Outcomes for Nitazoxanide Treatment in a Case Series of Patients with Primary Immunodeficiencies and Rubella Virus-Associated Granuloma.

PURPOSE: Nitazoxanide was recently reported as having in vitro effectiveness against the rubella virus. Immunodeficiency-related vaccine-derived rubella occurs in some patients who have an inherited immunodeficiency and who received the MMR vaccine. This study investigated the in vivo effectiveness of nitazoxanide therapy. METHODS: This is a retrospective analysis of seven patients treated with nitazoxanide as salvage therapy for immunodeficiency-related vaccine-derived rubella infection. The patients were recruited from an ongoing rubella detection surveillance project. RESULTS: Seven patients with persistent rubella were treated with nitazoxanide and one demonstrated significant clinical improvement. Two additional patients exhibited diminished viral capsid production with one patient having transient slowing of progression. The cohort overall generally had low T cell counts and had a high burden of comorbidities. There were three deaths. Two deaths were from PML and one was related to hematopoietic stem cell transplantation. CONCLUSIONS: Nitazoxanide has limited in vivo anti-viral effects for immunodeficiency-related vaccine-derived rubella. Most patients did not exhibit clinical improvement.

Both Sphingomyelin and Cholesterol in the Host Cell Membrane Are Essential for Rubella Virus Entry.

Rubella virus (RuV) causes a systemic infection, and transplacental fetal infection causes congenital rubella syndrome. In this study, we showed that treatment of cells with sphingomyelinase inhibited RuV infection. Assays using inhibitors of serine palmitoyl transferase and ceramide transport protein demonstrated the contribution of sphingomyelin (SM) to RuV infection. Compelling evidence for direct binding of RuV to lipid membranes at neutral pH was obtained using liposome coflotation assays. The absence of either SM or cholesterol (Chol) abrogated the RuV-liposome interaction. SM and Chol (SM/Chol) were also critical for RuV binding to erythrocytes and lymphoid cells. Removal of Ca2+ from the assay buffer or mutation of RuV envelope E1 protein Ca2+-binding sites abrogated RuV binding to liposomes, erythrocytes, and lymphoid cells. However, RuV bound to various nonlymphoid adherent cell lines independently of extracellular Ca2+ or SM/Chol. Even in these adherent cell lines, both the E1 protein Ca2+-binding sites and cellular SM/Chol were essential for the early stage of RuV infection, possibly affecting envelope-membrane fusion in acidic compartments. Myelin oligodendrocyte glycoprotein (MOG) has recently been identified as a cellular receptor for RuV. However, RuV bound to MOG-negative cells in a Ca2+-independent manner. Collectively, our data demonstrate that RuV has two distinct binding mechanisms: one is Ca2+ dependent and the other is Ca2+ independent. Ca2+-dependent binding observed in lymphoid cells occurs by the direct interaction between E1 protein fusion loops and SM/Chol-enriched membranes. Clarification of the mechanism of Ca2+-independent RuV binding is an important next step in understanding the pathology of RuV infection.IMPORTANCE Rubella has a significant impact on public health as infection during early pregnancy can result in babies being born with congenital rubella syndrome. Even though effective rubella vaccines are available, rubella outbreaks still occur in many countries. We studied the entry mechanism of rubella virus (RuV) and found that RuV binds directly to the host plasma membrane in the presence of Ca2+ at neutral pH. This Ca2+-dependent binding is specifically directed to membranes enriched in sphingomyelin and cholesterol and is critical for RuV infection. Importantly, RuV also binds to many cell lines in a Ca2+-independent manner. An unidentified RuV receptor(s) is involved in this Ca2+-independent binding. We believe that the data presented here may aid the development of the first anti-RuV drug.

The hUC-MSCs cell line CCRC-1 represents a novel, safe and high-yielding HDCs for the production of human viral vaccines.

MRC-5 represents the most frequent human diploid cells (HDCs)-type cell substrate in the production of human viral vaccines. However, early-passage MRC-5 is diminishing and, due to both technical and ethical issues, it is extremely difficult to derive novel HDCs from fetal lung tissues, which are the common sources of HDCs. Our previous studies suggested that human umbilical cord may represent an alternative but convenient source of new HDCs. Here, we established a three-tiered cell banking system of a hUC-MSC line, designated previously as Cell Collection and Research Center-1 (CCRC-1). The full characterization indicated that the banked CCRC-1 cells were free from adventitious agents and remained non-tumorigenic. The CCRC-1 cells sustained its rapid proliferation even at passage 30 and were susceptible to the infection of a wide spectrum of viruses. Interestingly, the CCRC-1 cells showed much higher production of EV71 or Rubella viruses than MRC-5 and Vero cells when growing in serum-free medium. More importantly, the EV71 vaccine produced from CCRC-1 cells induced immunogenicity while eliciting no detectable toxicities in the tested mice. Collectively, these studies further supported that CCRC-1, and likely other hUC-MSCs as well, may serve as novel, safe and high-yielding HDCs for the production of human viral vaccines.

Inhibition of rubella virus replication by the broad-spectrum drug nitazoxanide in cell culture and in a patient with a primary immune deficiency.

Persistent rubella virus (RV) infection has been associated with various pathologies such as congenital rubella syndrome, Fuchs's uveitis, and cutaneous granulomas in patients with primary immune deficiencies (PID). Currently there are no drugs to treat RV infections. Nitazoxanide (NTZ) is an FDA-approved drug for parasitic infections, and has been recently shown to have broad-spectrum antiviral activities. Here we found that empiric 2-month therapy with oral NTZ was associated in the decline/elimination of RV antigen from lesions in a PID patient with RV positive granulomas, while peginterferon treatment had no effect. In addition, we characterized the effects of NTZ on cell culture models of persistent RV infection. NTZ significantly inhibited RV replication in a primary culture of human umbilical vein endothelial cells (HUVEC) and Vero and A549 epithelial cell lines in a dose dependent manner with an average 50% inhibitory concentration of 0.35 µg/ml (1.1 µM). RV strains representing currently circulating genotypes were inhibited to a similar extent. NTZ affected early and late stages of infection by inhibiting synthesis of cellular and RV RNA and interfering with intracellular trafficking of the RV surface glycoproteins, E1 and E2. These results suggest a potential application of NTZ for the treatment of persistent rubella infections, but more studies are required.

Impact of the two-dose rubella vaccination regimen on incidence of rubella seronegativity in gravidae aged 25 years and younger.

OBJECTIVE: This study compared the incidence of rubella seronegativity among gravidae of 25 year-old and younger, between those born in Hong Kong after 1983 when the two-dose rubella vaccination was implemented, versus gravidae born before, to examine the impact of the two-dose regimen. METHODS: In this retrospective cohort study, the incidence of antenatal rubella seronegativity in our parturients managed in1997-2015 was analysed by their age from ≤16 to 25 years, and the effect of year of birth was determined adjusting for confounding factors including teenage status, obstetric history, anthropometric factors, and health parameters including anaemia, thalassaemia trait and hepatitis B carrier status. RESULTS: Among the 12743 gravidae, the 6103 gravidae born after 1983 had overall higher rubella seronegativity (9.1% versus 4.4%, OR 2.061, 95% CI 1.797-2.364), with significant difference (p = 0.006) and inverse correlation (p<0.001) with age, in contrast to the 6640 gravidae born in/before 1983 whom there was significant difference (p = 0.027) but a positive correlation (p = 0.008) with age. For each year of age, the former had significantly higher incidence of rubella seronegativity except for those of ≤16 years. Regression analysis confirmed that birth after 1983 was independently associated with rubella seronegativity (aOR 2.207, 95% CI 1.902-2.562). CONCLUSION: There was a significant trend between rubella seronegativity with age in young gravidae, but the pattern was opposite between gravidae born after versus in/before 1983, with the former having a higher incidence of seronegativity at all ages. Young women covered by the two-dose rubella immunisation programme have a paradoxically higher incidence of rubella seronegativity.

[Pre-pregnancy and early clinical analysis of TORCH infections detection].

OBJECTIVE: For the birth, to improve the quality of the population, to explore the prevention and treatment of early pregnancy TORCH infection, and treatment of patients with positive eugenics and guidance. METHODS: Enzyme-linked immunosorbent assay (ELISA) testing of all the objects in the peripheral blood-money pathogen-specific antibodies LgM. Person in charge of testing. In strict accordance with the instructions. Reagents from Shanghai magnolia biotechnology institute. RESULT: The total number of 319 cases of positive, with a total infection rate: 3.28%, TOX-IgM, RV-IgM, CMV-IgM, HSV (II)-IgM infection rate of 0.103 percent, 2.64 percent and 0.309 percent, 0.237 percent; 319 cases of TORCH infected persons are to receive treatment for the treatment of wrap, with a total negative rate of 97.49 percent. CONCLUSION: Detection of TORCH infections to ensure early diagnosis, early treatment and early prevention is necessary.

[Clinical and experimental study on effects of huanglan granule in inhibiting rubella virus].

OBJECTIVE: To explore the therapeutic effect and acting mechanism of Huanglan Granule (HLG) on rubella virus (RuV). METHODS: Sixty patients with positive RuV-IgM were randomly assigned to two groups equally, the treatment group was medicated by HLG (one dosage per day, containing milkvetch root, isatis root and basket fern, each 30 g), while the control group by ribavirin (0.2 g, three times per day) for 20 days. The negative conversion rate of RuV-IgM and the serum levels of interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-alpha) were observed before and after treatment. Moreover, the in vitro inhibitory activity of HLG against RuV Gos line on cultured Vero cells was determined by cytopathic inhibition method. RESULTS: The difference of negative conversion rate between the two groups after one course treatment was significant (86.7% vs 63.3%, P <0.05). However, it turned to insignificant after two courses of treatment (100% vs 86.7%, P >0.05). The serum level of IL-2 was lower and TNF-alpha was higher significantly in patients with positive RuV-IgM as compared with the normal range, and the two indexes returned to the normal range rapidly after HLG treatment. In vitro study showed that the inhibitory effect of HLG on RuV caused cellular change was evident. CONCLUSION: HLG has obvious inhibitory effect on RuV, both in vitro and in vivo, it can also raise the immunity of organism and thus it serves as a safe and effective Chinese medicine for treatment of active RuV infection.

Synthesis and biological evaluation of 4-alkylamino-6-(2-hydroxyethyl)-2-methylthiopyrimidines as new rubella virus inhibitors.

In the search for new chemotherapeutic agents useful against Rubella virus (RV) infections, a solution-phase parallel approach for the synthesis of a small library of 4-alkylamino-6-(2-hydroxyethyl)-2-methylthiopyrimidines has been set up, based on previous results from our research group. Biological evaluation of the newly synthesized compounds pointed out their interesting properties as anti-RV agents with IC(50) values in the micromolar range.

[Preliminary study on nasal spray of interferon alpha-2b used for prevention of rubella and measles virus infections].

OBJECTIVE: To evaluate the efficacy of the interferon alpha-2b nasal spray in prevention of rubella and measles virus infections. METHODS: The properly selected volunteer groups have been divided into interferon alpha-2b experimental and control group. The experimental group received interferon alpha-2b treatment by nasal spray for 2 days before the immunization, then both groups were challenged with rubella and measles attenuated live vaccine respectively through nasal spray. The sera from pre-immunization and 21 and 28 days after immunization were collected to test the IgG antibody titers. The influence on the viral antibody titer reflects the viral preventive effect by interferon alpha-2b. RESULTS: The antibody titer difference of measles virus between experimental and control group was 1.26 (21 day) and 2.96 (28 day), there were statistically difference between them; the difference of rubella virus was 0.95 (21 day) and 0.37 (28 day), but there were no statistically differences found. CONCLUSION: The preliminary results showed that the interferon alpha-2b can be used as prevention method for measles and rubella viral infections.

Effects of endocytosis inhibitory drugs on rubella virus entry into VeroE6 cells.

It has been suggested that infectious entry of rubella virus (RV) is conducted by receptor mediated endocytosis. To explore the cellular entry mechanism of RV, inhibitory effects of drugs affecting various endocytic pathways on RV entry into VeroE6 cells were analyzed. Results showed that RV infectious entry into VeroE6 cells is mediated by clathrin-dependent endocytosis and not by caveolae-mediated endocytosis. Moreover, chemical inhibition of macropinocytosis such as treatments of amiloride, actin and microtubule-disrupting drug significantly reduced RV infection. Considering that macropinocytosis is inducible endocytosis by cellular stimulations, clathrin-mediated endocytosis is likely to be a major route of RV infectious entry.

A new and efficient synthesis of substituted 6-[(2'-dialkylamino)ethyl] pyrimidine and 4-N,N-dialkyl-6-vinyl-cytosine derivatives and evaluation of their anti-rubella activity.

New 6-[(2'-dialkylamino)ethyl]-4(3H)-pyrimidinones were prepared by a multistep procedure starting from acetone dicarboxylic acid diethyl ester and urea derivatives. These compounds were used as starting materials to obtain 4-N,N-dialkyl-6-vinyl-pyrimidine derivatives by an unprecedented tandem C-6 side chain Hofmann-like elimination/C-4 pyrimidinone substitution. Among the new derivatives obtained, various compounds show anti-Rubella activity. The inhibition of HIV-1 Reverse Transcriptases (RT), from both wild type and modified viruses, is also reported.

[Experimental study on effect of recombined decoction on mumps virus].

OBJECTIVE: To evaluate possible inactivating effect of recombined decoction in on mumps virus. METHODS: By adopting tissue cell culturing technology, a group of viruses including the mumps virus, herpes simplex virus (type I, II), rubella virus, cytomegalovirus (CMV), herpes zoster virus, influenza virus, parainfluenza virus, adeno viruses, respiratory syneytial virus (RSV) were cultured. The cells infected with the viruses were treated with the decoction. RESULTS: The decoction showed remarkable inhibitory and killing effects on the mumps virus while had no obvious inhibitory and killing effects on host's cells, herpes simplex virus (type I, II), rubella virus, cytomegalovirus (CMV), herpes zoster virus, influenza virus, parainfluenza virus, adeno viruses, respiratory syneytial virus (RSV). CONCLUSIONS: The decoction had obvious inhibitory and killing effects on mumps virus during single layer cells culture.

Inhibition of rubella virus growth by Fungizone.

Fungizone added to agar overlay medium inhibited plaque formation in both size and number by rubella virus in rabbit kidney 13 cells. In the presence of 1 microg/ml of Fungizone, the diameter of the plaques was reduced to one half of that in the absence of the drug, and at 5 microg/ml, plaque formation was inhibited by 80%. When the drug was added to the culture medium, the growth of infectious virus was also inhibited with reduction in the synthesis of envelope glycoprotein E1 and capsid protein C in infected cells.

Inhibitory effect of a cyclic urea derivative on rubella virus replication.

1-(4-Morpholinomethyl)-tetrahydro-2(1H)-pyrimidinone (mopyridone) exhibited a marked activity against rubella virus (Judith and RA27/3 strains), a MIC50 value of 0.9 microM and selectivity ratio of 557.7 been found in the case of Judith strain. These data, in addition to the previous ones about its anti-alphavirus effects suggest the compound to be considered as a broad spectrum inhibitor of togavirus replication.

Synthesis and biological evaluation of 2-methoxy- and 2-methylthio-6-[2'-alkylamino)ethyl]-4(3H)-pyrimidinones with anti-rubella virus activity.

The synthesis of a new family of antiviral compounds, 2-methoxy-, and 2-methylthio-6-[(2'-alkylamino)ethyl]-4(3H)-pyrimidinones, has been accomplished. The activity of these agents against positive strand (rubella virus and sindbis virus) and negative strand (vesicular stomatitis virus) RNA viruses is reported. Some of these compounds are efficient and selective inhibitors of rubella virus.

Inactivation of human viruses by povidone-iodine in comparison with other antiseptics.

Inactivation of a range of viruses, such as adeno-, mumps, rota-, polio- (types 1 and 3), coxsackie-, rhino-, herpes simplex, rubella, measles, influenza and human immunodeficiency viruses, by povidone-iodine (PVP-I) and other commercially available antiseptics in Japan was studied in accordance with the standardized protocol in vitro. In these experiments, antiseptics such as PVP-I solution, PVP-I gargle, PVP-I cream, chlorhexidine gluconate, alkyldiaminoethyl-glycine hydrochloride, benzalkonium chloride (BAC) and benzethonium chloride (BEC) were used. PVP-I was effective against all the virus species tested. PVP-I drug products, which were examined in these experiments, inactivated all the viruses within a short period of time. Rubella, measles, mumps viruses and HIV were sensitive to all of the antiseptics, and rotavirus was inactivated by BAC and BEC, while adeno-, polio- and rhinoviruses did not respond to the other antiseptics. PVP-I had a wider virucidal spectrum, covering both enveloped and nonenveloped viruses, than the other commercially available antiseptics.

Effect of honey versus thyme on Rubella virus survival in vitro.

In this paper, we assess the antiviral properties of honey solutions and thyme extracts at varying concentrations. This was done by testing these solutions in vitro using monkey kidney cell cultures that were infected with the Rubella virus. Our results indicated that honey had good anti-Rubella activity, while thyme did not. These results may justify the continuing use of honey in traditional medicines from different ethnic communities worldwide and in some modern medications such as cough syrups.

Inhibition of herpes simplex, rabies and rubella viruses by lectins with different specificities.

A series of eleven lectins were investigated for their inhibiting activity towards herpes simplex type 1, rabies and rubella viruses. Herpes simplex virus adsorption was inhibited by Limulus polyphemus (LPA), Canavalia ensiformis (ConA) and wheat germ (WGA) agglutinins, whereas Bauhinia purpurea (BPA) and soybean agglutinins were more effective if present during viral replication; rabies virus attachment to susceptible cells was prevented by LPA and Narcissus pseudonarcissus (NPA) agglutinin whereas WGA and BPA were inhibitory at a later step; rubella virus multiplication was affected only after the attachment step by NPA, ConA and WGA.

Conformational change of rubella virus spike proteins induced by 2-mercaptoethanol.

Hemagglutinating (HA) activity of rubella virus was inactivated with 2-mercaptoethanol (2ME) in a dose-dependent manner. But even low concentrations of 2ME, which had little effect on HA activity by themselves, greatly increased the sensitivity of spike polypeptides to the subsequent trypsin treatment. Increased trypsin sensitivity was shown by an enhanced reduction of HA activity and an enhanced proteolytic removal of both E1 and E2 polypeptides from the surface of the virion. The findings indicate that 2ME causes an extensive disruption in the conformation of spikes composed of E1 and E2 polypeptides.

Rubella virus replication: effect of interferons and actinomycin D.

The effect of alpha and gamma interferon (IFN alpha, IFN gamma) and actinomycin D on the expression of wild type rubella virus in African green monkey kidney cells (Vero 76) was studied. Viral protein synthesis in the infected cells was significantly reduced upon treatment of the cells with IFN alpha or IFN gamma, which is accompanied by the reduction in the level of both the (+) stranded and the (-) stranded viral RNAs. The residual rubella viral RNA from interferon-treated cells, however, was structurally intact as judged by Northern blot analysis and in vitro translation. These results suggest that the effect of IFN alpha and IFN gamma on rubella viral protein synthesis is both at the transcriptional and the translational level. The effect of actinomycin D on rubella virus replication was found to be time-dependent. It is much more pronounced during the eclipse phase of the viral growth (first 4 h) than after 8 h at which time actinomycin D had lesser effect. A similar effect on rubella virus replication was observed when alpha-amanitin was used instead of actinomycin D. These results were taken to indicate that during the viral infection, host cell DNA directs the synthesis of a cellular factor(s) which is essential for the viral replication. When the synthesis of this cellular factor(s) is terminated at an early stage of viral infection by actinomycin D or by alpha-amanitin, viral replication is impaired.