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In December 2019, a novel coronavirus crossed species barriers to infect humans and was effectively transmitted from person to person, leading including vaccines and antiviral drugs that could prevent or limit the burden or transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global health priority. It is thus of utmost importance to assess possible therapeutic strategies against SARS-CoV-2 using experimental models that recapitulate aspects of the human disease. Here, we review available models currently being developed and used to study SARS-CoV-2 infection and highlight their application to screen potential therapeutic approaches, including repurposed antiviral drugs and vaccines. Each identified model provides a valuable insight into SARS-CoV-2 cellular tropism, replication kinetics, and cell damage that could ultimately enhance understanding of SARS-CoV-2 pathogenesis and protective immunity. Expected final online publication date for the Annual Review of Pharmacology and Toxicology, Volume 62 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Murine astrovirus 2 (MuAstV2) is a novel murine astrovirus recently identified in laboratory and wild mice. MuAstV2 readily transmits between immunocompetent mice yet fails to transmit to highly immunocompromised mouse strains—a unique characteristic when contrasted with other murine viruses including other astroviruses. We characterized the viral shedding kinetics and tissue tropism of MuAstV2 in immunocompetent C57BL/6NCrl mice and evaluated the apparent resistance of highly immunocompromised NOD- Prkdcem26Cd52Il2rgem26Cd22 /NjuCrl mice to MuAstV2 after oral inoculation. Temporal patterns of viral shedding were determined by serially measuring fecal viral RNA. Tissue tropism and viral load were characterized and quantified by using in-situ hybridization (ISH) targeting viral RNA. Cellular tropism was characterized by evaluating fluorescent colocalization of viral ISH with various immunohistochemical markers. We found a rapid increase of fecal viral RNA in B6 mice, which peaked at 5 d after inoculation (dpi) followed by cessation of shedding by 168 dpi. The small intestine had the highest percentage of hybridization (3.09% of tissue area) of all tissues in which hybridization occurred at 5 dpi. The thymus displayed the next highest degree of hybridization (2.3%) at 7 dpi, indicating extraintestinal viral spread. MuAstV2 RNA hybridization was found to colocalize with only 3 of the markers evaluated: CD3 (T cells), Iba1 (macrophages), and cytokeratin (enterocytes). A higher percentage of CD3 cells and Iba1 cells hybridized with MuAstV2 as compared with cytokeratin at 2 dpi (CD3, 59%; Iba1, 46%; cytokeratin, 6%) and 35 dpi (CD3, 14%; Iba1, 55%; cytokeratin, 3%). Neither fecal viral RNA nor viral hybridization was noted in NCG mice at the time points examined. In addition, mice of mixed genetic background were inoculated, and only those with a functioning Il2rg gene shed MuAstV2. Results from this study suggest that infection of, or interaction with, the immune system is required for infection by or replication of MuAstV2.

The numerous global outbreaks and continuous reassortments of highly pathogenic avian influenza (HPAI) A(H5N6/H5N8) clade 2.3.4.4 viruses in birds pose a major risk to the public health. We investigated the tropism and innate host responses of 5 recent HPAI A(H5N6/H5N8) avian isolates of clades 2.3.4.4b, e, and h in human airway organoids and primary human alveolar epithelial cells. The HPAI A(H5N6/H5N8) avian isolates replicated productively but with lower competence than the influenza A(H1N1)pdm09, HPAI A(H5N1), and HPAI A(H5N6) isolates from humans in both or either models. They showed differential cellular tropism in human airway organoids; some infected all 4 major epithelial cell types: ciliated cells, club cells, goblet cells, and basal cells. Our results suggest zoonotic potential but low transmissibility of the HPAI A(H5N6/H5N8) avian isolates among humans. These viruses induced low levels of proinflammatory cytokines/chemokines, which are unlikely to contribute to the pathogenesis of severe disease.

Although they globally cause viral gastroenteritis in children, astroviruses are understudied due to the lack of well-defined animal models. While murine astroviruses (muAstVs) chronically infect immunodeficient mice, a culture system and understanding of their pathogenesis is lacking. Here, we describe a platform to cultivate muAstV using air–liquid interface (ALI) cultures derived from mouse enteroids, which support apical infection and release. Chronic muAstV infection occurs predominantly in the small intestine and correlates with higher interferon-lambda (IFN-λ) expression. MuAstV stimulates IFN-λ production in ALI, recapitulating our in vivo findings. We demonstrate that goblet cells and enterocytes are targets for chronic muAstV infection in vivo, and that infection is enhanced by parasite co-infection or type 2 cytokine signaling. Depletion of goblet cells from ALI limits muAstV infection in vitro. During chronic infection, muAstV stimulates IFN-λ production in infected cells and induces ISGs throughout the intestinal epithelium in an IFN-λ-receptor-dependent manner. Collectively, our study provides insights into the cellular tropism and innate immune responses to muAstV and establishes an enteroid-based culture system to propagate muAstV in vitro.

COVID-19, caused by SARS-CoV-2, is a primarily pulmonary disease that can affect several organs, directly or indirectly. To date, there are many questions about the different pathological mechanisms. Here, we generate an approach to identify the cellular-level tropism of SARS-CoV-2 using human proteomics, virus-host interactions, and enrichment analysis. Through a network-based approach, the molecular context was visualized and analyzed. This procedure was also performed for SARS-CoV-1. We obtained proteomes and interactomes from 145 different cells corresponding to 57 different tissues. We discarded the cells without the proteins known for interacting with the virus, such as ACE2 or TMPRSS2. Of the remaining cells, a gradient of susceptibility to infection was observed. In addition, we identified proteins associated with the coagulation cascade that can be directly or indirectly affected by viral proteins. As a whole we identified 55 cells that could be potentially controlled by the virus, with different susceptibilities, mainly being pneumocytes, heart, kidney, liver, or small intestine cells. These results help to explain the molecular context and provide elements for possible treatments in the current situation. This strategy may be useful for other viruses, especially those with limited reported PPI, such as a new virus.

Malaria is one of the most life-threatening infectious diseases worldwide, caused by infection of humans with parasites of the genus Plasmodium. The complex life cycle of Plasmodium parasites is shared between two hosts, with infection of multiple cell types, and the parasite needs to adapt for survival and transmission through significantly different metabolic environments. Within the blood-stage alone, parasites encounter changing levels of key nutrients, including sugars, amino acids, and lipids, due to differences in host dietary nutrition, cellular tropism, and pathogenesis. In this review, we consider the mechanisms that the most lethal of malaria parasites, Plasmodium falciparum, uses to sense nutrient levels and elicit changes in gene expression during blood-stage infections. These changes are brought about by several metabolic intermediates and their corresponding sensor proteins. Sensing of distinct nutritional signals can drive P. falciparum to alter the key blood-stage processes of proliferation, antigenic variation, and transmission.

Severe acute respiratory syndrome coronavirus 2 did not replicate efficiently in 13 bat cell lines, whereas severe acute respiratory syndrome coronavirus replicated efficiently in kidney cells of its ancestral host, the Rhinolophus sinicus bat, suggesting different evolutionary origins. Structural modeling showed that RBD/RsACE2 binding may contribute to the differential cellular tropism.

The blacklegged tick, Ixodes scapularis, a species of significant importance to human and animal health, harbors an endosymbiont Rickettsia buchneri sensu stricto. The symbiont is largely restricted to the ovaries, but all life stages can harbor various quantities or lack R. buchneri entirely. The endosymbiont is cultivable in cell lines isolated from embryos of Ixodes ticks. Rickettsia buchneri most readily grows and is maintained in the cell line IRE11 from the European tick, Ixodes ricinus. The line was characterized by light and electron microscopy and used to analyze the growth dynamics of wildtype and GFPuv-expressing R. buchneri. qPCR indicated that the genome copy doubling time in IRE11 was &gt, 7 days. Measurements of fluorescence using a plate reader indicated that the amount of green fluorescent protein doubled every 11 days. Two 23S rRNA probes were tested via RNA FISH on rickettsiae grown in vitro and adapted to evaluate the tissue tropism of R. buchneri in field-collected female I. scapularis. We observed strong positive signals of R. buchneri in the ovaries and surrounding the nucleus of the developing oocytes. Tissue tropism in I. scapularis and in vitro growth dynamics strengthen the contemporary understanding of R. buchneri as a transovarially transmitted, non-pathogenic endosymbiont.

The human adenovirus phylogenetic tree is split across seven species (A–G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D—such as their cellular tropism, receptor usage, and in vivo biodistribution profile—remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)—a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications.

Adeno-associated virus (AAV) vectors have become an important tool for delivering therapeutic genes for a wide range of neurological diseases. AAV serotypes possess differential cellular tropism in the central nervous system. Although several AAV serotypes or mutants have been reported to transduce the brain efficiently, conflicting data occur across studies with the use of various rodent strains from different genetic backgrounds. Herein, we performed a systematic comparison of the brain transduction properties among five AAV serotypes (AAV2, 5, 7, 8, and 9) in two common rodent strains (C57BL/6J and FVB/N), following local intrastriatal injection of AAV vectors encoding enhanced green fluorescent protein (EGFP) driven by the CBh promoter. Important differences were found regarding overall cellular tropism and transduction efficiency, including contralateral transduction among the AAV serotypes and between the mouse strains. We have further found loss of NeuN-immunoreactivity and microglial activation from AAV transduction in the different mouse strains. The important strain-specific differences from our study suggest that the genetic background of the mouse may affect AAV serotype transduction properties in the brain. These data can provide valuable information about how to choose an effective AAV vector for clinical application and interpret the data obtained from preclinical studies and clinical trials.

Ex vivo organ cultures (EVOCs) are extensively used to study the cellular tropism and infectivity of different pathogens. In this study, we used ovine and porcine respiratory EVOCs to investigate the replication kinetics and cellular tropism of selected emerging reoviruses namely Pteropine orthoreovirus, an emerging bat-borne zoonotic respiratory virus, and atypical Bluetongue virus (BTV) serotypes which, unlike classical serotypes, do not cause Bluetongue, a major OIE-listed disease of ruminants. BTV failed to replicate in ovine EVOCs. Instead, PRV showed slight replication in porcine lower respiratory EVOCs and a more sustained replication in all ovine respiratory tissues. By confocal laser scanning microscopy, PRV was demonstrated to infect bronchiolar and type I pneumocytes of ovine tissues. Overall, respiratory EVOCs from different animal species, eventually obtained at slaughterhouse, are a useful tool for testing and preliminarily characterize novel and emerging viruses addressing the essential in vivo animal work. Further experiments are, indeed, warranted in order to characterize the pathogenesis and transmission of these emerging reoviruses.

The hemagglutinin (H) protein of measles viruses (MeV) mediates binding to the cellular receptors, CD46,human signaling lymphocyte activation molecule and nectin-4. Vaccine strains primarily contain H-proteins possessing MeV-H: Y481 and can utilize CD46. Reports suggest that a single amino acid change in MeV-H at position 481 in wild type strains renders them inefficient in utilizing CD46. The in-depth molecular mechanism by which substitutions at 481 and another reported critical residue position 546 affects CD46 binding affinity however remains elusive. We used molecular docking studies of CD46 with MeV-H possessing Y481 N/D to understand the in-depth molecular mechanism involved. It was found that loss in either of the hydrogen bond (H-bond) contacts (MeV-H:481–CD46:65, MeV-H:546–CD46:63) in the central contact region prevented efficient CD46 binding. Y481 N could form the specific H-bond, while G546S H-bond could be formed only in conjunction with Y481, revealing the significance of these residues in determining CD46 receptor binding potential. Elucidating the underlying molecular mechanism of receptor usage by the MeV has implications to understanding cellular tropism, viral pathogenesis and therapy.

Parvovirus B-19, a single human pathogenic member of the Parvoviridae family with it's small ssDNA and non-enveloped structure, is known to cause diseases in erythroid progenitor cells. But a wide range of clinical association of this virus with cells of non-erythroid origins has recently been discovered and many of those are being investigated for such association. Higher substitution rates in due course of evolution has been suggested for this cellular tropism and persistence. In this report, we have summarized the different disease manifestations of B-19 virus and have tried to find out a pattern of pathogenesis. Finally, we have focused on the vaccination strategies available against B-19 virus to correlate these with the mechanisms involved in various diseases caused by this virus.

Viral tropism, or the specificity of a particular virus to infect a certain cell type, is crucial in determining virus replication, viral spread, and ultimately host survival. Rabies, one of the deadliest known zoonotic diseases, is still causing 60.000 human deaths annually. Upon central nervous system (CNS) entry, neurotropic rabies virus (RABV) preserves the neural network by limiting apoptosis and inflammation. To date, we do not fully understand the factors determining RABV tropism and why glial cells are unable to clear RABV from the infected brain. Here, we compare susceptibilities and innate immune responses of CNS cell types towards infection with virulent dog RABV Tha and less virulent Th2P-4M in vitro, highlighting differences in cellular susceptibility and antiviral responses. Less virulent Th2P-4M bears mutations introduced in viral phosphoprotein (P-protein) and matrix protein (M-protein) thereby hindering viral immune evasion of the host nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Janus kinase (JAK) - signal transducer and activator of transcription protein (STAT) pathways. Our results reveal that human neural stem cell (hNSC)-derived neurons and astrocytes, in contrast to human iPSC-derived microglia, are highly susceptible to Tha and Th2P-4M infection in vitro. Surprisingly, Th2P-4M presents a stronger neurotropism in hNSC-derived CNS cultures compared to Tha suggesting that NF-κB- and JAK-STAT-mediated antiviral host responses are defining RABV replication and thereby its tropism. Further, we show that astrocyte-like (SVGp12) and microglia-like (HMC3) cells protect neuroblastoma cells (SK-N-SH) from Tha infection in vitro. Transcription profiles and quantification of intracellular protein levels revealed major differences in antiviral immune responses mediated by neurons, astrocytes (IFNB1, CCL5, CXCL10, IL1B, IL6, LIF), and microglia (CCL5, CXCL10, ISG15, MX1, IL6) upon virulent Tha infection. Overall, we provide evidence that RABV tropism depends on its capability to evade cell-type specific immune responses via P- and M-proteins.Author summaryRabies virus (RABV) neurotropism is widely reported as a unique feature of rabies, and still the exact mechanism underlying RABV susceptibility remains to be elucidated. Several receptors are known to accelerate RABV entry to the cell (1–4) and yet, none of them seems to be essential for successful infection (5,6) questioning a marked cellular tropism. Although RABV is classically reported as strictly neurotropic (7), recent studies report profound infection of glial cells in vivo depending on the viral strain and the infection route used (8,9). Here, we provide evidence that human neural stem cell (hNSC)-derived neurons (hiNeurons) and astrocytes (hiAstrocytes) are highly susceptible towards infection with the virulent field RABV strain Tha and less virulent Th2P-4M. In contrast, human iPSC-derived microglia-like cells (hiMicros) are resistant to viral replication in vitro. Whereas hiNeurons are immunologically quiescent upon Tha infection, fetal astrocytes and hiMicros establish strong antiviral responses. In contrast to Tha, Th2P-4M, which is unable to evade NF-κB and JAK-STAT pathways (10), shows a more profound neurotropism suggesting that cell-type specific responses shape RABV tropism. Hence, we conclude that viral evasion mechanisms mediated by P- and M-proteins partly determine Tha tropism of human CNS cell types in vitro.

Despite major advancements in gene therapy technologies, there are no approved gene therapies for diseases which predominantly effect the brain. Adeno-associated virus (AAV) vectors have emerged as the most effective delivery vector for gene therapy owing to their simplicity, wide spread transduction and low immunogenicity. Unfortunately, the blood–brain barrier (BBB) makes IV delivery of AAVs, to the brain highly inefficient. At IV doses capable of widespread expression in the brain, there is a significant risk of severe immune-mediated toxicity. Direct intracerebral injection of vectors is being attempted. However, this method is invasive, and only provides localized delivery for diseases known to afflict the brain globally. More advanced methods for AAV delivery will likely be required for safe and effective gene therapy to the brain. Each step in AAV delivery, including delivery route, BBB transduction, cellular tropism and transgene expression provide opportunities for innovative solutions to optimize delivery efficiency. Intra-arterial delivery with mannitol, focused ultrasound, optimized AAV capsid evolution with machine learning algorithms, synthetic promotors are all examples of advanced strategies which have been developed in pre-clinical models, yet none are being investigated in clinical trials. This manuscript seeks to review these technological advancements, and others, to improve AAV delivery to the brain, and to propose novel strategies to build upon this research. Ultimately, it is hoped that the optimization of AAV delivery will allow for the human translation of many gene therapies for neurodegenerative and other neurologic diseases.

The upper respiratory tract is compromised in the early period of COVID-19, but SARS-CoV-2 tropism at the cellular level is not fully defined. Unlike recent single-cell RNA-Seq analyses indicating uniformly low mRNA expression of SARS-CoV-2 entry-related host molecules in all nasal epithelial cells, we show that the protein levels are relatively high and that their localizations are restricted to the apical side of multiciliated epithelial cells. In addition, we provide evidence in patients with COVID-19 that SARS-CoV-2 is massively detected and replicated within the multiciliated cells. We observed these findings during the early stage of COVID-19, when infected ciliated cells were rapidly replaced by differentiating precursor cells. Moreover, our analyses revealed that SARS-CoV-2 cellular tropism was restricted to the nasal ciliated versus oral squamous epithelium. These results imply that targeting ciliated cells of the nasal epithelium during the early stage of COVID-19 could be an ideal strategy to prevent SARS-CoV-2 propagation.

The first step of viral infection requires interaction with the host cell. Before finding the specific receptor that triggers entry, the majority of viruses interact with the glycocalyx. Identifying the carbohydrates that are specifically recognized by different viruses is important both for assessing the cellular tropism and for identifying new antiviral targets. Advances in the tools available for studying glycan–protein interactions have made it possible to identify them more rapidly; however, it is important to recognize the limitations of these methods in order to draw relevant conclusions. Here, we review different techniques: genetic screening, glycan arrays, enzymatic and pharmacological approaches, and surface plasmon resonance. We then detail the glycan interactions of enterovirus D68 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlighting the aspects that need further clarification.

The spike protein of SARS-CoV-2 has been undergoing mutations and is highly glycosylated. It is critically important to investigate the biological significance of these mutations. Here, we investigated 80 variants and 26 glycosylation site modifications for the infectivity and reactivity to a panel of neutralizing antibodies and sera from convalescent patients. D614G, along with several variants containing both D614G and another amino acid change, were significantly more infectious. Most variants with amino acid change at receptor binding domain were less infectious, but variants including A475V, L452R, V483A, and F490L became resistant to some neutralizing antibodies. Moreover, the majority of glycosylation deletions were less infectious, whereas deletion of both N331 and N343 glycosylation drastically reduced infectivity, revealing the importance of glycosylation for viral infectivity. Interestingly, N234Q was markedly resistant to neutralizing antibodies, whereas N165Q became more sensitive. These findings could be of value in the development of vaccine and therapeutic antibodies.

Recently, emerging evidence has indicated that COVID-19 represents a major threat to older populations, but the underlying mechanisms remain unclear. The pathogen causing COVID-19 is acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infection depends on the key entry factors, angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). Recognizing the importance of ACE2 and TMPRSS2 for the cellular tropism of SARS-CoV-2, we analyzed and presented the landscape of cell-type identities for ACE2+ TMPRSS2+ cells across different human tissues and the age-related alterations in ACE2 and TMPRSS2 expression across different cell types. Additionally, most of the post-acute COVID-19 sequelae could attribute to the ACE2-expressing organ systems. Therefore, these SARS-CoV-2 tropism data should be an essential resource for guiding clinical treatment and pathological studies, which should draw attention toward the prioritization of COVID-19 research in the future. Notably, we discovered the age-related expression of ACE2 and TMPRSS2 in the immune-inflammatory stromal cells, implying the potential interplay between COVID-19, stromal cells, and aging. In this study, we developed a novel and practical analysis framework for mapping the cellular tropism of SARS-CoV-2. This approach was built to aid the identification of viral-specific cell types and age-related alterations of viral tropism, highlighting the power of single-cell RNA sequencing (scRNA-seq) to address viral pathogenesis systematically. With the rapid accumulation of scRNA-seq data and the continuously increasing insight into viral entry factors, we anticipate that this scRNA-seq-based approach will attract broader interest in the virus research communities.

Herpes simplex viruses (HSV-1 and HSV-2) contain up to 16 different proteins in their envelopes. Four of these, glycoproteins gB, gD, gH, and gL, are termed essential with regard to entry, whereas the rest are typically referred to as nonessential based on the entry phenotypes of the respective single genetic deletions., Herpes simplex viruses (HSV-1 and HSV-2) encode up to 16 envelope proteins, four of which are essential for entry. However, whether these four proteins alone are sufficient to dictate the broad cellular tropism of HSV-1 and the selection of different cell type-dependent entry routes is unknown. To begin addressing this, we previously pseudotyped vesicular stomatitis virus (VSV), lacking its native glycoprotein G, with only the four essential entry glycoproteins of HSV-1: gB, gH, gL, and gD. This novel VSVΔG-BHLD pseudotype recapitulated several important features of HSV-1 entry: the requirement for gB, gH, gL, gD, and a cellular receptor and sensitivity to anti-gB and anti-gH/gL neutralizing antibodies. However, due to the use of a single cell type in that study, the tropism of the VSVΔG-BHLD pseudotype was not investigated. Here, we show that the cellular tropism of the pseudotype is severely limited compared to that of wild-type HSV-1 and that its entry pathways differ from the native HSV-1 entry pathways. To test the hypothesis that other HSV-1 envelope proteins may contribute to HSV-1 tropism, we generated a derivative pseudotype containing the HSV-1 glycoprotein C (VSVΔG-BHLD-gC) and observed a gC-dependent increase in entry efficiency in two cell types. We propose that the pseudotyping platform developed here has the potential to uncover functional contributions of HSV-1 envelope proteins to entry in a gain-of-function manner.

Adenoviruses are powerful tools experimentally and clinically. To maximize efficacy, the development of serotypes with low preexisting levels of immunity in the population is desirable., The human adenovirus (HAdV) phylogenetic tree is diverse, divided across seven species and comprising over 100 individual types. Species D HAdV are rarely isolated with low rates of preexisting immunity, making them appealing for therapeutic applications. Several species D vectors have been developed as vaccines against infectious diseases, where they induce robust immunity in preclinical models and early phase clinical trials. However, many aspects of the basic virology of species D HAdV, including their basic receptor usage and means of cell entry, remain understudied. Here, we investigated HAdV-D49, which previously has been studied for vaccine and vascular gene transfer applications. We generated a pseudotyped HAdV-C5 presenting the HAdV-D49 fiber knob protein (HAdV-C5/D49K). This pseudotyped vector was efficient at infecting cells devoid of all known HAdV receptors, indicating HAdV-D49 uses an unidentified cellular receptor. Conversely, a pseudotyped vector presenting the fiber knob protein of the closely related HAdV-D30 (HAdV-C5/D30K), differing in four amino acids from HAdV-D49, failed to demonstrate the same tropism. These four amino acid changes resulted in a change in isoelectric point of the knob protein, with HAdV-D49K possessing a basic apical region compared to a more acidic region in HAdV-D30K. Structurally and biologically we demonstrate that HAdV-D49 knob protein is unable to engage CD46, while potential interaction with coxsackievirus and adenovirus receptor (CAR) is extremely limited by extension of the DG loop. HAdV-C5/49K efficiently transduced cancer cell lines of pancreatic, breast, lung, esophageal, and ovarian origin, indicating it may have potential for oncolytic virotherapy applications, especially for difficult to transduce tumor types. IMPORTANCE Adenoviruses are powerful tools experimentally and clinically. To maximize efficacy, the development of serotypes with low preexisting levels of immunity in the population is desirable. Consequently, attention has focused on those derived from species D, which have proven robust vaccine platforms. This widespread usage is despite limited knowledge in their basic biology and cellular tropism. We investigated the tropism of HAdV-D49, demonstrating that it uses a novel cell entry mechanism that bypasses all known HAdV receptors. We demonstrate, biologically, that a pseudotyped HAdV-C5/D49K vector efficiently transduces a wide range of cell lines, including those presenting no known adenovirus receptor. Structural investigation suggests that this broad tropism is the result of a highly basic electrostatic surface potential, since a homologous pseudotyped vector with a more acidic surface potential, HAdV-C5/D30K, does not display a similar pantropism. Therefore, HAdV-C5/D49K may form a powerful vector for therapeutic applications capable of infecting difficult to transduce cells.

Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a Cd300lf conditional knockout (CD300lf(F/F)) mouse to elucidate the cell tropism of persistent and nonpersistent strains of murine norovirus. Using this mouse model, we demonstrated that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoV(CR6)) in vivo. In contrast, the nonpersistent MNoV strain CW3 (MNoV(CW3)) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells (LysM Cre+) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells (CD19 Cre), neutrophils (Mrp8 Cre), and dendritic cells (CD11c Cre) did not affect MNoV(CW3) viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoV(CW3) to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoV(CR6); that myelomonocytic cells are a major, but not exclusive, target cell of MNoV(CW3); and that STAT1 signaling restricts the cellular tropism of MNoV(CW3). This study provides the first genetic system for studying the cell type-specific role of CD300lf in norovirus pathogenesis. IMPORTANCE Human noroviruses (HuNoVs) are a leading cause of gastroenteritis resulting in up to 200,000 deaths each year. The receptor and cell tropism of HuNoV in immunocompetent humans are unclear. We use murine norovirus (MNoV) as a model for HuNoV. We recently identified CD300lf as the sole physiologic receptor for MNoV. Here, we leverage this finding to generate a Cd300lf conditional knockout mouse to decipher the contributions of specific cell types to MNoV infection. We demonstrate that persistent MNoV(CR6) requires CD300lf expression on tuft cells. In contrast, multiple CD300lf+ cell types, dominated by myelomonocytic cells, are sufficient for nonpersistent MNoV(CW3) infection. CD300lf expression on epithelial cells, B cells, neutrophils, and dendritic cells is not critical for MNoV(CW3) infection. Mortality associated with the MNoV(CW3) strain in Stat1(−/−) mice does not require CD300lf expression on LysM+ cells, highlighting that both CD300lf receptor expression and innate immunity regulate MNoV cell tropism in vivo.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) has a highly restricted cellular tropism. In vivo, the virus primarily infects tissue-specific macrophages in the nose, lungs, tonsils, and pharyngeal lymphoid tissues. In vitro however, the MARC-145 cell line is one of the few PRRSV susceptible cell lines that are routinely used for in vitro propagation. Previously, several PRRSV non-permissive cell lines were shown to become susceptible to PRRSV infection upon expression of recombinant entry receptors (e.g., PK15Sn-CD163, PK15S10-CD163). In the present study, we examined the suitability of different cell lines as a possible replacement of primary pulmonary alveolar macrophages (PAM) cells for isolation and growth of PRRSV. The susceptibility of four different cell lines (PK15Sn-CD163, PK15S10-CD163, MARC-145, and MARC-145Sn) for the primary isolation of PRRSV from PCR positive sera (both PRRSV1 and PRRSV2) was compared with that of PAM. To find possible correlations between the cell tropism and the viral genotype, 54 field samples were sequenced, and amino acid residues potentially associated with the cell tropism were identified. Regarding the virus titers obtained with the five different cell types, PAM gave the highest mean virus titers followed by PK15Sn-CD163, PK15S10-CD163, MARC-145Sn, and MARC-145. The titers in PK15Sn-CD163 and PK15S10-CD163 cells were significantly correlated with virus titers in PAM for both PRRSV1 (p &lt, 0.001) and PRRSV2 (p &lt, 0.001) compared with MARC-145Sn (PRRSV1: p = 0.22 and PRRSV2: p = 0.03) and MARC-145 (PRRSV1: p = 0.04 and PRRSV2: p = 0.12). Further, a possible correlation between cell tropism and viral genotype was assessed using PRRSV whole genome sequences in a Genome-Wide-Association Study (GWAS). The structural protein residues GP2:187L and N:28R within PRRSV2 sequences were associated with their growth in MARC-145. The GP5:78I residue for PRRSV2 and the Nsp11:155F residue for PRRSV1 was linked to a higher replication on PAM. In conclusion, PK15Sn-CD163 and PK15S10-CD163 cells are phenotypically closely related to the in vivo target macrophages and are more suitable for virus isolation and titration than MARC-145/MARC-145Sn cells. The residues of PRRSV proteins that are potentially related with cell tropism will be further investigated in the future.

The pandemic spread of SARS-CoV-2, the etiological agent of COVID-19, represents a significant and ongoing international health crisis. A key symptom of SARS-CoV-2 infection is the onset of fever, with a hyperthermic temperature range of 38 to 41°C. Fever is an evolutionarily conserved host response to microbial infection and inflammation that can influence the outcome of viral pathogenicity and regulation of host innate and adaptive immune responses. However, it remains to be determined what effect elevated temperature has on SARS-CoV-2 tropism and replication. Utilizing a 3D air-liquid interface (ALI) model that closely mimics the natural tissue physiology and cellular tropism of SARS-CoV-2 infection in the respiratory airway, we identify tissue temperature to play an important role in the regulation of SARS-CoV-2 infection. We show that temperature elevation induces wide-spread transcriptome changes that impact upon the regulation of multiple pathways, including epigenetic regulation and lncRNA expression, without disruption of general cellular transcription or the induction of interferon (IFN)-mediated antiviral immune defences. Respiratory tissue incubated at temperatures >37°C remained permissive to SARS-CoV-2 infection but severely restricted the initiation of viral transcription, leading to significantly reduced levels of intraepithelial viral RNA accumulation and apical shedding of infectious virus. To our knowledge, we present the first evidence that febrile temperatures associated with COVID-19 inhibit SARS-CoV-2 replication. Our data identify an important role for temperature elevation in the epithelial restriction of SARS-CoV-2 that occurs independently of the induction of canonical IFN-mediated antiviral immune defences and interferon-stimulated gene (ISG) expression.