Epidemiological and molecular characterization of rubella virus isolated in São Paulo,Brazil during 1997–2004

Rubella virus (RV) infection during the early stages of pregnancy can lead to serious birth defects, known as the congenital rubella syndrome (CRS). In 2003, the Pan American Health Organization (PAHO) adopted a resolution calling for the elimination of rubella and the congenital rubella syndrome (CRS) in the Americas by the year 2010. Brazil will have implemented the recommended PAHO strategy for elimination and interruption of endemic rubella virus transmission. The characterization of genotypes during the final stages of rubella elimination is important for determining whether new rubella isolates represent endemic transmission or importations. Samples (blood, urine, cerebrospinal fluid, and throat swabs) collected from patients with symptoms suggestive of rubella infection in 1997–2004 were isolated in cell culture and genotyped. Twenty‐eight sequences were analyzed and two genotypes were identified: 1a and 1G. The information reported in this paper will contribute to understanding the molecular epidemiology of RV in São Paulo, Brazil. J. Med. Virol. 84:1831–1838, 2012. © 2012 Wiley Periodicals, Inc.     

Rubella vaccine: new horizon in prevention of congenital rubella syndrome in the India

Rubella is a contagious viral disease, which mainly affects the fetus, if the mother is infected in the 1st trimester of her pregnancy. All adolescent girls (aged 11 to 19 y) and women of childbearing age are at risk of developing rubella. This disease is mild and self-limiting, and incubation period is 2-3 weeks. Humans are the only hosts for rubella. Rubella infection during pregnancy may lead to abortions, stillbirth or congenital deformities (birth defects). Moreover it is surprising to know that over 200,000 babies are born with birth defects because of Rubella infection during pregnancy in the Indian sub-continent. The risk of fetal infection is highest in first trimester; the infection rate declines between 12-28 weeks, suggesting that the placenta may prevent transfer of virus but not completely. The incidence of defects is inversely related to the time of maternal infection. Rubella outbreaks have been reported from many countries in South East Asian region with congenital rubella syndrome (CRS) due to maternal rubella being on the increase in many countries. In India, although the endemicity of rubella is established, the majority of cases remain undiagnosed, being subclinical or clinically mild. Consequently, in spite of evidence of CRS in all States of India, no distinct policy has been envisaged for assessing the burden of rubella, and no control measures against this silent crippling disease are in place. The European Regional Committee of the World Health Organization has adopted the goals of "Elimination of CRS" in the Health for All programs. There is no treatment for rubella. Vaccination is the only way to prevent all these complications.     

The role of rubella-immunoblot and rubella-peptide-EIA for the diagnosis of the congenital rubella syndrome during the prenatal and newborn periods

Rubella infection during the first trimester of pregnancy can cause the congenital rubella syndrome (CRS). Patients with CRS were shown to have a decreased humoral and cellular immunity. It is not known whether asymptomatic newborns who had experienced intrauterine infection with rubella virus (RV) differ in their antibody response from newborns with CRS. In this study we compared both groups for a difference which might be a useful diagnostic criterion for CRS during the prenatal and newborn periods. We used the nonreducing Rubella-Immunoblot and the Rubella-IgG-Peptide-Enzyme Immunoassay (EIA) to determine the antibodies directed to rubella proteins E1, E2 and C. The results showed that only newborns with CRS who had experienced RV infection during the first 12 weeks of gestation showed significantly reduced levels of antibodies directed to both the linear RV E1 epitope (SP 15) and the topographic RV E2 epitope. Asymptomatic newborns infected mostly later than week 10 of gestation showed normal levels of antibodies. These data suggest that the lack of antibody response in CRS is linked to the immaturity of the fetal immune system during the first trimester of gestation. Rubella-IgG-Peptide-EIA and Rubella-Immunoblot should be used additionally for CRS diagnosis in the prenatal/newborn periods. These results may have an impact on the early treatment of late-onset symptoms of CRS patients. J. Med. Virol. 51:280–283, 1997. © 1997 Wiley-Liss, Inc.     

Molecular epidemiology of rubella viruses involved in congenital rubella infections in São Paulo,Brazil, between 1996 and 2009

Rubella virus (RV) infection during the early stages of pregnancy can lead to serious birth defects, known as congenital rubella syndrome (CRS). This retrospective study was conducted between 1996 and 2009 with surveillance specimens collected from patients suspected of congenital rubella infection (CRI) and CRS. The clinical samples (nine amminiotic fluid, eight urine, eight blood, one conception product, and one placenta) were sent for viral isolation and genotyping. Twenty‐seven sequences were analysed and four genotypes (1a, 1B, 1G, and 2B) were identified in São Paulo that were involved in congenital infection. To our knowledge, this study is the first report that describes genetic diversity of the circulating rubella strains involved in CRI. J Med. Virol. 85:2034–2041, 2013. © 2013 Wiley Periodicals, Inc.

     

Seroprevalence and incidence of rubella in and around Delhi (1988-2002)

National Institute of Communicable Diseases (NICD) has been engaged in rubella testing for serodiagnosis of the infection and screening for immunity status. The compiled and evaluated data of the work done on rubella testing for the past fifteen years has been presented here to show the trend and changing scenario of the disease in Delhi. Blood samples were from 7424 patients referred to NICD, Delhi for serodiagnosis of congenital Rubella syndrome (CRS) in malformed babies, in utero rubella infection in women and immunity status of pregnant women and women with bad obstetric history. They were tested for rubella IgG and/or rubella IgM antibodies using commercially available reagents and kits. The data from the 15 years of testing was then compiled and evaluated. From the available data it was seen that immunity status against rubella in childbearing age group of women increased steadily from 49% in 1988 to 87% in 2002. Reported cases of CRS at NICD are also on the decline over the time period. There is periodic indication of high incidence of rubella in the year 1988; 1991 and 1998 as the reported cases of acute rubella infection in childbearing age group is high during these years.     

Application of new assays for rapid confirmation and genotyping of isolates of rubella virus

Rubella virus (RV) isolation is recommended by the WHO Measles and Rubella Labnet for studying the etiology and epidemiology of rubella. However, the absence of cytopathologic effects (CPE) in many of the cell lines used commonly makes it difficult to confirm RV growth. In this study, two assays amplifying RV cDNA were developed and validated in order to confirm and genotype RV isolates after cell culture. A SYBR Green I-based real-time PCR (Rtime-SGE317) was established for initial rapid detection of RV in Vero cells and a nested PCR (PCR-E860) was used for amplifying further the 739 nt window of the E1 gene for the identification of RV genotype as recommended by the WHO. Sensitivities of the two assays were evaluated using eight RV isolates, two from infants with the congenital rubella syndrome (CRS) and six from patients with acute rubella. All the isolates had cycle threshold (C(t)) values <37 after the third passage, which is recommended as the cut-off for the confirmation of a viable RV isolate. Phylogenetic analysis based on the 739 nt window generated by the PCR-E860 showed that the eight RV isolates belonged to genotypes 1E, 1G, and 2B. The Rtime-SGE317 assay can be carried out in local public health laboratories, which would extend the molecular surveillance of rubella and contribute to the WHO goal of eradicating rubella worldwide.     

The present situation and limitation of antibody assays for diagnosis of rubella virus infection in pregnant women]

Rubella virus infection during early stages of pregnancy often results in a number of developmental disorders referred to as congenital rubella syndrome(CRS). Both clinical and laboratory diagnosis of suspect cases of CRS can be made with relative ease, particularly when expectant mothers show the typical rubella-specific rash. Serological diagnosis of CRS is accomplished using hemagglutination inhibition (HI) and enzyme-linked immunosorbent(IgM-EIA) assays. Antibody titers as determined by these assays are generally very high following acute apparent rubella infections, thus making serological diagnosis relatively easy in most cases. However, the detection of possible CRS cases can be hampered by clinically inapparent rubella infections during early pregnancy. As much as 30 percent of all acute rubella cases are inapparent infections, and there is the very real potential for such inapparent infections to occur during pregnancy, to result in fetal infections, and consequently to cause CRS. Detection of CRS becomes extremely difficult in such settings. Complicating CRS detection even more are rare rubella re-infections that might occur in early pregnancy, and unknown risk of fetal infection and CRS. In re-infection cases, HI antibody titer becomes elevated due to a secondary immune response, and IgM antibody is produced in a significant number of cases. To determine directly the fetal infection, virus genome detection was developed and applied clinically for the past decade. Using a combination of serological and genomic detection methods, the results of the investigation suggest that when rubella infection during early pregnancy occurs 1) there is a significant risk of fetal infection that results from acute apparent rubella infection, 2) there is a measurable risk of fetal infection resulting from inapparent infections as defined by HI antibody titers > or = 256 and with and IgM-EIA index > or = 7, and 3) high HI antibody titers with low IgM-EIA indices or no detectable IgM antibody in cases of inapparent rubella infections may represent rubella re-infections and result in a low risk of fetal infections.     

The Rubella Virus Putative Replicase Interacts with the Retinoblastoma Tumor Suppressor Protein

In utero fetal infection of rubella virus (RV), a positive-stranded RNA virus, frequently induces birth defects if contracted in the first trimester of pregnancy. The underlying mechanism of RV-induced birth defects is not known. Birth defects are also common in certain DNA viral infections such as human cytomegalovirus (HCMV). During HCMV infection, one of its proteins interacts with a cell growth regulatory protein, the retinoblastoma protein (Rb) and stimulates DNA synthesis which is associated with chromosomal damage and cellular mitotic arrest. These affects have been implicated in HCMV induced teratogenesis. Since RV and HCMV both cause teratogenesis, we postulated that during RV infection, a virus-encoded protein might interact with Rb and affect fetal cell growth. In the present study, we have identified a known Rb-binding motif, L×C×E (LPCAE) in the carboxy-terminal half of the putative replicase (NSP90) of RV and demonstrated that the C-terminal region specifically binds to GST-Rb in vitro. Further, by coimmunoprecipitating NSP90 and Rb using specific antibodies to respective proteins, we have confirmed that NSP90 specifically binds to Rb in vivo as well. In addition, RV replication was shown to be less in null-mutant (Rb^−/−) mouse embryonic fibroblast cells than in wild-type (Rb^+/+) cells, suggesting a possible physiological role for this interaction. Thus, in facilitating RV replication, binding of NSP90 to Rb potentially alters the cell growth regulatory property of Rb, and this could be one of the initial steps in RV-induced teratogenesis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Rubella prevalence and its transmission in children

Rubella is a major cause of birth defects among the TORCH group of agents causing congenital anomalies. Almost all the symptomatic infected infants have long-term neurological sequelae & many asymptomatic infants also develop deafness or psychomotor retardation later in life. In India need for rubella prevention & control is being recognized. Before formulating any kind of rubella vaccination policies, data on the burden of disease is important. Hence the prevalence of rubella in children and their transmission was evaluated. Paired sera of 146 babies with suspected intra uterine infection and their mothers from lower socioeconomic strata was tested for IgM antibodies by commercially available Enzyme immunoassay (EIA) kits. Congenital Rubella Syndrome (CRS) was confirmed in babies presenting with rubella compatible defects with positive IgM antibodies against rubella. It was seen that out of 146-paired samples evaluated, 15-paired samples (10.27%) were positive for IgM antibodies. The transmission rate of rubella virus from mother to child when the mother was infected was around 55.55% according to this study. CRS prevalence of 10.27% among symptomatic infants is significant as a large majority of rubella infection remains undetected and hence the actual burden of the disease may be higher. Since the disease is preventable by an effective vaccination, strategies for rubella immunization should be developed and enhanced.     

<Emphasis Type="Italic">Rubella virus</Emphasis> P90 associates with the cytokinesis regulatory protein Citron-K kinase and the viral infection and constitutive expression of P90 protein both induce cell cycle arrest following S phase in cell culture

Summary. In utero infection of developing fetus by Rubella virus (RV) causes cell division inhibition of critical precursor cells in organogenesis, CNS-associated birth defects and induction of apoptosis in cell culture. The underlying mechanisms of RV-induced congenital abnormalities are not known. Here, we identified a novel interaction between RV replicase P90 protein and a cytokinesis-regulatory protein, the Citron-K kinase (CK), in a yeast two-hybrid cDNA library screen. Aberrations in cytokinesis and subsequent apoptosis do occur in specific cell types when the CK gene is knocked out or, its regulatory function is perturbed. Our analysis found that full-length P90 binds CK and in RV-infected cells P90 colocalizes with CK in the cytoplasm. Furthermore, during RV infection as well as cellular expression of P90 alone, we identified a discrete subpopulation of cells containing 4N DNA content, indicating that these cells are arrested in the cell cycle following S phase, suggesting that cellular expression of viral P90 during RV infection perturbs cytokinesis. Previous reports by others established that RV infection leads to apoptosis in cell culture. These observations together taken to the fetal organogenesis level, favor the idea that RV P90, by binding to cellular CK, invokes cell cycle aberrations resulting in the cell- and organ-specific growth inhibition and programmed cell death during RV infection in utero, which commonly is referred to as RV-induced teratogenesis.     

Acute myelomonocytic leukaemia following atypical congenital rubella.

The child of a woman immunised against rubella presented at 5 months with developmental delay and recurrent infection; she was shown to have congenital rubella. At 15 months she developed acute myelomonocytic leukaemia (AMML). Rubella is difficult to diagnose after immunisation. AMML has not been previously described in association with congenital rubella, as far as is known.     

Prevalence of HI antibody titer against rubella virus to determine the effect of mass vaccination in Tehran.

BACKGROUND: Rubella is an infectious viral disease, has a worldwide distribution and is normally a mild childhood disease. Infection during early pregnancy may cause fetal death or congenital rubella syndrome. The highest risk of CRS is found in countries with high susceptibility rates among women of childbearing age. In many developed and some developing countries, large-scale rubella vaccination during the past decade has drastically reduced or practically eliminated rubella and CRS. Mass vaccination campaigns and Expanded Program of Immunization (EPI) have increased vaccine coverage in the world with a substantial impact on the reduction of rubella infections, such as CRS. OBJECTIVE: The present study was preformed to evaluate the immune status against rubella before and after the mass campaign vaccination on 22 December 2003. STUDY DESIGN: A total of 320 samples were collected from the healthy subjects before and after the vaccination and 80 paired sera were collected and tested for the presence of rubella antibody using HI test. RESULTS AND CONCLUSIONS: Based on the results, 98.1% of the population has gained anti-rubella antibody, compared with 92.2% before the vaccination. The data revealed that 98.75% of the paired subjects had rubella antibody after mass vaccination which is statistically significant.     

Evaluation of rubella IgM enzyme immunoassays.

BACKGROUND: Rubella virus generally causes a mild fever, rash illness similar in clinical presentation to infections by other viruses including measles and parvovirus B19. Rubella infections in pregnant women in the first trimester carry a high risk of congenital rubella syndrome (CRS) which can result in severe congenital defects in the infants. The goal of rubella immunization programs is therefore to eliminate CRS. The primary test for the laboratory confirmation of rubella is IgM serology. It is therefore important to evaluate currently available commercial rubella IgM immunoassays to ensure high quality rubella diagnostic testing. STUDY DESIGN: In this study, we compared the performance of seven commercial rubella IgM enzyme immunoassays (EIA) (Meddens, Denka Seiken, Behring, Wampole, Captia, Sigma and Abbott Axsym) using well-defined panels of sera from rubella and non-rubella/rash-illness cases. RESULTS: The Meddens, Denka Seiken, Behring and Wampole rubella IgM EIAs all performed similarly for sensitivity (range of 74.1-76.8%) and specificity (range of 93.9-96.1%). Relative to the other assays, the Axsym had a higher sensitivity (78.9%) but lower specificity (86.5%). The Captia assay had the lowest overall sensitivity (66.4%), while the Sigma assay had a lower specificity (85.6%) in relation to the other assays. CONCLUSIONS: In conclusion, the Meddens, Denka Seiken, Behring and Wampole rubella IgM EIAs are comparable in their overall performance with respect to sensitivity and specificity.     

Isolation and genotype analysis of rubella virus from a case of Guillain-Barré syndrome

Rubella virus (RV) infection has sporadically been linked to Guillain-Barré syndrome (GBS), but the association with RV has been based only on clinical and/or serological backgrounds. In the present case it was possible to isolate RV (genotype 1a) from cerebrospinal fluid and peripheral blood mononuclear cells of an 18-year-old woman diagnosed with GBS after clinical manifestations of rubella. This report contributes to confirm RV as one of the triggering pathogens of this peripheral nervous system disease.     

Evaluation of a commercial rubella IgM assay for use on oral fluid samples for diagnosis and surveillance of congenital rubella syndrome and postnatal rubella.

BACKGROUND: Clinical diagnosis (surveillance) of rubella is unreliable and laboratory confirmation is essential. Detection of virus specific IgM in serum is the most commonly used method. However, the use of serum necessitates the drawing of blood, either through venipuncture or finger/heel prick, which can be difficult in young babies. Oral fluid samples have proved useful as an alternative, less invasive sample for virus specific IgM detection however until recently no commercial rubella IgM tests were available, restricting the usefulness of this approach. OBJECTIVES: To evaluate the performance of the Microimmune Rubella IgM capture EIA using oral fluid samples from outbreaks as well as in cases of suspected congenital rubella syndrome (CRS). STUDY DESIGN: Paired serum and oral fluids were collected from cases during a rubella outbreak in three provinces in Turkey. Matched serum and oral fluid samples were collected from children with suspected CRS in an active surveillance programme at the Aravind Eye Hospital in South India. Serum samples were collected as part of the measles surveillance programme in Ethiopia. RESULTS: On serum samples the sensitivity and specificity of the Microimmune Rubella IgM capture EIA compared to Behring Enzygnost rubella IgM test was 96.9% (62/64; 95% CI 94.2-100%) and 100% (53/53; 95% CI 93.2-100%). On oral fluids compared to matched Behring results on serum the sensitivity was 95.5% (42/44; 95% CI 84.5-99.4%). The sensitivity and specificity of Microimmune Rubella IgM capture EIA on oral fluids from suspected CRS cases compared to serum results using Behring Enzygnost IgM assay was 100% (95% CI 84.5-100%) and 100% (95% CI 95.8-100.0%) respectively. CONCLUSION: Microimmune Rubella IgM capture EIA has adequate performance for diagnosis and surveillance of rubella in outbreak using either serum or oral fluid specimens.     

Secretion of rubella virions and virus-like particles in cultured epithelial cells.

Rubella virus (RV) is an enveloped RNA virus that causes systemic infections in humans. More importantly, first trimester in utero infection leads to a collection of devastating birth defects known as congenital rubella syndrome. Epithelial cells are the first line of defense against viruses and consequently, the polarity of virus secretion is an important factor affecting viral spread. As a first step toward understanding how RV interacts with epithelial cells, we have examined the release of RV-like particles and virions from polarized cells in culture. RV structural proteins were targeted to the Golgi complex and virus particle formation occurred on intracellular membranes in three different polarized epithelial cells. Polarized cells could be infected from the apical and basal membranes, indicating that receptors are not confined to one surface. The secretion of virus-like particles and infectious virions varied according to cell type. In two of the three polarized cell lines examined, virus was released primarily from the apical surface, but significant quantities were also secreted from the basolateral membrane. Release of virus from the apical surface may facilitate virus spread from person to person, whereas basolateral secretion could be important for establishing a systemic infection and/or crossing the placenta prior to fetal infection.