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Several animal models have been developed to study the pathophysiology of SARS-CoV-2 infection and to evaluate vaccines and therapeutic agents for this emerging disease. Similar to infection with SARS-CoV-1, infection of Syrian hamsters with SARS-CoV-2 results in moderate respiratory disease involving the airways and lung parenchyma but does not lead to increased mortality. Using a combination of immunohistochemistry and transmission electron microscopy, we showed that the epithelium of the conducting airways of hamsters was the primary target for viral infection within the first 5 days of infection, with little evidence of productive infection of pneumocytes. At 6 days postinfection, antigen was cleared but parenchymal damage persisted, and the major pathological changes resolved by day 14. These findings are similar to those previously reported for hamsters with SARS-CoV-1 infection. In contrast, infection of K18-hACE2 transgenic mice resulted in pneumocyte damage, with viral particles and replication complexes in both type I and type II pneumocytes together with the presence of convoluted or cubic membranes; however, there was no evidence of virus replication in the conducting airways. The Syrian hamster is a useful model for the study of SARS-CoV-2 transmission and vaccination strategies, whereas infection of the K18-hCE2 transgenic mouse results in lethal disease with fatal neuroinvasion but with sparing of conducting airways.

Equine herpesvirus type 1 (EHV-1) and EHV-4 are genetically and antigenically very similar, but their pathogenic potentials are strikingly different. The differences in pathogenicity between both viruses seem to be reflected in cellular host range: EHV-1 can readily be propagated in many cell types of multiple species, while EHV-4 entry and replication appear to be restricted mainly to equine cells. The clear difference in cellular tropism may well be associated with differences in the gene products involved in virus entry and/or spread from cell to cell. Here we show that (i) most of the EHV-1 permissive cell lines became resistant to EHV-1 expressing EHV-4 glycoprotein D (gD4) and the opposite was observed for EHV-4 harboring EHV-1 gD (gD1). (ii) The absence of integrins did not inhibit entry into and replication of EHV-1 in CHO-K1 or peripheral blood mononuclear cells (PBMC). Furthermore, integrin-negative K562 cells did not acquire the ability to bind to gD1 when αVβ3 integrin was overexpressed. (iii) PBMC could be infected with similar efficiencies by both EHV-1 and EHV-4 in vitro . (iv) In contrast to results for equine fibroblasts and cells of endothelial or epithelial origin, we were unable to block entry of EHV-1 or EHV-4 into PBMC with antibodies directed against major histocompatibility complex class I (MHC-I), a result that indicates that these viruses utilize a different receptor(s) to infect PBMC. Cumulatively, we provide evidence that efficient EHV-1 and EHV-4 entry is dependent mainly on gD, which can bind to multiple cell surface receptors, and that gD has a defining role with respect to cellular host range of EHV-1 and EHV-4.

Severe acute respiratory syndrome (SARS) is a new human infectious disease with significant morbidity and mortality. The disease has been shown to be associated with a new coronavirus (SARS-CoV). The clinical and epidemiological aspects of SARS have been described. Moreover, the viral genome of SARS-CoV has been fully sequenced. However, much of the biological behaviour of the virus is not known and data on the tissue and cellular tropism of SARS-CoV are limited. In this study, six fatal cases of SARS were investigated for the tissue and cellular tropism of SARS-CoV using an in-situ hybridization (ISH) technique. Among all the tissues studied, positive signals were seen in pneumocytes in the lungs and surface enterocytes in the small bowel. Infected pneumocytes were further confirmed by immunofluorescence-fluorescence in-situ hybridization (FISH) analysis. These results provide important information concerning the tissue tropism of SARS-CoV, which is distinct from previously identified human coronaviruses, and suggest the possible involvement of novel receptors in this infection. Whereas the lung pathology was dominated by diffuse alveolar damage, the gut was relatively intact. These findings indicated that tissue responses to SARS-CoV infection are distinct in different organs.

Supplemental Material, Combined_supplemental_materials-Odendaal_et_al for Ovine Fetal and Placental Lesions and Cellular Tropism in Natural Rift Valley Fever Virus Infections by Lieza Odendaal, Sarah J. Clift, Geoffrey T. Fosgate and A. Sally Davis in Veterinary Pathology

West Nile virus (WNV) is a mosquito-borne flavivirus that is neurotropic in humans, birds, and other animals. While adaptive immunity plays an important role in preventing WNV spread to the central nervous system (CNS), little is known about how alpha/beta interferon (IFN-α/β) protects against peripheral and CNS infection. In this study, we examine the virulence and tropism of WNV in IFN-α/β receptor-deficient (IFN- α/βR −/− ) mice and primary neuronal cultures. IFN-α/βR −/− mice were acutely susceptible to WNV infection through subcutaneous inoculation, with 100% mortality and a mean time to death (MTD) of 4.6 ± 0.7 and 3.8± 0.5 days after infection with 10 0 and 10 2 PFU, respectively. In contrast, congenic wild-type 129Sv/Ev mice infected with 10 2 PFU showed 62% mortality and a MTD of 11.9 ± 1.9 days. IFN-α/βR −/− mice developed high viral loads by day 3 after infection in nearly all tissues assayed, including many that were not infected in wild-type mice. IFN-α/βR −/− mice also demonstrated altered cellular tropism, with increased infection in macrophages, B cells, and T cells in the spleen. Additionally, treatment of primary wild-type neurons in vitro with IFN-β either before or after infection increased neuronal survival independent of its effect on WNV replication. Collectively, our data suggest that IFN-α/β controls WNV infection by restricting tropism and viral burden and by preventing death of infected neurons.

Measles virus (MV) is a member of the genus Morbillivirus in the family Paramyxoviridae. Clinical isolates of MV use signaling lymphocyte activating molecule (SLAM) as a cellular receptor. SLAM is mainly expressed on immune cells such as immature thymocytes, activated lymphocytes and mature dendritic cells. This distribution of SLAM can account for the lymphotropism of MV. On the other hand, laboratory strains of MV use CD46 as an alternative receptor, through amino acid change(s) in the receptor binding hemagglutinin protein. Recently, several reports imply the existence of the cellular receptor(s) other than SLAM and CD46. In this review, we discuss the receptor usage of MV and its adaptation to cultured cells.

Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLB-type and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions., Author summary Human astroviruses (HAstV) are understudied positive-strand RNA viruses that typically cause gastroenteritis mostly in children and the elderly, but more recent studies also implicate them in neurological disease in immunocompromised patients. To better understand these viruses, a physiologically relevant cell culture model that supports growth of all clades of HAstV would be highly beneficial. Herein, we demonstrated robust infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts from different patients and intestinal regions, making HIE a valuable model to study HAstV biology. Using this system, we identify for the first time that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes. Analysis of the antiviral host response to infection demonstrated that HIE respond to infection with a type I and III interferon response. This response reduced HAstV replication and when blocked resulted in increased infection. Establishment of the HIE system for HAstV research lays the foundation for future basic and translational discoveries.

Although HIV type 1 (HIV-1) cannot efficiently replicate in simian cells, the mechanism(s) involved in the restriction of virus tropism remain unclear. To investigate this, we have focused on the identification of human cellular factors that can influence the infectivity of HIV-1 derived from African green monkey producer cells. Whereas the infectivity of HIV-1 derived from such cells was only 10–15% of that of human cell-derived virus, expression of human topoisomerase I in the African green monkey cells resulted in a 5-fold increase of the infectivity of progeny HIV-1 virions. Replacement of glutamate-236 and asparagine-237 of human topoisomerase I with the corresponding residues (aspartate and serine, respectively) of the African green monkey enzyme abolished this enhancement of HIV-1 infectivity. This positive effect of human topoisomerase I expression in the African green monkey producer cells seemed to result from the promotion of HIV-1 cDNA synthesis. Thus, human topoisomerase I plays an important role in HIV-1 replication and infectivity, and differences in the species specificity of HIV-1 infection can at least in part be attributed to differences in topoisomerase I activities.

Purpose: Nanoparticles (NPs) carry important promises for the treatment of neurological diseases, such as glioblastoma multiform (GBM) and Parkinson's disease. Several methods have been developed to achieve higher NP concentrations in the brain, including local infusions using convection enhanced delivery (CED), focused ultrasound, and the use of surface targeting moieties specifically designed to increase the passage across the blood brain barrier (BBB). However, even when sufficient NP amounts are delivered to the targeted region, a better understanding of the interactions between the particles and the brain parenchyma will be necessary to reach clinical efficacy. This is particularly true for polymeric NPs, which behavior can be dramatically influenced by multiple factors such as their size and their surface properties. Here, we investigated the cellular fate of PLA-based nanoparticles of similar size, but bearing different surface modifications, following CED in the healthy brain and the tumor bearing brain. Methods: Four PLA-based NP formulations with different surface modifications (PLA, PLA-PEG, PLA-HPG and PLA-HPG-CHO) and similar size were obtained by emulsion or nanoprecipitation. CED of each formulation was performed in healthy or tumor bearing brain, and comparable volumes of distribution were obtained. 4 h and 24 h after infusion, brains were harvested and processed for flow cytometry analysis and immunohistochemical staining, to quantify particle internalization by neurons, astrocytes, microglia and tumor cells, when applicable. In vitro uptake studies were performed using relevant cell lines for neurons (N27 cells), astrocytes (TNC1), microglia (BV2) and tumors (RG2). Rate of association kinetics of different particles with these cells were derived from an uptake study and then correlated with in vivo internalization results. Finally, to evaluate how different NPs surface modifications and their different internalization patterns can influence survival benefits, the different particles were loaded with epothilone B (EB) and infused into rats bearing RG2 tumors via CED. Results: We observed that in the healthy brain, stealth NPs distributed evenly between neurons, astrocytes and microglia, while exhibiting the highest specificity towards tumor cells in the tumor bearing brain. Overall, the functionalization of PLA NPs with aldehyde groups allowed for an increased uptake by all cell types, in both healthy and tumor bearing brain. These NPs also presented an increased relative uptake by microglia cells in both environments, compared to stealth particles, suggesting the induction of an immune response. Rates of association of NPs in vitro varied significantly between particle types. We were then able to correlate the in vivo uptake of each particle and cell type with in vitro particle association rates with neurons, astrocytes and microglia in culture, demonstrating the possibility of predicting in vivo uptake using in vitro association rates. Finally, comparison of particles in a survival efficacy showed significant differences, highlighting the importance of uptake of NPs. Conclusions: This study demonstrates for the first time that NP surface modifications significantly influence the cellular tropism of NPs in the brain in vivo, and that in vitro association rates can be used to anticipate the different internalization patterns. These differences open the possibility of tuning surface properties to optimize cellular delivery and therapeutic outcome. Acknowledgements: This work is supported by a NIH/NCI R01 grant (#5R01CA149128-04). Citation Format: Eric Song, Alice Gaudin, Amanda R. King, Youngeun Seo, Paul Won, Heewon Suh, Yang Deng, Jiajia Cui, Gregory Tietjen, W Mark Saltzman. Surface chemistry governs cellular tropism of nanoparticles in the brain. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B46.

Combined_supplemental_materials-Odendaal_et_al for Lesions and Cellular Tropism of Natural Rift Valley Fever Virus Infection in Young Lambs by Lieza Odendaal, A. Sally Davis, Geoffrey T. Fosgate and Sarah J. Clift in Veterinary Pathology

A primary isolate (KMT) of human immunodeficiency virus type 1 (HIV-1) resistant to recombinant soluble CD4 (rsCD4) was isolated from an HIV-1-infected individual and grown in a T lymphoid cell line. KMT isolate passaged on CEM cells (KMT/CEM) was still resistant to rsCD4. The V1/V2 and V3 regions of the viral envelope glycoprotein are thought to be involved in various biological phenotypes. To determine the exact envelope region of the KMT isolate responsible for sensitivity to rsCD4 and cellular tropism, we performed sequence analysis of KMT and KMT/CEM isolates. Sequence analysis of the KMT isolate showed that the sequence of the V3 region was relatively homogeneous, whereas a considerable heterogeneity of the V1/V2 region was noted. In contrast, the sequences of the V1 to V3 regions were homogeneous in KMT/CEM isolates. Analysis of NL4-3-based recombinant viruses with amplified sequences of the V1 to V3 regions from KMT and KMT/CEM isolates showed that the V1/V2 region modulated the sensitivity to rsCD4. A change in resistance to rsCD4 by the V1/V2 region was associated with the ability of the isolate to replicate in macrophages and efficiently replicate in T lymphoid cell lines. A change to an isolate sensitive to rsCD4 was associated with reduced replication efficiency in T lymphoid cell lines. Our results suggest that the V1/V2 region is involved in modulating the sensitivity to rsCD4, macrophage tropism, and replication efficiency in T lymphoid cell lines.

The cellular tropism of the feline immunodeficiency virus (FIV) is affected by changes in variable region 3 (V3) of the surface (SU) envelope glycoprotein (Verschoor, E. J., et al., J. Virol. 69:4752-4757, 1995). By using high-dose DNA transfection, an FIV molecular clone with a non-CRFK-tropic V3 acquired the ability to replicate in CRFK cells. A single point mutation from a methionine to a threonine in the ectodomain of its transmembrane (TM) envelope glycoprotein was responsible for this change in viral tropism. This substitution is located in the putative SU interactive region, between the fusion peptide and the membrane-spanning region. Our results show that this region of the TM envelope glycoprotein constitutes an additional determinant for cell tropism.

Coinfections of human immunodeficiency virus (HIV), EBV, and HTLV or sperm proteins act synergistically to enhance infectivity and replication and expand cellular tropism. While some aspects of these synergisms are understood, others are not. We have found that membrane or surface proteins of CMV, HTLV, EBV and sperm proteins share large regions of similarity with the CD4 protein of T-helper lymphocytes. Since HIV uses CD4 as a receptor, it may bind to CD4 homologues on CMV, HTLV, EBV or sperm proteins. HIV could then "piggyback" with these viruses into cells with which it normally has no tropism. Similarly, HIV may expand the cellular tropism of CMV, or EBV. Such a piggyback mechanism may provide insight into the formation or presentation of CD4-like antigens from CMV, HTLV, EBV and sperm proteins with class II MHC-like antigens on HIV (gp160 and Nef proteins) and may break immunological tolerance, inducing the autoimmunity observed against both CD4+ and class II MHC+ T cells in AIDS patients.

Using recombinant and mutant viruses generated between two human immunodeficiency virus type 1 isolates that display differences in cell tropism and sensitivity to soluble CD4 neutralization, we show that these two properties of the virus are regulated by different mechanisms. Whereas there is an association between V3 loop conformation and a particular cellular tropism, soluble CD4 neutralization sensitivity appears to be determined by amino acid differences in the C2 domain of the envelope gp120 that modulate the stability of gp120-gp41 association. Our findings further illustrate the importance of functional interactions among different regions of the envelope gp120 in regulating the biological phenotypes of human immunodeficiency virus and suggest that additional probing of the V3 loop with monoclonal antibodies may identify specific structural features of this loop that determine cell tropism.

The long terminal repeats (LTRs) of human immunodeficiency virus type 1 (HIV-1) strains from the central nervous systems of four patients with AIDS and of an HIV-1 isolate which is highly macrophage-tropic were isolated by using the polymerase chain reaction. In transient transfection assays, these LTRs demonstrated no significant difference in basal or stimulated levels of transcription in any of a variety of cell lines tested, compared with expression directed from the LTR of a T-lymphocyte-tropic strain of HIV-1. Chimeric viruses were created with the LTRs of the macrophage-tropic and brain-derived viruses ligated to the viral backbone from a T-lymphocyte-tropic strain. No change in cellular tropism was demonstrated with these chimeric viruses. Thus, unlike the LTRs of some murine retroviruses, the LTR of HIV-1 does not appear to play a major role in determining cellular tropism.

Rift Valley fever (RVF) is a mosquito-borne disease that affects both ruminants and humans, with epidemics occurring more frequently in recent years in Africa and the Middle East, probably as a result of climate change and intensified livestock trade. Sheep necropsied during the 2010 RVF outbreak in South Africa were examined by histopathology and immunohistochemistry (IHC). A total of 124 sheep were available for study, of which 99 cases were positive for RVF. Multifocal-random, necrotizing hepatitis was confirmed as the most distinctive lesion of RVF cases in adult sheep. Of cases where liver, spleen, and kidney tissues were available, 45 of 70 had foci of acute renal tubular epithelial injury in addition to necrosis in both the liver and spleen. In some cases, acute renal injury was the most significant RVF lesion. Immunolabeling for RVFV was most consistent and unequivocal in liver, followed by spleen, kidney, lung, and skin. RVFV antigen-positive cells included hepatocytes, adrenocortical epithelial cells, renal tubular epithelial cells, macrophages, neutrophils, epidermal keratinocytes, microvascular endothelial cells, and vascular smooth muscle. The minimum set of specimens to be submitted for histopathology and IHC to confirm or exclude a diagnosis of RVFV are liver, spleen, and kidney. Skin from areas with visible crusts and lung could be useful additional samples. In endemic areas, cases of acute renal tubular injury should be investigated further if other more common causes of renal lesions have already been excluded. RVFV can also cause an acute infection in the testis, which requires further investigation.