Persistent Infection of Human Fetal Endothelial Cells with Rubella Virus

Cardiovascular abnormalities are the leading cause of neonatal death among patients with congenital rubella syndrome (CRS). Although persistence of rubella virus (RV) in fetal endothelium has been repeatedly suggested as a possible cause of cardiovascular birth defects, evidence of the permissiveness of fetal endothelial cells to RV is lacking. In this study we evaluated the ability of RV to infect and persist in primary fetal endothelial cells derived from human umbilical vein (HUVEC). We found that wild type (wt) low passage clinical RV productively infected HUVEC cultures without producing cytopathology or ultrastructural changes. RV did not inhibit host cell protein synthesis, cell proliferation, or interfere with the cell cycle. Persistently infected cultures were easily established at low and high multiplicities of infection (MOI) with both laboratory and wt clinical RV strains. However, synchronous infections of entire HUVEC monolayers were only observed with clinical RV strains. The release of infectious virions into media remained at consistently high levels for several subcultures of infected HUVEC. The results indicate that macrovascular fetal endothelial cells are highly permissive to RV and allow slow persistent RV replication. The findings provide more evidence for the suggestion that vascular pathologies in CRS are triggered by persistent rubella virus infection of the endothelium.     

Pregnant Women in and Around Dhaka City: Are Their Children at Risk of Developing Congenital Rubella Syndrome?

Rubella Virus (RUBV) is a common cause of childhood rash and fever in non-immunized populations, and its public health importance relates to teratogenic effects of primary rubella infection in women with early pregnancy. Infection of the fetus may lead to congenital rubella syndrome (CRS). This work aimed to assess the degree of risk associated in acquiring rubella virus infection by the women during pregnancy and developing CRS among their children in Bangladesh. The study population (n = 275) included pregnant mothers (15–38 years) from various socioeconomic backgrounds attending a women health care based hospital. All subjects were personally interviewed, clinically examined and a standardized questionnaire was filled up for each of them. From each participant 3 ml blood was taken and serum was separated. Commercially available ELISA kit was used for the qualitative and quantitative determination of IgM and IgG class antibodies against RUBV in collected serum samples. 209 women were found to contain detectable level of antiRUBV IgG antibodies, but did not possess IgM antibodies against rubella. Only 9% participants were vaccinated previously against rubella virus among the whole antenatal population studied. Ninety-two percent of these vaccinated pregnant women contained serum anti-rubella IgG antibody which was significantly (P = 0.05) higher than that of the nonvaccinated study population (75%). Pregnant women from lower middle and poor socioeconomic class had significantly (P = 0.05) more intra uterine growth retardation (IUGR) of fetus than the upper middle class. 20% of the women of child bearing age examined in this work were not yet exposed to RUBV and at risk of acquiring this virus during pregnancy and subsequently transmitting the virus to the fetus. Our work demonstrates rubella attack rate among antenatal population in Bangladesh as 14.5 in 1000 during pregnancy. A proper and reliable vaccination policy against rubella virus is not yet adopted at the national level in many developing countries including Bangladesh. This work identifies the requirement of detailed study for the identification of intrauterine rubella infection and its related influence on perinatal morbidity and mortality. Thorough epidemiological studies are also considered necessary prior to the development and acceptance of national immunization program against rubella virus in Bangladesh.     

Using Seroprevalence and Immunisation Coverage Data to Estimate the Global Burden of Congenital Rubella Syndrome, 1996-2010: A Systematic Review

Background

The burden of Congenital Rubella Syndrome (CRS) is typically underestimated in routine surveillance. Updated estimates are needed following the recent WHO position paper on rubella and recent GAVI initiatives, funding rubella vaccination in eligible countries. Previous estimates considered the year 1996 and only 78 (developing) countries.

Methods

We reviewed the literature to identify rubella seroprevalence studies conducted before countries introduced rubella-containing vaccination (RCV). These data and the estimated vaccination coverage in the routine schedule and mass campaigns were incorporated in mathematical models to estimate the CRS incidence in 1996 and 2000–2010 for each country, region and globally.

Results

The estimated CRS decreased in the three regions (Americas, Europe and Eastern Mediterranean) which had introduced widespread RCV by 2010, reaching <2 per 100,000 live births (the Americas and Europe) and 25 (95% CI 4–61) per 100,000 live births (the Eastern Mediterranean). The estimated incidence in 2010 ranged from 90 (95% CI: 46–195) in the Western Pacific, excluding China, to 116 (95% CI: 56–235) and 121 (95% CI: 31–238) per 100,000 live births in Africa and SE Asia respectively. Highest numbers of cases were predicted in Africa (39,000, 95% CI: 18,000–80,000) and SE Asia (49,000, 95% CI: 11,000–97,000). In 2010, 105,000 (95% CI: 54,000–158,000) CRS cases were estimated globally, compared to 119,000 (95% CI: 72,000–169,000) in 1996.

Conclusions

Whilst falling dramatically in the Americas, Europe and the Eastern Mediterranean after vaccination, the estimated CRS incidence remains high elsewhere. Well-conducted seroprevalence studies can help to improve the reliability of these estimates and monitor the impact of rubella vaccination.     

Simulations of rubella vaccination strategies in China

Many infants whose mothers have rubella infections during their first trimester of pregnancy have birth defects called congenital rubella syndrome (CRS). China does not routinely vaccinate against rubella in the public sector, but may need to start as its 'one child per couple' policy changes the population age distribution and the dynamics of rubella epidemiology, so that the incidence of rubella in pregnant women increases. Computer simulations with demographic transitions and rubella transmission dynamics predict that, with no or limited rubella vaccination, CRS incidence in China in the 30 years after 2020 will be more than twice the level in 2005. Comparisons of rubella vaccination strategies using computer simulations show that routine vaccination of over 80% of 1-year-old children would be effective in reducing total CRS cases in 2005-2051 and eliminating rubella in China by 2051. Routine immunizations at higher levels and the addition of early mass vaccinations of 2-14-year-old children and women of childbearing ages would further reduce total CRS cases and speed up the elimination of rubella.     

Burden of congenital rubella syndrome (CRS) in India based on data from cross-sectional serosurveys, 2017 and 2019–20

BackgroundIndia has set a goal to eliminate measles and rubella/Congenital Rubella Syndrome (CRS) by 2023. Towards this goal, India conducted nationwide supplementary immunization activity (SIA) with measles-rubella containing vaccine (MRCV) targeting children aged between 9 months to <15 years and established a hospital-based sentinel surveillance for CRS. Reliable data about incidence of CRS is necessary to monitor progress towards the elimination goal.MethodsWe conducted serosurveys in 2019–20 among pregnant women attending antenatal clinics of 6 hospitals, which were also sentinel sites for CRS surveillance, to estimate the prevalence of IgG antibodies against rubella. We systematically sampled 1800 women attending antenatal clinics and tested their sera for IgG antibodies against rubella. We used rubella seroprevalence data from the current survey and the survey conducted in 2017 among antenatal women from another 6 CRS surveillance sites to construct a catalytic models to estimate the incidence and burden of CRS.ResultThe seroprevalence of rubella antibodies was 82.3% (95% CI: 80.4–84.0). Rubella seropositivity did not differ by age group and educational status. Based on the constant and age-dependent force of infection models, we estimated that the annual incidence of CRS in India was 225.58 per 100,000 live births (95% CI: 217.49–232.41) and 65.47 per 100,000 live births (95% CI: 41.60–104.16) respectively. This translated to an estimated 14,520 (95% CI: 9,225–23,100) and 50,028 (95% CI: 48,234–51,543) infants with CRS every year based on age-dependent and constant force of infection models respectively.ConclusionsOur findings indicated that about one fifth of women in the reproductive age group in India were susceptible for rubella. The estimates of CRS incidence will serve as a baseline to monitor the impact of MRCV SIAs, as well progress towards the elimination goal of rubella/CRS.     

Letter: Spontaneous pelvic thrombosis in the postmenopausal woman.

An epidemic of rubella reached its peak in the Atlantic provinces in 1974, subsiding in early 1975. With the exception of Quebec the remainder of Canada showed a reverse trend, with a large increase in the numbers of cases reported in the first 41/2 months of 1975. The Halifax virus laboratory reported 106 serologically proven cases of rubella in 1974, 44 of them in pregnant women. In the aftermath of the epidemic many infants were born with the congenital rubella syndrome (CRS). A study carried out from Sept. 1, 1974 through Apr. 30, 1975 showed an 80% correlation between clinical diagnosis and the presence of rubella-specific IgM antibodies in 35 of these infants. Of the 23 infants in whom the diagnosis of CRS was made by laboratory or clinical findings or both, laboratory criteria were met in 20 (87.0%), clinical criteria in 19 (82.6%) and both laboratory and clinical criteria in 16 (69.6%).     

Balancing Evidence and Uncertainty when Considering Rubella Vaccine Introduction

Background

Despite a safe and effective vaccine, rubella vaccination programs with inadequate coverage can raise the average age of rubella infection; thereby increasing rubella cases among pregnant women and the resulting congenital rubella syndrome (CRS) in their newborns. The vaccination coverage necessary to reduce CRS depends on the birthrate in a country and the reproductive number, R₀, a measure of how efficiently a disease transmits. While the birthrate within a country can be known with some accuracy, R₀ varies between settings and can be difficult to measure. Here we aim to provide guidance on the safe introduction of rubella vaccine into countries in the face of substantial uncertainty in R₀.

Methods

We estimated the distribution of R₀ in African countries based on the age distribution of rubella infection using Bayesian hierarchical models. We developed an age specific model of rubella transmission to predict the level of R₀ that would result in an increase in CRS burden for specific birth rates and coverage levels. Combining these results, we summarize the safety of introducing rubella vaccine across demographic and coverage contexts.

Findings

The median R₀ of rubella in the African region is 5.2, with 90% of countries expected to have an R₀ between 4.0 and 6.7. Overall, we predict that countries maintaining routine vaccination coverage of 80% or higher are can be confident in seeing a reduction in CRS over a 30 year time horizon.

Conclusions

Under realistic assumptions about human contact, our results suggest that even in low birth rate settings high vaccine coverage must be maintained to avoid an increase in CRS. These results lend further support to the WHO recommendation that countries reach 80% coverage for measles vaccine before introducing rubella vaccination, and highlight the importance of maintaining high levels of vaccination coverage once the vaccine is introduced.     

Rubella virus and birth defects: molecular insights into the viral teratogenesis at the cellular level

BACKGROUND: In utero rubella virus (RV) infection of a fetus can result in birth defects that are often collectively referred to as congenital rubella syndrome (CRS). In extreme cases, fetal death can occur. In spite of the availability of a safe and effective vaccine against rubella, recent worldwide estimates are that more than 100,000 infants are born with CRS annually. RECENT PROGRESS: Recently, several significant findings in the field of cell biology, as well as in the RV replication and virus-cell interactions, have originated from the authors' laboratory, and other researchers have provided insights into RV teratogenesis. It has been shown that 1) an RV protein induces cell-cycle arrest by generating a subpopulation of tetraploid nuclei (i.e., 4N DNA) cells, perhaps representative of the tetraploid state following S phase in the cell cycle, due to its interaction with citron-K kinase (CK); 2) RV infection induces apoptosis in cell culture, and 3) CK functional perturbations lead to tetraploidy, followed by apoptosis, in specific cell types. CONCLUSIONS: Based on several similarities between known RV-associated fetal and cellular manifestations and CK deficiency-associated phenotypes, it is reasonable to postulate that P90-CK interaction in RV-infected cells interferes with CK function and induces cell-cycle arrest following S phase in a subpopulation, perhaps representative of tetraploid stage, which could lead to subsequent apoptosis in RV infection. Taking all these observations to the fetal organogenesis level, it is plausible that P90-CK interaction could perhaps be one of the initial steps in RV infection-induced apoptosis-associated fetal birth defects in utero.     

Monitoring congenital rubella embryopathy

BACKGROUND: Currently, all developed countries include rubella vaccination in their immunization programs, targeting the complete elimination of congenital rubella syndrome (CRS). In the underdeveloped world, where this severely disabling condition still exists, only a few countries have implemented vaccination policies, and almost no data on their effectiveness or on prevalence rates are available. The aims of the present work were to search for the best phenotype to be used as a sentinel for CRS in a large series of malformed newborns and to propose a CRS surveillance system, based only on clinical data. METHODS: A total of 43 infants diagnosed as having CRS were obtained from 19,184 multimalformed infants, ascertained by the Latin-American Collaborative Study of Congenital Malformations, World Health Organization (WHO) Collaborating Centre for the Prevention of Birth Defects (ECLAMC), over 3,883,165 consecutive births, between 1982 and 2003. They were distributed by country and the most frequent birth defects were identified. From the 19,184 multimalformed infants, all cases presenting the birth defects identified were selected. The sensitivity, specificity, and likelihood ratio (LR) in detecting CRS were determined for these birth defects, alone and in combination. The sample size of multimalformed infants required to detect different levels of increase in the rate of CRS was determined for three sentinel phenotypes. RESULTS: The rate of CRS was highest in Brazil. Based on the best possible combination of sensitivity, specificity, and LR, the dyad comprising eye anomalies and congenital heart defects was shown to be the most appropriate sentinel, with the lowest sample size required, to detect CRS in neonates. CONCLUSIONS: A surveillance system for CRS, based on clinical data in newborns, is being proposed, in an attempt to monitor ongoing vaccination policies, aimed at eliminating CRS in developing countries.     

Cryo-electron tomography of rubella virus

Rubella virus is the only member of the Rubivirus genus within the Togaviridae family and is the causative agent of the childhood disease known as rubella or German measles. Here, we report the use of cryo-electron tomography to examine the three-dimensional structure of rubella virions and compare their structure to that of Ross River virus, a togavirus belonging the genus Alphavirus. The ectodomains of the rubella virus glycoproteins, E1 and E2, are shown to be organized into extended rows of density, separated by 9 nm on the viral surface. We also show that the rubella virus nucleocapsid structure often forms a roughly spherical shell which lacks high density at its center. While many rubella virions are approximately spherical and have dimensions similar to that of the icosahedral Ross River virus, the present results indicate that rubella exhibits a large degree of pleomorphy. In addition, we used rotation function calculations and other analyses to show that approximately spherical rubella virions lack the icosahedral organization which characterizes Ross River and other alphaviruses. The present results indicate that the assembly mechanism of rubella virus, which has previously been shown to differ from that of the alphavirus assembly pathway, leads to an organization of the rubella virus structural proteins that is different from that of alphaviruses.     

Defectiveness of Interferon Production and of Rubella Virus Interference in a Line of African Green Monkey Kidney Cells (Vero)

Vero cells, a line of African green monkey kidney cells, failed to produce interferon when infected with Newcastle disease, Sendai, Sindbis, and rubella viruses, although the cells were sensitive to interferon. Further, infection of Vero cells with rubella virus did not result in interference with the replication of echovirus 11, Newcastle disease virus, or vesicular stomatitis virus, even in cultures where virtually every cell was infected with rubella virus. Under the same conditions, BSC-1 cells and other cells of primate origin produced interferon and showed rubella virus interference. The results indicate that the presence of rubella virus in the cell does not in itself exclude multiplication of other viruses and that rubella virus interference appears to be linked to the capability of the cell to produce interferon.     

风疹病毒松叶株衣壳蛋白C基因稳定性研究

目的研究风疹病毒(Rubella virus,RV)疫苗松叶株(Matsuba)衣壳蛋白(Capsid Protein)的基因稳定性及遗传特征,探讨其功能结构及生物学活性在病毒传代过程中的变化特点。方法利用RT-PCR方法扩增风疹病毒Matsuba株14、16、17、18、23代次C基因序列,测序后进行序列比对分析,并将各代次病毒的C基因与风疹病毒疫苗株Matsuba(GenBank登陆号:AB588193)及其他风疹病毒株C基因序列进行同源性分析。结果风疹病毒Mat-suba株传代病毒C基因在传代过程中核苷酸及氨基酸均未发生变异;各代次病毒与AB588193核苷酸及氨基酸序列完全一致,各关键功能区未发生变异;Matsuba株与18株风疹病毒核苷酸相似性在90.2%～99.9%之间。结论风疹病毒Matsuba疫苗株C基因遗传特性非常稳定,与生物学作用相关的区域未发生传代改变。从分子水平证明Matsuba疫苗株毒种及其生产的疫苗具有安全性。     

Specific binding of host cell proteins to the 3''-terminal stem-loop structure of rubella virus negative-strand RNA.

At the 5' end of the rubella virus genomic RNA, there are sequences that can form a potentially stable stem-loop (SL) structure. The complementary negative-strand equivalent of the 5'-end SL structure of positive-strand rubella virus RNA [5' (+) SL structure] is thought to serve as a promoter for the initiation of positive-strand synthesis. We screened the negative-strand equivalent of the 5' (+) SL structure (64 nucleotides) and the adjacent region of the negative-strand RNA for their ability to bind to host cell proteins. Specific binding to the 64-nucleotide-long potential SL structure of three cytosolic proteins with relative molecular masses of 97, 79, and 56 kDa was observed by UV-induced covalent cross-linking. There was a significant increase in the binding of the 97-kDa protein from cells upon infection with rubella virus. Altering the SL structure by deleting sequences in either one of the two potential loops abolished the binding interaction. The 56-kDa protein also appeared to bind specifically to an SL derived from the 3' end of positive-strand RNA. The 3'-terminal structure of rubella virus negative-strand RNA shared the same protein-binding activity with similar structures in alphaviruses, such as Sindbis virus and eastern equine encephalitis virus. A possible role for the host proteins in the replication of rubella virus and alphaviruses is discussed.     

Biological characterization of Campylobacter fetus lipopolysaccharides

Abstract Ten patients with chronic liver disease, seven healthy seropositive individuals with a remote history of rubella, and three patients with acute rubella were examined for serum levels of IgG subclasses and subclass antibodies against rubella virus structural proteins. One patient with AICAH had no detectable total or rubella specific IgG3 or IgG4. The liver disease patients were hypergammaglobulinemic and had greatly raised IgG1 levels. Patients with acute rubella lacked antibodies to the rubella virus E2 protein and showed no IgG4 antibody response. The liver disease patients showed a somewhat weaker IgG4 antibody response against the core (C) protein than healthy controls. However, differences are suggested within the subclasses in antibody reactivity against the individual rubella virus antigens. It is concluded that test systems that discriminate reactivities against individual antigens have to be used for characterization of viral antibody subclass profiles.     

Rubella and the Rubella Syndrome—New Epidemiologic and Virologic Observations

The importance of rubella lies in the 15 to 20 per cent incidence of damage to the fetus when infection occurs in the first trimester of pregnancy. The “rubella syndrome” appears as various combinations of congenital defects, chiefly cardiac anomalies, cataracts and impaired hearing.Now that the rubella virus has been isolated and grown in tissue culture, it is possible to study the spread of the disease, to determine apparent and inapparent infection rates and to investigate the nature of fetal infection. It has been found that the disease is a highly contagious one in the family setting, and that inapparent infections are more common than overt cases with rash. Infection of the fetus in the early weeks of intrauterine life may become chronic, and virus has been recovered from placenta and fetal specimens collected at induced abortions many weeks after the maternal disease. Infants born with the rubella syndrome are still shedding virus at birth and may continue to do so for at least several months.Gamma globulin, which is effective in preventing measles and hepatitis, has not been highly effective in the prevention of rubella when given to those exposed to the disease. Successful control of the rubella problem will depend upon the development of an active vaccine, which is a possibility now that the virus can be grown in tissue culture.     

The development and standardization of an antigen detection ELISA for rubella virus grown in rabbit cells

A double antibody sandwich ELISA for the detection of rubella virus antigen was developed and standardized. Commercially available antisera were chosen in order to make the assay readily available. Antigen detection gave an excellent correlation to titers obtained by examination of cytopathogenic effect (CPE, r = 0.986). Replication of rubella virus grown in rabbit cells was identified with CPE and positive ELISA appearing within a difference of +/- one day. ELISA provided an objective detection of rubella virus which is often difficult by the reading of CPE. The method was found to be both sensitive and reproducible and facilitated work in rubella virus control involving a large number of virus titrations.     

Studies on Rubella in Pregnancy: Report of the Public Health Laboratory Service Working Party on Rubella

In a further survey of women of child-bearing age neutralizing antibody to rubella virus was found in 80 to 96% of those sampled from different parts of the country.When assessment was restricted to susceptible pregnant contacts and rubella in the index case was confirmed by virus isolation immunoglobulin in the dosage used did not appear to give any appreciable protection.One instance of a confirmed second attack after known exposure to rubella among more than 100 immune contacts is recorded.The implications of all these findings are discussed.     

The relationship between rubella hemagglutination inhibition antibody (HIA) and rubella induced in vitro lymphocyte tritiated thymidine incorporation

The parameters of lymphocyte, rubella virus interaction were studied by means of tritiated thymidine incorporation and compared with the rubella HIA level. Stimulation of lymphocyte DNA synthesis was found to depend on antigen concentration and to be independent of the presence of viable virus. When compared to HIA titer, lymphocyte transformation was found to be detectable in the absence of antibody. It is suggested that lymphocyte transformation is a sensitive method to detect both prior exposure and cellular immune reactivity to rubella virus.     

2003～2007年中国风疹病毒基因特征分析

本研究用Vero细胞或Vero/SLAM细胞从我国10个省(直辖市、自治区,下同)2003～2007年风疹暴发和散发病例的咽拭子标本中分离到57株风疹病毒,用RT-PCR方法扩增了57株风疹病毒E1基因1 107个核苷酸的片段,并对该PCR产物进行序列测定和分析.结果提示,在基于WHO基因定型靶序列739个核苷酸片段构建的基因亲缘关系树上,其中55株风疹病毒株属于1E基因型,相对于其他国家的1E基因型,形成一个独立分支;另外2株风疹病毒属于2B基因型.57株风疹病毒大部分核苷酸的突变为无义突变,氨基酸序列高度保守,除了2株风疹病毒在E1蛋白血凝抑制和中和位点区域第212位氨基酸由Thr变为Ser,其他病毒株均无重要抗原位点的改变;所有我国已分离到的1E基因型风疹病毒在E1蛋白第338位氨基酸共享突变位点(Leu338→Phe338),而其他基因型以及其他国家的1E基因型风疹病毒在该位点均未发生突变,提示该氨基酸(Phe338)可能是我国1E基因型风疹病毒所特有.2003～2007年在我国10个省均分离到1E基因型,而2B基因型只在2006年从四川省的越南输入病例中分离到,提示1E为绝对优势基因型,2B基因型为输入基因型.与1979～1984年和1999～2002年我国流行的风疹基因型不同,发生了基因型的更替,近年我国风疹的流行是由1E基因型为主的风疹野病毒的多个传播链引起.     

Viremia in a Recipient of HPV-77 Rubella Virus Vaccine

A live rubella virus vaccine, HPV-77 (High Passage Virus - 77 tissue culture passages) was administered subcutaneously to eight rubella-susceptible children housed in an isolation ward. One blood specimen, taken on the tenth day after vaccination, from one of the eight vaccines, yielded a rubella virus. This virus had laboratory markers which were “vaccine-like.” To our knowledge, this represents the first isolation of rubella virus from the blood of a recipient of HPV-77 vaccine. However, the consistent antibody responses among vaccinees and the regular presence of rubella virus in their pharynges argue that viremia occurs in almost every susceptible recipient. The most logical explanation for the failure to document viremia in other recipients of HPV-77 vaccine is that the viremia is ordinarily low grade or transient or both.