Pathogenesis of Primary Foot-and-Mouth Disease Virus Infection in the Nasopharynx of Vaccinated and Non-Vaccinated Cattle

A time-course pathogenesis study was performed to compare and contrast primary foot-and-mouth disease virus (FMDV) infection following simulated-natural (intra-nasopharyngeal) virus exposure of cattle that were non-vaccinated or vaccinated using a recombinant adenovirus-vectored FMDV vaccine. FMDV genome and infectious virus were detected during the initial phase of infection in both categories of animals with consistent predilection for the nasopharyngeal mucosa. A rapid progression of infection with viremia and widespread dissemination of virus occurred in non-vaccinated animals whilst vaccinated cattle were protected from viremia and clinical FMD. Analysis of micro-anatomic distribution of virus during early infection by lasercapture microdissection localized FMDV RNA to follicle-associated epithelium of the nasopharyngeal mucosa in both groups of animals, with concurrent detection of viral genome in nasopharyngeal MALT follicles in vaccinated cattle only. FMDV structural and non-structural proteins were detected in epithelial cells of the nasopharyngeal mucosa by immunomicroscopy 24 hours after inoculation in both non-vaccinated and vaccinated steers. Co-localization of CD11c⁺/MHC II⁺ cells with viral protein occurred early at primary infection sites in vaccinated steers while similar host-virus interactions were observed at later time points in non-vaccinated steers. Additionally, numerous CD8⁺/CD3^- host cells, representing presumptive natural killer cells, were observed in association with foci of primary FMDV infection in the nasopharyngeal mucosa of vaccinated steers but were absent in non-vaccinated steers. Immunomicroscopic evidence of an activated antiviral response at primary infection sites of vaccinated cattle was corroborated by a relative induction of interferon -α, -β, -γ and -λ mRNA in micro-dissected samples of nasopharyngeal mucosa. Although vaccination protected cattle from viremia and clinical FMD, there was subclinical infection of epithelial cells of the nasopharyngeal mucosa that could enable shedding and long-term persistence of infectious virus. Additionally, these data indicate different mechanisms within the immediate host response to infection between non-vaccinated and vaccinated cattle.     

Epstein-Barr virus infection in ex vivo tonsil epithelial cell cultures of asymptomatic carriers

Epstein-Barr virus (EBV) is found frequently in certain epithelial pathologies, such as nasopharyngeal carcinoma and oral hairy leukoplakia, indicating that the virus can infect epithelial cells in vivo. Recent studies of cell lines imply that epithelial cells may also play a role in persistent EBV infection in vivo. In this report, we show the establishment and characterization of an ex vivo culture model of tonsil epithelial cells, a likely site for EBV infection in vivo. Primary epithelial-cell cultures, generated from tonsil explants, contained a heterogeneous mixture of cells with an ongoing process of differentiation. Keratin expression profiles were consistent with the presence of cells from both surface and crypt epithelia. A small subset of cells could be latently infected by coculture with EBV-releasing cell lines, but not with cell-free virus. We also detected viral-DNA, -mRNA, and -protein expression in cultures from EBV-positive tonsil donors prior to in vitro infection. We conclude that these cells were either already infected at the time of explantation or soon after through cell-to-cell contact with B cells replicating EBV in the explant. Taken together, these findings suggest that the tonsil epithelium of asymptomatic virus carriers is able to sustain EBV infection in vivo. This provides an explanation for the presence of EBV in naso- and oropharyngeal pathologies and is consistent with epithelial cells playing a role in the egress of EBV during persistent infection.     

The level of virus-specific T-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load

Porcine reproductive and respiratory syndrome virus (PRRSV) is the most important infectious disease agent of pigs worldwide, causing reproductive failure in pregnant sows and respiratory problems in nursing and growing pigs. PRRSV infection is characterized by a prolonged viremia of 30 or more days and an extended persistent infection of lymphoid tissues. To better understand the immunological basis for prolonged acute and persistent PRRSV infection, we have examined the cell-mediated immune (CMI) response throughout the course of infection and compared the results to the local distribution and abundance of PRRSV in infected tissues. PRRSV-specific T cells, enumerated by gamma interferon enzyme-linked immunospot assay, did not appear until 2 weeks after PRRSV inoculation, and their abundance exhibited substantial variation over time and among animals. In all cases the T-cell response was transient. High levels of viral RNA were present in lymphoid tissues of all animals in the acute phase of infection. Viral loads were decreased 1,000-fold or more in persistent infections, with the primary sites of persistence being tonsil, sternal lymph node, and inguinal lymph node. The abundance of virus-specific T cells in either acutely or persistently infected animals was highly variable and showed no correlation to the level of virus in lymphoid tissues. No significant difference in antigen-specific T-cell abundance was observed in secondary lymphoid tissues in either acute or persistent infection except for tonsil, in which the number of responding cells was extremely low. CD4(+)- and CD8(+)-T-cell frequencies did not change after PRRSV infection, though a decrease in gammadelta T cells was observed. Macrophages, the permissive cell type for PRRSV, were present in various levels in all tissue preparations and were not in proportion to local virus load. These findings indicate that a weak CMI response contributes to prolonged PRRSV infection and suggests that PRRSV suppresses T-cell recognition of infected macrophages. Thus, the slow but eventual resolution of PRRSV infection may be dependent on limiting permissive macrophages and on innate immune factors.     

Bovine plasmacytoid dendritic cells are the major source of type I interferon in response to foot-and-mouth disease virus in vitro and in vivo

Type I interferons (alpha/beta interferons [IFN-α/β]) are the main innate cytokines that are able to induce a cellular antiviral state, thereby limiting viral replication and disease pathology. Plasmacytoid dendritic cells (pDCs) play a crucial role in the control of viral infections, especially in response to viruses that have evolved mechanisms to block the type I IFN signal transduction pathway. Using density gradient separation and cell sorting, we have highly enriched a population of bovine cells capable of producing high levels of biologically active type I IFN. These cells represented less than 0.1% of the total lymphocyte population in blood, pseudoafferent lymph, and lymph nodes. Phenotypic analysis identified these cells as bovine pDCs (CD3(-) CD14(-) CD21(-) CD11c(-) NK(-) TCRδ(-) CD4(+) MHC II(+) CD45RB(+) CD172a(+) CD32(+)). High levels of type I IFN were generated by these cells in vitro in response to Toll-like receptor 9 (TLR-9) agonist CpG and foot-and-mouth disease virus (FMDV) immune complexes. In contrast, immune complexes formed with UV-inactivated FMDV or FMDV empty capsids failed to elicit a type I IFN response. Depletion of CD4 cells in vivo resulted in levels of type I IFN in serum early during FMDV infection that were significantly lower than those for control animals. In conclusion, pDCs interacting with immune-complexed virus are the major source of type I interferon production during acute FMDV infection in cattle.     

Differential cytokeratin and glycoconjugate expression by the surface and crypt epithelia of human palatine tonsils

Human palatine tonsils are clinically important due to their susceptibility to tonsillitis and association with other local and systemic diseases. Paradoxically, the tonsils function as antigen sampling sites of the mucosal immune system and, consequently, the tonsil epithelia perform both protective and antigen sampling roles. These epithelia are divided into stratified squamous epithelium overlying the tonsil surface and crypt epithelium lining the tonsil crypts, the latter of which includes reticular areas which are infiltrated by lymphocytes and are responsible for antigen sampling. In this study we characterised cytokeratin and glycoconjugate expression by healthy epithelia of human palatine tonsils. We identified pan-epithelial tonsil markers and also demonstrated that the surface and reticular crypt epithelia are differentiated by the expression of multiple cytokeratins. The latter finding supports the hypothesis that these epithelia undergo alternate differentiation pathways and possess different functional roles. In addition, we identified cell subpopulations in the tonsil epithelia which may represent distinct cell subtypes including specialised antigen sampling cells. These findings establish a basis for future studies to investigate histochemical changes in tonsil epithelia that are associated with or predispose to local and/or systemic disease.     

In vivo natural killer cell depletion during primary simian immunodeficiency virus infection in rhesus monkeys

The contribution of natural killer (NK) cells to the immune containment of human immunodeficiency virus infection remains undefined. To directly assess the role of NK cells in an AIDS animal model, we depleted rhesus monkeys of >88% of CD3(-) CD16(+) CD159a(+) NK cells at the time of primary simian immunodeficiency virus (SIV) infection by using anti-CD16 antibody. During the first 11 days following SIV inoculation, when NK cell depletion was most profound, a trend toward higher levels of SIV replication was noted in NK cell-depleted monkeys compared to those in control monkeys. However, this treatment did not result in significant changes in the overall levels or kinetics of plasma viral RNA or affect the SIV-induced central memory CD4(+) T-lymphocyte loss. These findings are consistent with a limited role for cytotoxic CD16(+) NK cells in the control of primary SIV viremia.     

Viremia and nasal and rectal shedding of rotavirus in gnotobiotic pigs inoculated with Wa human rotavirus

Respiratory symptoms with rotavirus shedding in nasopharyngeal secretions have been reported in children with and without gastrointestinal symptoms (Zheng et al., 1991, J. Med. Virol. 34:29-37). To investigate if attenuated and virulent human rotavirus (HRV) strains cause upper respiratory tract infections or viremia in gnotobiotic pigs, we inoculated them with attenuated or virulent HRV intranasally, intravenously, or orally or via feeding tube (gavage) and assayed virus shedding. After oral or intranasal inoculation with attenuated HRV, the pigs remained asymptomatic, but 79 to 95% shed virus nasally and 5 to 17% shed virus rectally. After inoculation by gavage, no pigs shed virus nasally or rectally, but all pigs seroconverted with antibodies to HRV. No viremia was detected through postinoculation day 10. Controls inoculated intranasally with nonreplicating rotavirus-like particles or mock inoculated did not shed virus. In contrast, 100% of pigs inoculated with virulent HRV (oral, intranasal, or gavage) developed diarrhea, shed virus nasally and rectally, and had viremia. The infectivity of sera from the viremic virulent HRV-inoculated pigs was confirmed by inoculating gnotobiotic pigs orally with pooled HRV-positive serum. Serum-inoculated pigs developed diarrhea and fecal and nasal virus shedding and seroconverted with serum and intestinal HRV antibodies. Pigs inoculated intravenously with serum or intestinal contents from the viremic virulent HRV-inoculated pigs developed diarrhea, virus shedding, and viremia, similar to the orally inoculated pigs. This study provides new evidence that virulent HRV causes transient viremia and upper respiratory tract infection in addition to gastrointestinal infection in gnotobiotic pigs, confirming previous reports of rotavirus antigenemia (Blutt et al., Lancet 362:1445-1449, 2003). Our data also suggest that intestinal infection might be initiated from the basolateral side of the epithelial cells via viremia. Additionally, virus shedding patterns indicate a different pathogenesis for attenuated versus virulent HRV.     

Selection of classical swine fever virus with enhanced pathogenicity reveals synergistic virulence determinants in E2 and NS4B

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious disease of pigs. There are numerous CSFV strains that differ in virulence, resulting in clinical disease with different degrees of severity. Low-virulent and moderately virulent isolates cause a mild and often chronic disease, while highly virulent isolates cause an acute and mostly lethal hemorrhagic fever. The live attenuated vaccine strain GPE(-) was produced by multiple passages of the virulent ALD strain in cells of swine, bovine, and guinea pig origin. With the aim of identifying the determinants responsible for the attenuation, the GPE(-) vaccine virus was readapted to pigs by serial passages of infected tonsil homogenates until prolonged viremia and typical signs of CSF were observed. The GPE(-)/P-11 virus isolated from the tonsils after the 11th passage in vivo had acquired 3 amino acid substitutions in E2 (T830A) and NS4B (V2475A and A2563V) compared with the virus before passages. Experimental infection of pigs with the mutants reconstructed by reverse genetics confirmed that these amino acid substitutions were responsible for the acquisition of pathogenicity. Studies in vitro indicated that the substitution in E2 influenced virus spreading and that the changes in NS4B enhanced the viral RNA replication. In conclusion, the present study identified residues in E2 and NS4B of CSFV that can act synergistically to influence virus replication efficiency in vitro and pathogenicity in pigs.     

Foot-and-Mouth Disease Virus Replicates Only Transiently in Well-Differentiated Porcine Nasal Epithelial Cells

Three-dimensional (3D) porcine nasal mucosal and tracheal mucosal epithelial cell cultures were developed to analyze foot-and-mouth disease virus (FMDV) interactions with mucosal epithelial cells. The cells in these cultures differentiated and polarized until they closely resemble the epithelial layers seen in vivo. FMDV infected these cultures predominantly from the apical side, primarily by binding to integrin αvβ6, in an Arg-Gly-Asp (RGD)-dependent manner. However, FMDV replicated only transiently without any visible cytopathic effect (CPE), and infectious progeny virus could be recovered only from the apical side. The infection induced the production of beta interferon (IFN-β) and the IFN-inducible gene Mx1 mRNA, which coincided with the disappearance of viral RNA and progeny virus. The induction of IFN-β mRNA correlated with the antiviral activity of the supernatants from both the apical and basolateral compartments. IFN-α mRNA was constitutively expressed in nasal mucosal epithelial cells in vitro and in vivo. In addition, FMDV infection induced interleukin 8 (IL-8) protein, granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES mRNA in the infected epithelial cells, suggesting that it plays an important role in modulating the immune response.Foot-and-mouth disease is an economically important disease caused by foot-and-mouth disease virus (FMDV), a picornavirus, belonging to the genus Aphthovirus and family Picornaviridae. Field strains of FMDV infect cells by attaching to integrin receptors through a highly conserved Arg-Gly-Asp (RGD) tripeptide motif which is located on a surface-exposed loop of VP1 (29). A number of different species of RGD-binding integrin (αvβ1, αvβ3, αvβ6, and αvβ8) have been reported to serve as receptors for infection (8, 22-24). However, αvβ6 is believed to function as the principal receptor for infection in the host (33). FMDV is highly infectious and can become established in susceptible animals by inhalation of relatively small levels of airborne virus (predominantly cattle) or ingestion of contaminated material (primarily pigs) (5). Apart from direct infection through injured skin or abrasion, the epithelial cells of the oral and upper respiratory tract mucosal surfaces are likely to be the first point of contact with the virus. In animals, FMDV undergoes early and rapid replication at the predilection sites in the pharynx and tonsil (5). This is followed by a viremic phase lasting 4 or 5 days, during which the virus spreads to other epithelial tissues and establishes itself at other secondary sites of replication (5). At these secondary sites, vesicles normally form, and the vesicles rupture to produce classical ragged-edge erosions. Although nasal mucosa has been suggested as a primary site of FMDV replication (26), there has been no evidence to support this claim (4). FMDV RNA can be detected in the nasal mucosa and trachea of pigs up to 4 days after infection; however, pathological signs of infection are not observed at these sites (3; P. Dash, unpublished observation).Pigs are known to be more resistant to aerosol infection than cattle, though they excrete larger amounts of virus once infection is established (1, 2, 17, 38). The reason for the apparent reduced susceptibility to aerosol infection of pigs is unclear. Here we have investigated the interaction of FMDV with pig nasal and tracheal epithelial cells using polarized, differentiated three-dimensional (3D) cultures. The results provide insight into the possible reasons why pigs are relatively resistant to FMDV by aerosol challenge.     

Expression and function of tight junctions in the crypt epithelium of human palatine tonsils.

The human palatine tonsils have surface and crypt stratified epithelium and may be initiated via the epithelium to mount immune responses to various presenting antigens. Here we investigated the expression and function of tight junctions in the epithelium of human palatine tonsils from patients with tonsillar hypertrophy or recurrent tonsillitis. Occludin, ZO-1, JAM-1, and claudin-1, -3, -4, -7, -8, and -14 mRNAs were detected in tonsillar hypertrophy. Occludin and claudin-14 were expressed in the uppermost layer of the tonsil surface epithelium, whereas ZO-1, JAM-1, and claudin-1, -4, and -7 were found throughout the epithelium. In the crypt epithelium, claudin-4 was preferentially expressed in the upper layers. In freeze-fracture replicas, short fragments of continuous tight junction strands were observed but never formed networks. In the crypt epithelium of recurrent tonsillitis, the tracer was leaked from the surface regions where occludin and claudin-4 disappeared. Occludin, ZO-1, JAM-1, and claudin-1, -3, -4, and -14, but not claudin-7, mRNAs were decreased in recurrent tonsillitis compared with those of tonsillar hypertrophy. These studies suggest unique expression of tight junctions in human palatine tonsillar epithelium, and the crypt epithelium may possess an epithelial barrier different from that of the surface epithelium.     

Novel viral disease control strategy: adenovirus expressing alpha interferon rapidly protects swine from foot-and-mouth disease

We have previously shown that replication of foot-and-mouth disease virus (FMDV) is highly sensitive to alpha/beta interferon (IFN-alpha/beta). In the present study, we constructed recombinant, replication-defective human adenovirus type 5 vectors containing either porcine IFN-alpha or IFN-beta (Ad5-pIFNalpha or Ad5-pIFNbeta). We demonstrated that cells infected with these viruses express high levels of biologically active IFN. Swine inoculated with 10(9) PFU of a control Ad5 virus lacking the IFN gene and challenged 24 h later with FMDV developed typical signs of foot-and-mouth disease (FMD), including fever, vesicular lesions, and viremia. In contrast, swine inoculated with 10(9) PFU of Ad5-pIFNalpha were completely protected when challenged 24 h later with FMDV. These animals showed no clinical signs of FMD and no viremia and did not develop antibodies against viral nonstructural proteins, suggesting that complete protection from infection was achieved.     

Activation of Kaposi's sarcoma-associated herpesvirus lytic gene expression during epithelial differentiation

The oral cavity has been identified as the major site for the shedding of infectious Kaposi's sarcoma-associated herpesvirus (KSHV). While KSHV DNA is frequently detected in the saliva of KSHV seropositive persons, it does not appear to replicate in salivary glands. Some viruses employ the process of epithelial differentiation for productive viral replication. To test if KSHV utilizes the differentiation of oral epithelium as a mechanism for the activation of lytic replication and virus production, we developed an organotypic raft culture model of epithelium using keratinocytes from human tonsils. This system produced a nonkeratinized stratified squamous oral epithelium in vitro, as demonstrated by the presence of nucleated cells at the apical surface; the expression of involucrin and keratins 6, 13, 14, and 19; and the absence of keratin 1. The activation of KSHV lytic-gene expression was examined in this system using rKSHV.219, a recombinant virus that expresses the green fluorescent protein during latency from the cellular EF-1alpha promoter and the red fluorescent protein (RFP) during lytic replication from the viral early PAN promoter. Infection of keratinocytes with rKSHV.219 resulted in latent infection; however, when these keratinocytes differentiated into a multilayered epithelium, lytic cycle activation of rKSHV.219 occurred, as evidenced by RFP expression, the expression of the late virion protein open reading frame K8.1, and the production of infectious rKSHV.219 at the epithelial surface. These findings demonstrate that KSHV lytic activation occurs as keratinocytes differentiate into a mature epithelium, and it may be responsible for the presence of infectious KSHV in saliva.     

Identification of short hairpin RNA targeting foot-and-mouth disease virus with transgenic bovine fetal epithelium cells

Background

Although it is known that RNA interference (RNAi) targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV), it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge.

Principal Finding

Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2), VP3 (RNAi-VP3), or VP4 (RNAi-VP4) of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID₅₀ of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h.

Conclusion

RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.     

Pathogenesis of a Thai strain of white spot syndrome virus (WSSV) in juvenile, specific pathogen-free Litopenaeus vannamei

White spot syndrome virus (WSSV) causes disease and mortality in cultured and wild shrimp. A standardized WSSV oral inoculation procedure was used in specific pathogen-free (SPF) Litopenaeus vannamei (also called Penaeus vannamei) to determine the primary sites of replication (portal of entry), to analyze the viral spread and to propose the cause of death. Shrimp were inoculated orally with a low (10(1.5) shrimp infectious dose 50% endpoint [SID50]) or a high (10(4) SID50) dose. Per dose, 6 shrimp were collected at 0, 6, 12, 18, 24, 36, 48 and 60 h post inoculation (hpi). WSSV-infected cells were located in tissues by immunohistochemistry and in hemolymph by indirect immunofluorescence. Cell-free hemolymph was examined for WSSV DNA using 1-step PCR. Tissues and cell-free hemolymph were first positive at 18 hpi (low dose) or at 12 hpi (high dose). With the 2 doses, primary replication was found in cells of the foregut and gills. The antennal gland was an additional primary replication site at the high dose. WSSV-infected cells were found in the hemolymph starting from 36 hpi. At 60 hpi, the percentage of WSSV-infected cells was 36 for the epithelial cells of the foregut and 27 for the epithelial cells of the integument; the number of WSSV-infected cells per mm2 was 98 for the gills, 26 for the antennal gland, 78 for the hematopoietic tissue and 49 for the lymphoid organ. Areas of necrosis were observed in infected tissues starting from 48 hpi (low dose) or 36 hpi (high dose). Since the foregut, gills, antennal gland and integument are essential for the maintenance of shrimp homeostasis, it is likely that WSSV infection leads to death due to their dysfunction.     

Bovine type III interferon significantly delays and reduces the severity of foot-and-mouth disease in cattle

Interferons (IFNs) are the first line of defense against viral infections. Although type I and II IFNs have proven effective to inhibit foot-and-mouth disease virus (FMDV) replication in swine, a similar approach had only limited efficacy in cattle. Recently, a new family of IFNs, type III IFN or IFN-λ, has been identified in human, mouse, chicken, and swine. We have identified bovine IFN-λ3 (boIFN-λ3), also known as interleukin 28B (IL-28B), and demonstrated that expression of this molecule using a recombinant replication-defective human adenovirus type 5 (Ad5) vector, Ad5-boIFN-λ3, exhibited antiviral activity against FMDV in bovine cell culture. Furthermore, inoculation of cattle with Ad5-boIFN-λ3 induced systemic antiviral activity and upregulation of IFN-stimulated gene expression in the upper respiratory airways and skin. In the present study, we demonstrated that disease could be delayed for at least 6 days when cattle were inoculated with Ad5-boIFN-λ3 and challenged 24 h later by intradermolingual inoculation with FMDV. Furthermore, the delay in the appearance of disease was significantly prolonged when treated cattle were challenged by aerosolization of FMDV, using a method that resembles the natural route of infection. No clinical signs of FMD, viremia, or viral shedding in nasal swabs was found in the Ad5-boIFN-λ3-treated animals for at least 9 days postchallenge. Our results indicate that boIFN-λ3 plays a critical role in the innate immune response of cattle against FMDV. To this end, this work represents the most successful biotherapeutic strategy so far tested to control FMDV in cattle.     

Selective lymphocyte depletion during the early stage of the immune response to foot-and-mouth disease virus infection in swine

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious vesicular disease of cloven-hoofed animals. In the present study we use FMDV serotype C infection of swine to determine, by analytical techniques, the direct ex vivo visualization of virus-infected immune cells during the first 17 days of infection. We report, for the first time, that FMDV C-S8c1 can infect T and B cells at short periods of time postinoculation, corresponding with the peak of the viremia. There is a significant lymphopenia that involves CD3(+) CD4(-) CD8(+/-), CD3(+) CD4(-) CD8(+)Tc, and CD3(+) CD4(+) CD8(+) memory Th but not CD3(+) CD4(+) CD8(-) na?ve Th lymphocytes. In addition, a profound depletion of the vast majority of peripheral T cells in lymph nodes and spleen is observed. This selective depletion of T cells is not due mainly to in situ death via apoptosis as visualized by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) technique. Thus, early infection of T cells by FMDV may be the main cause of the observed T-cell depletion. Importantly, this lack of T cells is reflected in a reduced response to mitogen activation, which in many cases is totally eliminated. These data suggest a mechanism by which the virus causes a transient immunosuppression, subvert the immune systems, and spreads. These results have important implications for our understanding of early events in the development of a robust immune response against FMDV.     

Concomitant primary infection of the midgut epithelial cells and the hemocytes of Trichoplusia ni by Autographa californica nucleopolyhedrovirus

We have constructed a modified Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) to express the green fluorescent protein (GFP) under the polyhedrin promoter and used it to study the infection process of AcMNPV in Trichoplusia ni larvae. T. ni larvae that ingested the virus showed localized expression of GFP in the midgut epithelial cells and the hemocytes at 12 h post infection (hpi). The presence of GFP-related fluorescence in the midgut columnar cells indicated that the virus was not only replicating, but also synthesizing the late viral proteins. Studies using the transmission electron microscope showed that the virus infected the midgut columnar cells. At the same time a proportion of the parental virus travelled through the midgut epithelial layer, possibly utilizing the plasma membrane reticular system, entered the hemocoel and infected the hemocytes. This resulted in the simultaneous infection of the midgut epithelial cells and the hemocytes. Subsequently, the budded virus (BV) released from the infected hemocytes into the hemolymph caused secondary infection within the tracheal epithelial cells. The virus then rapidly spread through the tracheal system allowing the infection of a variety of other tissues such as the epidermis and the fat body.