Krupple-like factor 4 (KLF4) methylation signature in host cell in active viral keratitis with epithelial manifestation.

HSV1 presents as epithelial or stromal keratitis or keratouveitis and can lead to sight-threatening complications. KLF4, a critical transcription factor, and regulator of cell growth and differentiation, is essential in corneal epithelium stratification and homeostasis. Here, we want to understand the epigenetic modification specifically the methylation status of KLF4 in epithelium samples of HSV1 keratitis patients. After obtaining consent, epithelial scrapes were collected from 7 patients with clinically diagnosed HSV1 keratitis and 7 control samples (patients undergoing photorefractive keratectomy). Genomic DNA was isolated from the collected samples using the Qiagen DNeasy Kit. Subsequently, bisulfite modification was performed. The bisulphite-modified DNA was then subjected to PCR amplification using specific primers designed to target the KLF4, ACTB gene region, allowing for the amplification of methylated and unmethylated DNA sequences. The amplified DNA products were separated and visualized on a 3% agarose gel. KLF4 hypermethylation was found in 6 out of 7 (85.71%) eyes with viral keratitis, while 1 eye showed hypomethylation compared to PRK samples. Out of these 6, there were 2 each of epithelial dendritic keratitis, epithelial geographical keratitis, and neurotrophic keratitis. The patient with hypomethylated KLF4 had a recurrent case of HSV1 keratitis with multiple dendrites and associated vesicular lesions of the lip along with a history of fever. KLF4 hypermethylation in most viral keratitis cases indicated the under functioning of KLF4 and could indicate a potential association between KLF4 hypermethylation and the development or progression of HSV1 keratitis.

Molecular imaging on ACE2-dependent transocular infection of coronavirus.

A transocular infection has been proved as one of the main approaches that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) invades the body, and angiotensin-converting enzyme 2 (ACE2) plays a key role in this procedure. Dynamic and quantitative details on virus distribution are lacking for virus prevention and drug design. In this study, a radiotraceable pseudovirus packed with an enhanced green fluorescent protein (EGFP) gene, 125 I-CoV, was prepared and inoculated in the unilateral eye of humanized ACE2 (hACE2) mice or ACE2-knockout (ACE2-KO) mice. Single-photon emission computed tomography/computed tomography images were acquired at multiple time points to exhibit ACE2-dependent procedures from invasion to clearance. Positron emission tomography (PET) and western blot were performed to quantify ACE2 expression and verify the factors affecting transocular infection. For the transocular infection of coronavirus (CoV), the renin-angiotensin-aldosterone system (RAAS), lungs, intestines, and genital glands were the main targeted organs. Due to the specific anchor to ACE2-expressed host cells, virus concentrations in genital glands, liver, and lungs ranked the top three most and stabilized at 3.75 ± 0.55, 3.30 ± 0.25, and 2.10 ± 0.55% inoculated dose (ID)/mL at 48 h post treatment. Meanwhile, ACE2-KO mice had already completed the in vivo clearance. In consideration of organ volumes, lungs (14.50 ± 3.75%ID) and liver (10.94 ± 0.71%ID) were the main in-store reservoirs of CoV. However, the inoculated eye (5.52 ± 1.85%ID for hACE2, 5.24 ± 1.45%ID for ACE2-KO, p > 0.05) and the adjacent brain exhibited ACE2-independent virus infection at the end of 72 h observation, and absolute amount of virus played a key role in host cell infection. These observations on CoV infection were further manifested by infection-driven intracellular EGFP expression. ACE2 PET revealed an infection-related systematic upregulation of ACE2 expression in the organs involved in RAAS (e.g., brain, lung, heart, liver, and kidney) and the organ that was of own local renin-angiotensin system (e.g., eye). Transocular infection of CoV is ACE2-dependent and constitutes the cause of disturbed ACE2 expression in the host. The brain, genital glands, and intestines were of the highest unit uptake, potentially accounting for the sequelae. Lungs and liver were of the highest absolute amount, closely related to the respiratory diffusion and in vivo duplication. ACE2 expression was upregulated in the short term after infection with CoV. These visual and quantitative results are helpful to fully understanding the transocular path of SARS-CoV-2 and other CoVs.

Absence of Severe Acute Respiratory Syndrome-Coronavirus-2 RNA in Human Corneal Tissues.

PURPOSE: To examine corneal tissue for severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) positivity regarding implications for tissue procurement, processing, corneal transplantation, and ocular surgery on healthy patients. We performed quantitative reverse transcription-polymerase chain reaction qRT-PCR-testing for SARS-CoV-2 RNA on corneal stroma and endothelium, bulbar conjunctiva, conjunctival fluid swabs, anterior chamber fluid, and corneal epithelium of coronavirus disease 2019 (COVID-19) postmortem donors. METHODS: Included in this study were 10 bulbi of 5 COVID-19 patients who died because of respiratory insufficiency. Informed consent and institutional review board approval was obtained before this study (241/2020BO2). SARS-CoV-2 was detected by using a pharyngeal swab and bronchoalveolar lavage. Tissue procurement and tissue preparation were performed with personal protective equipment (PPE) and the necessary protective measures. qRT-PCR-testing was performed for each of the abovementioned tissues and intraocular fluids. RESULTS: The qRT-PCRs yielded no viral RNA in the following ocular tissues and intraocular fluid: corneal stroma and endothelium, bulbar-limbal conjunctiva, conjunctival fluid swabs, anterior chamber fluid, and corneal epithelium. CONCLUSIONS: In this study, no SARS-CoV-2-RNA was detected in conjunctiva, anterior chamber fluid, and corneal tissues (endothelium, stroma, and epithelium) of COVID-19 donors. This implicates that the risk for SARS-CoV-2 infection using corneal or conjunctival tissue is very low. However, further studies on a higher number of COVID-19 patients are necessary to confirm these results. This might be of high importance for donor tissue procurement, processing, and corneal transplantation.

Association of TLR2 Gene Polymorphisms with Presumed Viral-Induced Anterior Uveitis in male Han Chinese.

The purpose of this study was to investigate whether single nucleotide polymorphisms (SNPs) of TLR2, TLR3, TLR4 and TLR9 genes are associated with susceptibility to presumed viral-induced anterior uveitis (PVIAU) and Posner-Schlossman syndrome (PSS). A case-control study was performed in 205 PVIAU patients and 1007 healthy controls. A total of 15 SNPs were genotyped by MassARRAY platform and iPLEX Gold Assay. Data were analyzed using a Chi-square (χ2) test and Fisher's exact calibration test. Two hundred and three PSS patients served as an extra control to investigate whether there were similar genetic factors between PVIAU and PSS in the context of these tested SNPs in TLR genes. The results showed that the frequency of TLR2/rs7656411 GG genotype and G allele were significantly higher in PVIAU patients as compared with healthy controls (P = 1.10 × 10-4, corrected P value [Pc] = 4.93 × 10-3, odds ratio [OR] = 1.848; P = 3.57 × 10-4, Pc = 1.07 × 10-2, OR = 1.478, respectively). Gender stratification analysis showed a significantly increased frequency of the G allele in male patients (P = 7.46 × 10-5, Pc = 2.24 × 10-3, OR = 1.894). A weak correlation was found between two SNPs (rs3804100 and rs5743705) of the TLR2 gene with PSS. However, after Bonferroni correction, statistical significance was lost. This study shows that the polymorphisms of TLR2/rs7656411 are positively associated with PVIAU in male Chinese patients. PVIAU and PSS have a different genetic background in the context of the tested SNPs.

Interferon-stimulated gene 15 (ISG15) restricts Zika virus replication in primary human corneal epithelial cells.

PURPOSE: Zika virus (ZIKV) has emerged as an important human pathogen causing ocular complications. There have been reports of the shedding of ZIKV in human as well as animal tears. In this study, we investigated the infectivity of ZIKV in corneal epithelial cells and their antiviral immune response. METHODS: Primary human corneal epithelial cells (Pr. HCECs) and an immortalized cell line (HUCL) were infected with two different strains of ZIKV (PRVABC59 & BeH823339) or dengue virus (DENV, serotypes 1-4). Viral infectivity was assessed by immunostaining of viral antigen and plaque assay. qRT-PCR and immunoblot analyses were used to assess the expression of innate inflammatory and antiviral genes. Supplementation of recombinant ISG15 (rISG15) and gene silencing approaches were used to elucidate the role of ISG15 in corneal antiviral defense. RESULTS: Pr. HCECs, but not the HUCL cells, were permissive to both ZIKV strains and specifically to DENV3 infection. ZIKV induced the expression of viral recognition receptors (TLR3, RIG-I, &MDA5), and genes involved in inflammatory (CXCL10 & CCL5) and antiviral (IFNs, MX1, OAS2, ISG15) responses in Pr. HCECs. Furthermore, ZIKV infection caused Pr. HCECs cell death, as evidenced by TUNEL staining. Silencing of ISG15 increased ZIKV infectivity while supplementation with rISG15 reduced ZIKV infection by direct inactivation of ZIKV and inhibiting its entry. CONCLUSIONS: Our study demonstrates for the first time, that ZIKV can readily infect and replicate in Pr. HCECs. Therefore, ZIKV may persist in the cornea and pose the potential risk of transmission via corneal transplantation.

Interaction between the cellular E3 ubiquitin ligase SIAH-1 and the viral immediate-early protein ICP0 enables efficient replication of Herpes Simplex Virus type 2 in vivo.

Herpes Simplex Virus type 2 (HSV-2) is a neurotropic human pathogen. Upon de novo infection, the viral infected cell protein 0 (ICP0) is immediately expressed and interacts with various cellular components during the viral replication cycle. ICP0 is a multifunctional regulatory protein that has been shown to be important for both efficient viral replication and virus reactivation from latency. In particular, as previously demonstrated in transfected tissue culture models, ICP0 interacts with the cellular E3 ubiquitin ligase SIAH-1, which targets ICP0 for proteasomal degradation. However, the consequence of this virus-host interaction during the establishment of HSV-2 infection in vivo has not yet been elucidated. Here we confirmed that ICP0 of HSV-2 interacts with SIAH-1 via two conserved PxAxVxP amino acid binding motifs. We also demonstrate in vitro that a SIAH-1 binding-deficient HSV-2 strain, constructed by homologous recombination technology, exhibits an attenuated growth curve and impaired DNA and protein synthesis. This attenuated phenotype was also confirmed in an in vivo ocular infection mouse model. Specifically, viral load of the SIAH-1 binding-deficient HSV-2 mutant was significantly reduced in the trigeminal ganglia and brain stem at day 5 and 7 post infection. Our findings indicate that the interplay between ICP0 and SIAH-1 is important for efficient HSV-2 replication in vivo, thereby affecting viral dissemination kinetics in newly infected organisms, and possibly revealing novel targets for antiviral therapy.

Transcriptional Profile and Integrative Analyses of Long Noncoding RNAs in Primary Human Corneal Epithelial Cells in Response to HSV-1 Infection.

PURPOSE: Long noncoding RNAs (lncRNAs) have been demonstrated to have important regulatory functions in diverse cellular processes; however, the role of lncRNAs in the pathogenesis of herpes simplex keratitis (HSK) remains poorly understood. METHODS: Primary human corneal epithelial cells (HCECs) were infected with herpes simplex virus-1 (HSV-1) and the total RNAs extracted from both the infected group and the mock-infected group subjected to microarray analysis to identify the differential expression of lncRNAs and mRNAs. We also performed bioinformatic analysis including gene ontology (GO) analysis, pathway analysis and co-expression network analysis. RESULTS: Compared with mock-infected group, the expression of thousands of lncRNAs and mRNAs were significantly changed, and the microarray results were validated by qRT-PCR. The most enriched GOs targeted by up-regulated transcripts were defense response, intrinsic component of plasma membrane and cytokine activity，and the most enriched GOs targeted by the down-regulated transcripts were cellular metabolic process, intracellular part and poly (A) RNA binding. Pathway analysis indicated that the most correlated pathways for up- and down-regulated transcripts were cytokine-cytokine receptor interaction and RNA transport, respectively. CONCLUSIONS: Our study identified the genome-wide profile of lncRNAs and mRNAs expression in primary corneal epithelial cells with HSV-1 infection. These transcriptomic data together with subsequent bioinformatic analysis will provide us with novel clue to the insight into molecular mechanism and potential therapeutic targets of HSK. Further studies are expected to verify the potentially functional genes and pathways and explore the critical lncRNAs. ABBREVIATIONS: Long noncoding RNAs: lncRNAs; herpes simplex virus-1: HSV-1; herpes simplex virus keratitis: HSK; human corneal epithelial cells: HCECs.

Genotypic Detection of Epstein Barr Virus from Clinically Suspected Viral Retinitis Patients in a Tertiary Eye Care Centre, India.

PURPOSE: To evaluate the diagnostic value of PCR on aqueous humour for detection and genotyping of Epstein Bar Virus in patients with viral retinitis. METHODS: 70 AH samples were collected from 20 HIV positive patients with clinically suspected viral retinitis and 25 patients with serpignous choroiditis and 25 AH from patients undergoing cataract surgery. PCR was performed to screen HHV-1 to HHV-5, Mtb and Toxoplasma gondii. Genotype prevalence was confirmed by phylogenetic analysis targetig EBV. RESULTS: EBV was detected in 17 (37.7%) samples. Genotyping to subtype EBV, revealed the circulation of only one subtype (Type 1). PCR results for other infective agents were negative except for the presence of CMV in 5 (11.1%) AH. CONCLUSION: The application of PCR to detect genotypes can be used as an epidemiological tool for clinical management. To our knowledge this is the first report on genotyping of EBV performed on intra ocular samples.

Delivery of herpes simplex virus to retinal ganglion cell axon is dependent on viral protein Us9.

PURPOSE: How herpes simplex virus (HSV) is transported from the infected neuron cell body to the axon terminal is poorly understood. Several viral proteins are candidates for regulating the process, but the evidence is controversial. We compared the results of Us9 deletions in two HSV strains (F and NS) using a novel quantitative assay to test the hypothesis that the viral protein Us9 regulates the delivery of viral DNA to the distal axon of retinal ganglion cells in vivo. We also deleted a nine-amino acid motif in the Us9 protein of F strain (Us9-30) to define the role of this domain in DNA delivery. METHODS: The vitreous chambers of murine eyes were infected with equivalent amounts of F or NS strains of HSV. At 3, 4, or 5 days post infection (dpi), both optic tracts (OT) were dissected and viral genome was quantified by qPCR. RESULTS: At 3 dpi, the F strain Us9- and Us9-30 mutants delivered less than 10% and 1%, respectively, of the viral DNA delivered after infection with the Us9R (control) strain. By 4 and 5 dpi, delivery of viral DNA had only partially recovered. Deletion of Us9 in NS-infected mice has a less obvious effect on delivery of new viral DNA to the distal OT. By 3 dpi the NS Us9-strain delivered 22% of the DNA that was delivered by the NS wt, and by 4 and 5 dpi the amount of Us9-viral DNA was 96% and 81%, respectively. CONCLUSIONS: A highly conserved acidic cluster within the Us9 protein plays a critical role for genome transport to the distal axon. The transport is less dependent on Us9 expression in the NS than in the F strain virus. This assay can be used to compare transport efficiency in other neurotropic viral strains.

Membrane nanotubes in myeloid cells in the adult mouse cornea represent a novel mode of immune cell interaction.

Membrane nanotubes (MNTs) are newly discovered cellular extensions that are either blind-ended or can connect widely separated cells. They have predominantly been investigated in cultured isolated cells, however, previously we were the first group to demonstrate the existence of these structures in vivo in intact mammalian tissues. We previously demonstrated the frequency of both cell-cell or bridging MNTs and blind-ended MNTs was greatest between major histocompatibility complex (MHC) class II(+) cells during corneal injury or TLR ligand-mediated inflammation. The present study aimed to further explore the dynamics of MNT formation and their size, presence in another tissue, the dura mater, and response to stress factors and an active local viral infection of the murine cornea. Confocal live cell imaging of myeloid-derived cells in inflamed corneal explants from Cx(3)cr1(GFP) and CD11c(eYFP) transgenic mice revealed that MNTs form de novo at a rate of 15.5 µm/min. This observation contrasts with previous studies that demonstrated that in vitro these structures originate from cell-cell contacts. Conditions that promote formation of MNTs include inflammation in vivo and cell stress due to serum starvation ex vivo. Herpes simplex virus-1 infection did not cause a significant increase in MNT numbers in myeloid cells in the cornea above that observed in injury controls, confirming that corneal epithelium injury alone elicits MNT formation in vivo. These novel observations extend the currently limited understanding of MNTs in live mammalian tissues.

Overexpression of human HMW FGF-2 but not LMW FGF-2 reduces the cytotoxic effect of lentiviral gene transfer in human corneal endothelial cells.

PURPOSE: Recently, insertion of immuno-modulatory or anti-apoptotic genes into corneal endothelial cells (HCECs) came into focus. Basic FGF-2 occurs in one secreted (low molecular weight, LMW, 18 kD) and four nuclear (high molecular weight, HMW, 22-34 kD) isoforms. HMW isoforms are known differentiation and survival factors, while LMW FGF-2 is a known mitogen. The effect of FGF-2 overexpression of each of the five known isoforms on HCEC cell survival after lentiviral gene transfer in different culture media was investigated. METHODS: Cells were transduced with lentiviral vectors encoding for each of the five FGF-2 isoforms. Transduction efficiency and expression of individual FGF-2 isoforms was assessed by marker gene transfer and western blotting. Primary HCECs were cultured and transduced in four different media previously described for HCEC cultivation or corneal organ cultivation. Cytotoxic effect of virus infection and a possible rescue effect of FGF-2 overexpression were determined by resazurin conversion assay. RESULTS: Transduction with FGF-2 encoding lentiviral vectors resulted in overexpression of the respective isoform in all tested cell populations. Western blotting after total cell lysis proved nuclear localization of transgenic HMW isoforms. Overexpression of HMW FGF-2-especially 34 kD FGF-2-reduced lentiviral cytotoxicity, while overexpression of LMW FGF-2 aggravated viral cytotoxicity. CONCLUSIONS: Cytotoxicity of lentiviral gene transfer in corneal endothelial cells may be reduced by using bicistronic vectors that encode for the target gene and the 34-kD isoform of human FGF-2. Such cotransduction of a survival factor may increase cell survival after gene transfer, thereby improving gene therapeutic approaches.

Anti-heparan sulfate peptides that block herpes simplex virus infection in vivo.

Heparan sulfate (HS) and its highly modified form, 3-O-sulfated heparan sulfate (3-OS HS), contribute strongly to herpes simplex virus type-1 (HSV-1) infection in vitro. Here we report results from a random M13-phage display library screening to isolate 12-mer peptides that bind specifically to HS, 3-OS HS, and block HSV-1 entry. The screening identified representative candidates from two-different groups of anti-HS peptides with high positive charge densities. Group 1, represented by G1 peptide (LRSRTKIIRIRH), belongs to a class with alternating charges (XRXRXKXXRXRX), and group 2, represented by G2 peptide (MPRRRRIRRRQK), shows repetitive charges (XXRRRRXRRRXK). Viral entry and glycoprotein D binding assays together with fluorescent microscopy data indicated that both G1 and G2 were potent in blocking HSV-1 entry into primary cultures of human corneal fibroblasts and CHO-K1 cells transiently expressing different glycoprotein D receptors. Interestingly, G2 peptide isolated against 3-OS HS displayed wider ability to inhibit entry of clinically relevant strains of HSV-1 and some divergent members of herpesvirus family including cytomegalovirus and human herpesvirus-8. To identify functional residues within G1 and G2, we performed point mutations and alanine-scanning mutagenesis. Several arginine and a lysine residues were needed for anti-HSV-1 activity, suggesting the importance of the positively charged residues in virus-cell binding and virus-induced membrane fusion. In vivo administration of G1 or G2 peptide as a prophylactic eye drop completely blocked HSV-1 spread in the mouse cornea as evident by immunohistochemistry. This result also highlights an in vivo significance of HS and 3-OS HS during ocular herpes infection.

Increased expression of hepcidin and toll-like receptors 8 and 10 in viral keratitis.

PURPOSE: Antimicrobial peptides (AMPs) and toll-like receptors (TLRs) form part of the "chemical barrier" of the ocular surface to microbes. Evidence suggests that pathogen recognition by TLR releases AMPs, altering AMP-TLR profiles in pathological states. This study investigated ocular surface expression of AMP-TLRs in health and disease. METHODS: Complementary DNA from conjunctival and corneal impression cytology samples was used for semiquantitative and quantitative polymerase chain reactions, to determine gene expression of 6 AMPs and TLRs-1-10, in healthy subjects and patients with bacterial (n = 6), viral (n = 6), Acanthamoeba (n = 3), or dry eye (n = 7) diseases. RESULTS: Semiquantitative polymerase chain reaction showed variable AMP expression within groups and some expression patterns between groups, increased levels of LEAP (liver-expressed antimicrobial peptide)-1/hepcidin in viral disease, LEAP-2 in dry eye, and human beta defensin 3 in bacterial disease. There was no significant variability in TLR expression. Quantitative polymerase chain reaction showed significantly higher expression of LEAP-1 (P = 0.002) and TLR-8 (P = 0.023) and TLR-10 (P = 0.014) in viral keratitis and LEAP-2 (P = 0.034) in dry eye, versus controls. CONCLUSIONS: Increased expression of LEAP-1 and TLRs 8 and 10 in viral keratitis is novel; TLR-10 has not previously had a documented ligand. LEAP-2 may have a role in dry eye. Further studies will help to improve the understanding of these diseases and yield novel therapeutic interventions.

Characterization of cytokine gene expression associated with noninfectious human immunodeficiency virus retinopathy in human autopsy eyes.

PURPOSE: The purpose of this study was to determine the cytokine-related pathogenesis of human immunodeficiency virus retinopathy in human autopsy eyes. METHODS: Fresh autopsy eyes were procured from clinically diagnosed patients with acquired immunodeficiency syndrome who had died as a result of disease-related complications; eyes were immediately immersed in RNAlater. Clean 2-mm trephines were used to punch individual pathologic retina in areas of cotton-wool spots and control punches. Total RNA was extracted using the TRIzol extraction protocol, and the optimal density of the RNA was measured at an optical density of 260 nm. [Delta]Ct (cytokine) values were calculated using the comparative cytokine analysis method. The results are expressed as a mean fold modulation and as a statistical comparison of Ct values controlling for retinal areas without a lesion in the same eye. RESULTS: The fold modulations and the statistical comparisons of the cytokines studied in tissues from cotton-wool spots and control retina, respectively, regulated on activation normal T cell expressed and secreted (RANTES), macrophage inflammatory protein 1beta, macrophage inflammatory protein 1alpha (5.32x, P = 0.04), and Bcl-2-associated X protein (1.24x, P = 0.05) had a marked elevation of fold modulation and were statistically significant compared with control tissue. Interleukin-8 (1.09x, P = 0.18), interleukin-4, and interleukin-10 (2.7x, P = 0.30) were not significantly expressed in cotton-wool spots. CONCLUSION: Certain inflammatory human immunodeficiency virus-associated and apoptotic cytokines are expressed in cotton-wool spots in eyes with human immunodeficiency virus retinopathy.

Role of pseudorabies virus Us3 protein kinase during neuronal infection.

The pseudorabies virus (PRV) Us3 gene is conserved among the alphaherpesviruses and encodes a serine/threonine protein kinase that is not required for growth in standard cell lines. In this report, we used a compartmented culture system to investigate the role of PRV Us3 in viral replication in neurons, in spread from neurons to PK15 cells, and in axon-mediated spread of infection. We also examined the role of Us3 in neuroinvasion and virulence in rodents. Us3 null mutants produce about 10-fold less infectious virus from neurons than wild-type virus and have no discernible phenotypes for axonal targeting of viral components in cultured peripheral nervous system neurons. After eye infection in rodents, Us3 null mutants were slightly attenuated for virulence, with a delayed onset of symptoms compared to the wild type or a Us3 null revertant. While initially delayed, the symptoms increased in severity until they approximated those of the wild-type virus. Us3 null mutants were neuroinvasive, spreading in both efferent and afferent circuits innervating eye tissues.

Analysis of HLA class I and class II gene polymorphisms in Japanese patients with human T-cell lymphotropic virus type 1-associated uveitis.

PURPOSE: To investigate the immunogenetic background of human T-cell lymphotropic virus type 1 (HTLV-1)-associated uveitis (HAU) that presents immune-mediated reactive changes in the uvea. METHODS: HLA class I and class II genes were studied in 51 patients with HAU, 192 asymptomatic HTLV-1 carriers, and 266 HTLV-1-seronegative controls using a high-resolution method of HLA DNA typing. The HLA alleles of HAU were compared with those of HTLV-1 carriers and healthy controls. RESULTS: We identified 62 distinct alleles of HLA-A, HLA-Cw, and HLA-B and 49 distinct alleles of HLA-DRB1 and HLA-DQB1 in patients with HAU, asymptomatic HTLV-1 carriers, and healthy controls. The relative frequencies of these HLA alleles did not differ among the three groups. CONCLUSION: The results suggest that HLA class I and class II genes do not contribute to susceptibility to HAU.

DNA microarray analysis of the uninoculated eye following anterior chamber inoculation of HSV-1.

PURPOSE: To use DNA microarray to analyze the expression patterns of genes in the uninoculated eye following uniocular anterior chamber inoculation of HSV-1. METHODS: On Day 9 following inoculation of 2 x 10( 4) PFU of HSV-1 (KOS strain) or an equivalent volume of tissue culture medium into one anterior chamber of BALB/c mice, the uninoculated eyes were enucleated, pooled, and total RNA was isolated. cDNA was synthesized from the total RNA. The gene expression patterns were inferred based on the hybridization intensities of the probes on the cDNA array. The hybridization signals were globally normalized and filtered. The data were analyzed using hierarchical and gene tree clustering algorithms. Additional uninoculated eyes collected on Day 9 p.i. were stained for F4/80 and CD19. RESULTS: Compared with the uninoculated eye of control mice, 3800 genes were upregulated at least twofold in the contralateral eye of HSV-1-infected mice. Among the 10 most upregulated genes, T cell-specific protein, MHC II antigen A, and MHC II k region locus 2 were upregulated 179-, 164-, and 162-fold, respectively. Ten T-cell receptor-related genes, 61 cytokine and chemokine genes, and 16 MHC genes were upregulated. Furthermore, 11 immunoglobulin and B cell genes and 11 macrophage-related genes were also upregulated. F4/80+ and CD19+ cells were observed on Day 9 p.i. CONCLUSIONS: The DNA microarray results support the idea that T cells and immunomodulatory factors (cytokines, chemokines) are likely to be involved in HSV-1 retinitis. These results also suggest that B cells and/or macrophages play a role in the pathogenesis of HSV-1 retinitis.

Family and population effects on disease resistance in a reptile.

Despite its importance in evolutionary biology, studies of the pattern of disease resistance in natural populations are rare. In this paper, we report patterns of infection of a viral eye disease in juvenile Swedish common lizards (Lacerta vivipara). Females were sampled at random from natural populations immediately prior to parturition with equal exposure of pathogens for all lizards once in captivity. No causative agents could be found that linked risk of disease to maternal/interfollicular transfer of pathogens. The results show that a major factor influencing offspring susceptibility is family identity, suggesting heritable variation in pathogen resistance. Our interpopulation comparison provides additional support for a link between genetics and disease resistance. Lizards in northern Sweden were not only more susceptible to the disease but were also more health compromised once infected, with relatively more reduced growth rate and increased mortality than lizards from the south. This scenario suggests that southern lizards have been under selection for resistance to this pathogen, whereas northern lizards have not, or at least not to the same degree. Thus, this study confirms the importance of genetic (family) effects on pathogen resistance with variation in this trait among natural populations.