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Molecular differences in the envelope glycoproteins of human immunodeficiency virus type 1 and simian immunodeficiency virus (SIV) determine virus infectivity and cellular tropism. To examine how these properties contribute to productive infection in vivo, rhesus macaques were inoculated with strains of single-cycle SIV (scSIV) engineered to express three different envelope glycoproteins with full-length (TM open ) or truncated (TM stop ) cytoplasmic tails. The 239 envelope uses CCR5 for infection of memory CD4 + T cells, the 316 envelope also uses CCR5 but has enhanced infectivity for primary macrophages, and the 155T3 envelope uses CXCR4 for infection of both naive and memory CD4 + T cells. Separate groups of six rhesus macaques were inoculated intravenously with mixtures of TM open and TM stop scSIV mac 239, scSIV mac 316, and scSIV mac 155T3. A multiplex real-time PCR assay specific for unique sequence tags engineered into each virus was then used to measure viral loads for each strain independently. Viral loads in plasma peaked on day 4 for each strain and were resolved below the threshold of detection within 4 to 10 weeks. Truncation of the envelope cytoplasmic tail significantly increased the peak of viremia for all three envelope variants and the titer of SIV-specific antibody responses. Although peak viremias were similar for both R5- and X4-tropic viruses, clearance of scSIV mac 155T3 TM stop was significantly delayed relative to the other strains, possibly reflecting the infection of a CXCR4 + cell population that is less susceptible to the cytopathic effects of virus infection. These studies reveal differences in the peaks and durations of a single round of productive infection that reflect envelope-specific differences in infectivity, chemokine receptor specificity, and cellular tropism.

Background Several subtypes of HIV-1 circulate in infected people worldwide, including subtype B in the United States and subtype C in Africa and India. To understand the biological properties of HIV-1 subtype C, including cellular tropism, virus entry, replication efficiency and cytopathic effects, we reciprocally inserted our previously characterized envelope V3–V5 regions derived from 9 subtype C infected patients from India into a subtype B molecular clone, pNL4-3. Equal amounts of the chimeric viruses were used to infect T-lymphocyte cell lines (A3.01 and MT-2), coreceptor cell lines (U373-MAGI-CCR5/CXCR4), primary blood T-lymphocytes (PBL) and monocyte-derived macrophages (MDM). Results We found that subtype C envelope V3–V5 region chimeras failed to replicate in T-lymphocyte cell lines but replicated in PBL and MDM. In addition, these chimeras were able to infect U373MAGI-CD4+-CCR5+ but not U373MAGI-CD4+-CXCR4+ cell line, suggesting CCR5 coreceptor utilization and R5 phenotypes. These subtype C chimeras were unable to induce syncytia in MT-2 cells, indicative of non-syncytium inducing (NSI) phenotypes. More importantly, the subtype C envelope chimeras replicated at higher levels in PBL and MDM compared with subtype B chimeras and isolates. Furthermore, the higher levels subtype C chimeras replication in PBL and MDM correlated with increased virus entry in U373MAGI-CD4+-CCR5+. Conclusion Taken together, these results suggest that the envelope V3 to V5 regions of subtype C contributed to higher levels of HIV-1 replication compared with subtype B chimeras, which may contribute to higher viral loads and faster disease progression in subtype C infected individuals than other subtypes as well as rapid HIV-1 subtype C spread in India.

Nipah (NiV) and Hendra (HeV) viruses are members of the newly defined Henipavirus genus of the Paramyxoviridae. Nipah virus (NiV) is an emergent paramyxovirus that causes fatal encephalitis in up to 70% of infected patients, and there is increasing evidence of human‐to‐human transmission. NiV is designated a priority pathogen in the NIAID Biodefense Research Agenda, and could be a devastating agent of agrobioterrorism if used against the pig farming industry. Endothelial syncytium is a pathognomonic feature of NiV infections, and is mediated by the fusion (F) and attachment (G) envelope glycoproteins. This review summarizes what is known about the pathophysiology of NiV infections, and documents the identification of the NiV receptor. EphrinB2, the NiV and HeV receptor, is expressed on endothelial cells and neurons, consistent with the known cellular tropism for NiV. We discuss how the identification of the henipahvirus receptor sheds light on the pathobiology of NiV infection, and how it will spur the rational development of effective therapeutics. In addition, ephrinB3, a related protein, can serve as an alternative receptor, and we suggest that differential usage of ephrinB2 versus B3 may explain the variant pathogenic profiles observed between NiV and HeV. Thus, identifying the NiV receptors opens the door for a more comprehensive analysis of the envelope–receptor interactions in NiV pathobiology. Finally, we also describe how galectin‐1 (an innate immune defense lectin) can interact with specific N‐glycans on the Nipah envelope fusion protein, underscoring the potential role that innate immune defense mechanisms may play against emerging pathogens.

Publisher Summary This chapter discusses the body of knowledge accrued over the last two decades on the biology of human herpesvirus-6 (HHV-6). HHV-6 has a restricted range of susceptible species, essentially limited to humans and selected nonhuman primates. Antibodies to HHV-6 or to a closely related simian herpesvirus have been demonstrated in monkeys and a simian HHV-6 homolog has been recently identified in mandrill and drill monkeys as well as chimpanzees. The biological features of HHV-6 have been extensively characterized using in vitro models, while in vivo studies are still limited, owing to the lack of suitable and easily accessible animal model systems. A major factor in determining the complexity of the biology of HHV-6 is the existence of two viral variants or subgroups, defined HHV-6A and 6B, which exhibit different biological features and possibly also reflected in the different disease associations described in vivo . The finding that both HHV-6 variants use the ubiquitous molecule CD46 as a membrane receptor is compatible with a broad cellular tropism. Productive infection, however, is limited to a small range of cells most likely by intracellular restriction factors acting beyond the viral entry step. The study of the pathogenesis of HHV-6 infection has been significantly hampered by the lack of physiologically relevant models. The systems most commonly used to grow HHV-6 in vitro , such as cord blood- and peripheral blood-derived mononuclear cells cultured in suspension, require the cells to be maximally stimulated with polyclonal activators in order to sustain HHV-6 replication, a condition that is unlikely to occur in vivo.

The parvoviruses are a family of small, single-stranded DNA viruses that have a wide cellular tropism and broad host range, causing infection in invertebrate and vertebrate species from insects to mammals. Although many are important veterinary pathogens, there is only one proven human pathogen in the family, the human parvovirus B19. The virus is most commonly referred to as parvovirus B19, or simply B19. A new genus and name have been proposed for this virus: erythrovirus B19, 1 based on its cellular tropism for erythroid lineage cells and to distinguish it from the other mammalian parvoviruses.

Publisher Summary This chapter reviews properties of the genome of the human herpesvirus-6 (HHV-6) with some updates includingthe general properties of HHV-6 and the closely related HHV-7 and their genomes, besides their complete genomic sequences. There are two strain groups for HHV-6, the prototypes are strain U1102 for HHV-6 variant A (HHV-6A) and strain Z29 for variant B (HHV-6B). HHV-6A strain U1102 was identified and first characterized in the UK and HHV-6B strain Z29 in the USA; both genomes are sequenced. Both HHV-6 and HHV-7 are T lymphotropic and neurotropic. Furthermore, it is likely that in vivo , the lytic and latent cell types that HHV-6 infects are highly specific subsets. Both HHV-6 and HHV-7 have a cellular tropism for T lymphocytes as shown in vivo during viremia from acute infection as well as in vitro . Infection and lytic replication results in a characteristic cytopathic effect of large cells (cytomegalia) and ballooning cells. For HHV-6, some differences upon the serial passage of strain Z29 in culture have been observed including an expansion of repetitive sequences from the origin of lytic replication, terminal direct repeats, het region, and other rearrangements are possible.

Although processing of the hepatitis C virus (HCV) polyprotein and characterization of each of its viral proteins have been described in detail, analysis of the structure and assembly of HCV particles has been hampered by the lack of a robust cell culture system to support efficient replication of HCV. In this study, we generated HCV-like particles (HCV-LP) using a recombinant baculovirus encoding structural and a part of non-structural proteins in a human hepatoma cell line. The HCV-LP exhibited a buoyant density of 1.17 g/ml in CsCl equilibrium gradient and particles of 40 to 50 nm in diameter. Binding of the HCV-LP to human hepatoma cells was partially inhibited by the treatment with anti-hCD81 antibody, in contrast to the hCD81-independent binding of HCV-LP produced in insect cells. These results indicate that HCV-LP generated in different types of cells exhibit different cellular tropism for binding to target cells.

We identified a postentry restriction, termed Lv2, which determines the cellular tropism of two related human immunodeficiency virus type 2 (HIV-2) isolates and is dependent on the sequence of the capsid (CA) and envelope (Env) proteins. To explain the reliance on both CA and Env, we proposed that restrictive Envs deliver susceptible capsids to a compartment where Lv2 is active whereas nonrestrictive Envs deliver capsids into a compartment where Lv2 is either absent or less active. To test this model, we used compounds that affect endocytic pathways (ammonium chloride, bafilomycin A1, hypertonic sucrose) or lipid rafts (methyl-β-cyclodextrin) to treat restrictive cells and show that restricted virus can be rescued from Lv2 if a lipid-raft-dependent, pH-independent endocytic pathway is inhibited. Furthermore, viral entry into HeLa/CD4 cells containing a tailless CD4 receptor, located outside lipid rafts, was fully permissive. Finally, we show that a variety of primary HIV-1 and HIV-2 viruses are susceptible to Lv2. Thus, we show that the route of entry, determined by the viral envelope, can influence cellular tropism by avoiding intracellular blocks to infection.

With the discovery that changes in the biological properties of HIV-1 correlate with the progression to disease, it became more and more important to develop assays to distinguish between the viral phenotypes. In this chapter, it is described how the biological phenotype of HIV-1 with regard to cellular tropism can be determined on primary monocyte-derived macrophages, established T-cell lines: MT2, SupT1, and H9, and promonocytic cell lines: U937, HL-60, and THP-1.

Truncation of the envelope cytoplasmic tail has enabled FIV, SIV, and some laboratory HIV-1 strains to acquire broader cellular tropism and enhanced fusogenicity. Here we have characterized a primary CD4-independent HIV-1 isolate (92UG046-T8) with a truncated cytoplasmic tail that was able to infect and induce syncytia in primary lymphocytes from human, chimpanzee, and monkey, as well as CD4-negative cell lines from human and monkey. Increased syncytia were also noticeable with 293 cells expressing the cloned envelope from the 92UG046-T8 isolate suggesting envelope-mediated cellular fusion. Except pooled serum from HIV-1-infected individuals, monoclonal anti-envelope antibodies or antibodies/antagonists against CD4, CXCR4, and CCR5 were not able to prevent infection by the 92UG046-T8 isolate. This is the first report showing a primary HIV-1 variant with truncated cytoplasmic tail which is highly fusogenic and can infect a broad range of cells from human and non-human origins. In vivo evolution of similar HIV-1 mutants may have important implications in AIDS pathogenesis.

Although HHV-6A and -6B are known to replicate preferably in human T-lymphocytes, in vitro infection of several other cell types has been described. Also, the finding that both variants use the ubiquitous molecule CD46 as a membrane receptor fully supports the possibility of a broad cellular tropism. However, productive infection, which requires complete progression through the viral replication cycle, depends on multiple cellular processes. Our studies were aimed at determining the differences in replication efficiency according to the cell type infected and at relating these differences to the sequential transcriptional events preceding DNA replication. A strong expression of immediate-early, early, and late genes was only seen in the T-lymphoblastoma lines. In the other cell lines, there was no clear correlation between the level of transcription and the final outcome of replication. Finally, we investigated the cytopathic effects of HHV-6 on different cell types of neural origin (oligodendrocytes, astrocytes, and neurons) in greater detail, and found that although all three sustained HHV-6 replication, HHV-6A was more neurovirulent than HHV-6B. This was confirmed in primary human oligodendrocyte cultures.

The staphylococcal superantigen-like proteins (SSLs) are a family of polymorphic paralogs encoded in the S taphylococcus aureus genome whose function is unknown. The crystal structure of SSL7 was determined and compared to that of SSL5 and that of a classical superantigen, streptococcal pyrogenic exotoxin. Although the overall architecture of the superantigen family is retained in both SSL7 and SSL5, there are significant differences in the structures which suggest that the characteristic major histocompatibility complex binding site of superantigens has been lost. To complement these data, the abilities of SSL7 and a closely related paralog, SSL9, to interact with cells of the immune system were investigated. In populations of human white blood cells, both SSLs interacted selectively with monocytes via specific saturable but separate binding sites, which led to rapid uptake of the SSLs. In addition, SSLs were rapidly taken up by dendritic cells, but not by macrophages, into the same endosomal compartment as dextran. The ability of these secreted proteins to target antigen-presenting cells may enhance a misplaced antibody response against the proteins, which may facilitate bacterial colonization rather than contribute to host protection. Like classical superantigens, therefore, SSLs may distract the host's immune system, but they may do so via entirely different molecular mechanisms.

Following single viruses on their journey through a host cell provides valuable insights that help to understand the infection process. Fluorescent labeling techniques together with high-resolution microscopic imaging allows tracking of single viruses on living cells with high temporal and spatial accuracy.Influenza A viruses were fluorescently labeled without interfering with their biological activity. Single-virus trajectories were recorded on the surface and inside living cells of different permissive cell lines. By using conventional computational methods we extracted the position of viruses within each image frame. In addition, we have applied a recently developed mathematical tool to further analyze a special type of recurrent trajectory obtained at the surface of living cells. using this approach we can identify and characterize microdomains on the cell surface. We show that the viruses are surprisingly mobile and display a variety of different types of movement, which depend on the studied cell line. The cell surface was characterized by scanning electron microscopy (SEM) and revealed a unique topography that also strongly depends on the cell type and the cells growth state.By comparing trajectories obtained from two permissive cell lines we were able to correlate the distinctive behavior of single viruses with the cell surface ultrastructure. We used cells that are infected to a different degree and combine this with virus uptake and intracellular fusion kinetics. This allows us to draw a comprehensive picture of the first steps of virus infection. Further we can draw conclusions about the infectivity and could explain why some cells are permissive while others are not.

The process of virus entry accomplishes the delivery of the viral genetic information into the cell so that replication can take place. Entry of enveloped viruses into mammalian cells requires that the virus attach to the host cell surface through an interaction between an envelope component and a cellular molecule that serves as a receptor. After attachment, the virus must cross the plasma membrane during a phase of the life cycle termed penetration. For enveloped viruses, a fusion reaction occurs between the viral envelope in either the endosomal membrane or the plasma membrane. In simple viral systems that have one or two envelope glycoproteins, generally, a single, specific interaction between a viral protein and a cellular constituent is necessary and sufficient to result in infection. The herpesviruses, including human cytomegalovirus (HCMV), are structurally much more complex and often exhibit broad, diverse cellular tropism. Thus, all evidence to date points to the involvement of multiple cellular and viral proteins with overlapping or compensatory mechanisms possibly responsible for entry into specialized cell types.

A reliable detection system for HCV antigens in liver tissue may be used to identify the HCV cellular tropism and subcellular sites of viral replication Also, it can be used to study the relationship between viral expression and disease activity. Finally, it can facilitate the study of host-viral interactions at the cellular level (1).

Biological membrane fusion is an important part of many cellular processes and is a critical step in the entry of enveloped viruses, such as HIV-1, into cells. For HIV-1 to infect cells, the virus must bind to the cell surface, after which the viral Env protein must be triggered to undergo a conformational change that mediates membrane fusion. Cell-surface binding has long been known to occur via a high-affinity interaction between Env and CD4. However, the cell-surface molecules responsible for triggering the fusion-inducing conformational change in the Env protein have been only recently identified, permitting the study of HIV-1 Env-mediated membrane fusion in much greater detail (for review, see 1). These molecules, termed coreceptors, have been shown to be members of the nine-transmembrane domain receptor family. The most important HIV-1 coreceptors are the chemokine receptors CCR5 and CXCR4 (2-7), although at least nine other chemokine receptors or orphan receptors have been shown to support cellular entry for subsets of HIV-1 or SIV strains (3,5,8-11). The ability of a given virus strain to utilize particular chemokine receptors is a major determinant of cellular tropism. Thus, it is desirable to identify the receptors used by virus strains in a rapid, quantitative, and reproducible manner.

Heterologous lentiviral vectors (LVs) represent a way to address safety concerns in the field of gene therapy by decreasing the possibility of genetic recombination between vector and packaging constructs and the generation of replication-competent viruses. Using described LVs based on human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus MAC251 (SIV MAC251 ), we asked whether heterologous virion particles in which trans -acting factors belonged to HIV-1 and cis elements belonged to SIV MAC251 (HIV- siv ) would behave as parental homologous vectors in all cell types. To our surprise, we found that although the heterologous HIV- siv vector was as infectious as its homologous counterpart in most human cells, it was defective in the transduction of dendritic cells (DCs) and, to a lesser extent, macrophages. In DCs, the main postentry defect was observed in the formation of two-long-terminal-repeat circles, despite the fact that full-length proviral DNA was being synthesized and was associated with the nucleus. Taken together, our data suggest that heterologous HIV- siv vectors display a cell-dependent infectivity defect, most probably at a post-nuclear entry migration step. As homologous HIV and SIV vectors do transduce DCs, we believe that these results underscore the importance of a conserved interaction between cis elements and trans -acting viral factors that is lost or suboptimal in heterologous vectors and essential only in the transduction of certain cell types. For gene therapy purposes, these findings indicate that the cellular tropism of LVs can be modulated not only through the use of distinct envelope proteins or tissue-specific promoters but also through the specific combinatorial use of packaging and transfer vector constructs.

We have studied infectivity and neutralization of X4, R5, and R5X4 tropic HIV-1 mutants, which are lacking N-linked glycosylation sites for glycans g13, g14, g15, and g17 in the V3 loop region of gp120. X4-tropic NL4-3 mutants lacking combinations of g14/15 or g15/17 showed markedly higher infectivity in CXCR4-specific infection. The role of g15 in CCR5-specific infection was investigated using viruses with high (NL-918, R5-monotropic), medium (NL-991, R5-monotropic), and low (NL-952, R5X4-dualtropic) CCR5-specific infectivity. For NL-991, a reduction of infectivity on GHOST-CCR5 cells was observed for a mutant lacking g15. For NL-952 mutants all lacking g15, a complete loss of CCR5-specificity was observed and NL-952 was shifted from R5X4 to X4 tropism. For all mutants of NL4-3, NL-991, and NL-952, where the lack of g15 markedly influenced infectivity or coreceptor usage, neutralization was enhanced. In contrast, NL-918 mutants with or without g15 showed no difference in neutralization and no difference in GHOST-CCR5 infection rates. Thus, for viruses with a low or medium CCR5-specificity the role of g15 for changing CCR5-usage and sensitivity to neutralization was more significant than for viruses with high infection rates on GHOST-CCR5 cells. Our data demonstrate that V3 glycans play an important role in the usage of CXCR4 and CCR5. The lack of g15 was relevant for a more efficient use of CXCR4, whereas interaction with CCR5 was facilitated in the presence of g15. This study also demonstrates that glycan g15 is involved in blocking of neutralizing antibodies and shifting HIV tropism from R5X4 to X4.

The development of a targeted adenoviral vector, which can be delivered systemically, is one of the major challenges facing cancer gene therapy. The virus is readily cleared from the bloodstream, can be neutralised by pre-existing antibodies, and has a permissive cellular tropism. Clinical studies using the ONYX virus have shown limited efficacy, but there are several hurdles to overcome to achieve an effective tumor-specific systemic therapy. In this review, we have summarized the various strategies used to overcome the limitations of adenoviral-mediated gene delivery.

The use of adenovirus (Ad) as an efficient and versatile vector for in vivo tumor therapy requires the modulation of its cellular tropism. We previously developed a method to genetically alter the tropism of Ad5 fibers by replacing the fiber knob domain by an extrinsic trimerization motif and a new cellular ligand. However, fibers carrying complex ligands such as single-chain antibody fragments did not assemble into functional pentons in vitro in the presence of penton base, and failed to be rescued into infectious virions because of their inability to fold correctly within the cytoplasm of Ad-infected cells. Here we show that the coding sequence for a disulfide bond-independent three-helix bundle scaffold Z, derived from domain B of Staphylococcal protein A and capable of binding to the Fc portion of immunoglobulin (Ig) G1, could be incorporated into modified knobless Ad fiber gene constructs with seven shaft repeats. These fiber gene constructs could be rescued into viable virions that were demonstrated to enter 293 cells engineered for IgG Fc surface expression but not unmodified 293 cells, via a mechanism that could be specifically blocked with soluble Fc target protein. However, the tropism modified viruses showed a slightly impaired cellular entry and a lower infectivity than wildtype (WT) virus. In addition, we generated recombinant fibers containing an IgA binding Affibody ligand, derived from combinatorial specificity-engineering of the Z domain scaffold. Such fiber constructs also showed the expected target specific binding, indicating that the affibody protein class is ideally suited for genetic engineering of Ad tropism.

Viruses use specific cell surface receptors to bind to and subsequently gain entry into their host cells. Some retroviruses such as HIV-1 and HIV-2 utilize one receptor for high-affinity binding (CD4), and a separate coreceptor to mediate fusion of the viral envelope with the cell membrane (CCR5 or CXCR4). The identification of these receptors explains the cellular tropism of HIV, and hence its pathogenesis leading to immune deficiency (T-helper cell depletion), the wasting syndrome (macrophage infection), and dementia (microglia infection). HIV can infect cells by membrane fusion at the cell surface and by receptor-mediated endocytosis. Knowledge of the HIV receptors has led to practical developments such as inhibitory drugs, reasons for genetic resistance to infection, and should inform the judicious choice of candidate vaccines.

Contributions of humoral and cellular immunity in controlling neurotropic mouse hepatitis virus persistence within the CNS were determined in B cell-deficient JHD and syngeneic H-2d B cell+ Ab-deficient mice. Virus clearance followed similar kinetics in all mice, confirming initial control of virus replication by cellular immunity. Nevertheless, virus reemerged within the CNS of all Ab-deficient mice. In contrast to diminished T cell responses in H-2b B cell-deficient μMT mice, the absence of B cells or Ab in the H-2d mice did not compromise expansion, recruitment into the CNS, or function of virus-specific CD4+ and CD8+ T cells. The lack of B cells and lymphoid architecture thus appears to manifest itself on T cell responses in a genetically biased manner. Increasing viral load did not enhance frequencies or effector function of virus-specific T cells within the CNS, indicating down-regulation of T cell responses. Although an Ab-independent antiviral function of B cells was not evident during acute infection, the presence of B cells altered CNS cellular tropism during viral recrudescence. Reemerging virus localized almost exclusively to oligodendroglia in B cell+ Ab-deficient mice, whereas it also replicated in astrocytes in B cell-deficient mice. Altered tropism coincided with distinct regulation of CNS virus-specific CD4+ T cells. These data conclusively demonstrate that the Ab component of humoral immunity is critical in preventing virus reactivation within CNS glial cells. B cells themselves may also play a subtle role in modulating pathogenesis by influencing tropism.

Publisher Summary This chapter focuses on the chemokine response to viral infection of the central nervous system (CNS) with an emphasis on the functional significance of chemokine expression as it relates to both host defense and neuropathology. Available evidence demonstrates clearly that viral infection of the CNS results in a dramatic increase in chemokine gene expression. Moreover, production of chemokines in response to infection is highly focused within areas of viral replication early in the disease process and areas of viral RNA persistence during the chronic stages of disease. Resident glial cells are capable of generating a robust chemokine response following viral infection in the absence of inflammatory cells suggesting that this response may reflect an innate CNS response against viral infection analogous to the response of phagocytic cells in the periphery. Differences in virus and cellular tropism are likely explanations for the slight differences in chemokine profiles and duration of expression observed in the different models. The non-ELR CXC chemokine CXCL10 is often the predominant chemokine expressed early following viral infection suggesting an important role as a sentinel molecule in initiating neuroinflammation. Based on the studies presented in this chapter, it is clear that targeting chemokines during either acute or chronic viral-induced CNS disease may offer exciting new insights into potentially novel interventional mechanisms in treating human neuroinflammatory diseases.

Human cytomegalovirus (HCMV) continues to be a significant cause of morbidity and mortality in organ transplant recipients despite the availability of antiviral therapy. Considerable controversy exists regarding the use of granulocyte-colony stimulating factor (G-CSF) mobilized blood products from HCMV seropositive donors during stem cell transplantation (SCT) and in patients receiving granulocyte transfusions to treat neutropenia. In order to understand mechanisms of HCMV transmission to patients receiving G-CSF mobilized blood products, we generated a novel NOD-scid IL2Rγcnull humanized mouse model in which HCMV establishes a latent infection in human hematopoietic lineage cells. In this model, G-CSF induces the reactivation of latent HCMV in monocytes/macrophages that have migrated into organ tissues. These results suggest that the use of G-CSF mobilized blood products from seropositive donors pose an elevated risk for HCMV transmission to recipients.

The present study sought to determine how usage of coreceptors by human immunodeficiency virus type 1 dictates cell tropism and depletion of CD4 + T cells in human lymphoid tissues cultured ex vivo. We found that coreceptor preferences control the marked, preferential depletion of coreceptor-expressing CD4 + lymphocytes. In addition, there was a strong, but not absolute, preference shown by CXCR4-using strains for lymphocytes and by CCR5-using strains for macrophages.

1399 H IV-2CBL24 (CBL-24) W AS ISOLA T ED from an asymptomatic individual and characterized. 1 The cellular tropism of this isolate was restricted to cord blood mononuclear cells (CBMCs) and Molt-4 cl.8 T cells. Replication was not seen in other T cell lines (H9, C8166, CEM, and U937).1 However, herpesvirus saimiri-im mortalized T lymphocytes (HVS T cells) support CBL-24 replication. 2 A persistently productive infection was established and at 8–10 weeks postinfection (wpi), when the p26 concentration was high ( $ 1000 ng/ml), HVS T cell-passaged CBL-24 induced syncytia in C8166 and CEM-SS T cell lines. Selection of host range mutants in HIV-1/2 has been reported in several continuous cell lines, and so we investigated this observation further. The original isolate, designated passage zero (P0-CBL-24), was obtained from CBMC/Molt-4 cl.8 cocultures. Passage 1 (P1-CBL-24) was obtained after feeding cocultures with uninfected Molt-4 cl.8 cells. Passages 2 (P2-CBL-24) and 3 (P3CBL-24) were obtained by further infections of Molt-4 cl.8. Proviral DNA was extracted at each passage. The env gene sequence of P0-CBL-24 (GenBank U05353), referred to here as the “parent sequence,” has been reported. 3 P3-CBL-24 was used in our previous study.2 We now report on the cellular tropism of P2-CBL-24, P3CBL-24, and HVS T cell-passaged CBL-24 (Kesting-CBL-24 and CB23-CBL-24) in a panel of 11 cell lines and pooled peripheral blood mononuclear cells (PBMCs) that had (CD8 2 ) or had not (CD8 1 ) been depleted of CD8-expressin g cells.2 The cell tropism of HVS T cell CBL-24 isolates was determined after short-term (3 wpi) and extended (11 wpi) passage. The cell lines were inoculated with p26 at 20, 200, or 2000 ng/ml as determined by p26 enzyme-linked immunosorbent assay (ELISA) (Murex, Dartford, UK). CBL-24 phenotype/ cell tropism was assessed by microscopic examination and p26 ELISA. After an inoculum of 20 or 200 ng of p26 per milliliter, similar results were obtained. P2-CBL-24 and P3-CBL-24 replicated in 10 of 11 cell lines, and in CD8 1 and CD8 2 PBMCs (Table 1). However, syncytia were seen in five cell lines only (H9, C8166, Molt-4 cl.8, PM-1, and MT-2), and in CD81 and CD8 2 PBMCs. In contrast, short-term passaged HVS T cell isolates, Kesting-CBL-24 and CB23-CBL-24, replicated in CD8 1 and CD8 2 PBMCs but in only five and six cell lines respectively, with syncytium formation in C8166, Molt-4 cl.8, PM-1, and MT-2. Results were similar, after extended passage (11 weeks) in HVS T cells, except that at a p26 concentration of 200 ng/ml CB23-CBL-24 appeared to lose its tropism for H9. However, by increasing the virus inoculum (p26 at 2000 ng/ml), CBL-24 replicated with syncytia in 7 of 11 cell lines, including CEMSS. These results were reproducible. We could not test the cell tropism of short-term passaged HVS T cell isolates at 2000 ng/ml because the virus titer was not high enough. Clearly, CBL-24 tropism was restricted after passage in HVS T cell lines when compared with Molt-4 cl.8 isolates, but no phenotype/ tropism differences were observed between the two HVS T cell isolates when the T cell panel was inoculated with p26 at 2000 ng/ml. The env genes of these isolates were sequenced to look for substitutions that might explain the results. DNA was prepared from cells infected with the six isolates shown in Table 1. env genes were amplified by nested polymerase chain reaction (PCR), using the primers H2POLF (CARGGAGTAGTRGAAGCAATGAA) and H2LTRR (TGCTTCTAAYTGGCAGCTTTATT) and primers H2ENVF (GACTCCGCGGAAAACTAAGRCTCATYCGTCTYCTGCAYCAGA) and H2ENVR (TTGGTGTGTCCGGAACYCCYACTAGGTCATC), as described. 3 Regions corresponding to nucleotides (nt) 4499–9671 and nt 6096–8869 of HIV-2ROD were amplified. Purified env genes were sequenced directly on an ABI-377 sequencer using dye terminator cycle sequencing Ready Reaction kits (Perkin-Elmer, Foster City, CA) and a bank of primers.3 Sequences were analyzed using GCG and GDE programs (Fig. 14). The most significant changes in all six sequences, compared

Bovine immunodeficiency-like virus (BIV) was first isolated from an animal showing transient leucocytosis, lymphadenopathy, lesions in the central nervous system and progressive weakness and emaciation. Similar signs are observed in other immunosuppressive lentiviral infections. BIV, like other lentiviruses, has been isolated from peripheral blood mononuclear cells and lymphoid tissue of infected animals. However, the in vivo cellular tropism of BIV remains unclear although initial studies indicate that BIV may be pantropic, infecting T cells, B cells and monocytes similar to some of the immunodeficiency-causing lentiviruses. PCR, Southern blot hybridisation, cell culture and reverse transcriptase assays were used to demonstrate the presence of BIV proviral DNA and the production of infectious virus in CD2+, WC1+, B cells and monocytes during the acute stages of infection. Western immunoblot assays were used to assess the development of antibody responses towards the virus.

Adeno associated virus (AAV) has been quite successfully adapted as a gene therapy vector and is currently in clinical trials for a variety of disorders. The AAV family includes multiple serotypes that vary in capsid amino acid composition, resulting in different profiles of target tropism. As the number of different serotypes have expanded, so have the number of potential therapeutic targets for AAV based treatment. However, identifying and screening novel serotypes is not an efficacious strategy for generating de novo vectors with high target specificity. By chimerizing the serotype AAV1 and AAV2 capsids, we are able to delineate the surface domains which play a critical role in regulating vector efficacy. While chimerized vectors include the sequences necessary for binding to the primary receptor for AAV2, heparin, conferring heparin-binding to AAV1 alone is insufficient to recapitulate the target profile of AAV2. Our studies further demonstrate restrictions in the combinations of capsid surface amino acids. Therefore, capsid modification must take into consideration both primary receptor targeting as well as effects on viral formation and function. Prior attempts to retarget using display libraries have inserted random peptide sequences at permissible sites in the capsid. We opted instead to modify the AAV1 capsid by directly substituting a nonapeptide sequence centered on the heparin binding site. The library was screened against non-permissive cell lines resulting in two novel vectors. Clone DL2 bound heparin and its activity could be blocked by competition with heparin whereas DL1 has no heparin affinity. When compared to wild type AAV1 and AAV2, both clones have unique patterns of cellular tropism. Efficacy was enhanced for both above that of wild type AAV1 from which the library was created. Interestingly, when the novel nonapeptide sequences were placed into an AAV2 backbone, recombinant particles could be generated with equal efficacy as AAV2, but the particles were nonfunctional, providing further evidence of restrictions to capsid sequence combinations. As the in vitro data suggests that DL1 and DL2 are both significantly more efficient at transducing a myoblasts than wild type AAV the clones were tested in vivo by directly injecting muscle. When assayed for transgene activity 6 weeks later, DL2 showed markedly greater activity than both wild type viruses and DL1. Thus, our studies demonstrate the capability of substitutive display libraries to generate novel AAV based vectors for gene therapy with greater efficacy and more restricted tropism.

The level of human immunodeficiency virus (HIV) replication in patients reflects a balance between stimulatory and inhibitory host factors (particularly endogenous cytokines). New information concerning the cellular co-receptors for HIV and the cellular tropism of different strains of virus will advance our understanding of HIV-induced pathogenesis and suggests new therapeutic and preventive strategies.

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.

The recent emergence of a novel human coronavirus (HCoV-EMC) in the Middle East raised considerable concerns, as it is associated with severe acute pneumonia, renal failure, and fatal outcome and thus resembles the clinical presentation of severe acute respiratory syndrome (SARS) observed in 2002 and 2003. Like SARS-CoV, HCoV-EMC is of zoonotic origin and closely related to bat coronaviruses. The human airway epithelium (HAE) represents the entry point and primary target tissue for respiratory viruses and is highly relevant for assessing the zoonotic potential of emerging respiratory viruses, such as HCoV-EMC. Here, we show that pseudostratified HAE cultures derived from different donors are highly permissive to HCoV-EMC infection, and by using reverse transcription (RT)-PCR and RNAseq data, we experimentally determined the identity of seven HCoV-EMC subgenomic mRNAs. Although the HAE cells were readily responsive to type I and type III interferon (IFN), we observed neither a pronounced inflammatory cytokine nor any detectable IFN responses following HCoV-EMC, SARS-CoV, or HCoV-229E infection, suggesting that innate immune evasion mechanisms and putative IFN antagonists of HCoV-EMC are operational in the new host. Importantly, however, we demonstrate that both type I and type III IFN can efficiently reduce HCoV-EMC replication in HAE cultures, providing a possible treatment option in cases of suspected HCoV-EMC infection., IMPORTANCE A novel human coronavirus, HCoV-EMC, has recently been described to be associated with severe respiratory tract infection and fatalities, similar to severe acute respiratory syndrome (SARS) observed during the 2002-2003 epidemic. Closely related coronaviruses replicate in bats, suggesting that, like SARS-CoV, HCoV-EMC is of zoonotic origin. Since the animal reservoir and circumstances of zoonotic transmission are yet elusive, it is critically important to assess potential species barriers of HCoV-EMC infection. An important first barrier against invading respiratory pathogens is the epithelium, representing the entry point and primary target tissue of respiratory viruses. We show that human bronchial epithelia are highly susceptible to HCoV-EMC infection. Furthermore, HCoV-EMC, like other coronaviruses, evades innate immune recognition, reflected by the lack of interferon and minimal inflammatory cytokine expression following infection. Importantly, type I and type III interferon treatment can efficiently reduce HCoV-EMC replication in the human airway epithelium, providing a possible avenue for treatment of emerging virus infections.

CD4 is the predominant cell membrane protein that binds human immunodeficiency virus type 1 (HIV-1) gp120 and facilitates HIV-1 infection, but other membrane-associated molecules may be involved in determining HIV-1 cellular infection. Our prior work had suggested that CD44, the transmembrane receptor for hyaluronan, might play a role in the infection of mononuclear phagocytes with HIV-1. In the present work, we have used cells of the CD4-positive, CD44-negative human T-lymphoblast cell line Jurkat to study the role of CD44 in HIV-1 infection and tropism. Cells were transfected with cDNA for the standard (S, or hematopoietic) CD44 isoform CD44S or the epithelial isoform CD44E. The resultant lines expressed appropriate CD44S or CD44E mRNA and protein. While the parent Jurkat cells, those transfected with vector alone, and those transfected with CD44E could be productively infected with only the lymphocytotropic strain HIV-1-LAI, cells transfected with CD44S were rendered susceptible to productive infection with the monocytotropic strains HIV-1-BaL and HIV-1-ADA. Also, CD44S-transfected cells displayed higher levels of infection with HIV-1-LAI than did the other transfected Jurkat cells. The transfected cell line cells all had comparable growth rates and expressed similar levels of the membrane antigens CD4, CD7, major histocompatibility complex (MHC) class I, MHC class II, and CD11a, while levels of CD3 were slightly higher in cells transfected with vector alone and in one of the clones transfected with CD44S. Hyaluronan binding was increased in cells transfected with either CD44S or CD44E. Mouse NIH 3T3 fibroblasts transfected with human CD4, human CD44S, or both human CD4 and CD44S displayed the appropriate antigens, but they could not be productively infected with lymphocytotropic or monocytotropic strains of HIV-1. The results indicate that in human leukocytes, CD44S is an important determinant of HIV-1 productive infection and may be involved in viral cellular tropism.

International audience; To establish the in vivo cellular tropism of human T-cell leukemia virus type II (HTLV-II) in peripheral blood, subpopulations of mononuclear cells isolated from patients with a history of drug abuse and with high proviral load were analyzed by polymerase chain reaction for the presence of the proviral sequences. After purification of cellular subsets by immunomagnetic fractionation of blood cells of an infected patient, HTLV-II DNA was detected in CD4+ and CD8+ T-cells as well as in CD19+ B-cells. A positive PCR signal was obtained for purified B-cells also at limiting dilutions. This observation was confirmed by purifying the B-cell fraction by a two-step immunomagnetic procedure from the peripheral blood of another patient with very high HTLV-II copy number and quantifying the B-cell proviral load by means of competitive PCR. A proviral copy number of 90/100 B-cells was found, demonstrating that the great majority of these cells were infected by HTLV-II in this subject. The results indicate that HTLV-II has a broad host range in some infected individuals, showing an enlargement of cellular tropism to B lymphocytes and suggesting that this expression is associated with an increase in proviral load.

Differences in kinetics of infection, cellular tropism, and cytopathology of SIV and HIV appear to depend on both viral and host factors. We investigated the role of critical CD4 structures from African green monkeys (AGM) a natural SIV host, from pig-tailed macaques (PT) an unnatural SIV host, and from humans, as well as the role of species-specific cellular factors involved in the tropism, kinetics of infection, and cytopathic effects of several SIV and HIV-1. Critical regions of the PT macaque and AGM CD4 genes (V1, V1J1, and V1J1V2J2) were stably expressed as chimeras with the human CD4 gene in human (HeLa and 293) and macaque (CMMT) cell lines. CD4 expressing cell lines were used for infection studies with cell-free SIVsm, SIVmac, SIVsmmPBj, SIVagm, and HIV-1. Results show that both PT CD4 and AGM CD4 supported infection with comparable infection kinetics by all SIV or HIV-1 strains tested. Although structural analysis predicted a major change in secondary structure of AGM CD4/CDR-3, these structural changes did not influence the degree of syncytia formation induced by several SIV and HIV-1. However, the cell line used to express the CD4 gene appeared to be a critical determinant of infection. Thus, SlV strains did not infect human cell lines regardless of the CD4 expressed in these cells. In contrast, HIV-1 did not infect any macaque cell line. This study demonstrates that the differences in CD4 structure among different primate species are clearly not responsible for differences in SIV and HIV infection kinetics, tropism, and cytopathology. However, species-specific factor(s), presumably expressed on the cell surface, markedly influences the ability of SIV or HIV to infect cells expressing CD4.

modified live virus vaccine strain of EAV, we engineered a panel of five chimeric viruses by exchanging the Nterminal ectodomains and/or full-lengths of the two major envelope proteins (GP5 and M) from PRRSV. The growth characteristics and plaque morphology of EAV/ PRRSV chimeric viruses were studied using equine endothelial cells (EECs). The cellular tropism of EAV/PRRSV chimeric viruses were tested in different EAV-susceptible cell lines (EEC, RK-13, BHK-21, and MARC-145), as well as in PRRSV-susceptible cells (porcine alveolar macrophages [PAM] and MARC-145) to confirm the cellular tropism and infectivity of the progeny virus. Virus replication in various cell lines was determined by IFA staining with MAb 3E2 (a-N of EAV) and polyclonal pig anti-PRRSV sera. Of the five chimeric viruses, the recombinant viruses expressing the N-terminal ectodomain of PRRSV GP5 alone or M alone or together (GP5ecto, Mecto and GP5&Mecto, respectively) in the EAV backbone were viable and genetically stable. Compared to the parental virus, these three chimeric viruses produced lower titers and smaller plaque sizes indicating that they have a compromised phenotype. The three chimeric viruses could only infect EAV-susceptible cell lines but not PRRSVsusceptible cells. The two major envelope proteins may not be determining factors in the cellular tropism of EAV and other arteriviruses. These findings further support the recent findings that the minor envelope proteins GP2, GP3, GP4 and E are the critical proteins in mediating cellular tropism.

Background HTLV-1 predominantly transforms CD4+T cells in vitro and induces leukemia and neurological disease in vivo, whereas HTLV-2 shows a preference for CD8+T cell transformation in vitro with limited in vivo pathology. To better understand cellular tropism of HTLV-1 and HTLV-2 early after in vivo infection we determined proviral load and gene expression kinetics of these viruses in purified CD4+ and CD8+T cells from newly infected rabbits.

No predictive parameters of in utero or perinatal vertical transmission of HIV to newborns are known at present. Vertical transmission may be related to several biological parameters of maternal HIV infection: (1) immunological parameters (neutralizing antibodies); (2) the concentration of viral particles and/or infected cells; and (3) the selection of HIV subspecies of particular cellular tropism. The present study was designed to examine the relationship between cellular viral burden and transmission, and between maternal viral burden and CD4+ cell count and clinical status at delivery.We investigated mother-to-infant HIV-1 transmission at delivery in a cohort of 51 pairs of mothers and newborns. Twelve infants were HIV-infected, as determined by successive polymerase chain reaction and culture determinations within the first 6 months of life, and nine of these were diagnosed as HIV-infected during the first week of life. We determined peripheral blood mononuclear cell proviral DNA burden using a quantitative polymerase chain reaction assay. Polymerase chain reaction was performed in the HIV-1 gag gene, using [32P]-end-labelled primers. External standard DNA samples were from the 85-14 F2 cell line, which contains a unique defective proviral DNA genome.There was a linear relationship between the logarithms of c.p.m. and the number of HIV-1 DNA copies.We have previously reported that the number of HIV provirus copies in maternal blood cells is related to transmission of the virus. Quantification of the HIV provirus by polymerase chain reaction may be used as a predictive parameter of vertical transmission if accompanied by an exhaustive clinical and biological follow-up during pregnancy.

Background Gene-based therapy is a new paradigm for the treatment of Parkinson disease (PD) and offers considerable promise for precise targeting and flexibility to impact multiple pathobiological processes for which small molecule agents are not available. Some success has been achieved utilizing adeno-associated virus for this approach, but it is likely that the characteristics of this vector system will ultimately create barriers to progress in clinical therapy. Adenovirus (Ad) vector overcomes limitations in payload size and targeting. The cellular tropism of Ad serotype 5 (Ad5)–based vectors is regulated by the Ad attachment protein binding to its primary cellular receptor, the coxsackie and adenovirus receptor (CAR). Many clinically relevant tissues are refractory to Ad5 infection due to negligible CAR levels but can be targeted by tropism-modified, CAR-independent forms of Ad. Our objective was to evaluate the role of CAR protein in transduction of dopamine (DA) neurons in vivo. Methodology/Principal Findings Ad5 was delivered to the substantia nigra (SN) in wild type (wt) and CAR transgenic animals. Cellular tropism was assessed by immunohistochemistry (IHC) in the SN and striatal terminals. CAR expression was assessed by western blot and IHC. We found in wt animals, Ad5 results in robust transgene expression in astrocytes and other non-neuronal cells but poor infection of DA neurons. In contrast, in transgenic animals, Ad5 infects SNc neurons resulting in expression of transduced protein in their striatal terminals. Western blot showed low CAR expression in the ventral midbrain of wt animals compared to transgenic animals. Interestingly, hCAR protein localizes with markers of post-synaptic structures, suggesting synapses are the point of entry into dopaminergic neurons in transgenic animals. Conclusions/Significance These findings demonstrate that CAR deficiency limits infection of wild type DA neurons by Ad5 and provide a rationale for the development of tropism-modified, CAR-independent Ad-vectors for use in gene therapy of human PD.

A method for rapidly producing helper-free murine leukemia virus (MLV) without using packaging cell lines is described. Viruses bearing ecotropic or amphotropic MLV or Rous sarcoma virus envelope glycoprotein and containing various retroviral vector genomes have been prepared with titers 30 to 40-fold higher than those produced by transient transfection of standard packaging cells. This system can be used to alter the cellular tropism of MLV by incorporating other envelope glycoproteins and to prepare retroviral vector stocks without establishing stable producer cell lines. This method will be particularly useful for preparing viruses that encode toxic proteins and for the rapid analysis of panels of mutant envelope glycoproteins.

We established persistent infection with a strain of human immunodeficiency type 1, HTLV-IIIB, in a ++promyelo-monocytic cell line, ML-1 (CD4 antigen nearly negative and CD4 mRNA negative), and a promonocytic cell line, THP-1 (CD4 antigen positive). Different reactions of giant cell formation were found after cocultivation of infected and uninfected cells of ML-1, HL-60, THP-1 and U-937 cell lines with uninfected and infected MOLT4 (a T-lymphoma cell line). Factors affecting the cellular tropism of human immunodeficiency virus were discussed.

In vitro exposure of human blood monocyte-derived macrophages to T-cell tropic human immunodeficiency virus (HIV) isolates fails to establish a productive viral infection. Several studies have shown that such preferential HIV-1 replication in T cells or in mononuclear phagocytes (HIV tropism) may be determined by distinct viral characteristics. In the present study it was demonstrated that transforming growth factor-beta (TGF-beta), a factor known to be produced by platelets, macrophages, and other cells present at a wound site, can act as a mediator in overcoming the lymphocytotropic restriction of several well-characterized viral isolates of HIV-1 (i.e., LAV, Z84, pLAI, NY5). Macrophages infected with these isolates show cytopathic changes comparable to those seen upon infection with the monocytotropic isolate ADA. To achieve this effect with TGF-beta, the factor must be present after the infection period. The emerging virus retains its original cellular tropism. Based on these observations the authors propose a role for TGF-beta in the establishment and progression of HIV infection and disease.

Human immunodeficiency viruses (HIVs) isolated from infected individuals show genetic and biological diversity. To delineate the genetic determinants underlying specific biological characteristics such as rate of replication and cellular tropism, generation of hybrid HIV using viruses which exhibit distinct biological feature is essential. We have used three different infectious HIV proviral DNAs, designated pZ6, pHXB2 and pARV, derived from HIVZr6, HIVHTLV-IIIB and HIVSF-2 strains, respectively, to construct hybrid HIV. Proviral DNAs differed in their ability to direct the synthesis of viral particles upon transfection into cells and the viruses derived from the molecular clones exhibited different cellular tropism. Three different methods were utilized to generate hybrid HIV, including construction of hybrid proviral DNA using molecular techniques, intracellular ligation of viral DNA fragments and the homologous recombination approach. The chimeric proviral DNAs with exchanges involving only the long terminal repeat (LTR) region indicated that LTR does not exert influence on the overall level of virus production despite extensive differences in the U3 region of the LTR. Regarding the cellular tropism of HIV, the virus derived from pHXB2 productively infected CEMx174 cells. On the other hand, pARV-derived virus did not show productive infection of CEMx174 cells. The hybrid HIV containing the 3'-end of the genome from pARV and the 5'-end of the genome from pHXB2 was effective in infecting CEMx174 cells. However, the converse hybrid containing the 5'-pARV and the 3'-pHXB2 was not effective in infecting CEMx174 cells. These results suggest that differences in the genes outside of env and nef may play a role in the ability of virus to infect a certain cell type.

MHV3 is an hepatotropic Coronavirus. The injection of this virus into susceptible.BALB/c mice causes a mortal fulminating hepatitis. Several viral genes have been implicated as determinants of MHV virulence, including the nucleocapsid (5), E2 (4, 6) and polymerase genes (7). The E2 glycoprotein plays a central role in determining cellular tropism and MHV virulence. It is responsible for the attachment of the virus to the cell and point mutations in the E2 gene could thus modify virus pathogenicity and the target organ. The selection of antibody “escape mutants” has been used by several laboratories to confirm the importance of E2 as a viral determinant of virulence (8). In the case of the MHV3 strain, no results have so far been reported. We have selected MHV3 variants by virtue of their resistance to neutralization by monoclonal antibodies. Two of the mutants selected were less pathogenic than the parental strain. This difference in the biological effect may be associated with a modification in organ tropism and failure to recognize viral receptors on certain hepatic cells.

Background: Conditionally replicative adenoviruses (CRAds) are widely studied tools for cancer gene therapy. The utility of CRAds is based on their ability to destroy tumor by oncolysis. Unfortunately, systemic adenoviral delivery into tumors and metastases is currently inefficient due to liver sequestration of intravenous virus and a neutralizing antibody response that hinders readministration. One potential solution for improving tumor targeting might be to engineer cells as viral carriers, thereby combining cellular tropism for tumors with the oncolytic effect provided by CRAds.

Tissue culture of cataractous lens epithelium shows deterioration of cellular tropism, anisomorphism, anisometry, and formation of large intracytoplasmic vacuoles with deterioration of the lysosomal system, the vacuolated cells showing no tendency to form fibers.