Hantaan (pathogenic) viruses. We measured the level of RNA transcripts for genes representing antiviral, proinflammatory, anti-inflammatory, and metabolic pathways from 12 to 72 h with time points every 12 h. Gene expression analysis in conjunction with mathematical modeling revealed a similar profile for all three viruses in terms of upregulated genes that partake in interferon signaling (TLR3, IRF7, IFNB1), host immune cell recruitment (CXCL10, CXCL11, and CCL5), and host immune response modulation (IDO1). We examined secreted protein levels of IFN-ß, CXCL10, CXCL11, CCL5, and IDO in two male and two female primary HLMVEC donors at 48 and 60 h post infection. All three viruses induced similar levels of CCL5, CXCL10, and CXCL11 within a particular donor, and the levels were similar in three of the four donors. All three viruses induced different protein secretion levels for both IFN-ß and IDO and secretion levels differed between donors. In conclusion, we show that there was no difference in the transcriptional profiles of key genes in primary HLMVECs following infection by pathogenic and nonpathogenic hantaviruses, with protein secretion levels being more donor-specific than virus-specific.

Deep spatial profiling of Venezuelan equine encephalitis virus reveals increased genetic diversity amidst neuroinflammation and cell death during brain infection.

Venezuelan equine encephalitis virus (VEEV) causes a febrile illness that can progress to neurological disease with the possibility of death in human cases. The evaluation and optimization of therapeutics that target brain infections demands knowledge of the host's response to VEEV, the dynamics of infection, and the potential for within-host evolution of the virus. We hypothesized that selective pressures during infection of the brain may differ temporally and spatially and so we investigated the dynamics of the host response, viral transcript levels, and genetic variation of VEEV TC-83 in eight areas of the brain in mice over 7 days post-infection (dpi). Viral replication increased throughout the brain until 5-6 dpi and decreased thereafter with neurons as the main site of viral replication. Low levels of genetic diversity were noted on 1 dpi and were followed by an expansion in the genetic diversity of VEEV and nonsynonymous (Ns) mutations that peaked by 5 dpi. The pro-inflammatory response and the influx of immune cells mirrored the levels of virus and correlated with substantial damage to neurons by 5 dpi and increased activation of microglial cells and astrocytes. The prevalence and dynamics of Ns mutations suggest that the VEEV is under selection within the brain and that progressive neuroinflammation may play a role in acting as a selective pressure. IMPORTANCE Treatment of encephalitis in humans caused by Venezuelan equine encephalitis virus (VEEV) from natural or aerosol exposure is not available, and hence, there is a great interest to address this gap. In contrast to natural infections, therapeutic treatment of infections from aerosol exposure will require fast-acting drugs that rapidly penetrate the blood-brain barrier, engage sites of infection in the brain and mitigate the emergence of drug resistance. Therefore, it is important to understand not only VEEV pathogenesis, but the trafficking of the viral population within the brain, the potential for within-host evolution of the virus, and how VEEV might evolve resistance.

Upper Respiratory Infection Drives Clinical Signs and Inflammatory Responses Following Heterologous Challenge of SARS-CoV-2 Variants of Concern in K18 Mice.

The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence of several variants of concern (VOC) with increased immune evasion and transmissibility. This has motivated studies to assess protection conferred by earlier strains following infection or vaccination to each new VOC. We hypothesized that while NAbs play a major role in protection against infection and disease, a heterologous reinfection or challenge may gain a foothold in the upper respiratory tract (URT) and result in a self-limited viral infection accompanied by an inflammatory response. To test this hypothesis, we infected K18-hACE2 mice with SARS-CoV-2 USA-WA1/2020 (WA1) and, after 24 days, challenged with WA1, Alpha, or Delta. While NAb titers against each virus were similar across all cohorts prior to challenge, the mice challenged with Alpha and Delta showed weight loss and upregulation of proinflammatory cytokines in the URT and lower RT (LRT). Mice challenged with WA1 showed complete protection. We noted increased levels of viral RNA transcripts only in the URT of mice challenged with Alpha and Delta. In conclusion, our results suggested self-limiting breakthrough infections of Alpha or Delta in the URT, which correlated with clinical signs and a significant inflammatory response in mice.

Dissecting Phenotype from Genotype with Clinical Isolates of SARS-CoV-2 First Wave Variants.

The emergence and availability of closely related clinical isolates of SARS-CoV-2 offers a unique opportunity to identify novel nonsynonymous mutations that may impact phenotype. Global sequencing efforts show that SARS-CoV-2 variants have emerged and then been replaced since the beginning of the pandemic, yet we have limited information regarding the breadth of variant-specific host responses. Using primary cell cultures and the K18-hACE2 mouse, we investigated the replication, innate immune response, and pathology of closely related, clinical variants circulating during the first wave of the pandemic. Mathematical modeling of the lung viral replication of four clinical isolates showed a dichotomy between two B.1. isolates with significantly faster and slower infected cell clearance rates, respectively. While isolates induced several common immune host responses to infection, one B.1 isolate was unique in the promotion of eosinophil-associated proteins IL-5 and CCL11. Moreover, its mortality rate was significantly slower. Lung microscopic histopathology suggested further phenotypic divergence among the five isolates showing three distinct sets of phenotypes: (i) consolidation, alveolar hemorrhage, and inflammation, (ii) interstitial inflammation/septal thickening and peribronchiolar/perivascular lymphoid cells, and (iii) consolidation, alveolar involvement, and endothelial hypertrophy/margination. Together these findings show divergence in the phenotypic outcomes of these clinical isolates and reveal the potential importance of nonsynonymous mutations in nsp2 and ORF8.

Positive-strand RNA viruses-a Keystone Symposia report.

Positive-strand RNA viruses have been the cause of several recent outbreaks and epidemics, including the Zika virus epidemic in 2015, the SARS outbreak in 2003, and the ongoing SARS-CoV-2 pandemic. On June 18-22, 2022, researchers focusing on positive-strand RNA viruses met for the Keystone Symposium "Positive-Strand RNA Viruses" to share the latest research in molecular and cell biology, virology, immunology, vaccinology, and antiviral drug development. This report presents concise summaries of the scientific discussions at the symposium.

Screening of febrile patients with suspected malaria from the Brazilian Amazon for virus infection.

Arthropod-borne viruses (arboviruses) are a significant public health threat, especially in tropical and subtropical regions. More than 150 arboviruses can cause febrile illness following infection in humans. The Brazilian Amazon region has the highest number of arboviruses detected worldwide. In addition to arboviruses, malaria, caused by Plasmodium vivax, is endemic in the Amazon. Patients with malaria and arboviral disease frequently show similar clinical presentation and laboratory findings, making the diagnosis of the cause of the infection challenging. The aim of this study was to evaluate the potential for viral infections in patients with suspected malaria but without Plasmodium infection in the Brazilian Amazon. We recruited 200 subjects with suspected malaria in Manaus, Brazil. First, we tested for arboviruses in serum samples from 124 of the 200 participants using an arbovirus DNA microarray platform, which did not detect any virus. Then, we mixed the serum samples of the other 76 participants in 10 pools and subjected them to next-generation sequencing. Analysis of the sequencing data revealed the presence of only one arbovirus (Zika virus) in one sample pool. This analysis also detected the presence of primate erythroparvovirus 1 and pegivirus C. These results suggest that arboviruses are not the most frequent viral infections in patients with suspected malaria but without Plasmodium infection in the metropolitan region of Manaus. Implementation of specific viral surveillance tests will help in the early detection of viruses with epidemic potential.

Time-Dependent Increase in Susceptibility and Severity of Secondary Bacterial Infections During SARS-CoV-2.

Secondary bacterial infections can exacerbate SARS-CoV-2 infection, but their prevalence and impact remain poorly understood. Here, we established that a mild to moderate infection with the SARS-CoV-2 USA-WA1/2020 strain increased the risk of pneumococcal (type 2 strain D39) coinfection in a time-dependent, but sex-independent, manner in the transgenic K18-hACE2 mouse model of COVID-19. Bacterial coinfection increased lethality when the bacteria was initiated at 5 or 7 d post-virus infection (pvi) but not at 3 d pvi. Bacterial outgrowth was accompanied by neutrophilia in the groups coinfected at 7 d pvi and reductions in B cells, T cells, IL-6, IL-15, IL-18, and LIF were present in groups coinfected at 5 d pvi. However, viral burden, lung pathology, cytokines, chemokines, and immune cell activation were largely unchanged after bacterial coinfection. Examining surviving animals more than a week after infection resolution suggested that immune cell activation remained high and was exacerbated in the lungs of coinfected animals compared with SARS-CoV-2 infection alone. These data suggest that SARS-CoV-2 increases susceptibility and pathogenicity to bacterial coinfection, and further studies are needed to understand and combat disease associated with bacterial pneumonia in COVID-19 patients.

Data-driven models for replication kinetics of Orthohantavirus infections.

The Hantaviridae constitute a family of viruses harbored by mice, rats, shrews, voles, moles and bats. Intriguingly, only viruses harbored by mice and rats may cause disease in humans with up to 40% case fatality rate in the Americas. Transmission of virus from rodents to humans occurs via the respiratory route and results in replication of the virus in the microvascular endothelial cells of the lung or kidney. Understanding the replication kinetics of these viruses in various cell types and how replication is abrogated by the host is critical to the development of effective therapeutics for treatment for which there are none. We formulate several new ordinary differential equation (ODE) models to examine the replication kinetics of Prospect Hill orthohantavirus (PHV). The models are distinguished by the distribution of the viral replication delay. A new threshold, RGE, the genome equivalent replication number, is defined in terms of the model parameters. New final density relations are derived that associate RGE to the asymptotic number of virions in each model. All models are fit to real time (qRT)-PCR data of genomic RNA from PHV released from Vero E6 cells over 192 h. A sensitivity analysis of the parameters is performed and models are tested for best fit. Our findings provide a basis for future research into formulating more complex mathematical models for evaluation of the replication of hantaviruses in various cell types and sources.

Time-Dependent Increase in Susceptibility and Severity of Secondary Bacterial Infection during SARS-CoV-2 Infection.

Secondary bacterial infections can exacerbate SARS-CoV-2 infection, but their prevalence and impact remain poorly understood. Here, we established that a mild to moderate SARS-CoV-2 infection increased the risk of pneumococcal coinfection in a time-dependent, but sexindependent, manner in the transgenic K18-hACE mouse model of COVID-19. Bacterial coinfection was not established at 3 d post-virus, but increased lethality was observed when the bacteria was initiated at 5 or 7 d post-virus infection (pvi). Bacterial outgrowth was accompanied by neutrophilia in the groups coinfected at 7 d pvi and reductions in B cells, T cells, IL-6, IL-15, IL-18, and LIF were present in groups coinfected at 5 d pvi. However, viral burden, lung pathology, cytokines, chemokines, and immune cell activation were largely unchanged after bacterial coinfection. Examining surviving animals more than a week after infection resolution suggested that immune cell activation remained high and was exacerbated in the lungs of coinfected animals compared with SARS-CoV-2 infection alone. These data suggest that SARS-CoV-2 increases susceptibility and pathogenicity to bacterial coinfection, and further studies are needed to understand and combat disease associated with bacterial pneumonia in COVID-19 patients.

Common Themes in Zoonotic Spillover and Disease Emergence: Lessons Learned from Bat- and Rodent-Borne RNA Viruses.

Rodents (order Rodentia), followed by bats (order Chiroptera), comprise the largest percentage of living mammals on earth. Thus, it is not surprising that these two orders account for many of the reservoirs of the zoonotic RNA viruses discovered to date. The spillover of these viruses from wildlife to human do not typically result in pandemics but rather geographically confined outbreaks of human infection and disease. While limited geographically, these viruses cause thousands of cases of human disease each year. In this review, we focus on three questions regarding zoonotic viruses that originate in bats and rodents. First, what biological strategies have evolved that allow RNA viruses to reside in bats and rodents? Second, what are the environmental and ecological causes that drive viral spillover? Third, how does virus spillover occur from bats and rodents to humans?

Prevalence of Hantaviruses Harbored by Murid Rodents in Northwestern Ukraine and Discovery of a Novel Puumala Virus Strain.

In Europe, two species of hantaviruses, Puumala orthohantavirus (PUUV) and Dobrava orthohantavirus (DOBV), cause hemorrhagic fever with renal syndrome in humans. The rodent reservoirs for these viruses are common throughout Ukraine, and hence, the goal of this study was to identify the species and strains of hantaviruses circulating in this region. We conducted surveillance of small rodent populations in a rural region in northwestern Ukraine approximately 30 km from Poland. From the 424 small mammals captured, we identified nine species, of which the most abundant were Myodes glareolus, the bank vole (45%); Apodemus flavicollis, the yellow-necked mouse (29%); and Apodemus agrarius, the striped field mouse (14.6%) Using an indirect immunofluorescence assay, 15.7%, 20.5%, and 33.9% of the sera from M. glareolus, A. glareolus, and A. flavicollis were positive for hantaviral antibodies, respectively. Additionally, we detected antibodies to the hantaviral antigen in one Microtus arvalis, one Mus musculus, and one Sorex minutus. We screened the lung tissue for hantaviral RNA using next-generation sequencing and identified PUUV sequences in 25 small mammals, including 23 M. glareolus, 1 M. musculus, and 1 A. flavicollis, but we were unable to detect DOBV sequences in any of our A. agrarius specimens. The percent identity matrix and Bayesian phylogenetic analyses of the S-segment of PUUV from 14 M. glareolus lungs suggest the highest similarity (92-95% nucleotide or 99-100% amino acid) with the Latvian lineage. This new genetic information will contribute to future molecular surveillance of human cases in Ukraine.

Mixed Effects of Habitat Degradation and Resources on Hantaviruses in Sympatric Wild Rodent Reservoirs within a Neotropical Forest.

Understanding the ecology of rodent-borne hantaviruses is critical to assessing the risk of spillover to humans. Longitudinal surveys have suggested that hantaviral prevalence in a given host population is tightly linked to rodent ecology and correlates with changes in the species composition of a rodent community over time and/or habitat composition. We tested two hypotheses to identify whether resource addition and/or habitat composition may affect hantavirus prevalence among two sympatric reservoir hosts in a neotropical forest: (i) increased food resources will alter the rodent community and thus hantaviral prevalence; and (ii) host abundance and viral seroprevalence will be associated with habitat composition. We established a baseline of rodent-virus prevalence in three grid pairs of distinct habitat compositions and subjected one grid of each pair to resource augmentation. Increased rodent species diversity was observed on grids where food was added versus untreated control grids during the first post-treatment sampling session. Resource augmentation changed species community composition, yet it did not affect the prevalence of hantavirus in the host population over time, nor was there evidence of a dilution effect. Secondly, we show that the prevalence of the virus in the respective reservoir hosts was associated with habitat composition at two spatial levels, independent of resource addition, supporting previous findings that habitat composition is a primary driver of the prevalence of hantaviruses in the neotropics.

Corrigendum: Amplicon-Based, Next-Generation Sequencing Approaches to Characterize Single Nucleotide Polymorphisms of Orthohantavirus Species.

[This corrects the article DOI: 10.3389/fcimb.2020.565591.].

Seroprevalence of Old World Hantaviruses and Crimean Congo Hemorrhagic Fever Viruses in Human Populations in Northwestern Ukraine.

In Ukraine, a retrospective review of clinical case reports by public health officials suggest that human cases of febrile illnesses associated with hemorrhage may be due to infections of Crimean-Congo hemorrhagic fever virus (CCHFV) and Old World hantaviruses. In a serosurvey of 966 healthy individuals in the Lviv Oblast, Ukraine, bordering Poland, we found that 1.6% showed cross-reactivity to hantaviral antigens by an immunofluorescence assay (IFA) and 1.7% of the study participants had antibodies cross-reactive to CCHFV by enzyme-linked immunosorbent assay (ELISA). Demographic variables and history of exposures obtained through questionnaires were assessed by logistic regression models for association with seroprevalence for both viruses with no significant risk factors found. Analysis of spatial distribution identified two clusters of samples positive for antibodies to both hantaviruses and CCHFV, which, however, were not statistically significant (p > 0.05). In general, the study results suggest that the population of the study area is exposed to hantaviruses and CCHFV. Further surveillance for respective pathogens in Ukraine is warranted and prospective surveillance of febrile patients with unidentified febrile illness.

Cryofixation of Inactivated Hantavirus-Infected Cells as a Method for Obtaining High-Quality Ultrastructural Preservation for Electron Microscopic Studies.

Hantaviruses rewire the host cell and induce extensive membrane rearrangements for their replication and the morphogenesis of the virion. Transmission electron microscopy (TEM) is a powerful technique for imaging these pathological membrane changes especially when combined with large volume electron tomography. Excellent preservation of membrane structure can be obtained when chemical fixation is combined with cryofixation via high pressure freezing making the samples amenable to serial-section tomographic reconstruction. Taking advantage of this, we have optimized a hybrid method that employs aldehyde fixation, a step that is essential for virus inactivation, followed by high-pressure freezing for ultrastructural study of Hantaan (HTN) and Andes (AND) virus infected Vero E6 cells. HTNV and ANDV are two species of the Orthohantavirus, from the Old and New World, respectively, and the causative agents of hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome in humans. We applied the method for the qualitative assessment of the perturbation of the endomembrane system induced by HTNV and ANDV in infected vs. mock-infected cells. Screening of serial-sections revealed consistency of membrane preservation across large volumes indicating potential of these samples for tomographic studies. Images revealed large-scale perturbations of the endomembrane system following HTNV-infection that included the dilation of the rough endoplasmic reticulum and fragmentation of the Golgi apparatus. Infected cells exhibited a tendency to accumulate large numbers of vacuoles that were especially apparent in ANDV. In summary, our hybrid method provides a path for the study of BSL-3 pathogens using cutting edge 3D-imaging technologies.

Amplicon-Based, Next-Generation Sequencing Approaches to Characterize Single Nucleotide Polymorphisms of Orthohantavirus Species.

Whole-genome sequencing (WGS) of viruses from patient or environmental samples can provide tremendous insight into the epidemiology, drug resistance or evolution of a virus. However, we face two common hurdles in obtaining robust sequence information; the low copy number of viral genomes in specimens and the error introduced by WGS techniques. To optimize detection and minimize error in WGS of hantaviruses, we tested four amplification approaches and different amplicon pooling methods for library preparation and examined these preparations using two sequencing platforms, Illumina MiSeq and Oxford Nanopore Technologies MinION. First, we tested and optimized primers used for whole segment PCR or one kilobase amplicon amplification for even coverage using RNA isolated from the supernatant of virus-infected cells. Once optimized we assessed two sources of total RNA, virus-infected cells and supernatant from the virus-infected cells, with four variations of primer pooling for amplicons, and six different amplification approaches. We show that 99-100% genome coverage was obtained using a one-step RT-PCR reaction with one forward and reverse primer. Using a two-step RT-PCR with three distinct tiling approaches for the three genomic segments (vRNAs), we optimized primer pooling approaches for PCR amplification to achieve a greater number of aligned reads, average depth of genome, and genome coverage. The single nucleotide polymorphisms identified from MiSeq and MinION sequencing suggested intrinsic mutation frequencies of ~10-5-10-7 per genome and 10-4-10-5 per genome, respectively. We noted no difference in the coverage or accuracy when comparing WGS results with amplicons amplified from RNA extracted from infected cells or supernatant of these infected cells. Our results show that high-throughput diagnostics requiring the identification of hantavirus species or strains can be performed using MiSeq or MinION using a one-step approach. However, the two-step MiSeq approach outperformed the MinION in coverage depth and accuracy, and hence would be superior for assessment of genomes for epidemiology or evolutionary questions using the methods developed herein.

Cocirculation of Two Orthohantavirus Species in Small Mammals of the Northwestern Ukraine.

Rodent-borne hantaviruses have been reported in many of the countries surrounding Ukraine; however, to date we have no knowledge of the viral strains circulating in Ukraine within reservoirs such as the striped field mouse (Apodemus agrarius), the yellow-necked field mouse (Apodemus flavicollis), and the bank vole (Myodes glareolus). To determine the prevalence of hantaviruses in Ukraine, we captured 1,261 mammals, of which 1,109 were rodents, in 58 field sites within the province of Volyn in western Ukraine. Foci of the striped field mouse tended to occur in the eastern and southern parts of the province, whereas the bank vole were clustered in western and northern regions. The striped field mouse and bank vole had detectable serum antibodies to Puumala virus (PUUV) or Dobrava virus (DOBV) antigens at 7% or 2%, respectively, using an indirect immunofluorescence assay. Antibody prevalence among the bank vole males and females was equivalent, whereas for the striped field mouse, the prevalence among males was 5% versus 1% for females. In two bank vole specimens, we were able to detect partial nucleotide sequences that showed identity to PUUV. In summary, this study suggests that two human pathogens, PUUV and DOBV, cocirculate in the bank vole and the striped field mouse, respectively, in Ukraine. Future studies will focus on new rodent collections that will enable obtaining the complete genome sequences of the PUUV and DOBV strains circulating in Ukraine to provide guidance on the design of optimal molecular diagnostics that can enable insight into the potential contribution of hantaviruses to human disease in Ukraine.

A new inactivation method to facilitate cryo-EM of enveloped, RNA viruses requiring high containment: A case study using Venezuelan Equine Encephalitis Virus (VEEV).

The challenges associated with operating electron microscopes (EM) in biosafety level 3 and 4 containment facilities have slowed progress of cryo-EM studies of high consequence viruses. We address this gap in a case study of Venezuelan Equine Encephalitis Virus (VEEV) strain TC-83. Chemical inactivation of viruses may physically distort structure, and hence to verify retention of native structure, we selected VEEV strain TC-83 to develop this methodology as this virus has a 4.8 Šresolution cryo-EM structure. In our method, amplified VEEV TC-83 was concentrated directly from supernatant through a 30 % sucrose cushion, resuspended, and chemically inactivated with 1 % glutaraldehyde. A second 30 % sucrose cushion removed any excess glutaraldehyde that might interfere with single particle analyses. A cryo-EM map of fixed, inactivated VEEV was determined to a resolution of 7.9 Å. The map retained structural features of the native virus such as the icosahedral symmetry, and the organization of the capsid core and the trimeric spikes. Our results suggest that our strategy can easily be adapted for inactivation of other enveloped, RNA viruses requiring BSL-3 or BSL-4 for cryo-EM. However, the validation of inactivation requires the oversight of Biosafety Committee for each Institution.

Diverse Morphology and Structural Features of Old and New World Hantaviruses.

To further understanding of the structure and morphology of the Orthohantavirus, family Hantaviridae, we have employed cryo-electron microscopy (cryo-EM) for three New World hantaviruses: Andes (ANDV), Sin Nombre (SNV), and Black Creek Canal (BCCV). Building upon our prior cryo-EM and cryo-tomography study of the Old World hantavirus, Hantaan virus (HTNV), we have expanded our studies to examine the entire virion population present in cell culture supernatant. Hence, in contrast to the prior cryo-EM/ET studies in which we used a polyethylene precipitation, a sucrose gradient, and a sucrose cushion, we used two sucrose cushions. We inactivated the material after the first cushion. We tested the method using HTNV which has a known cryo-EM structure and observed equivalent results. Therefore, we used this method to assess the particle distribution of the New World hantaviruses by cryo-EM. Cryo-EM images showed a diverse range of sizes and morphologies for the New World viruses that we classified as round, tubular, and irregular. Strikingly, BCCV virions were mostly tubular. These first cryo-EM images of the New World Orthohantavirus confirm prior EM observations that noted tubular projections of SNV at the plasma membrane during virion morphogenesis but were not confirmed. These findings underscore the need for further investigation of virion morphogenesis of the Orthohantavirus.

A Student's Guide to Giant Viruses Infecting Small Eukaryotes: From Acanthamoeba to Zooxanthellae.

The discovery of infectious particles that challenge conventional thoughts concerning "what is a virus" has led to the evolution a new field of study in the past decade. Here, we review knowledge and information concerning "giant viruses", with a focus not only on some of the best studied systems, but also provide an effort to illuminate systems yet to be better resolved. We conclude by demonstrating that there is an abundance of new host-virus systems that fall into this "giant" category, demonstrating that this field of inquiry presents great opportunities for future research.