Epithelial Tropism Research Articles

ORF Nodule Complicated by Erythema Multiforme: About 2 Cases

ORF nodule is a disease caused by a parapoxvirus ovis with epithelial tropism which is transmitted to humans from sick animals, sheep and goats, suffering from contagious ovine pustular dermatosis ecthyma [1]. Common complications of ORF infection have been reported, including secondary bacterial infection, lymphadenopathy, lymphangitis, more rarely erythema multiforme and bullous pemphigoid [2]. We describe here two cases of ORF nodule with secondary erythema multiforme.

Modified Banff Criteria in Assessing SARS-CoV-2-Associated Renal Pathology: An Autopsy Study.

Introduction SARS-CoV-2 is an epitheliotropic viral agent with epithelial tropism. Although the clinical significance and severity of affection is the most pronounced in the respiratory system, other organs and systems are also infected and, hence affected, such as the central nervous system, gastrointestinal tract, cardiovascular, and urinary systems. Herein, we set out to evaluate the presence and degree of morphological changes within the renal parenchyma and its relation to disease outcome. Materials and methods A retrospective non-clinical approach was utilized for the means of the study. All patients with real-time reverse transcriptase-polymerase chain reaction proven infection, subject to an autopsy performed in a period of two calendar years, were included in the study. Kidney tissue histopathology samples were analyzed using a modified Banff criteria system for acute onset and chronic changes. The results were compared for statistical significance with overall patient survival from symptom onset to death. Furthermore, SARS-CoV-2 viral presence was evaluated in renal structures by means of immunohistochemistry. Results A total of 40 patients were included in the study. Immunohistochemistry showed viral presence within a myriad of renal structured - endothelial cells, tubular cells, and podocytes. Modified Banff criteria showed significant acute changes within the parenchyma, including endotheliitis, glomerulitis, mesangial matrix expansion, tubulitis, capillaritis, arteritis, thrombosis (including thrombotic microangiopathy in four patients), and hemorrhages. Individual cases also presented with signs of rhabdomyolysis - myoglobulin casts. Signs of chronic injury were also present in most patients. However, when calculated as scores, neither acute nor chronic changes showed a correlation with time from symptom onset to death. Conclusion The results of the present study show both viral presence and a myriad of induced changes in the contents of SARS-CoV-2 infection within the renal parenchyma. The lack of correlation with the degree of changes, when compared to survival, is an encouraging fact that the changes are unlikely to play a role in direct tanatogenesis while having the potential to manifest as chronic kidney disease in the future.

Different Mechanisms Are Utilized by Coronavirus Transmissible Gastroenteritis Virus To Regulate Interferon Lambda 1 and Interferon Lambda 3 Production.

Type III interferons (IFN-λ) are shown to be preferentially produced by epithelial cells, which provide front-line protection at barrier surfaces. Transmissible gastroenteritis virus (TGEV), belonging to the genus Alphacoronavirus of the family Coronaviridae, can cause severe intestinal injuries in porcine, resulting in enormous economic losses for the swine industry, worldwide. Here, we demonstrated that although IFN-λ1 had a higher basal expression, TGEV infection induced more intense IFN-λ3 production in vitro and in vivo than did IFN-λ1. We explored the underlying mechanism of IFN-λ induction by TGEV and found a distinct regulation mechanism of IFN-λ1 and IFN-λ3. The classical RIG-I-like receptor (RLR) pathway is involved in IFN-λ3 but not IFN-λ1 production. Except for the signaling pathways mediated by RIG-I and MDA5, TGEV nsp1 induces IFN-λ1 and IFN-λ3 by activating NF-κB via the unfolded protein responses (UPR) PERK-eIF2α pathway. Furthermore, functional domain analysis indicated that the induction of IFN-λ by the TGEV nsp1 protein was located at amino acids 85 to 102 and was dependent on the phosphorylation of eIF2α and the nuclear translocation of NF-κB. Moreover, the recombinant TGEV with the altered amino acid motif of nsp1 85-102 was constructed, and the nsp1 (85-102sg) mutant virus significantly reduced the production of IFN-λ, compared with the wild strain. Compared to the antiviral activities of IFN-λ1, the administration of IFN-λ3 showed greater antiviral activity against TGEV infections in IPEC-J2 cells. In summary, our data point to the significant role of IFN-λ in the host innate antiviral responses to coronavirus infections within mucosal organs and in the distinct mechanisms of IFN-λ1 and IFN-λ3 regulation. IMPORTANCE Coronaviruses cause infectious diseases in various mammals and birds and exhibit an epithelial cell tropism in enteric and respiratory tracts. It is critical to explore how coronavirus infections modulate IFN-λ, a key innate cytokine against mucosal viral infection. Our results uncovered the different processes of IFN-λ1 and IFN-λ3 production that are involved in the classical RLR pathway and determined that TGEV nsp1 induces IFN-λ1 and IFN-λ3 production by activating NF-κB via the PERK-eIF2α pathway in UPR. These studies highlight the unique regulation of antiviral defense in the intestine during TGEV infection. We also demonstrated that IFN-λ3 induced greater antiviral activity against TGEV replication than did IFN-λ1 in IPEC-J2 cells, which is helpful in finding a novel strategy for the treatment of coronavirus infections.

Murine Cytomegalovirus MCK-2 Facilitates In Vivo Infection Transfer from Dendritic Cells to Salivary Gland Acinar Cells.

The cytomegaloviruses (CMVs) spread systemically via myeloid cells and demonstrate broad tissue tropism. Human CMV (HCMV) UL128 encodes a component of the virion pentameric complex (PC) that is important for entry into epithelial cells and cell-cell spread in vitro. It possesses N-terminal amino acid sequences similar to those of CC chemokines. While the species specificity of HCMV precludes confirmation of UL128 function in vivo, UL128-like counterparts in experimental animals have demonstrated a role in salivary gland infection. How they achieve this has not been defined, although effects on monocyte tropism and immune evasion have been proposed. By tracking infected cells following lung infection, we show that although the UL128-like protein in mouse CMV (MCMV) (designated MCK-2) facilitated entry into lung macrophages, it was dispensable for subsequent viremia mediated by CD11c+ dendritic cells (DCs) and extravasation to the salivary glands. Notably, MCK-2 was important for the transfer of MCMV infection from DCs to salivary gland acinar epithelial cells. Acinar cell infection of MCMVs deleted of MCK-2 was not rescued by T-cell depletion, arguing against an immune evasion mechanism for MCK-2 in the salivary glands. In contrast to lung infection, peritoneal MCMV inoculation yields mixed monocyte/DC viremia. In this setting, MCK-2 again promoted DC-dependent infection of salivary gland acinar cells, but it was not required for monocyte-dependent spread to the lung. Thus, the action of MCK-2 in MCMV spread was specific to DC-acinar cell interactions. IMPORTANCE Cytomegaloviruses (CMVs) establish myeloid cell-associated viremias and persistent shedding from the salivary glands. In vitro studies with human CMV (HCMV) have implicated HCMV UL128 in epithelial tropism, but its role in vivo is unknown. Here, we analyzed how a murine CMV (MCMV) protein with similar physical properties, designated MCK-2, contributes to host colonization. We demonstrate that MCK-2 is dispensable for initial systemic spread from primary infection sites but within the salivary gland facilitates the transfer of infection from dendritic cells (DCs) to epithelial acinar cells. Virus transfer from extravasated monocytes to the lungs did not require MCK-2, indicating a tissue-specific effect. These results provide new information about how persistent viral tropism determinants operate in vivo.

Clinicopathologic correlations of COVID-19–related cutaneous manifestations with special emphasis on histopathologic patterns

Skin is one of target organs affected by the novel coronavirus SARS-CoV-2, and in response to the current COVID-19 pandemic, a fast body of literature has emerged on related cutaneous manifestations. Current perspective is that the skin is not only a bystander of the general cytokines storm with thrombophilic multiorgan injury, but it is directly affected by the epithelial tropism of the virus, as confirmed by the detection of SARS‐CoV‐2 in endothelial cells and epithelial cells of epidermis and eccrine glands. In contrast with the abundance of epidemiologic and clinical reports, histopathologic characterization of skin manifestations is limited. Without an adequate clinicopathologic correlation, nosology of clinically similar conditions is confusing, and effective association with COVID-19 remains presumptive. Several patients with different types of skin lesions, including the most specific acral chilblains-like lesions, showed negative results at SARS-CoV-2 nasopharyngeal and serologic sampling. The aim of this review is to provide an overview of what has currently been reported worldwide, with a particular emphasis on microscopic patterns of the skin manifestations in patients exposed to or affected by COVID-19. Substantial breakthroughs may occur in the near future from more skin biopsies, improvement of immunohistochemistry studies, RNA detection of SARS-CoV-2 strain by real-time polymerase chain reaction-based assay, and electron microscopic studies.

Assessing Gammapapillomavirus infections of mucosal epithelia with two broad-spectrum PCR protocols

BackgroundHuman papillomaviruses (HPVs) have been divided into mucosal and cutaneous types according to their primary epithelial tissue tropism. However, recent studies showed the presence of several cutaneous types in mucosal lesions and healthy mucosa from different anatomical sites.MethodsHere, the HPV prevalence and type-specific distribution were assessed in a variety of mucosal samples from 435 individuals using a combination of two established broad-spectrum primer systems: Gamma-PV PCR and CUT PCR.ResultsOverall HPV prevalence in anal canal swabs, cervical cancer biopsies, genital warts and oral swabs was 85, 47, 62 and 4%, respectively. In anal canal swabs, Alpha-PVs were most frequently found (59%), followed by Gamma- (37%) and Beta-PVs (4%). The prevalence and persistence of HPV infection in the anal canal of 226 individuals were further explored. Overall HPV, Gamma-PVs and multiple HPV infections were significantly higher in men vs. women (p = 0.034, p = 0.027 and p = 0.003, respectively); multiple HPV infections were more common in individuals ≤40 years (p = 0.05), and significantly higher prevalence of Gamma-PVs and multiple HPV infections was observed in HIV-1-positive vs. HIV-1-negative individuals (p = 0.003 and p = 0.04, respectively). Out of 21 patients with follow-up anal swabs, only one persistent infection with the same type (HPV58) was detected.ConclusionsOur findings suggest that Gamma-PVs (except species Gamma-6) are ubiquitous viruses with dual muco-cutaneous tissue tropism. Anal canal Gamma-PV infections may be associated with sexual behavior and the host immune status. This study expands the knowledge on Gamma-PVs’ tissue tropism, providing valuable data on the characteristics of HPV infection in the anal canal.

Virulence beneath the fleece; a tale of foot-and-mouth disease virus pathogenesis in sheep

Foot-and-mouth disease virus (FMDV) is capable of infecting all cloven-hoofed domestic livestock species, including cattle, pigs, goats, and sheep. However, in contrast to cattle and pigs, the pathogenesis of FMDV in small ruminants has been incompletely elucidated. The objective of the current investigation was to characterize tissue- and cellular tropism of early and late stages of FMDV infection in sheep following three different routes of simulated natural virus exposure. Extensive post-mortem harvest of tissue samples at pre-determined time points during early infection (24 and 48 hours post infection) demonstrated that tissues specifically susceptible to primary FMDV infection included the paraepiglottic- and palatine tonsils, as well as the nasopharyngeal mucosa. Additionally, experimental aerosol inoculation of sheep led to substantial virus replication in the lungs at 24-48 hours post-inoculation. During persistent infection (35 days post infection), the paraepiglottic- and palatine tonsils were the only tissues from which infectious FMDV was recovered. This is strikingly different from cattle, in which persistent FMDV infection has consistently been located to the nasopharyngeal mucosa. Analysis of tissue sections by immunomicroscopy revealed a strict epithelial tropism during both early and late phases of infection as FMDV was consistently localized to cytokeratin-expressing epithelial cells. This study expands upon previous knowledge of FMDV pathogenesis in sheep by providing detailed information on the temporo-anatomic distribution of FMDV in ovine tissues. Findings are discussed in relation to similar investigations previously performed in cattle and pigs, highlighting similarities and differences in FMDV pathogenesis across natural host species.

Mystery eye: Human adenovirus and the enigma of epidemic keratoconjunctivitis.

Known to occur in widespread outbreaks, epidemic keratoconjunctivitis (EKC) is a severe ocular surface infection with a strong historical association with human adenovirus (HAdV). While the conjunctival manifestations can vary from mild follicular conjunctivitis to hyper-acute, exudative conjunctivitis with formation of conjunctival membranes, EKC is distinct as the only form of adenovirus conjunctivitis in which the cornea is also involved, likely due to the specific corneal epithelial tropism of its causative viral agents. The initial development of a punctate or geographic epithelial keratitis may herald the later formation of stromal keratitis, and manifest as subepithelial infiltrates which often persist or recur for months to years after the acute infection has resolved. The chronic keratitis in EKC is associated with foreign body sensation, photophobia, glare, and reduced vision. However, over a century since the first clinical descriptions of EKC, and over 60 years since the first causative agent, human adenovirus type 8, was identified, our understanding of this disorder remains limited. This is underscored by a current lack of effective diagnostic tools and treatments. In part, stasis in our knowledge base has been encouraged by the continued acceptance, and indeed propagation of, inaccurate paradigms pertaining to disease etiology and pathogenesis, particularly with regard to mechanisms of innate and adaptive immunity within the cornea. Owing to its often persistent and medically refractory visual sequelae, reconsideration of key aspects of EKC disease biology is warranted to identify new treatment targets to curb its worldwide socioeconomic burden.

LANGERHANS CELLS IN THE EPITHELIUM OF UNICYSTIC AMELOBLASTOMAS

Background and Objectives Langerhans cells (LC's) are specialized dendritic cells known to colonize epithelial lined surfaces. Few studies relating to the distribution of LC's in odontogenic tumors and especially ameloblastomas are available and the influence of inflammation on the presence of these cells in odontogenic tumors is unclear. This study investigate the number of LC's in a series of unicystic ameloblastoma using two immunohistochemical stains, Langerin and S100. The association between the presence of LC's and the degree of inflammation was also investigated. Methods Formalin fixed, paraffin imbedded tissue blocks of thirty cases of unicystic ameloblastoma were retrieved from the archives of the department of Oral Pathology. A 4µm tissue sections in each case was stained with S100 and Langerin antibodies respectively. The average number of LC's/1mm of cyst wall were calculated from 10mm of cyst or the entire epithelial lining if less was available. The nature and density of inflammation was scored and compared to the number of LC's present. Results LC's were detected in 21 (70%) and 15 (50%) unicystic ameloblastomas stained with Langerin and S100 antibodies respectively. A statistically significant difference was noted in the number of LC's on Langerin (mean = 0.66/mm) compared to S100 (mean = 0.31/mm) (P=0,014). 26/30 cases (86.67%) were associated with inflammation distributed either diffusely (60.00%) or focally (26.67%) in the wall. The degree or type of inflammation did not have any influence on the presence or numbers of LC. Conclusion LC's are present in the epithelial lining of the majority of unicystic ameloblastomas irrespective of the type or degree of inflammation present in the wall. Their presence may be due to their epithelial tropism or as part of the normal anti-tumour immuno-surveillance. The exact role of LC's and what attracts them should be investigated on molecular level.

OR14I1 is a receptor for the human cytomegalovirus pentameric complex and defines viral epithelial cell tropism

A human cytomegalovirus (HCMV) pentameric glycoprotein complex (PC), gH-gL-UL128-UL130-UL131A, is necessary for viral infection of clinically relevant cell types, including epithelial cells, which are important for interhost transmission and disease. We performed genome-wide CRISPR/Cas9 screens of different cell types in parallel to identify host genes specifically required for HCMV infection of epithelial cells. This effort identified a multipass membrane protein, OR14I1, as a receptor for HCMV infection. This olfactory receptor family member is required for HCMV attachment, entry, and infection of epithelial cells and is dependent on the presence of viral PC. OR14I1 is required for AKT activation and mediates endocytosis entry of HCMV. We further found that HCMV infection of epithelial cells is blocked by a synthetic OR14I1 peptide and inhibitors of adenylate cyclase and protein kinase A (PKA) signaling. Identification of OR14I1 as a PC-dependent HCMV host receptor associated with epithelial tropism and the role of the adenylate cyclase/PKA/AKT-mediated signaling pathway in HCMV infection reveal previously unappreciated targets for the development of vaccines and antiviral therapies.

Inclusion of Antibodies to Cell Culture Media Preserves the Integrity of Genes Encoding RL13 and the Pentameric Complex Components During Fibroblast Passage of Human Cytomegalovirus.

Propagation of human cytomegalovirus (CMV) in cultured cells results in genetic adaptations that confer improved growth in vitro and significant attenuation in vivo. Mutations in RL13 arise quickly, while mutations in the UL128-131A locus emerge later during fibroblast passage and disrupt formation of a glycoprotein complex that is important for entry into epithelial and endothelial cells. As CMV replicates in the context of host antibodies in vivo, we reasoned that antibodies might mitigate the accumulation of adaptive mutations during cell culture passage. To test this, CMV in infant urine was used to infect replicate fibroblast cultures. One lineage was passaged in the absence of CMV-hyperimmuneglobulin (HIG) while the other was passaged with HIG in the culture medium. The former lost epithelial tropism and acquired mutations disrupting RL13 and UL131A expression, whereas the latter retained epithelial tropism and both gene loci remained intact after 22 passages. Additional mutations resulting in single amino acid changes also occurred in UL100 encoding glycoprotein M, UL102 encoding a subunit of the helicase/primase complex, and UL122 encoding the Immediate Early 2 protein. An epitheliotropic RL13+/UL131A+ virus was isolated by limiting dilution in the presence of HIG and expanded to produce a working stock sufficient to conduct cell tropism experiments. Thus, production of virus stocks by culture in the presence of antibodies may facilitate in vitro experiments using viruses that are genetically more authentic than previously available.

Expression of HPV-induced DNA Damage Repair Factors Correlates With CIN Progression.

Human papillomaviruses (HPVs) are DNA viruses with epithelial tropism. High-risk types of HPV are the causative agents of the majority of cervical cancers and are responsible for a number of other anogenital as well as oropharyngeal cancers. The life cycle of HPV is closely linked to the differentiation state of its host cell and is dependent on the activation of specific pathways of the DNA damage response. Several proteins from the ataxia telangiectasia mutated and the ataxia telangiectasia mutated and Rad3-related DNA repair pathways, which are essential for maintaining genomic stability in cells, are upregulated in HPV-positive cells and are required for viral replication. Our studies examine the expression of 5 such DNA repair factors-pCHK2, pCHK1, FANCD2, BRCA1, and H2AX-in cervical specimens from patients diagnosed with low-grade, intermediate-grade, or high-grade lesions. The percentage of cells expressing pCHK2, pCHK1, FANCD2, and BRCA1 is significantly higher in high-grade squamous intraepithelial lesions compared with that of either low-grade squamous intraepithelial lesions or normal tissue, particularly in differentiated cell layers. In addition, the distribution of this staining throughout the epithelium is altered with increasing lesion grade. This study characterizes the expression of pCHK2, pCHK1, FANCD2, H2AX and BRCA1 during cervical cancer progression and provides additional insight into the role of these DNA damage response proteins in viral transformation.

Comparison of vaccination with rhesus CMV (RhCMV) soluble gB with a RhCMV replication-defective virus deleted for MHC class I immune evasion genes in a RhCMV challenge model

A human cytomegalovirus (HCMV) vaccine to prevent infection and/or reduce disease associated with congenital infection or visceral disease in transplant recipients is a high priority, but has remained elusive. We created a disabled infectious single cycle rhesus CMV (RhCMV) deleted for glycoprotein L (gL) and the MHC class I immune evasion genes Rh178 and Rh182-189, and restored its epithelial cell tropism by inserting the Rh128-131A genes. The resulting virus, RhCMVRΔgL/178/182–189, was used to vaccinate rhesus monkeys intramuscularly and was compared with vaccination of animals with soluble RhCMV glycoprotein B (gB) in alum/monophosphoryl lipid A or with PBS as a control. At 4 weeks after the second vaccination, an increased frequency of RhCMV-specific CD8 T cells was detected in animals vaccinated with the RhCMVRΔgL/178/182–189 vaccine compared to animals vaccinated with soluble gB. In contrast, monkeys vaccinated with soluble gB had 20-fold higher gB antibody titers than animals vaccinated with RhCMVRΔgL/178/182–189. Titers of neutralizing antibody to RhCMV infection of fibroblasts were higher in animals vaccinated with gB compared with RhCMVRΔgL/178/182–189. Following vaccination, monkeys were challenged subcutaneously with RhCMV UCD59, a low passage virus propagated in monkey kidney epithelial cells. All animals became infected after challenge; however, the frequency of RhCMV detection in the blood was reduced in monkeys vaccinated with soluble gB compared with those vaccinated with RhCMVRΔgL/178/182–189. The frequency of challenge virus shedding in the urine and saliva and the RhCMV copy number shed at these sites was not different in animals vaccinated with RhCMVRΔgL/178/182–189 or soluble gB compared with those that received PBS before challenge. Although the RhCMVRΔgL/178/182–189 vaccine was superior in inducing cellular immunity to RhCMV, it induced lower titers of neutralizing antibody and antibody to gB than the soluble gB vaccine; after challenge, animals vaccinated with soluble gB had a lower frequency of virus detection in the blood than those vaccinated with RhCMVRΔgL/178/182–189.

Characterization of a new genotype of Betapapillomavirus HPV 17 through L1, E7, E7 and LCR sequences

Human papillomavirus (HPV) exhibits epithelial and mucosal tropism. HPV type 17 belongs to the Betapapillomavirus genus and molecular cloning experiments have identified two subtypes (17a and 17b) isolated from epidermodysplasia verruciformis (EV). HPV subtypes are characterized by dissimilarities from 2 to 10% at the nucleotide level from their referenced HPV. The aim of this study was to characterize the L1, E6, E7 and LCR sequences from an isolate, which was recovered from the oral mucosa of an asymptomatic 63 year-old woman. The whole late gene 1 (L1) was amplified using several sets of primers. The complete early genes 6 and 7 (E6, E7) and the long control region (LCR) were amplified using specific primers. Potential binding sites for transcriptional factors within the LCR were also investigated. Within these sets, the DNA sequence was altered at 91 positions (68 in L1, 13 in E6, 8 in E7, and 2 in LCR sequences). L1 analysis showed high dissimilarity compared with the HPV 17 prototype, reaching 4% of nucleotide substitutions and leading to aprobable third 17 subtype. The E6 oncoprotein presented the highest modification among the sequences studied, with four amino acid changes in comparison with the prototype isolate. One amino acid was modified at aposition 62 (S-T), azinc-binding domain (CxxC(C)29 CxxC). Our findings provide data on genetic variations seen in this genotype, reaching to dichotomic branching and pointing to an evolutionary process.

Differences in Growth Properties among Two Human Cytomegalovirus Glycoprotein O Genotypes.

Glycoprotein O (gO) of the human cytomegalovirus (HCMV) is the critical subunit of the envelope trimer gH/gL/gO as it interacts with platelet-derived growth factor alpha receptor upon fibroblast entry, and triggers gB-mediated fusion for fibroblast and epithelial cell infection. Eight genotypes (GT) of the highly polymorphic gO gene are described, yet it is unclear whether the distinct GTs differ in their function. Thus, we aimed to elucidate potential functional differences between two highly diverse gO GTs in an otherwise genomically identical HCMV strain. Therefore, resident gO GT1c sequence of strain TB40-BAC4-luc was entirely replaced by gO GT4 of strain Towne and both, GT1c and GT4 viruses, were investigated for their growth properties in fibroblasts and epithelial cells. In addition, two conserved gO cysteines involved in gH/gL/gO stabilization were mutated to serine either in GT1c (C218S and C343S) or GT4 (C216S and C336S) and their effects on cell-free infectivity were assessed. GT4 viruses displayed a significantly enhanced epithelial cell tropism and this resulted in higher virus release upon replication in epithelial cells when compared to GT1c viruses. Further, when the two cysteines were individually mutated in gO GT1c no impairment in cell-free infectivity was observed. This, however, was in sharp contrast to gO GT4, in which both of the corresponding cysteine mutations led to a substantial reduction in cell-free infectivity which was even more pronounced upon mutation of GT4-C336 than of GT4-C216. In conclusion, these findings provide evidence that the two highly diverse gO genotypes, GT1c and GT4, differ in their functional properties as revealed by their different infection capacities for epithelial cells and by their different responsiveness to mutation of strictly conserved cysteine residues. Thus, it is likely that the gO heterogeneity influences cell-free infectivity of HCMV also in vivo which may have important implications for virus host transmission.

Cytomegalovirus UL128 homolog mutants that form a pentameric complex produce virus with impaired epithelial and trophoblast cell tropism and altered pathogenicity in the guinea pig

Guinea pig cytomegalovirus (GPCMV) encodes a homolog pentameric complex (PC) for specific cell tropism and congenital infection. In human cytomegalovirus, the PC is an important antibody neutralizing target and GPCMV studies will aid in the development of intervention strategies. Deletion mutants of the C-terminal domains of unique PC proteins (UL128, UL130 and UL131 homologs) were unable to form a PC in separate transient expression assays. Minor modifications to the UL128 homolog (GP129) C-terminal domain enabled PC formation but viruses encoding these mutants had altered tropism to renal and placental trophoblast cells. Mutation of the presumptive CC chemokine motif encoded by GP129 was investigated by alanine substitution of the CC motif (codons 26–27) and cysteines (codons 47 and 62). GP129 chemokine mutants formed PC but GP129 chemokine mutant viruses had reduced epitropism. A GP129 chemokine mutant virus pathogenicity study demonstrated reduced viral load to target organs but highly extended viremia.

A Luciferase Gene Driven by an Alphaherpesviral Promoter Also Responds to Immediate Early Antigens of the Betaherpesvirus HCMV, Allowing Comparative Analyses of Different Human Herpesviruses in One Reporter Cell Line.

Widely used methods for quantification of human cytomegalovirus (HCMV) infection in cell culture such as immunoblotting or plaque reduction assays are generally restricted to low throughput and require time-consuming evaluation. Up to now, only few HCMV reporter cell lines have been generated to overcome these restrictions and they are afflicted with other limitations because permanently expandable cell lines are normally not fully permissive to HCMV. In this work, a previously existing epithelial cell line hosting a luciferase gene under control of a Varicella-zoster virus promoter was adopted to investigate HCMV infection. The cells were susceptible to different HCMV strains at infection efficiencies that corresponded to their respective degree of epithelial cell tropism. Expression of early and late viral antigens, formation of nuclear inclusions, release of infectious virus progeny, and focal growth indicated productive viral replication. However, viral release and spread occurred at lower levels than in primary cell lines which appears to be due to a malfunction of virion morphogenesis during the nuclear stage. Expression of the luciferase reporter gene was specifically induced in HCMV infected cultures as a function of the virus dose and dependent on viral immediate early gene expression. The level of reporter activity accurately reflected infection efficiencies as determined by viral antigen immunostaining, and hence could discriminate the cell tropism of the tested virus strains. As proof-of-principle, we demonstrate that this cell line is applicable to evaluate drug resistance of clinical HCMV isolates and the neutralization capacity of human sera, and that it allows comparative and simultaneous analysis of HCMV and human herpes simplex virus type 1. In summary, the permanent epithelial reporter cell line allows robust, rapid and objective quantitation of HCMV infection and it will be particularly useful in higher throughput analyses as well as in comparative analyses of different human herpesviruses.

Cross-Species Rhesus Cytomegalovirus Infection of Cynomolgus Macaques.

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

Selection Pressure in the Human Adenovirus Fiber Knob Drives Cell Specificity in Epidemic Keratoconjunctivitis.

Viruses adapt various survival strategies to gain entry into target host cells. Human adenovirus (HAdV) types are associated with distinct disease conditions, yet evidence for connections between genotype and cellular tropism is generally lacking. Here, we provide a structural and evolutionary basis for the association between specific genotypes within HAdV species D and epidemic keratoconjunctivitis, a severe ocular surface infection. We find that HAdV-D fiber genes of major EKC pathogens, specifically the fiber knob gene region, share a distinct phylogenetic clade. Deeper analysis of the fiber gene revealed that evolutionary pressure at crucial amino acid sites has a significant impact on its structural conformation, which is likely important in host cell binding and entry. Specific amino acids in hot spot residues provide a link to ocular cell tropism and possibly to corneal pathogenesis.

Host Immune Responses Associated with Clearance or Persistence of Human Papillomavirus Infections

Specific types of human papillomavirus (HPV) are involved in a number of human cancers, including cervical cancer. With strict host epithelial cell tropism, HPV avoids immune detection using both passive and active mechanisms resulting in persistent, often asymptomatic infections with very limited and tightly regulated viral gene expression linked closely to the natural pathway of host epithelial cell maturation. With a focus on genital HPV infections, this review summarizes some of the host and viral factors that may influence HPV clearance, persistence, and latency and discusses factors that could favor progression to cervical disease.