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Many viruses initiate infection by binding to sialoglycan receptors at the cell surface. Binding to such receptors comes at a cost, however, as the sheer abundance of sialoglycans e.g. in mucus, may immobilize virions to non-functional decoy receptors. As a solution, sialoglycan-binding as well as sialoglycan-cleavage activities are often present in these viruses, which for paramyxoviruses are combined in the hemagglutinin-neuraminidase (HN) protein. The dynamic interactions of sialoglycan-binding paramyxoviruses with their receptors are thought to be key determinants of species tropism, replication and pathogenesis. Here we used biolayer interferometry to perform kinetic analyses of receptor interactions of animal and human paramyxoviruses (Newcastle disease virus, Sendai virus, and human parainfluenza virus 3). We show that these viruses display strikingly different receptor interaction dynamics, which correlated with their receptor-binding and -cleavage activities and the presence of a second sialic acid binding site. Virion binding was followed by sialidase-driven release, during which virions cleaved sialoglycans until a virus-specific density was reached, which was largely independent of virion concentration. Sialidase-driven virion release was furthermore shown to be a cooperative process and to be affected by pH. We propose that paramyxoviruses display sialidase-driven virion motility on a receptor-coated surface, until a threshold receptor density is reached at which virions start to dissociate. Similar motility has previously been observed for influenza viruses and is likely to also apply to sialoglycan-interacting embecoviruses. Analysis of the balance between receptor-binding and -cleavage increases our understanding of host species tropism determinants and zoonotic potential of viruses.

Enteroviruses (EVs) are among the most prevalent viruses worldwide. They are characterized by a high genetic and phenotypic diversity, being able to cause a plethora of symptoms. EV-D68, a respiratory EV, and EV-D94, an enteric EV, represent an interesting paradigm of EV tropism heterogeneity. They belong to the same species, but display distinct phenotypic characteristics and in vivo tropism. Here, we used these two viruses as well as relevant 3D respiratory, intestinal and neural tissue culture models, to highlight key distinctive features of enteric and respiratory EVs. We emphasize the critical role of temperature in restricting EV-D68 tissue tropism. Using transcriptomic analysis, we underscore fundamental differences between intestinal and respiratory tissues, both in the steady-state and in response to infection. Intestinal tissues present higher cell proliferation rate and are more immunotolerant than respiratory tissues. Importantly, we highlight the different strategies applied by EV-D94 and EV-D68 towards the host antiviral response of intestinal and respiratory tissues. EV-D68 strongly activates antiviral pathways while EV-D94, on the contrary, barely induces any host defense mechanisms. In summary, our study provides an insightful characterization of the differential pathogenesis of EV-D68 and EV-D94 and the interplay with their main target tissues.

Trypanosoma brucei is a protozoan parasite that causes human and animal African trypanosomiases (HAT and AAT). In the mammalian host, the parasite lives entirely extracellularly, in both the blood and interstitial spaces in tissues. Although most T. brucei research has focused on the biology of blood- and central nervous system (CNS)-resident parasites, a number of recent studies have highlighted parasite reservoirs in the dermis and adipose tissue, leading to a renewed interest in tissue-resident parasite populations. In light of this renewed interest, work describing tissue-resident parasites can serve as a valuable resource to inform future investigations of tissue-resident T. brucei. Here, we review this body of literature, which describes infections in humans, natural hosts, and experimental animal models, providing a wealth of information on the distribution and biology of extravascular parasites, the corresponding immune response in each tissue, and resulting host pathology. We discuss the implications of these studies and future questions in the study of extravascular T. brucei.

Despite 25 years of research, the basic virology of Kaposi Sarcoma Herpesviruses (KSHV) in B lymphocytes remains poorly understood. This study seeks to fill critical gaps in our understanding by characterizing the B lymphocyte lineage-specific tropism of KSHV. Here, we use lymphocytes derived from 40 human tonsil specimens to determine the B lymphocyte lineages targeted by KSHV early during de novo infection in our ex vivo model system. We characterize the immunological diversity of our tonsil specimens and determine that overall susceptibility of tonsil lymphocytes to KSHV infection varies substantially between donors. We demonstrate that a variety of B lymphocyte subtypes are susceptible to KSHV infection and identify CD138+ plasma cells as a highly targeted cell type for de novo KSHV infection. We determine that infection of tonsil B cell lineages is primarily latent with few lineages contributing to lytic replication. We explore the use of CD138 and heparin sulfate proteoglycans as attachment factors for the infection of B lymphocytes and conclude that they do not play a substantial role. Finally, we determine that the host T cell microenvironment influences the course of de novo infection in B lymphocytes. These results improve our understanding of KSHV transmission and the biology of early KSHV infection in a naïve human host, and lay a foundation for further characterization of KSHV molecular virology in B lymphocyte lineages., Author summary KSHV infection is associated with cancer in B cells and endothelial cells, particularly in the context of immune suppression. Very little is known about how KSHV is transmitted and how it initially establishes infection in a new host. Saliva is thought to be the primary route of person-to-person transmission for KSHV, making the tonsil a likely first site for KSHV replication in a new human host. Our study examines KSHV infection in B cells extracted from the tonsils of 40 human donors in order to determine what types of B cells are initially targeted for infection and examine how the presence (or absence) of other immune cells influence the initial stages of KSHV infection. We found that a variety of B cell subtypes derived from tonsils can be infected with KSHV. Interestingly, plasma cells (mature antibody-secreting B cells) were a highly targeted cell type. These results lay the foundation for further studies into the specific biology of KSHV in different types of B cells, an effort that may help us ultimately discover how to prevent the establishment of infection in these cells or reveal new ways to halt the progression of B cell cancers associated with KSHV infection.

Field isolates of foot-and-mouth disease viruses (FMDVs) utilize integrin-mediated cell entry but many, including Southern African Territories (SAT) viruses, are difficult to adapt to BHK-21 cells, thus hampering large-scale propagation of vaccine antigen. However, FMDVs acquire the ability to bind to cell surface heparan sulphate proteoglycans, following serial cytolytic infections in cell culture, likely by the selection of rapidly replicating FMDV variants. In this study, fourteen SAT1 and SAT2 viruses, serially passaged in BHK-21 cells, were virulent in CHO-K1 cells and displayed enhanced affinity for heparan, as opposed to their low-passage counterparts. Comparative sequence analysis revealed the fixation of positively charged residues clustered close to the icosahedral 5-fold axes of the virus, at amino acid positions 83–85 in the βD-βE loop and 110–112 in the βF-βG loop of VP1 upon adaptation to cultured cells. Molecular docking simulations confirmed enhanced binding of heparan sulphate to a model of the adapted SAT1 virus, with the region around VP1 arginine 112 contributing the most to binding. Using this information, eight chimeric field strain mutant viruses were constructed with additional positive charges in repeated clusters on the virion surface. Five of these bound heparan sulphate with expanded cell tropism, which should facilitate large-scale propagation. However, only positively charged residues at position 110–112 of VP1 enhanced infectivity of BHK-21 cells. The symmetrical arrangement of even a single amino acid residue in the FMD virion is a powerful strategy enabling the virus to generate novel receptor binding and alternative host-cell interactions., Author summary Foot-and-mouth disease (FMD) is a major threat for world economies. Local farmers in endemic areas relying on livestock for their livelihoods are affected by FMD outbreaks. Improved vaccines are essential for effective control and should be produced based on currently circulating field viruses. Current vaccines are produced by chemical inactivation of virus grown in large-scale production cultures of BHK-21 cells. However, not all field viruses can be adapted to cell cultures. The significance of our research is in identifying amino acid residues responsible for enhancing cell culture adaptation of FMD field viruses. This study engineered chimeric viruses containing the antigenic region of a field strain and novel cell culture receptor binding sites. This facilitated rapid virus amplification within a few passages in BHK-21 cells to create master virus seed stocks, circumventing the need for isolation on primary cell lines prior to further adaptation.

Sexual transmission and persistence of Zika virus (ZIKV) in the testes pose new challenges for controlling virus outbreaks and developing live-attenuated vaccines. It has been shown that testicular infection of ZIKV is initiated in the testicular interstitium, followed by spread of the virus in the seminiferous tubules. This leads to testicular damage and/or viral dissemination into the epididymis and eventually into semen. However, it remains unknown which cell types are targeted by ZIKV in the testicular interstitium, and what is the specific order of infectious events leading to ZIKV invasion of the seminiferous tubules. Here, we demonstrate that interstitial leukocytes expressing mir-511-3p microRNA are the initial targets of ZIKV in the testes, and infection of mir-511-3p-expressing cells in the testicular interstitium is necessary for downstream infection of the seminiferous tubules. Mir-511-3p is expressed concurrently with CD206, a marker of lineage 2 (M2) macrophages and monocyte derived dendritic cells (moDCs). Selective restriction of ZIKV infection of CD206-expressing M2 macrophages/moDCs results in the attenuation of macrophage–associated inflammatory responses in vivo and prevents the disruption of the Sertoli cell barrier in vitro. Finally, we show that targeting of viral genome for mir-511-3p significantly attenuates early ZIKV replication not only in the testes, but also in many peripheral organs, including spleen, epididymis, and pancreas. This incriminates M2 macrophages/moDCs as important targets for visceral ZIKV replication following hematogenous dissemination of the virus from the site of infection., Author summary Zika virus (ZIKV) has recently gained global media attention as a major emerging pathogen. Unique among the flaviviruses, ZIKV can be transmitted sexually as well as through the canonical arthropod route. Transmission through sexual contact raises new challenges for controlling ZIKV outbreaks, and infection of the testes poses a unique challenge for the development of live attenuated vaccines against ZIKV. In this manuscript, we utilized the microRNA targeting technique to demonstrate that testicular macrophages/DCs are a critical first target for ZIKV, and infection of these cells is required for passage of the virus through the Sertoli cell barrier and subsequent infection of the seminiferous tubules. Our investigations are the first to clearly show the sequence of infection events required for testicular infection that would subsequently lead to sexual transmission of ZIKV.

Post-transcriptional regulation via small regulatory RNAs (sRNAs) has been implicated in diverse regulatory processes in bacteria, including virulence. One class of sRNAs, termed trans-acting sRNAs, can affect the stability and/or the translational efficiency of regulated transcripts. In this study, we utilized a collaborative approach that employed data from infection with the Borrelia burgdorferi Tn library, coupled with Tn-seq, together with borrelial sRNA and total RNA transcriptomes, to identify an intergenic trans-acting sRNA, which we designate here as ittA for infectivity-associated and tissue-tropic sRNA locus A. The genetic inactivation of ittA resulted in a significant attenuation in infectivity, with decreased spirochetal load in ear, heart, skin and joint tissues. In addition, the ittA mutant did not disseminate to peripheral skin sites or heart tissue, suggesting a role for ittA in regulating a tissue-tropic response. RNA-Seq analysis determined that 19 transcripts were differentially expressed in the ittA mutant relative to its genetic parent, including vraA, bba66, ospD and oms28 (bba74). Subsequent proteomic analyses also showed a significant decrease of OspD and Oms28 (BBA74) proteins. To our knowledge this is the first documented intergenic sRNA that alters the infectivity potential of B. burgdorferi., Author summary Lyme disease is a tick-borne infection mediated by the spirochetal bacterium, Borrelia burgdorferi, that is responsible for greater than 300,000 infections in the United States per year. As such, additional knowledge regarding how this pathogen modulates its regulatory armamentarium is needed to understand how B. burgdorferi establishes and maintains infection. The identification and characterization of small, non-coding RNA molecules in living systems, designated as sRNAs, has recalibrated how we view post-transcriptional regulation. Recently, over 1,000 sRNAs were identified in B. burgdorferi. Despite the identification of these sRNAs, we do not understand how they affect infectivity or B. burgdorferi pathogenesis related outcomes. Here, we characterize the ittA B. burgdorferi sRNA and show that it is essential for optimal infection using murine experimental infection as our readout. We also track the effect of this sRNA on the transcriptional and proteomic profile as the first step in providing mechanistic insight into how this important sRNA mediates its regulatory effect.

Zika virus (ZIKV) is a unique flavivirus with high tropism to the testes. ZIKV can persist in human semen for months and can cause testicular damage in male mice. However, the mechanisms through which ZIKV enters the testes remain unclear. In this study, we revealed that matrix metalloproteinase 9 (MMP9) was upregulated by ZIKV infection in cell culture and in A129 mice. Furthermore, using an in vitro Sertoli cell barrier model and MMP9-/- mice, we found that ZIKV infection directly affected the permeability of the blood-testis barrier (BTB), and knockout or inhibition of MMP9 reduced the effects of ZIKV on the Sertoli cell BTB, highlighting its role in ZIKV-induced disruption of the BTB. Interestingly, the protein levels of MMP9 were elevated by ZIKV nonstructural protein 1 (NS1) in primary mouse Sertoli cells (mSCs) and other cell lines. Moreover, the interaction between NS1 and MMP9 induced the K63-linked polyubiquitination of MMP9, which enhanced the stability of MMP9. The upregulated MMP9 level led to the degradation of essential proteins involved in the maintenance of the BTB, such as tight junction proteins (TJPs) and type Ⅳ collagens. Collectively, we concluded that ZIKV infection promoted the expression of MMP9 which was further stabilized by NS1 induced K63-linked polyubiquitination to affect the TJPs/ type Ⅳ collagen network, thereby disrupting the BTB and facilitating ZIKV entry into the testes., Author summary Zika virus (ZIKV) is a flavivirus that shows high tropism to the testes and can persist in human semen for a long period. However, the entry mechanism of ZIKV into the testes has remained unclear. Here, we explored the mechanisms underlying matrix metalloproteinase 9 (MMP9)-modulated ZIKV infection in mice. We showed that MMP9 was upregulated by ZIKV infection both in vivo and in vitro. ZIKV infection affected the permeability of the blood-testis barrier (BTB) through MMP9 mediated degradation of TJPs and type Ⅳ collagens that are critically involved in the maintenance of the BTB. Additionally, the interaction between MMP9 and ZIKV NS1 induced the K63-linked polyubiquitination of MMP9, which enhanced the stability and function of MMP9. Overall, our findings provided important insights into the mechanisms through which MMP9 disrupted the BTB and promoted ZIKV entry into the testes.

Members of the Old World Arenaviruses primarily utilize α-dystroglycan (α-DAG1) as a cellular receptor for infection. Mutations within the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) reduce or abrogate the binding affinity to α-DAG1 and thus influence viral persistence, kinetics, and cell tropism. The observation that α-DAG1 deficient cells are still highly susceptible to low affinity variants, suggests the use of an alternative receptor(s). In this study, we used a genome-wide CRISPR Cas9 knockout screen in DAG1 deficient 293T cells to identify host factors involved in α-DAG1-independent LCMV infection. By challenging cells with vesicular stomatitis virus (VSV), pseudotyped with the GP of LCMV WE HPI (VSV-GP), we identified the heparan sulfate (HS) biosynthesis pathway as an important host factor for low affinity LCMV infection. These results were confirmed by a genetic approach targeting EXTL3, a key factor in the HS biosynthesis pathway, as well as by enzymatic and chemical methods. Interestingly, a single point mutation within GP1 (S153F or Y155H) of WE HPI is sufficient for the switch from DAG1 to HS binding. Furthermore, we established a simple and reliable virus-binding assay, using directly labelled VSV-GP by intramolecular fusion of VSV-P and mWasabi, demonstrating the importance of HS for virus attachment but not entry in Burkitt lymphoma cells after reconstitution of HS expression. Collectively, our study highlights the essential role of HS for low affinity LCMV infection in contrast to their high affinity counterparts. Residual LCMV infection in double knockouts indicate the use of (a) still unknown entry receptor(s)., Author summary Lymphocytic choriomeningitis virus (LCMV) contributed to the fundamental understanding of immunological processes due to characteristics such as persistent and immunosuppressive infection. However, not all strains of LCMV share the same traits. Differences in tissue tropism and the course of disease led to intensive characterization of distinct LCMV variants. Point mutations within the glycoprotein of LCMV were identified that reduce or abrogate the binding affinity to its host receptor α-Dystroglycan (α-DAG1), leading to the classification of low and high affinity LCMV variants. α-DAG1-independent infection and altered tissue tropism suggested the use of an alternative, unknown receptor by low affinity LCMV variants. Applying a genome-wide knockout screen and comparing different LCMV strains by genetic, enzymatic, and chemical approaches, we identified heparan sulfate proteoglycans (HSPG) as alternative receptors favoured by low affinity LCMV variants. These findings improve the understanding of receptor usage by different LCMV variants and explain their distinct characteristics. Furthermore, residual LCMV infection of double knockout cells indicate a major role of α-DAG1 and HSPG as attachment factors for high and low affinity LCMV variants, respectively, as well as the use of (a) still unknown entry receptor(s).

Pathogens possess the ability to adapt and survive in some host species but not in others–an ecological trait known as host tropism. Transmitted through ticks and carried mainly by mammals and birds, the Lyme disease (LD) bacterium is a well-suited model to study such tropism. Three main causative agents of LD, Borrelia burgdorferi, B. afzelii, and B. garinii, vary in host ranges through mechanisms eluding characterization. By feeding ticks infected with different Borrelia species, utilizing feeding chambers and live mice and quail, we found species-level differences in bacterial transmission. These differences localize on the tick blood meal, and specifically complement, a defense in vertebrate blood, and a polymorphic bacterial protein, CspA, which inactivates complement by binding to a host complement inhibitor, Factor H (FH). CspA selectively confers bacterial transmission to vertebrates that produce FH capable of allele-specific recognition. CspA is the only member of the Pfam54 gene family to exhibit host-specific FH-binding. Phylogenetic analyses revealed convergent evolution as the driver of such uniqueness, and that FH-binding likely emerged during the last glacial maximum. Our results identify a determinant of host tropism in Lyme disease infection, thus defining an evolutionary mechanism that shapes host-pathogen associations., Author summary The evolution of pathogens in response to a host environment often leads to a specificity of these pathogens to survive in particular hosts, known as host tropism. However, the mechanisms of host tropism remain unclear. The Lyme disease (LD) bacterium is an ideal model to study such mechanisms because the causative agent of this disease (bacteria species) are transmitted by ticks and carried by multiple vertebrates, including mammals and birds. We found that different LD bacteria species vary in their ability to survive in mice and quail, and in ticks fed on human or quail blood after transmission. We also showed that such a variation is dependent on whether tick blood meals have functional complement, an innate defense in vertebrate blood. We attributed such species-to-species differences of transmission to the polymorphisms of a bacterial complement inactivating protein, CspA: CspA, by inactivating vertebrate complement in tick blood meals in a species-specific manner, allows bacterial transmission to selective vertebrates. We further demonstrated that this CspA-mediated bacterium species-specific complement evasion arose through convergent evolution, as an evolutionary mechanism to permit such vertebrate-bacteria interaction. The mechanisms defined in this multi-disciplinary work provide new insights into the host-pathogen interactions.

Human noroviruses (HuNoVs) are the most common cause of foodborne illness, with a societal cost of $60 billion and 219,000 deaths/year. The lack of robust small animal models has significantly hindered the understanding of norovirus biology and the development of effective therapeutics. Here we report that HuNoV GI and GII replicate to high titers in zebrafish (Danio rerio) larvae; replication peaks at day 2 post infection and is detectable for at least 6 days. The virus (HuNoV GII.4) could be passaged from larva to larva two consecutive times. HuNoV is detected in cells of the hematopoietic lineage and the intestine, supporting the notion of a dual tropism. Antiviral treatment reduces HuNoV replication by >2 log10, showing that this model is suited for antiviral studies. Zebrafish larvae constitute a simple and robust replication model that will largely facilitate studies of HuNoV biology and the development of antiviral strategies., Author summary Human norovirus (HuNoV) is the number one agent of viral gastroenteritis worldwide. It can infect people of all age groups, resulting in 700 million infections and 219,000 deaths each year. Outbreaks of acute HuNoV gastroenteritis occur often, but chronic infections also happen in people with immune deficiencies. Despite its clinical relevance, studying the virus has been a decades-long challenge because no simple and efficient cultivation system existed. This has recently started to change; we here contribute with an important step forward by establishing an in vivo model system to study HuNoV replication using zebrafish larvae. We inject a HuNoV in the yolk (food reserve) of 3-day-old zebrafish larvae, which results in high virus titers up to 6 days after inoculation. We could detect the virus in the tissues of the infected larvae by a variety of techniques including histology, which showed us in which organs the virus is present. Importantly, by adding an antiviral to the water in which the larvae swim, we could significantly reduce the virus levels in the larvae. This means that testing small molecules and developing the first antiviral therapy for HuNoV will be much easier from hereon.

Since the initial report of the novel Coronavirus Disease 2019 (COVID-19) emanating from Wuhan, China, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally. While the effects of SARS-CoV-2 infection are not completely understood, there appears to be a wide spectrum of disease ranging from mild symptoms to severe respiratory distress, hospitalization, and mortality. There are no Food and Drug Administration (FDA)-approved treatments for COVID-19 aside from remdesivir; early efforts to identify efficacious therapeutics for COVID-19 have mainly focused on drug repurposing screens to identify compounds with antiviral activity against SARS-CoV-2 in cellular infection systems. These screens have yielded intriguing hits, but the use of nonhuman immortalized cell lines derived from non-pulmonary or gastrointestinal origins poses any number of questions in predicting the physiological and pathological relevance of these potential interventions. While our knowledge of this novel virus continues to evolve, our current understanding of the key molecular and cellular interactions involved in SARS-CoV-2 infection is discussed in order to provide a framework for developing the most appropriate in vitro toolbox to support current and future drug discovery efforts.

The frequent overexpression of CD46 in malignant tumors has provided a basis to use vaccine-lineage measles virus (MeV) as an oncolytic virotherapy platform. However, widespread measles seropositivity limits the systemic deployment of oncolytic MeV for the treatment of metastatic neoplasia. Here, we report the development of MeV-Stealth, a modified vaccine MeV strain that exhibits oncolytic properties and escapes antimeasles antibodies in vivo. We engineered this virus using homologous envelope glycoproteins from the closely-related but serologically non-cross reactive canine distemper virus (CDV). By fusing a high-affinity CD46 specific single-chain antibody fragment (scFv) to the CDV-Hemagglutinin (H), ablating its tropism for human nectin-4 and modifying the CDV-Fusion (F) signal peptide we achieved efficient retargeting to CD46. A receptor binding affinity of ~20 nM was required to trigger CD46-dependent intercellular fusion at levels comparable to the original MeV H/F complex and to achieve similar antitumor efficacy in myeloma and ovarian tumor-bearing mice models. In mice passively immunized with measles-immune serum, treatment of ovarian tumors with MeV-Stealth significantly increased overall survival compared with treatment with vaccine-lineage MeV. Our results show that MeV-Stealth effectively targets and lyses CD46-expressing cancer cells in mouse models of ovarian cancer and myeloma, and evades inhibition by human measles-immune serum. MeV-Stealth could therefore represent a strong alternative to current oncolytic MeV strains for treatment of measles-immune cancer patients., Author summary Vaccine strains of the measles virus (MeV) have been shown to be promising anti-cancer agents because of the frequent overexpression of the host-cell receptor CD46 in human malignancies. However, anti-MeV antibodies in the human population severely restrict the use of MeV as an oncolytic agent. Here, we engineered a neutralization-resistant MeV vaccine, MeV-Stealth, by replacing its envelope glycoproteins with receptor-targeted glycoproteins from wild-type canine distemper virus. By fully-retargeting the new envelope to the receptor CD46, we found that in mouse models of ovarian cancer and myeloma MeV-Stealth displayed oncolytic properties similar to the parental MeV vaccine. Furthermore, we found that passive immunization with measles-immune human serum did not eliminate the oncolytic potency of the MeV-Stealth, whereas it did destroy the potency of the parental MeV strain. The virus we here report may be considered a suitable oncolytic agent for the treatment of MeV-immune patients.

The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains., Author summary SARS-CoV-2 uses its spike (S) protein to enter target cells. Unlike other similar coronaviruses, the nascent S protein has a polybasic cleavage motif and is cleaved by the host protease. We have identified SARS-CoV-2 variants with mutations at the cleavage motif of S protein (S gene mutants) which undergo inefficient proteolytic cleavage, generate smaller plaques, and infect fewer cell lines. Notably, S gene mutants emerged rapidly through SARS-CoV-2 propagation in Vero cells. Since Vero cells are commonly used for SARS-CoV-2 propagation, it is a very real possibility that researchers have performed experiments, screened antivirals, and developed vaccines using SARS-CoV-2 S gene mutants without realizing.

Prions are pathogens formed from abnormal conformers (PrPSc) of the host-encoded cellular prion protein (PrPC). PrPSc conformation to disease phenotype relationships extensively vary among prion strains. In particular, prions exhibit a strain-dependent tropism for lymphoid tissues. Prions can be composed of several substrain components. There is evidence that these substrains can propagate in distinct tissues (e.g. brain and spleen) of a single individual, providing an experimental paradigm to study the cause of prion tissue selectivity. Previously, we showed that PrPC expression levels feature in prion substrain selection in the brain. Transmission of sheep scrapie isolates (termed LAN) to multiple lines of transgenic mice expressing varying levels of ovine PrPC in their brains resulted in the phenotypic expression of the dominant sheep substrain in mice expressing near physiological PrPC levels, whereas a minor substrain replicated preferentially on high expresser mice. Considering that PrPC expression levels are markedly decreased in the spleen compared to the brain, we interrogate whether spleen PrPC dosage could drive prion selectivity. The outcome of the transmission of a large cohort of LAN isolates in the spleen from high expresser mice correlated with the replication rate dependency on PrPC amount. There was a prominent spleen colonization by the substrain preferentially replicating on low expresser mice and a relative incapacity of the substrain with higher-PrPC level need to propagate in the spleen. Early colonization of the spleen after intraperitoneal inoculation allowed neuropathological expression of the lymphoid substrain. In addition, a pair of substrain variants resulting from the adaptation of human prions to ovine high expresser mice, and exhibiting differing brain versus spleen tropism, showed different tropism on transmission to low expresser mice, with the lymphoid substrain colonizing the brain. Overall, these data suggest that PrPC expression levels are instrumental in prion lymphotropism., Author summary The cause of prion phenotype variation among prion strains remains poorly understood. In particular, prions replicate in a strain-dependent manner in the spleen. This can result in prion asymptomatic carriers. Based on our previous observations that dosage of the prion precursor (PrP) determined prion substrain selection in the brain, we examine whether PrP levels in the spleen could drive prion replication in this tissue, due to the low levels of the protein. We observe that the prion substrain with higher PrP need for replication does barely replicate in the spleen, while the component with low PrP need replicates efficiently. In addition, other human co-propagating prions with differing spleen and brain tropism showed different tropism on transmission to mice expressing low PrP levels, with the lymphoid substrain colonizing the brain. PrPC expression levels may thus be instrumental in prion tropism for the lymphoid tissue. From a diagnostic point of view, given the apparent complexity of prion diseases with respect to prion substrain composition, these data advocate to type extraneural tissues or fluids for a comprehensive identification of the circulating prions in susceptible mammals.

A unique glycoprotein is expressed on the virus envelope of human herpesvirus 6B (HHV-6B): the complex gH/gL/gQ1/gQ2 (hereafter referred to as the HHV-6B tetramer). This tetramer recognizes a host receptor expressed on activated T cells: human CD134 (hCD134). This interaction is essential for HHV-6B entry into the susceptible cells and is a determinant for HHV-6B cell tropism. The structural mechanisms underlying this unique interaction were unknown. Herein we solved the interactions between the HHV-6B tetramer and the receptor by using their neutralizing antibodies in molecular and structural analyses. A surface plasmon resonance analysis revealed fast dissociation/association between the tetramer and hCD134, although the affinity was high (KD = 18 nM) and comparable to those for the neutralizing antibodies (anti-gQ1: 17 nM, anti-gH: 2.7 nM). A competition assay demonstrated that the anti-gQ1 antibody competed with hCD134 in the HHV-6B tetramer binding whereas the anti-gH antibody did not, indicating the direct interaction of gQ1 and hCD134. A single-particle analysis by negative-staining electron microscopy revealed the tetramer's elongated shape with a gH/gL part and extra density corresponding to gQ1/gQ2. The anti-gQ1 antibody bound to the tip of the extra density, and anti-gH antibody bound to the putative gH/gL part. These results highlight the interaction of gQ1/gQ2 in the HHV-6B tetramer with hCD134, and they demonstrate common features among viral ligands of the betaherpesvirus subfamily from a macroscopic viewpoint., Author summary Primary infection of human herpesvirus 6B (HHV-6B) with fever and roseola occurs for almost all children, and HHV-6B remains in the host as a latent infection. The reactivation of HHV-6B (especially in patients after hematopoietic stem-cell transplantation) occasionally causes severe encephalitis, which is a public health concern worldwide. HHV-6B's unique gH/gL/gQ1/gQ2 complex (a tetramer expressed on the viral envelope) recognizes the cellular receptor, i.e., human CD134 (hCD134), which is essential for the entry of the virus into the host's cells. The interaction between HHV-6B tetramer and hCD134 is therefore one of the determinants of HHV-6B-specific cell tropism. This article sheds light on the molecular and structural interactions between HHV-6B tetramer and its host receptor along with their neutralizing antibodies, and their affinities, competition, and binding modes based on the HHV-6B tetramer structure are described. Our findings provide molecular and structural bases for a comprehensive understanding of these interactions and relationships.

Lyme disease, caused by Borrelia burgdorferi, B. afzelii and B. garinii, is a chronic, multi-systemic infection and the spectrum of tissues affected can vary with the Lyme disease strain. For example, whereas B. garinii infection is associated with neurologic manifestations, B. burgdorferi infection is associated with arthritis. The basis for tissue tropism is poorly understood, but has been long hypothesized to involve strain-specific interactions with host components in the target tissue. OspC (outer surface protein C) is a highly variable outer surface protein required for infectivity, and sequence differences in OspC are associated with variation in tissue invasiveness, but whether OspC directly influences tropism is unknown. We found that OspC binds to the extracellular matrix (ECM) components fibronectin and/or dermatan sulfate in an OspC variant-dependent manner. Murine infection by isogenic B. burgdorferi strains differing only in their ospC coding region revealed that two OspC variants capable of binding dermatan sulfate promoted colonization of all tissues tested, including joints. However, an isogenic strain producing OspC from B. garinii strain PBr, which binds fibronectin but not dermatan sulfate, colonized the skin, heart and bladder, but not joints. Moreover, a strain producing an OspC altered to recognize neither fibronectin nor dermatan sulfate displayed dramatically reduced levels of tissue colonization that were indistinguishable from a strain entirely deficient in OspC. Finally, intravital microscopy revealed that this OspC mutant, in contrast to a strain producing wild type OspC, was defective in promoting joint invasion by B. burgdorferi in living mice. We conclude that OspC functions as an ECM-binding adhesin that is required for joint invasion, and that variation in OspC sequence contributes to strain-specific differences in tissue tropism displayed among Lyme disease spirochetes., Author summary Infection by different Lyme disease bacteria is associated with different manifestations, such as cardiac, neurologic, or, in the case of B. burgdorferi, the major cause of Lyme disease in the U.S., joint disease. The basis for these differences is unknown, but likely involve strain-specific interactions with host components in the target tissue. The sequence of the outer surface lipoprotein OspC varies with the strains, and we found that this variation influences the spectrum of host extracellular matrix components recognized. Infection of mice with strains that are identical except for ospC revealed that OspC variants that differ in binding spectrum promote infection of different tissues. A strain producing OspC invaded and colonized the joint in living animals, but an altered OspC protein incapable of binding tissue components did not. Thus, tissue-binding by OspC is critical for infection and joint invasion, and OspC variation directly influences tissue tropism.

Microfold (M) cell host-pathogen interaction studies would benefit from the visual analysis of dynamic cellular and microbial interplays. We adapted a human in vitro M cell model to physiological bacterial infections, expression of fluorescent localization reporters and long-term three-dimensional time-lapse microscopy. This approach allows following key steps of M cell infection dynamics at subcellular resolution, from the apical onset to basolateral epithelial dissemination. We focused on the intracellular pathogen Shigella flexneri, classically reported to transcytose through M cells to initiate bacillary dysentery in humans, while eliciting poorly protective immune responses. Our workflow was critical to reveal that S. flexneri develops a bimodal lifestyle within M cells leading to rapid transcytosis or delayed vacuolar rupture, followed by direct actin motility-based propagation to neighboring enterocytes. Moreover, we show that Listeria monocytogenes, another intracellular pathogen sharing a tropism for M cells, disseminates in a similar manner and evades M cell transcytosis completely. We established that actin-based M cell-to-enterocyte spread is the major dissemination pathway for both pathogens and avoids their exposure to basolateral compartments in our system. Our results challenge the notion that intracellular pathogens are readily transcytosed by M cells to inductive immune compartments in vivo, providing a potential mechanism for their ability to evade adaptive immunity., Author summary Microfold (M) epithelial cells are important for the onset of infections and induction of immune responses in many mucosal diseases. We extended a human in vitro M cell model to apical infections, expression of fluorescent host and microbial reporters and real-time fluorescence microscopy. Focusing on the human intracellular pathogen S. flexneri, responsible for bacillary dysentery, this workflow allowed us to uncover that the bacterium can subvert the immunological sampling function of M cells by promoting a cytosolic lifestyle and spreading directly to neighboring enterocytes. This mechanism was shared with the etiologic agent of listeriosis, the intracellular pathogen L. monocytogenes and allowed both pathogens to avoid exposure to underlying immune compartments. These results may provide a mechanism for the ability of intracellular pathogens to evade adaptive immunity in vivo, emphasizing the importance of advanced studies of M cell host-pathogen interactions to understand early steps of mucosal invasion and their consequences on immunity.

Murine norovirus (MNoV) is an important model of human norovirus (HNoV) and mucosal virus infection more broadly. Viral receptor utilization is a major determinant of cell tropism, host range, and pathogenesis. The bona fide receptor for HNoV is unknown. Recently, we identified CD300lf as a proteinaceous receptor for MNoV. Interestingly, its paralogue CD300ld was also sufficient for MNoV infection in vitro. Here we explored whether CD300lf is the sole physiologic receptor in vivo and whether HNoV can use a CD300 ortholog as an entry receptor. We report that both CD300ld and CD300lf are sufficient for infection by diverse MNoV strains in vitro. We further demonstrate that CD300lf is essential for both oral and parenteral MNoV infection and to elicit anti-MNoV humoral responses in vivo. In mice deficient in STAT1 signaling, CD300lf is required for MNoV-induced lethality. Finally, we demonstrate that human CD300lf (huCD300lf) is not essential for HNoV infection, nor does huCD300lf inhibit binding of HNoV virus-like particles to glycans. Thus, we report huCD300lf is not a receptor for HNoV., Author summary Human norovirus is the leading cause of non-bacterial gastroenteritis causing up to 200,000 deaths each year. How human norovirus enters cells is unknown. Because human norovirus is difficult to grow in the laboratory and in small animals, we use mouse or murine norovirus as a model system. We recently discovered that murine norovirus can use the either CD300ld or CD300lf as a receptor in vitro. We also showed that CD300lf deficient mice were resistant to oral challenge with a single virus strain. Here we determined that CD300lf is essential for infection of diverse murine norovirus strains in cell lines and in mice with normal immune systems demonstrating it’s the primary physiologic receptor for diverse murine norovirus strains independent of infection route. Finally, we demonstrated that human CD300lf is not the elusive proteinaceous receptor for human norovirus.

Species-dependent variation in proteins that aid or limit virus replication determines the ability of lentiviruses to jump between host species. Identifying and overcoming these differences facilitates the development of animal models for HIV-1, including models based on chimeric SIVs that express HIV-1 envelope (Env) glycoproteins, (SHIVs) and simian-tropic HIV-1 (stHIV) strains. Here, we demonstrate that the inherently poor ability of most HIV-1 Env proteins to use macaque CD4 as a receptor is improved during adaptation by virus passage in macaques. We identify a single amino acid, A281, in HIV-1 Env that consistently changes during adaptation in macaques and affects the ability of HIV-1 Env to use macaque CD4. Importantly, mutations at A281 do not markedly affect HIV-1 Env neutralization properties. Our findings should facilitate the design of HIV-1 Env proteins for use in non-human primate models and thus expedite the development of clinically relevant reagents for testing interventions against HIV-1., Author summary Understanding the interactions between viruses and their hosts allows manipulation of primate lentiviruses and the generation of better animal models for HIV/AIDS. Species-dependent differences in cellular proteins that play key roles in virus replication, such as the primary HIV-1 receptor CD4, can limit virus tropism. Our data reveal how adaptation in macaques improves the ability of HIV-1 envelope glycoproteins to use macaque CD4. Moreover, we identify a single amino acid in the HIV-1 envelope glycoprotein CD4 binding site that improves macaque CD4 use by most HIV-1 envelope proteins tested and allows viruses expressing these proteins to replicate efficiently in macaque cells without compromising their sensitivity to various antibodies. These findings should facilitate the development and preclinical evaluation of HIV-1 Env directed prophylactic and therapeutic interventions.

Herpesvirus gH/gL envelope glycoprotein complexes are key players in virus entry as ligands for host cell receptors and by promoting fusion of viral envelopes with cellular membranes. Human cytomegalovirus (HCMV) has two alternative gH/gL complexes, gH/gL/gO and gH/gL/UL128,130,131A which both shape the HCMV tropism. By studying binding of HCMV particles to fibroblasts, we could for the first time show that virion gH/gL/gO binds to platelet-derived growth factor-α (PDGFR-α) on the surface of fibroblasts and that gH/gL/gO either directly or indirectly recruits gB to this complex. PDGFR-α functions as an entry receptor for HCMV expressing gH/gL/gO, but not for HCMV mutants lacking the gH/gL/gO complex. PDGFR-α-dependent entry is not dependent on activation of PDGFR-α. We could also show that the gH/gL/gO—PDGFR-α interaction starts the predominant entry pathway for infection of fibroblasts with free virus. Cell-associated virus spread is either driven by gH/gL/gO interacting with PDGFR-α or by the gH/gL/UL128,130,131A complex. PDGFR-α-positive cells may thus be preferred first target cells for infections with free virus which might have implications for the design of future HCMV vaccines or anti-HCMV drugs., Author summary The identification of cellular receptors recognized by viral glycoproteins promoting entry is central for understanding virus pathogenesis and transmission for any virus. Although the roles of alternative gH/gL complexes of HCMV in cell tropism and virus spread have been extensively studied in cell culture, transfer to HCMV tropism in vivo is a controversial issue. Our characterization of the PDGFR-α –gH/gL/gO interaction offers an explanation for the tropism of HCMV for cells and tissues with high levels of surface PDGFR-α in vivo. Discrepant findings, when similar cell types were analyzed in culture, may retrospectively be attributed to a culture-dependent loss or up-regulation of PDGFR-α protein levels. Our finding that the PDGFR-α—gH/gL/gO interaction starts the predominant entry pathway for infection with free virus moves the gH/gL/gO complex in the center of interest for vaccines designed to prevent horizontal or vertical transmission and also for the development of CMV vaccine or gene therapy vectors.

Zika virus (ZIKV), an emerging flavivirus, has recently spread explosively through the Western hemisphere. In addition to symptoms including fever, rash, arthralgia, and conjunctivitis, ZIKV infection of pregnant women can cause microcephaly and other developmental abnormalities in the fetus. We report herein the results of ZIKV infection of adult rhesus macaques. Following subcutaneous infection, animals developed transient plasma viremia and viruria from 1-7 days post infection (dpi) that was accompanied by the development of a rash, fever and conjunctivitis. Animals produced a robust adaptive immune response to ZIKV, although systemic cytokine response was minimal. At 7 dpi, virus was detected in peripheral nervous tissue, multiple lymphoid tissues, joints, and the uterus of the necropsied animals. Notably, viral RNA persisted in neuronal, lymphoid and joint/muscle tissues and the male and female reproductive tissues through 28 to 35 dpi. The tropism and persistence of ZIKV in the peripheral nerves and reproductive tract may provide a mechanism of subsequent neuropathogenesis and sexual transmission.

Herpes simplex virus (HSV) entry into the cells requires glycoproteins gD, gH/gL and gB, activated in a cascade fashion by conformational modifications induced by cognate receptors and intermolecular signaling. The receptors are nectin1 and HVEM (Herpes virus entry mediator) for gD, and αvβ6 or αvβ8 integrin for gH. In earlier work, insertion of a single chain antibody (scFv) to the cancer receptor HER2 (human epidermal growth factor receptor 2) in gD, or in gH, resulted in HSVs specifically retargeted to the HER2-positive cancer cells, hence in highly specific non-attenuated oncolytic agents. Here, the scFv to HER2 was inserted in gB (gBHER2). The insertion re-targeted the virus tropism to the HER2-positive cancer cells. This was unexpected since gB is known to be a fusogenic glycoprotein, not a tropism determinant. The gB-retargeted recombinant offered the possibility to investigate how HER2 mediated entry. In contrast to wt-gB, the activation of the chimeric gBHER2 did not require the activation of the gD and of gH/gL by their respective receptors. Furthermore, a soluble form of HER2 could replace the membrane-bound HER2 in mediating virus entry, hinting that HER2 acted by inducing conformational changes to the chimeric gB. This study shows that (i) gB can be modified and become the major determinant of HSV tropism; (ii) the chimeric gBHER2 bypasses the requirement for receptor-mediated activation of other essential entry glycoproteins., Author summary Herpes simplex virus encodes an entry apparatus made of the glycoproteins gD, gH/gL and gB. gD is the major determinant of HSV tropism. Receptor-induced modifications to gD and gH/gL activate in a cascade fashion gB, the conserved fusogenic glycoprotein across the Herpesviridae family. In herpesviruses other than HSV, but not in HSV, gB also contributes to determine the virus tropism. We took advantage of retargeting studies to investigate the process of HSV glycoprotein activation, and the specific roles played by the glycoproteins. When a heterologous ligand is engineered in gB, the virus tropism is retargeted to the ligand receptor. gB becomes the major determinant of HSV tropism, and does not any longer need the receptor-mediated activation of glycoproteins gD and gH/gL.

Prion strains are characterized by strain-specific differences in neuropathology but can also differ in incubation period, clinical disease, host-range and tissue tropism. The hyper (HY) and drowsy (DY) strains of hamster-adapted transmissible mink encephalopathy (TME) differ in tissue tropism and susceptibility to infection by extraneural routes of infection. Notably, DY TME is not detected in the secondary lymphoreticular system (LRS) tissues of infected hosts regardless of the route of inoculation. We found that similar to the lymphotropic strain HY TME, DY TME crosses mucosal epithelia, enters draining lymphatic vessels in underlying laminae propriae, and is transported to LRS tissues. Since DY TME causes disease once it enters the peripheral nervous system, the restriction in DY TME pathogenesis is due to its inability to establish infection in LRS tissues, not a failure of transport. To determine if LRS tissues can support DY TME formation, we performed protein misfolding cyclic amplification using DY PrPSc as the seed and spleen homogenate as the source of PrPC. We found that the spleen environment can support DY PrPSc formation, although at lower rates compared to lymphotropic strains, suggesting that the failure of DY TME to establish infection in the spleen is not due to the absence of a strain-specific conversion cofactor. Finally, we provide evidence that DY PrPSc is more susceptible to degradation when compared to PrPSc from other lymphotrophic strains. We hypothesize that the relative rates of PrPSc formation and clearance can influence prion tropism., Author summary Strain specific distribution of prions throughout the infected host are observed in both naturally occurring and experimentally induced prion diseases. The distribution of prions in the host can influence prion shedding and transmission (e.g. iatrogenic prion transmission). The mechanism(s) responsible for strain tropism are unknown. Here we show that entry and transport of prions to lymphoid tissue are not influenced by the prion strain. However, we show that lymphotropic prion strains have a higher rate of PrPSc formation and a lower rate of prion degradation compared to a non-lymphotropic prion strain. We hypothesize that the relative rates of PrPSc formation and clearance is one of potentially several mechanisms that can determine prion strain tropism.

I entered the prion field in the early 1990s, when the nature of the infectious agent was still in dispute. At one of the very first scientific meetings I attended, a spirited argument broke out between the proponents of a viral etiology and the proponents of the protein-only (prion) etiology. One of the strong arguments made that day against the protein-only hypothesis was the observation that prion diseases displayed complex biological properties, such as strain diversity, which, at the time, were thought to be due to differences in the pathogen’s nucleic acid genome. How a protein-only agent, without a nucleic acid genome, could possess such complex biological properties was a mystery and went against biological dogma. At the time, I was a graduate student at the University of Wisconsin, and I was strongly influenced by the groundbreaking work of Howard Temin. This single conference and the knowledge that some of the basic tenants of science might need to be refined had a profound impact on my career. I have focused much of the last 25 years on understanding how prions accomplish complex biological functions in the absence of a nucleic acid genome. Today we know that prion diseases do not have a viral etiology and are indeed caused by an infectious conformation (designated PrPSc) of the normal host-encoded prion protein, PrPC. Prion strains are operationally defined as a phenotype of disease under a fixed set of agent and host parameters. Differences in the distribution and relative intensity of spongiform degeneration in select areas of the central nervous system are the favored criteria to distinguish strains. My early work examined interspecies transmission and adaptation of prions. What we found was that when a single prion strain was inoculated into a new host species, more than one strain would emerge. Moreover, changes in the conformation of PrPSc were predictive of which strain would emerge, lending support to the hypothesis that the conformation of PrPSc encodes strain diversity. These studies also indicated that strain interference, in which one strain interferes with the emergence of another, was an important parameter of prion adaptation to a new host species. The ability to target PrPSc from two different strains to the same identifiable population of neurons has been essential to understanding some of the basic parameters of strain interference. This was accomplished at the start of a long-standing collaboration with a neuroscientist, Anthony (Tony) Kincaid at Creighton University. We were able to determine, in fine detail, where strain interference occurs and gain important insight into the mechanism of interference. More recent work with Tony has investigated initial prion entry and subsequent transport in the host. We have shown that different prion strains cross epithelia, are transported in blood, and spread in the nervous systems with remarkably similar temporal and spatial patterns of spread. This work suggests, in a broad sense, that strain-specific tropism of prions is quite unlike viral tropism, which is influenced by the distribution of viral receptors. Instead, prions appear to be transported to a variety of cells, and the balance between replication, clearance, and toxicity may govern prion tropism. Another unexpected property of prions is that they can persist and remain infectious even after years in the environment. A chance phone call from an environmental engineer from the University of Nebraska, Shannon Bartelt-Hunt, has led to a fruitful collaboration and a series of important observations regarding this issue. Working with Shannon, we have identified that the soil type, the matrix that contains prions, and the strain of prion influences the binding of prions to soil. Additionally, this work demonstrated that weathering can partially inactivate prions and that PrPSc bound to surfaces has a strong influence on this process. I have been fortunate to collaborate with many talented investigators, and I have found that interdisciplinary collaborations have moved this work forward in interesting and sometimes unexpected directions. Prion research has provided the groundwork for important observations in other areas of biology. In yeast and fungi, there are numerous reports of two conformations of the same protein having different cellular functions. Importantly, the self-propagating (prion) conformation of the protein can alter the normal conformation of the protein into a misfolded prion conformation, as is seen in prion diseases of animals. Unlike prion diseases in animals, which are uniformly fatal, yeast prions can serve a useful function to the cell and can be thought of as a protein-based epigenetic mode of inheritance. Recent work suggests that other protein misfolding diseases share much in common with prion diseases. For example, the formation of amyloid in Alzheimer’s disease and Lewy bodies in Parkinson’s disease are thought to occur by a prion-like mechanism. Moreover, the spread of neuropathology in Parkinson’s disease and amyotrophic lateral sclerosis follows neuroanatomical pathways that are strikingly similar to those that we have reported in our studies of prion diseases. Also, the formation of α-synuclein fibrils with distinct biochemical properties that result in distinct patterns of pathology in Parkinson’s disease is reminiscent of what we and others have reported with prion strains. These are some of the unexpected and potentially significant findings of prion disease research that have implications for human health that extend far beyond the traditional boundaries of this relatively rare class of neurodegenerative diseases. Image 1 Jason C. Bartz.

In human cytomegalovirus (HCMV), tropism to epithelial and endothelial cells is dependent upon a pentameric complex (PC). Given the structure of the placenta, the PC is potentially an important neutralizing antibody target antigen against congenital infection. The guinea pig is the only small animal model for congenital CMV. Guinea pig cytomegalovirus (GPCMV) potentially encodes a UL128-131 HCMV PC homolog locus (GP128-GP133). In transient expression studies, GPCMV gH and gL glycoproteins interacted with UL128, UL130 and UL131 homolog proteins (designated GP129 and GP131 and GP133 respectively) to form PC or subcomplexes which were determined by immunoprecipitation reactions directed to gH or gL. A natural GP129 C-terminal deletion mutant (aa 107–179) and a chimeric HCMV UL128 C-terminal domain swap GP129 mutant failed to form PC with other components. GPCMV infection of a newly established guinea pig epithelial cell line required a complete PC and a GP129 mutant virus lacked epithelial tropism and was attenuated in the guinea pig for pathogenicity and had a low congenital transmission rate. Individual knockout of GP131 or 133 genes resulted in loss of viral epithelial tropism. A GP128 mutant virus retained epithelial tropism and GP128 was determined not to be a PC component. A series of GPCMV mutants demonstrated that gO was not strictly essential for epithelial infection whereas gB and the PC were essential. Ectopic expression of a GP129 cDNA in a GP129 mutant virus restored epithelial tropism, pathogenicity and congenital infection. Overall, GPCMV forms a PC similar to HCMV which enables evaluation of PC based vaccine strategies in the guinea pig model., Author Summary Congenital CMV is a leading cause of mental retardation and deafness in newborns. An effective vaccine against congenital CMV remains an elusive goal. HCMV encodes a pentameric glycoprotein complex (PC) necessary for tropism to epithelial, endothelial and myeloid cells. Given the structure of the placenta, the viral PC is considered important for congenital infection and potentially an important neutralizing antibody vaccine target antigen. The guinea pig, with a placenta structure similar to humans, is the only small animal model for congenital CMV. In this paper, GPCMV is shown to encode a homolog PC which enables epithelial tropism on a newly established cell line. It is likely that the GPCMV PC improves virus tropism to various cell types as PC positive virus has improved virus pathogenicity and congenital infection in vivo. This study lays important foundations for development of a PC based intervention strategy against congenital CMV in this model.

West Nile virus (WNV) is transmitted to vertebrate hosts by mosquitoes as they take a blood meal. The amount of WNV inoculated by mosquitoes as they feed on a live host is not known. Previous estimates of the amount of WNV inoculated by mosquitoes (101.2–104.3 PFU) were based on in vitro assays that do not allow mosquitoes to probe or feed naturally. Here, we developed an in vivo assay to determine the amount of WNV inoculated by mosquitoes as they probe and feed on peripheral tissues of a mouse or chick. Using our assay, we recovered approximately one-third of a known amount of virus inoculated into mouse tissues. Accounting for unrecovered virus, mean and median doses of WNV inoculated by four mosquito species were 104.3 PFU and 105.0 PFU for Culex tarsalis, 105.9 PFU and 106.1 PFU for Cx. pipiens, 104.7 PFU and 104.7 PFU for Aedes japonicus, and 103.6 PFU and 103.4 PFU for Ae. triseriatus. In a direct comparison, in vivo estimates of the viral dose inoculated by Cx. tarsalis were approximately 600 times greater than estimates obtained by an in vitro capillary tube transmission assay. Virus did not disperse rapidly, as >99% of the virus was recovered from the section fed or probed upon by the mosquito. Furthermore, 76% (22/29) of mosquitoes inoculated a small amount of virus (∼102 PFU) directly into the blood while feeding. Direct introduction of virus into the blood may alter viral tropism, lead to earlier development of viremia, and cause low rates of infection in co-feeding mosquitoes. Our data demonstrate that mosquitoes inoculate high doses of WNV extravascularly and low doses intravascularly while probing and feeding on a live host. Accurate estimates of the viral dose inoculated by mosquitoes are critical in order to administer appropriate inoculation doses to animals in vaccine, host competence, and pathogenesis studies., Author Summary Since it was first introduced into the United States in 1999, West Nile virus (WNV) has caused significant disease in humans, horses, and other animals. WNV is transmitted to humans and other vertebrate hosts by female mosquitoes as they take a blood meal. Currently, the amount of virus inoculated by mosquitoes while feeding on live hosts is unknown, and accurate estimates are critical so that appropriate challenge doses can be used in vaccine and viral pathogenesis studies. Here, we use a novel technique to determine the dose of WNV inoculated by mosquitoes as they probe and feed on the peripheral tissues of live animals. We found that mosquitoes inoculate high doses of virus into host tissues; these doses are 10 to 1,000 times higher than previous estimates obtained with assays that do not allow mosquitoes to probe or feed naturally. We also found that mosquitoes inoculate low doses of virus directly into the blood while blood feeding. Direct introduction of virus into the blood may alter viral tropism and cause low rates of infection in co-feeding mosquitoes. Our study provides new insights into the transmission of an emerging viral pathogen and the interaction of virus with its mosquito vector and vertebrate host.

The host lymphatic network represents an important conduit for pathogen dissemination. Indeed, the lethal human pathogen group A streptococcus has a predilection to induce pathology in the lymphatic system and draining lymph nodes, however the underlying basis and subsequent consequences for disease outcome are currently unknown. Here we report that the hyaluronan capsule of group A streptococci is a crucial virulence determinant for lymphatic tropism in vivo, and further, we identify the lymphatic vessel endothelial receptor-1 as the critical host receptor for capsular hyaluronan in the lymphatic system. Interference with this interaction in vivo impeded bacterial dissemination to local draining lymph nodes and, in the case of a hyper-encapsulated M18 strain, redirected streptococcal entry into the blood circulation, suggesting a pivotal role in the manifestation of streptococcal infections. Our results reveal a novel function for bacterial capsular polysaccharide in directing lymphatic tropism, with potential implications for disease pathology., Author Summary Pathogens are known to invade the host not only via the systemic circulation but also via the lymphatic network, however the mechanisms underlying the latter route and the consequences for disease outcome have not been well studied. The important human pathogen, group A streptococcus, is responsible for a number of clinical syndromes affecting both the lymphatic vessels and draining lymph nodes, such as lymphangitis and lymphadenitis. How such pathologies are orchestrated, and their significance in the development of serious infection are currently unknown. In this study, we show that the hyaluronan capsule secreted by group A streptococcus is critical for bacterial spread to draining lymph nodes, and we demonstrate that this occurs as a result of a specific interaction with the lymphatic vessel endothelial receptor-1. Genetic deletion or functional blockade of this receptor prevented streptococcal transit to draining lymph nodes in a murine model of infection, which in turn enhanced bacterial spread into the blood circulation. Together these results define a novel interaction between the group A streptococcal capsule and the lymphatic endothelial receptor-1 as a critical axis in the establishment of lymphatic tropism for this pathogen, with clear implications for disease severity in the host.

Varicella zoster virus (VZV), a human alphaherpesvirus, causes varicella during primary infection. VZV reactivation from neuronal latency may cause herpes zoster, post herpetic neuralgia (PHN) and other neurologic syndromes. To investigate VZV neuropathogenesis, we developed a model using human dorsal root ganglia (DRG) xenografts in immunodeficient (SCID) mice. The SCID DRG model provides an opportunity to examine characteristics of VZV infection that occur in the context of the specialized architecture of DRG, in which nerve cell bodies are ensheathed by satellite glial cells (SGC) which support neuronal homeostasis. We hypothesized that VZV exhibits neuron-subtype specific tropism and that VZV tropism for SGC contributes to VZV-related ganglionopathy. Based on quantitative analyses of viral and cell protein expression in DRG tissue sections, we demonstrated that, whereas DRG neurons had an immature neuronal phenotype prior to implantation, subtype heterogeneity was observed within 20 weeks and SGC retained the capacity to maintain neuronal homeostasis longterm. Profiling VZV protein expression in DRG neurons showed that VZV enters peripherin+ nociceptive and RT97+ mechanoreceptive neurons by both axonal transport and contiguous spread from SGC, but replication in RT97+ neurons is blocked. Restriction occurs even when the SGC surrounding the neuronal cell body were infected and after entry and ORF61 expression, but before IE62 or IE63 protein expression. Notably, although contiguous VZV spread with loss of SGC support would be predicted to affect survival of both nociceptive and mechanoreceptive neurons, RT97+ neurons showed selective loss relative to peripherin+ neurons at later times in DRG infection. Profiling cell factors that were upregulated in VZV-infected DRG indicated that VZV infection induced marked pro-inflammatory responses, as well as proteins of the interferon pathway and neuroprotective responses. These neuropathologic changes observed in sensory ganglia infected with VZV may help to explain the neurologic sequelae often associated with zoster and PHN., Author Summary Varicella zoster virus (VZV) causes varicella; herpes zoster results from VZV reactivation and is associated with post herpetic neuralgia (PHN). We hypothesized that VZV exhibits neuron-subtype specific tropism and that VZV tropism for satellite glial cells (SGC) results in loss of SGC functions that support neurons and contributes to VZV-related ganglionopathy. Using human DRG xenografts in SCID mice, we demonstrated that initial VZV access to neuronal cell bodies occurs by the axonal route, followed by axonal and contiguous spread between neuron-satellite cell complexes. VZV replication is restricted in mechanoreceptive neurons compared to nociceptive neurons. Despite restricted infection, mechanoreceptive neurons were selectively depleted in association with SGC loss following acute DRG infection. VZV infection of DRG triggers release of pro-inflammatory cytokines that cause neuronal damage. These observations may help to explain the neurologic sequelae often associated with herpes zoster and PHN.

Microbial tropism, the infection of specific cells and tissues by a microorganism, is a fundamental aspect of host-microbe interactions. The intracellular bacteria Wolbachia have a peculiar tropism for the stem cell niches in the Drosophila ovary, the microenvironments that support the cells producing the eggs. The molecular underpinnings of Wolbachia stem cell niche tropism are unknown. We have previously shown that the patterns of tropism in the ovary show a high degree of conservation across the Wolbachia lineage, with closely related Wolbachia strains usually displaying the same pattern of stem cell niche tropism. It has also been shown that tropism to these structures in the ovary facilitates both vertical and horizontal transmission, providing a strong selective pressure towards evolutionary conservation of tropism. Here we show great disparity in the evolutionary conservation and underlying mechanisms of stem cell niche tropism between male and female gonads. In contrast to females, niche tropism in the male testis is not pervasive, present in only 45% of niches analyzed. The patterns of niche tropism in the testis are not evolutionarily maintained across the Wolbachia lineage, unlike what was shown in the females. Furthermore, hub tropism does not correlate with cytoplasmic incompatibility, a Wolbachia-driven phenotype imprinted during spermatogenesis. Towards identifying the molecular mechanism of hub tropism, we performed hybrid analyses of Wolbachia strains in non-native hosts. These results indicate that both Wolbachia and host derived factors play a role in the targeting of the stem cell niche in the testis. Surprisingly, even closely related Wolbachia strains in Drosophila melanogaster, derived from a single ancestor only 8,000 years ago, have significantly different tropisms to the hub, highlighting that stem cell niche tropism is rapidly diverging in males. These findings provide a powerful system to investigate the mechanisms and evolution of microbial tissue tropism., Author Summary Microbes evolve to infect structures favoring their transmission in host populations. A large fraction of insects are infected with Wolbachia bacteria. Usually Wolbachia are transmitted the same way we inherit our mitochondria, via the eggs from the mother. In fruit flies, to favor maternal transmission, Wolbachia infect the microenvironment containing the egg producing stem cells, called the “stem cell niche”. Targeting of the stem cell niche is evolutionary conserved in female fruit flies, observed in all Wolbachia strains analyzed to date. Remarkably, in males, we find many Wolbachia strains not infecting the stem cell niche present in the testis. We report a surprising diversity in stem cell niche infection in males, contrasting with extreme conservation in females. We further show that even closely related Wolbachia strains in D. melanogaster display rapidly evolving patterns of stem cell niche targeting in males. Understanding the molecular mechanisms driving these differences will identify sex specific features of stem cell niche biology. Because Wolbachia promote insect resistance against human diseases transmitted by mosquitos, Wolbachia are becoming a valuable tool in the control of several diseases, including Dengue and malaria. Knowledge emerging from this research will also provide novel tools towards Wolbachia based strategies of disease control.

The picornaviruses coxsackievirus A24 variant (CVA24v) and enterovirus 70 (EV70) cause continued outbreaks and pandemics of acute hemorrhagic conjunctivitis (AHC), a highly contagious eye disease against which neither vaccines nor antiviral drugs are currently available. Moreover, these viruses can cause symptoms in the cornea, upper respiratory tract, and neurological impairments such as acute flaccid paralysis. EV70 and CVA24v are both known to use 5-N-acetylneuraminic acid (Neu5Ac) for cell attachment, thus providing a putative link between the glycan receptor specificity and cell tropism and disease. We report the structures of an intact human picornavirus in complex with a range of glycans terminating in Neu5Ac. We determined the structure of the CVA24v to 1.40 Å resolution, screened different glycans bearing Neu5Ac for CVA24v binding, and structurally characterized interactions with candidate glycan receptors. Biochemical studies verified the relevance of the binding site and demonstrated a preference of CVA24v for α2,6-linked glycans. This preference can be rationalized by molecular dynamics simulations that show that α2,6-linked glycans can establish more contacts with the viral capsid. Our results form an excellent platform for the design of antiviral compounds to prevent AHC., Author Summary Coxsackievirus A24 variant (CVA24v) and enterovirus 70 (EV70) are responsible for several outbreaks of a highly contagious eye disease called acute hemorrhagic conjunctivitis (AHC). These viruses represent a limited set of human picornaviruses that use glycan receptors for cell attachment. Until now no data has been available about the binding site of these glycan receptors. We therefore determined the structure of the entire virus capsid in its unbound state and also together with several glycan receptor mimics and could establish the structure of the receptor binding site. CVA24v recognizes the receptor at a solvent exposed site on the virus shell by interactions with a single capsid protein VP1. Moreover, we identified a glycan motif favoured for CVA24v binding and confirmed this preference biochemically and by in silico simulations. Our results form a solid basis for structure-based development of drugs to treat CVA24v-caused AHC.

Apicomplexan parasites are the causative agents of diseases that include malaria, toxoplasmosis, and coccidiosis. These obligate intracellular parasites have evolved to use a conserved mechanism for host-cell invasion. The apicomplexan phylum is defined by the presence of micronemes and rhoptries, which are distinct organelles located at the apical end of the parasite. These organelles secrete molecules necessary for host-cell invasion [1]. Apicomplexan parasites can invade disparate cell types, including hepatocytes, erythrocytes, lymphocytes, macrophages, and cells lining the digestive tract. Unlike viruses and intracellular bacteria, apicomplexans actively invade host cells without relying on host uptake pathways. As such, host-cell sensing and subsequent invasion are driven entirely by the parasite in a dynamic and rapid process. Intracellular residence protects the parasite from immune attack and enables parasite replication prior to host-cell lysis and subsequent invasion of neighboring host cells. The repertoire of ligand-receptor complexes utilized by parasites for entry into host cells is diverse. Some interactions occur through cell-specific receptors resulting in high-affinity interactions, while others occur through multiple lower-affinity interactions via surface moieties found on several cell types. Receptor-specific and general cell binding may explain host-cell tropism of different pathogens, although additional factors are important. There is growing evidence that multimeric assembly of parasite ligands and host surface molecules strengthens the host-pathogen interactions necessary for invasion. We discuss recent work that has advanced our knowledge of the assembly of adhesive complexes from two critical apicomplexan pathogens and highlight areas of research that require further investigation.

It is well established that RNA viruses exhibit higher rates of spontaneous mutation than DNA viruses and microorganisms. However, their mutation rates vary amply, from 10−6 to 10−4 substitutions per nucleotide per round of copying (s/n/r) and the causes of this variability remain poorly understood. In addition to differences in intrinsic fidelity or error correction capability, viral mutation rates may be dependent on host factors. Here, we assessed the effect of the cellular environment on the rate of spontaneous mutation of the vesicular stomatitis virus (VSV), which has a broad host range and cell tropism. Luria-Delbrück fluctuation tests and sequencing showed that VSV mutated similarly in baby hamster kidney, murine embryonic fibroblasts, colon cancer, and neuroblastoma cells (approx. 10−5 s/n/r). Cell immortalization through p53 inactivation and oxygen levels (1–21%) did not have a significant impact on viral replication fidelity. This shows that previously published mutation rates can be considered reliable despite being based on a narrow and artificial set of laboratory conditions. Interestingly, we also found that VSV mutated approximately four times more slowly in various insect cells compared with mammalian cells. This may contribute to explaining the relatively slow evolution of VSV and other arthropod-borne viruses in nature., Author Summary RNA viruses show high rates of spontaneous mutation, a feature that profoundly influences viral evolution, disease emergence, the appearance of drug resistances, and vaccine efficacy. However, RNA virus mutation rates vary substantially and the factors determining this variability remain poorly understood. Here, we investigated the effects of host factors on viral replication fidelity by measuring the viral mutation rate in different cell types and under various culturing conditions. To carry out these experiments we chose the vesicular stomatitis virus (VSV), an insect-transmitted mammalian RNA virus with an extremely wide cellular and host tropism. We found that the VSV replication machinery was robust to changes in cellular physiology driven by cell immortalization or shifts in temperature and oxygen levels. In contrast, VSV mutated significantly more slowly in insect cells than in mammalian cells, a finding may help us to understand why arthropod-borne viruses tend to evolve more slowly than directly transmitted viruses in nature.

Tumour mutations corrupt cellular pathways, and accumulate to disrupt, dysregulate, and ultimately avoid mechanisms of cellular control. Yet the very changes that tumour cells undergo to secure their own growth success also render them susceptible to viral infection. Enhanced availability of surface receptors, disruption of antiviral sensing, elevated metabolic activity, disengagement of cell cycle controls, hyperactivation of mitogenic pathways, and apoptotic avoidance all render the malignant cell environment highly supportive to viral replication. The therapeutic use of oncolytic viruses (OVs) with a natural tropism for infecting and subsequently lysing tumour cells is a rapidly progressing area of cancer research. While many OVs exhibit an inherent degree of tropism for transformed cells, this can be further promoted through pharmacological interventions and/or the introduction of viral mutations that generate recombinant oncolytic viruses adapted to successfully replicate only in a malignant cellular environment. Such adaptations that augment OV tumour selectivity are already improving the therapeutic outlook for cancer, and there remains tremendous untapped potential for further innovation.

The high rates of RNA virus evolution are generally attributed to replication with error-prone RNA-dependent RNA polymerases. However, these long-term nucleotide substitution rates span three orders of magnitude and do not correlate well with mutation rates or selection pressures. This substitution rate variation may be explained by differences in virus ecology or intrinsic genomic properties. We generated nucleotide substitution rate estimates for mammalian RNA viruses and compiled comparable published rates, yielding a dataset of 118 substitution rates of structural genes from 51 different species, as well as 40 rates of non-structural genes from 28 species. Through ANCOVA analyses, we evaluated the relationships between these rates and four ecological factors: target cell, transmission route, host range, infection duration; and three genomic properties: genome length, genome sense, genome segmentation. Of these seven factors, we found target cells to be the only significant predictors of viral substitution rates, with tropisms for epithelial cells or neurons (P, Author Summary RNA viruses are the fastest evolving human pathogens, making their treatment and control difficult. Compared to DNA viruses, RNA viruses replicate with much lower fidelity, which can explain why RNA viruses evolve significantly faster than most DNA viruses. However, there is tremendous variation among the evolutionary rates of different RNA viruses, which is not explained by variation in mutation rates. Here we present a survey of mammalian RNA virus rates of evolution, and a comprehensive comparison of these rates to different properties of virus genomic architecture and ecology. We found that cell tropism is the most significant predictor of long-term rates of mammalian RNA virus evolution. For instance, viruses targeting epithelial cells evolve significantly faster than viruses that target neurons. Our results provide mechanistic insight into why viruses that infect respiratory and gastrointestinal epithelia have been difficult to control.

Little is known about how the mode of respiratory virus transmission determines the dynamics of primary infection and protection from reinfection. Using non-invasive imaging of murine parainfluenza virus 1 (Sendai virus) in living mice, we determined the frequency, timing, dynamics, and virulence of primary infection after contact and airborne transmission, as well as the tropism and magnitude of reinfection after subsequent challenge. Contact transmission of Sendai virus was 100% efficient, phenotypically uniform, initiated and grew to robust levels in the upper respiratory tract (URT), later spread to the lungs, grew to a lower level in the lungs than the URT, and protected from reinfection completely in the URT yet only partially in the lungs. Airborne transmission through 7.6-cm and 15.2-cm separations between donor and recipient mice was 86%–100% efficient. The dynamics of primary infection after airborne transmission varied between individual mice and included the following categories: (a) non-productive transmission, (b) tracheal dominant, (c) tracheal initiated yet respiratory disseminated, and (d) nasopharyngeal initiated yet respiratory disseminated. Any previous exposure to Sendai virus infection protected from mortality and severe morbidity after lethal challenge. Furthermore, a higher level of primary infection in a given respiratory tissue (nasopharynx, trachea, or lungs) was inversely correlated with the level of reinfection in that same tissue. Overall, the mode of transmission determined the dynamics and tropism of primary infection, which in turn governed the level of seroconversion and protection from reinfection. These data are the first description of the dynamics of respiratory virus infection and protection from reinfection throughout the respiratory tracts of living animals after airborne transmission. This work provides a basis for understanding parainfluenza virus transmission and protective immunity and for developing novel vaccines and non-pharmaceutical interventions., Author Summary Parainfluenza viruses are highly contagious, a leading cause of acute respiratory infection (ARI) in children, often reoccurring, and currently controlled by non-pharmaceutical interventions. We tracked infection and reinfection of a prototypic murine parainfluenza virus after contact or airborne transmission. Our studies reveal that the mode of transmission determines the dynamics of primary infection. Additionally, higher levels of protection from reinfection are induced in individual respiratory tissues by higher levels of primary infection in those same tissues. Natural infection after either contact or airborne transmission tends to initiate in the URT, but not the lungs. Complete protection from infection in the URT was afforded by URT-biased, non-pathogenic infection after low-dose intranasal vaccination. Overall, the data suggest that parainfluenza virus transmission may be effectively controlled by handwashing, disinfection of surfaces, and environmental control of short-range transmission, in addition to the development of live attenuated vaccines that target the URT.

Toxoplasma gondii is an intracellular parasite that transitions from acute infection to a chronic infective state in its intermediate host via encystation, which enables the parasite to evade immune detection and clearance. It is widely accepted that the tissue cyst perimeter is highly and specifically decorated with glycan modifications; however, the role of these modifications in the establishment and persistence of chronic infection has not been investigated. Here we identify and biochemically and biologically characterize a Toxoplasma nucleotide-sugar transporter (TgNST1) that is required for cyst wall glycosylation. Toxoplasma strains deleted for the TgNST1 gene (Δnst1) form cyst-like structures in vitro but no longer interact with lectins, suggesting that Δnst1 strains are deficient in the transport and use of sugars for the biosynthesis of cyst-wall structures. In vivo infection experiments demonstrate that the lack of TgNST1 activity does not detectably impact the acute (tachyzoite) stages of an infection or tropism of the parasite for the brain but that Δnst1 parasites are severely defective in persistence during the chronic stages of the infection. These results demonstrate for the first time the critical role of parasite glycoconjugates in the persistence of Toxoplasma tissue cysts., Author Summary The Toxoplasma tissue cyst is essential to the persistence of the parasite during the chronic infection of an immunocompetent host. While significant efforts have been made to identify molecular factors that trigger and sustain parasite encystation, the role of the glycoconjugates that decorate the cyst wall has received little attention. Here we identify and characterize a bona fide nucleotide-sugar transporter, TgNST1, whose activity is required for the proper assembly of cyst wall glycoconjugates. We found that deletion of TgNST1 interferes with glycosylation during both the tachyzoite and bradyzoite stages of infection, and we observed substantial defects in the ability of Δnst1 parasites to maintain chronic infection. Surprisingly, Δnst1 parasites were not significantly defective in acute infection of mice, and showed wild type levels and migration rates to the brain. These results highlight the important role of cyst-wall glycosylation in parasite persistence during chronic infection, and suggest that drugs targeting nucleotide-sugar transporters and other enzymes required for glycosylation, perhaps in combination with drugs targeting other pathways, might be useful to prevent the establishment of chronic parasite infection.

We have reported that properties of prion strains may change when propagated in different environments. For example, when swainsonine-sensitive 22L prions were propagated in PK1 cells in the presence of swainsonine, drug-resistant variants emerged. We proposed that prions constitute quasi- populations comprising a range of variants with different properties, from which the fittest are selected in a particular environment. Prion populations developed heterogeneity even after biological cloning, indicating that during propagation mutation-like processes occur at the conformational level. Because brain-derived 22L prions are naturally swainsonine resistant, it was not too surprising that prions which had become swa sensitive after propagation in cells could revert to drug resistance. Because RML prions, both after propagation in brain or in PK1 cells, are swainsonine sensitive, we investigated whether it was nonetheless possible to select swainsonine-resistant variants by propagation in the presence of the drug. Interestingly, this was not possible with the standard line of PK1 cells, but in certain PK1 sublines not only swainsonine-resistant, but even swainsonine-dependent populations (i.e. that propagated more rapidly in the presence of the drug) could be isolated. Once established, they could be passaged indefinitely in PK1 cells, even in the absence of the drug, without losing swainsonine dependence. The misfolded prion protein (PrPSc) associated with a swainsonine-dependent variant was less rapidly cleared in PK1 cells than that associated with its drug-sensitive counterpart, indicating that likely structural differences of the misfolded PrP underlie the properties of the prions. In summary, propagation of prions in the presence of an inhibitory drug may not only cause the selection of drug-resistant prions but even of stable variants that propagate more efficiently in the presence of the drug. These adaptations are most likely due to conformational changes of the abnormal prion protein., Author Summary Prions consist of PrPSc, an aggregated conformer of the host protein PrPC. PrPSc multiplies by catalyzing the conformational conversion of PrPC into a likeness of itself. Prions present as distinct strains that have the same primary amino acid sequence but differ in their conformation. Many distinct mouse-derived prions strains, for example RML, 22L or Me7, have been isolated. Prions can adapt to their environment. We investigated whether propagation of swainsonine-sensitive RML prions in the presence of the drug would yield swainsonine-resistant variants. Interestingly, this was not possible with a standard line of neuroblastoma cells, but in certain sublines not only swainsonine-resistant, but even swainsonine-dependent populations (i.e. that propagated more rapidly in the presence than in the absence of the drug) could be isolated. Once established, they could be propagated “indefinitely” in PK1 cells, even in the absence of the drug, without losing swainsonine dependence. In summary, our paper shows that propagation of prions in the presence of an inhibitory drug may not only cause the selection of drug-resistant prions but even of stable prion variants that propagate more efficiently in the presence of the drug. These adaptations are most likely due to conformational changes of the abnormal prion protein.

Diversification of antiretroviral factors during host evolution has erected formidable barriers to cross-species retrovirus transmission. This phenomenon likely protects humans from infection by many modern retroviruses, but it has also impaired the development of primate models of HIV-1 infection. Indeed, rhesus macaques are resistant to HIV-1, in part due to restriction imposed by the TRIM5α protein (rhTRIM5α). Initially, we attempted to derive rhTRIM5α-resistant HIV-1 strains using two strategies. First, HIV-1 was passaged in engineered human cells expressing rhTRIM5α. Second, a library of randomly mutagenized capsid protein (CA) sequences was screened for mutations that reduced rhTRIM5α sensitivity. Both approaches identified several individual mutations in CA that reduced rhTRIM5α sensitivity. However, neither approach yielded mutants that were fully resistant, perhaps because the locations of the mutations suggested that TRIM5α recognizes multiple determinants on the capsid surface. Moreover, even though additive effects of various CA mutations on HIV-1 resistance to rhTRIM5α were observed, combinations that gave full resistance were highly detrimental to fitness. Therefore, we employed an ‘assisted evolution’ approach in which individual CA mutations that reduced rhTRIM5α sensitivity without fitness penalties were randomly assorted in a library of viral clones containing synthetic CA sequences. Subsequent passage of the viral library in rhTRIM5α-expressing cells resulted in the selection of individual viral species that were fully fit and resistant to rhTRIM5α. These viruses encoded combinations of five mutations in CA that conferred complete or near complete resistance to the disruptive effects of rhTRIM5α on incoming viral cores, by abolishing recognition of the viral capsid. Importantly, HIV-1 variants encoding these CA substitutions and SIVmac239 Vif replicated efficiently in primary rhesus macaque lymphocytes. These findings demonstrate that rhTRIM5α is difficult to but not impossible to evade, and doing so should facilitate the development of primate models of HIV-1 infection., Author Summary Retroviruses such as HIV-1 often exhibit limited capacity to infect species other than their natural hosts. This phenomenon is partly due to the existence of antiviral proteins that protect against infection by viruses that have not adapted to a particular species. For example, the resistance of rhesus macaques, the monkey species most commonly used in medical research, to HIV-1 infection is partly attributable to the vulnerability of HIV-1 to TRIM5α. Rhesus macaque TRIM5α (rhTRIM5α) blocks HIV-1 infection by recognition of the viral capsid following its entry into the cell, and it has proven difficult to derive HIV-1 strains that are resistant to rhTRIM5α. However, by devising an ‘assisted evolution’ approach, we identified particular combinations of mutations that render HIV-1 resistant to rhTRIM5α. These mutations enable HIV-1 to evade rhTRIM5α by abolishing recognition of the capsid. Notably, introduction of rhTRIM5α-resistant capsids into an HIV-1 that was also engineered to avoid the rhesus macaque APOBEC3 antiviral proteins, allowed efficient HIV-1 replication in rhesus macaque lymphocytes. These discoveries have the potential to advance the development of rhesus macaque models of HIV-1 infection.

Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV has spread very rapidly to many European countries. Here, we developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell culture and “synthetic” SBV replicates in vitro as efficiently as wild type virus. We developed an experimental mouse model to study SBV infection and showed that this virus replicates abundantly in neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced acute lesions are useful in understanding the progression from vacuolation to porencephaly and extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage. Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a biological SBV clone that after passage in cell culture displays increased virulence in mice. We also found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to study the biology and pathogenesis of SBV., Author Summary Schmallenberg virus (SBV) was discovered in Germany (near the town of Schmallenberg) in November 2011 and since then has been found to be the cause of malformations and stillbirths in ruminants. SBV has spread very rapidly to many European countries including the Netherlands, Belgium, France and the United Kingdom. Very little is known about the biological properties of this virus and there is no vaccine available. In this study (i) we developed an approach (called reverse genetics) that allows the recovery of “synthetic” SBV under laboratory conditions; (ii) we developed a mouse model of infection for SBV; (iii) we showed that SBV replicates in neurons of experimentally infected mice similar to naturally infected lambs and calves; (iv) we developed viral mutants that are not as pathogenic as the original virus due to the inability to counteract the host cell defenses; and v) we identified mutations that are associated with increased virulence. This work provides the experimental tools to understand how this newly emerged virus causes disease in ruminants. In addition, it will now be possible to manipulate the SBV genome in order to develop highly effective vaccines.

Cytomegaloviruses (CMV) are highly species-specific due to millennia of co-evolution and adaptation to their host, with no successful experimental cross-species infection in primates reported to date. Accordingly, full genome phylogenetic analysis of multiple new CMV field isolates derived from two closely related nonhuman primate species, Indian-origin rhesus macaques (RM) and Mauritian-origin cynomolgus macaques (MCM), revealed distinct and tight lineage clustering according to the species of origin, with MCM CMV isolates mirroring the limited genetic diversity of their primate host that underwent a population bottleneck 400 years ago. Despite the ability of Rhesus CMV (RhCMV) laboratory strain 68–1 to replicate efficiently in MCM fibroblasts and potently inhibit antigen presentation to MCM T cells in vitro, RhCMV 68–1 failed to productively infect MCM in vivo, even in the absence of host CD8+ T and NK cells. In contrast, RhCMV clone 68–1.2, genetically repaired to express the homologues of the HCMV anti-apoptosis gene UL36 and epithelial cell tropism genes UL128 and UL130 absent in 68–1, efficiently infected MCM as evidenced by the induction of transgene-specific T cells and virus shedding. Recombinant variants of RhCMV 68–1 and 68–1.2 revealed that expression of either UL36 or UL128 together with UL130 enabled productive MCM infection, indicating that multiple layers of cross-species restriction operate even between closely related hosts. Cumulatively, these results implicate cell tropism and evasion of apoptosis as critical determinants of CMV transmission across primate species barriers, and extend the macaque model of human CMV infection and immunology to MCM, a nonhuman primate species with uniquely simplified host immunogenetics., Author Summary Nonhuman primates are excellent models of HCMV infection and immunity, and their CMVs closely resemble HCMV in genomic organization and coding potential. Here, we expand the existing models by isolating new full-length, non-human primate CMV strains that more closely resemble primary HCMV isolates. In addition, we identify determinants needed for recombinant clones of RhCMV to infect MCM both in vitro and in vivo. Given their simplified immunogenetics, these results enable the use of MCM to deepen our understanding of CMV immunology.

Influenza Virus (IV) pneumonia is associated with severe damage of the lung epithelium and respiratory failure. Apart from efficient host defense, structural repair of the injured epithelium is crucial for survival of severe pneumonia. The molecular mechanisms underlying stem/progenitor cell mediated regenerative responses are not well characterized. In particular, the impact of IV infection on lung stem cells and their regenerative responses remains elusive. Our study demonstrates that a highly pathogenic IV infects various cell populations in the murine lung, but displays a strong tropism to an epithelial cell subset with high proliferative capacity, defined by the signature EpCamhighCD24lowintegrin(α6)high. This cell fraction expressed the stem cell antigen-1, highly enriched lung stem/progenitor cells previously characterized by the signature integrin(β4)+CD200+, and upregulated the p63/krt5 regeneration program after IV-induced injury. Using 3-dimensional organoid cultures derived from these epithelial stem/progenitor cells (EpiSPC), and in vivo infection models including transgenic mice, we reveal that their expansion, barrier renewal and outcome after IV-induced injury critically depended on Fgfr2b signaling. Importantly, IV infected EpiSPC exhibited severely impaired renewal capacity due to IV-induced blockade of β-catenin-dependent Fgfr2b signaling, evidenced by loss of alveolar tissue repair capacity after intrapulmonary EpiSPC transplantation in vivo. Intratracheal application of exogenous Fgf10, however, resulted in increased engagement of non-infected EpiSPC for tissue regeneration, demonstrated by improved proliferative potential, restoration of alveolar barrier function and increased survival following IV pneumonia. Together, these data suggest that tropism of IV to distal lung stem cell niches represents an important factor of pathogenicity and highlight impaired Fgfr2b signaling as underlying mechanism. Furthermore, increase of alveolar Fgf10 levels may represent a putative therapy to overcome regeneration failure after IV-induced lung injury., Author Summary Influenza Virus (IV) pneumonia causes disruption of the alveolar epithelial barrier, leading to edema formation which severely affects gas exchange. Recovery after IV-induced acute lung injury includes not only innate immune responses to clear virus infection, but also tissue renewal processes to restore the alveolar barrier. We demonstrate that highly pathogenic IV particularly infect cells of the epithelial stem/progenitor cell niche, which significantly impairs repair processes within the damaged lung, as demonstrated by transplantation experiments using intrapulmonary delivery of infected versus non-infected stem cells into IV-injured mice. Analyses of the underlying virus-host interactions reveal that IV infection of epithelial stem/progenitor cells results in blockade of a pathway involving Wnt/β-catenin and fibroblast growth factor receptor 2b (Fgfr2b) activation within a regenerative epithelial-mesenchymal cell signaling network. Our data therefore suggest that tropism of IV to the distal lung stem cell niche represents an important factor of pathogenicity. Therapeutic intrapulmonary application of excess Fgf10 to induce Fgfr2b signaling in the non-infected stem cell niche was found to counteract IV-induced repair failure and to restore barrier function, representing a promising treatment to foster epithelial cell regeneration after IV-induced injury.

Infectious disease burden remains high [1]. An improved knowledge of host–microorganism interactions is key to delineating disease progression and developing new control and treatment modalities. Microorganisms may be pathogenic (able to cause disease) or non-pathogenic (unable to cause disease). Disease severity is influenced by host and pathogen factors; in particular, pathogens may express different virulence factors that modulate disease severity and progression. Within this context, the interaction of the pathogen with organ and tissue niches is especially important. Tropism refers to the ability of a given pathogen to infect a specific location. Organ or tissue tropism reflects the ability of a given pathogen to infect a specific organ or sets of organs. Some pathogens are broadly tropic, infecting all or most organs, while others are restricted to a given tissue or even to certain tissue niches. From this point of view, the ability of a pathogen to infect specific organs may vary over the course of the disease and could be active, pathogen-mediated, or passive, requiring, for example, a prior skin break or vector bite. Within tissue niches, intracellular pathogens may also preferentially infect specific organelles or intracellular sites. This article will focus on tissue tropism and its relationship to pathogenesis with examples from Staphylococcus aureus bacteria, Trypanosoma brucei protozoan parasites, and the influenza virus.