Infectious agents in congenital cataract in a tertiary care referral center in North India.

Congenital cataract has the potential for inhibiting early visual development. Intrauterine infections with Rubella virus, Herpes simplex virus (HSV) and Toxoplasma gondii plays an important role in the development of congenital cataract. The study included 120 children under the age of 6 years presenting with congenital cataract and diagnosed using serology and polymerase chain reaction (PCR). The IgM positivity for rubella, HSV, T. gondii was found to be 5.8%, 1.6% and 8.3% respectively. The overall PCR positivity was found to be 40(33.3%), 25 (20.8%) and 39 (32.5%) for rubella, HSV and T. gondii with mean copy number of 1599 copies/µL; 1716 copies/µL and 1503 copies/µL respectively. Infective etiology significantly contributes to the causation of congenital cataract particularly for rubella virus which is a potentially eradicable disease. This study provides an epidemiological data for rubella, HSV and T. gondii in children with congenital cataract and highlights the need to introduce rubella vaccine in the National Immunization Programme of India.

A strange endocytobiont revealed as largest virus.

A lot of endocytobionts (or endosymbionts) have been discovered within free-living amoebae in recent years. In this article the results of a long lasting effort to derive valuable data about an extraordinary spore-like infectious microorganism (endocytobiont, endosymbiont) within host amoebae (Acanthamoeba sp.) recently isolated from the contact lens case of a patient with keratitis, are presented. It took some time until this endocytobiont could be attributed to the genus Pandoravirus following a publication of two other pandoraviruses isolated from aquatic environments. Consequently the molecular biological investigation led to the taxonomic affiliation of the endocytobiont with the genus Pandoravirus and to the description of a new Pandoravirus species, Pandoravirus inopinatum after whole-genome sequencing in 2015. The fact that it was isolated from a contact lens container of a keratitis patient gives another dimension to these findings showing paradigmatically, how readily these 'new' giant viruses get to humans.

[Intraocular infection with herpesviruses and antiviral immunity in patients with age-related macular degeneration].

Polymerase chain reaction revealed Herpesviridae (Herpes simplex, Cytomegalovirus) in the lens substance in 46 patients operated on for age-related cataract. Thirty-two patients of them had age-related macular degeneration. Herpes simplex was found in 6 (18.8%) of the patients with age-related macular degeneration. At the same time there were signs of lacrimal fluid antiviral immunity imbalance with decreased IFN-gamma and TNF-alpha and increased IFN-alpha. The authors consider it advisable to perform antivirus treatment in patients with the above abnormality, preventing the active forms of herpetic infection.

Laboratory confirmation of congenital rubella syndrome in infants: an eye hospital based investigation.

A total of 190 specimens from South Indian children aged 0-59 months with ocular anomalies consistent with suspected congenital rubella syndrome (CRS) were investigated. Twenty-six of the 65 infants (40%) were confirmed as CRS by detection of rubella specific IgM. Rubella RNA was detected in 41 samples from 26 infants by both real-time and block based PCR. The PCR results correlated well with the presence of anti-rubella IgM/IgG (23/27 cases with rubella IgM were PCR positive). Whereas, only 17 of 26 infants met the WHO CRS case definition. Amongst the various specimens tested from the sero-confirmed cases (n = 27), a high percentage of positives were detected in lens (92%) and oral fluid (60%) specimens, when compared to other samples. The quantification of viral load by real-time PCR demonstrated higher copy number of virus in lens samples of 0-11 months infants. The rubella viruses were characterized and revealed the circulation of genotype 2B in three South Indian states. The integrated analysis of clinical manifestations, serological and molecular data in the study has generated baseline information of rubella infection and CRS in infants with ocular anomalies.

Nested reverse transcription polymerase chain reaction for the detection of rubella virus in clinical specimens.

BACKGROUND & OBJECTIVE: Rubella virus (RV) is one of the leading causes of childhood blindness in India. In this study we applied an optimized nested reverse transcription polymerase chain reaction (nRT-PCR) to detect RV in clinical specimens. METHODS: nRT-PCR was optimized using total RNA extracted from standard strain of RV using nested sets of primers specific for E1 open reading frame. nRT-PCR was applied onto 30 lens aspirates and corresponding peripheral blood leucocytes of 30 infants with congenital (29)/ developmental (01) cataract. Serology for anti-RV IgG and IgM antibodies was done. RV isolation was attempted using Vero and SIRC cell cultures. RESULTS: Optimized nRT-PCR was specific for RV and sensitive to detect 10 fg of RV RNA. Among 30 patients, nRT-PCR detected presence of RV in lens aspirates of 6 (20%) and 4 corresponding leucocytes. RV was isolated from 3 (10%) lens aspirates (nRT-PCR positive) of the 30 patients. Sera of these 6 patients showed presence of anti-RV IgG and IgM in one, only anti-RV IgG in 3 others and none in the other two. Of the remaining 24 patients, anti-RV IgG was detected in 3 and no anti-RV IgM antibodies in others. INTERPRETATION & CONCLUSION: Findings of our study showed that the nRT-PCR was a more sensitive and rapid technique to detect RV from lens aspirates compared to conventional methods of virus isolation and serology.

Congenital rubella cataract: a timely reminder in the new millennium?

Maternal infection with rubella in the first trimester is an important cause of congenital cataract. Any injury affecting the foetus following maternal rubella infection in the phase of organogenesis results in congenital defects collectively termed as congenital rubella syndrome (CRS). Although rubella embryopathy is a less common cause for congenital cataract than in the past, it is still seen. The number of cases reduced to one in 1997 after which there were no new cases till 2002. However, there have been two new cases of CRS in 2003. Herein another one in early 2004 is reported. Outbreaks of CRS will continue until the percentage of susceptible individuals is reduced to a minimum through immunization. The majority of rubella cases in Australia are confined to young female immigrants, many coming for marriage. We must continue to immunize children, identify and immunize vaccine failures and susceptible women before they become pregnant, and to screen pregnant women so they can be vaccinated after delivery.

Inhibition of human lens epithelial B-3 cell proliferation by adenovirus-mediated transfer of antisense c-myc construct.

PURPOSE: To investigate the effects of adenovirus-mediated transfer of antisense c-myc construct on human lens epithelial B-3 (HLE B-3) cell proliferation, apoptosis and cell cycle. METHODS: HLE B-3 cell cultures were transduced with replication-defective adenovirus bearing either a nuclear-targeted beta-galactosidase (Ad-lacZ) or an antisense c-myc construct (Ad-AS-myc). The presence of beta-galactosidase activity in the transduced cultures was detected by immunohistochemical X-Gal staining, while c-myc mRNA and protein expression levels were evaluated by RT-PCR and Western blot analysis. HLE B-3 cell proliferation within 96 h after the transduction was analyzed by cell counting and MTT colorimetric assay. Apoptosis and cell cycle of the HLE-B3 cells were examined by flow-cytometric analysis. RESULTS: The mean transduction efficiency was 80% for HLE B-3 cells. Downregulation of c-myc mRNA and protein expression was noticed at 48, 96 and 144 h after the transduction with Ad-AS-myc. Cytostatic effects of Ad-AS-myc in HLE B-3 cells were obvious within 96 h after the transduction. An increased incidence of apoptosis and G1-phase arrest was identified in the Ad-AS-myc-transduced HLE B-3 cells. CONCLUSIONS: HLE B-3 cells were successfully transduced with adenovirus-mediated antisense c-myc construct. Ad-AS-myc transduction could significantly inhibit cell proliferation and induce cell apoptosis and G1-phase arrest in HLE B-3 cells. It may provide a novel approach for prevention of posterior capsular opacification.

Evaluation of fibroblast growth factor signaling during lens fiber cell differentiation.

PURPOSE: Previous studies have implicated members of the fibroblast growth factor (FGF) and insulin-like growth factor (IGF) families as stimulators of lens fiber cell differentiation in rodent and chicken embryo lenses, respectively. In the present study, the role of FGFs in fiber cell differentiation and epithelial cell proliferation in chicken embryos was examined. METHODS: Lenses were injected on embryonic day (E)3 with replication-defective retroviruses that express full-length or truncated FGF receptor (FGFR)-1 or a secreted form of FGF1. Lens epithelial explants were cultured in defined medium or medium supplemented with FGFs or vitreous humor, in the presence or absence of the FGF receptor antagonist SU5402. Explants were also cultured in vitreous humor that had been depleted of heparin-binding growth factors. Cell elongation was measured optically and protein accumulation by densitometry and Western blot analysis. RESULTS: Lens fiber cell differentiation was not inhibited in cells infected with virus expressing truncated FGFR1. Epithelial cells infected with virus encoding a secreted form of FGF1 did not differentiate into ectopic fiber cells. Viral transduction of FGFR1, truncated FGFR1, or FGF1 did not appreciably alter the proliferation of lens epithelial cells. Bovine vitreous humor stimulated chicken embryo lens epithelial cells to elongate and express markers of lens fiber cell differentiation. Bovine vitreous humor, but not FGF2, protected lens epithelial cells from apoptosis. Depleting vitreous humor of heparin-binding growth factors or treatment of lens cells with SU5402 did not inhibit the initial, rapid phase of lens cell elongation. Both treatments, used separately or together, reduced but did not prevent the expression of later markers of fiber cell differentiation. CONCLUSIONS: Fiber differentiation factors that are not members of the FGF family are present in chicken and mammalian vitreous humor. The factors that stimulate fiber cell differentiation in avian and mammalian eyes are similar.

The association of rubella virus in congenital cataract - a hospital-based study in India.

BACKGROUND: The association of rubella virus (RV) with congenital cataract has been well established. Since the data on association of RV with congenital cataract in India are scanty, a study was done based on virus isolation from lens aspirates in patients undergoing therapeutic lensectomy and serology. OBJECTIVE: To determine the incidence of the association of rubella virus with congenital cataract. STUDY DESIGN: The lens aspirates collected during the 9-year period (from 1990 to 1998), from 70 children up to 12 years of age with congenital cataract were processed for the isolation of rubella virus by conventional viral isolation culture method using BHK-21 and Vero cell lines. Identification of the virus was confirmed by immunofluorescence using human anti-rubella virus specific hyperimmune serum. Serum samples were collected from 55 out of these 70 children and the presence of antibodies to RV was detected by ELISA test. RESULTS: RV was isolated from lens aspirates in seven (10%) out of the 70 children with congenital cataract. Of the 55 sera tested, 22 had both anti-rubella IgM and IgG antibodies, in 13 only anti-RV IgG antibodies, in seven only IgM antibodies and the rest of the 13 samples did not have detectable levels of rubella antibodies. Among the children who had IgM antibodies, 12 (24.5%) were below the age of 6 months. CONCLUSION: It can be concluded based on virus isolation that 10% of patients with congenital cataract were due to rubella infection and the detection of 24.5% anti-RV IgM antibodies in children below 6 months old shows the possible association of rubella virus with congenital cataract.

Herpes simplex virus DNA in the lens one year after an episode of retinitis.

The present report describes a case where HSV was detected by polymerase chain reaction (PCR) in the lens cortical material removed during cataract surgery one year after resolution of retinal inflammation in a patient with ARN.

Three-dimensional organization of primary lens fiber cells.

PURPOSE: To visualize the three-dimensional organization of primary lens fiber cells. METHODS: The gene for Green Fluorescent Protein (GFP) was introduced into the lens vesicle using two different vector systems: a replication deficient adenovirus or an expression plasmid. Injected embryos were allowed to develop for several days and then were examined by confocal microscopy. RESULTS: Injection of either vector resulted in GFP expression in primary fiber cells. GFP-expressing cells were heterogeneous in shape and length. Some regions of the fibers were varicose, with diameters >10 microm; regions between the varicosities were often extremely thin, with diameters of <2 microm. No differences in the morphologies of GFP-expressing cells were noted between adenovirus and plasmid-injected lenses, suggesting that the irregular, undulating, appearance of the primary fibers was not the result of viral infection. Three-dimensional reconstruction of primary fiber cells revealed that, by E6, the posterior tips of the fibers had detached from the lens capsule. The anterior fiber tips remained in contact with the overlying epithelium for 1 to 2 additional days, demonstrating that the formation of the anterior and posterior sutures was asynchronous. CONCLUSIONS: The three-dimensional cellular organization of GFP-expressing cells is consistent with previous analyses of fiber cell morphology in the embryonic nucleus of adult human and bovine lenses. The present data confirm that the disorganized appearance of primary fiber cells observed in adult lenses is largely a reflection of developmental processes rather than a consequence of aging.

Retroviral expression of connexins in embryonic chick lens.

PURPOSE: To develop an in vivo model system in which exogenous proteins can be expressed in embryonic chick lens and to further understand the function of connexin-mediated gap junction intercellular communication in lens cell biology. METHODS: RCAS(A) is a replication-competent chicken retrovirus that infects dividing cells. Retroviral constructs were prepared containing alkaline phosphatase (AP) and FLAG-tagged connexins. Chick lenses were infected in situ by injecting virus into the lumen of lens vesicles at stage 18, cultures were taken at various periods. The lenses were then dissected, and the expressed proteins were visualized by AP histochemical examination and immunostaining. RESULTS: Twenty-four hours after infection, alkaline phosphatase could be seen in epithelia and fibers. As lens fiber maturation progressed, however, the alkaline phosphatase staining was lost as the fibers matured, presumably because of the proteolytic removal of the enzyme. By 72 hours, alkaline phosphatase staining could still be observed in epithelial cells and in differentiating fibers in the bow region but not in the mature lens fibers. FLAG-tagged exogenous lens connexins were also abundantly expressed by viral infection. The exogenous connexins were localized at the cell surfaces in junctional maculae and showed the same cell-type specific distribution as that of their endogenous connexin counterparts. CONCLUSIONS: An in vivo model system has been developed in the chick that provides opportunities to study the expression of wild-type and mutant proteins during lens differentiation. Expression of wild-type connexins has revealed that the characteristic distribution of the three different lens connexins is maintained even when expression is driven by a viral promoter.

Use of PCR for prenatal and postnatal diagnosis of congenital rubella.

A reverse transcription-nested PCR assay (RT-PCR) was evaluated for diagnosis of congenitally acquired rubella in utero and during infancy. RT-PCR was compared with virus isolation for retrospective detection of rubella virus in placental and fetal tissues obtained after termination of pregnancy following primary rubella or rubella virus reinfection. Concordant results were obtained for 85% of samples; rubella virus RNA was detected by RT-PCR alone in four samples, and rubella virus was detected by isolation alone in two samples. Samples were also obtained for prenatal diagnosis of congenital infection; rubella virus RNA was detected in three of seven chorionic villus samples and one of three amniotic fluid samples by RT-PCR, while rubella virus was isolated in only one chorionic villus sample. To demonstrate that the RNA extracted from chorionic villus samples contained amplifiable RNA, a nested RT-PCR was used to detect keratin mRNA. Rubella virus was detected in placenta in two cases in which the fetus was uninfected, and there was no evidence of rubella virus in the placenta from one case in which the fetus was infected. Thus, detection of rubella virus in chorionic villus samples by RT-PCR may not always correctly predict fetal rubella virus infection. RT-PCR was successfully used for the diagnosis of congenitally acquired rubella in infancy. Rubella virus RNA was detected in cyropreserved or formalin-fixed lens aspirates obtained from infants in India with serologically confirmed congenital rubella but not in samples from controls with inherited cataract.

Terminal dissemination of rabies virus in selected rat tissues.

Fifty rats were divided into 3 groups and challenged via the foot pad route with a fixed and 2 different strains of street rabies virus in order to study the dissemination of the virus and the affinity for certain tissues of the rat. The incubation period for rats inoculated with fixed rabies is shorter than with street virus, being 5 to 7 days compared with 10 to 12 days. Rats inoculated with the fixed strain were less aggressive and irritable than rats inoculated with street virus. The fixed strain demonstrated a greater affinity for the tissues studied as compared to the street strains of virus. Both the fixed and street strains revealed a low affinity for the parotid gland since no virus could be demonstrated in 14 of 20 in the fixed virus group and 27 of 30 in the street virus group.