The effects of co-infection with human parvovirus B19 and Plasmodium falciparum on type and degree of anaemia in Ghanaian children

Objective:To determin the extent to which parvovirus B19(B19V)and co-infection of B19V and malaria contribute to risk of anaemia in children.Methods:B19V DNA and malaria parasites were screened for 234 children at the PMI,Children's Hospital in Accra.The role of B19V and coinfection with B19V and malaria in anaemia was evaluated by analysing full blood cell counts,malaria and B19V DNA results from these children.Results:The prevalence of B19V,malaria and co-infection with B19V and malaria was 4.7%,41.9%and 2.6%,respectively.Malaria posed a greater risk in the development of mild anaemia compared to severe anaemia(OR=5.28 vrs3.15)whereas B19V posed a higher risk in the development of severe anaemia compared to mild anaemia(OR=4.07 vrs 1.00)from a non-anaemic child.Persons with co-infection with B19V and malaria had 2.23 times the risk(95%CI=0.40-12.54)of developing severe anaemia should they already have a mild anaemia.The degree of anaemia was about three times affected by coinfection(Pillai's trace=0.551,P=0.001)as was affected by malaria alone(Pillai's trace=0.185,P=0.001).B19V alone did not significantly affect the development of anaemia in a non-anaemic child.Microcytic anaemia was associated with B19V and co-infection with B19V and malaria more than normocytic normochromic anaemia.Conclusions:B19V was associated with malaria in cases of severe anaemia.The association posed a significant risk for exacerbation of anaemia in mild anaemic children.B19V and co-infection with B19V and malaria may be associated with microcytic anaemia rather than normocytic normochromic anaemia as seen in cases of B19V infection among persons with red cell abnormalities.

Molecular phenotypes of human parvovirus B19 in patients with myocarditis

AIM:To investigate molecular phenotypes of myocardial B19V-infection to determine the role of B19V in myocarditis and dilated cardiomyopathy(DCM).METHODS:Endomyocardial biopsies(EMBs) from 498 B19V-positive patients with myocarditis and DCMwere analyzed using molecular methods and functional experiments.EMBs were obtained from the University Hospitals of Greifswald and Tuebingen and additionally from 36 German cardiology centers.Control tissues were obtained at autopsy from 34 victims of accidents,crime or suicide.Identification of mononuclear cell infiltrates in EMBs was performed using immunohistological staining.Anti-B19V-IgM and anti-B19V-IgG were analyzed by enzyme-linked immunosorbent assay(ELISA).B19V viral loads were determined using in-house quantitative real-time polymerase chain reaction(PCR).For B19V-genotyping a new B19V-genotype-specific restriction fragment length polymorphism(RFLP)-PCR was established.B19V-genotyping was verified by direct DNAsequencing and sequences were aligned using BLAST and BioEdit software.B19V P6-promoter and HHV6-U94-transactivator constructs were generated for cell culture experiments.Transfection experiments were conducted using human endothelial cells 1.Luciferase reporter assays were performed to determine B19Vreplication activity.Statistical analysis and graphical representation were calculated using SPSS and Prism5 software.RESULTS:The prevalence of B19V was significantly more likely to be associated with inflammatory cardiomyopathy(iCMP) compared to uninflamed DCM(59.6% vs 35.3%)(P < 0.0001).The detection of B19V-mRNA replication intermediates proved that replication of B19V was present.RFLP-PCR assays showed that B19V-genotype 1(57.4%) and B19V-genotype 2(36.7%) were the most prevalent viral genotypes.B19V-genotype 2 was observed more frequently in EMBs with iCMP(65.0%) compared to DCM(35%)(P = 0.049).Although there was no significant difference in gender-specific B19V-loads,women were more frequently infected with B19V-genotype 2(44.6%) than men(36.0%)(P = 0.0448).Coinfection with B19V and other cardiotropic viruses was found in 19.2% of tissuesamples and was associated with higher B19V viral load compared to B19V-monoinfected tissue(P = 0.0012).The most frequent coinfecting virus was human herpes virus 6(HHV6,16.5%).B19V-coinfection with HHV6 showed higher B19V-loads compared to B19V-monoinfected EMBs(P = 0.0033),suggesting that HHV6 had transactivated B19V.In vitro experiments confirmed a 2.4-fold increased B19V P6-promoter activity by the HHV6 U94-transactivator.CONCLUSION:The finding of significantly increased B19V loads in patients with histologically proven cardiac inflammation suggests a crucial role of B19V-genotypes and reactivation of B19V-infection by HHV6-coinfection in B19V-associated iCMP.Our findings suggest that B19V-infection of the human heart can be a causative event for the development of an endothelial cell-mediated inflammatory disease and that this is related to both viral load and genotype.

Acute encephalitis and encephalopathy associated with human parvovirus B19 infection in children

Reports of neurologic manifestations of human parvovirus B19(B19) infection have been on the rise. Acute encephalitis and encephalopathy is the most common, accounting for 38.8% of total B19-associated neurological manifestations. To date, 34 children with B19encephalitis and encephalopathy have been reported, which includes 21 encephalitis and 13 encephalopathy cases. Ten(29%) were immunocompromised and 17(39%) had underlying diseases. Fever at the onset of disease and rash presented in 44.1% and 20.6% of patients, respectively. Neurological manifestations include alteration of consciousness occurred in all patients, seizures in 15(44.1%) patients, and focal neurologic signs in 12(35.3%) patients. Anemia and pleocytosis in cerebrospinal fluid(CSF) occurred in 56.3% and 48.1% of patients, respectively. Serum Anti-B19 Ig M(82.6%) and CSF B19 DNA(90%) were positive in the majority of cases. Some patients were treated with intravenous immunoglobulins and/or steroids, although an accurate evaluation of the efficacy of these treatment modalities cannot be determined. Nineteen(57.6%) patients recovered completely, 11(33.3%) patients had some neurological sequelae and 3(8.8%) patients died. Although the precise pathogenesis underlying the development of B19 encephalitis and encephalopathy is unclear, direct B19 infection or NS1 protein of B19 toxicity in the brain, and immune-mediated brain injuries have been proposed.

Porcine Parvovirus Inducing Autophagy to Benefit Its Replication

Porcine parvovirus(PPV) is one of the major causes of reproductive failure in pigs, which poses a great threat to the pig breeding industry and results in tremendous economic losses worldwide. Autophagy is the biological process of cell self-defense and self-protection. Despite many viruses can cause cell autophagy, when they enter cell or copied, the relationship between autophagy and PPV infection has not been reported. In this study, impact of autophagy after swine testicular(ST) cells infected by PPV was studied. Autophagy was demonstrated by the effective replication of PPV through transmission electron microscopy, immunofluorescence and western blot analysis. Moreover, autophagy was confirmed to benefit PPV replication by real-time fluorescence quantitative PCR and determination of median tissue culture infective dose(TCID). For the first time, the complex interaction between PPV infection and autophagy was explored in this study. It indicated that PPV could induce autophagy in ST cells, which in turn facilitated its own replication, which might be one of the mechanisms of the virus infection. These findings could facilitate the study of the pathogenesis of PPV infection and provide new insight into the development of effective therapeutic strategies.

Seroprevalence of parvovirus B19 antibodies and evidence of viremia among Nigerian patients with sickle cell anemia

Clinical, biochemical and molecular evidence for the sickle cell anemia （SCA） crisis in Nigerian patients arising from parvovirus b19 infection remains inadequate. This study determined the prevalence and correlates of antiparvovirus b19 antibodies in a population of SCA patients and non-SCA healthy controls in Lagos, Nigeria. In this prospective cross-sectional study, we enrolled 73 confirmed SCA patients from 5 district hospitals in Lagos and 81 sex and age-matched non-SCA healthy controls. Serum sample from each study participant was screened for anti-parvovirus b19 by ELISA and PCR techniques. Standard biomedical assays were also done. Anti-parvovirus b19 IgM and IgG antibodies were detected in 22 （14.3%） and 97 （62.9%） of the 154 sera screened, 13 （17.8%） and 45 （61.6%） in SCA patients; 9 （11.1%） and 52 （64.2%） in non-SCA controls. The overall seronegativity rate was 19.5%. Parvovirus B19 DNA was found in 2 （11.1%） of the 18 IgM seropositive SCA serum samples screened. On the whole, parvovirus b19 infection was more commonly asymptomatic in non-SCA controls but caused significant elevation in liver enzymes in infected SCA patients （P 〈 0.05）. The risk of acute parvovirus b19 infection increased 65 times during unsteady state among the SCA patients. Although no deaths of infected patients were recorded during the study, age below 12 years, hospitalization and overcrowded environment were risk factors for infection. We conclude that parvovirus b19 is common in SCA patients, incurring greater susceptibility to infections.

Aluminium-magnesium silicate inhibits parvovirus and cures infected dogs

Ability of a synthetic Aluminium-Magnesium Silicate [AMS] to inhibit activities of canine parvovirus [CPV] was investigated in vitro and in vivo. Five samples of CPV isolated in Nigeria, were each incubated with equal amount of a synthetic AMS on a volume to weight [v/w] basis, for one hour and then centrifuged. Viral titres of the supernatants were tested by the haemagglutination [HA] test and their mean titre compared with mean titre of portions of same viral samples, not incubated with the AMS. Also, five puppies and five adult dogs infected with the parvovirus isolates were treated by dosing each with 400 mg/kg of a drug formulation that has 12% AMS per os for seven days. As control, five puppies and five adult dogs from same class as the experimental dogs were similarly infected but were not treated. Incubating parvovirus with AMS reduced its load from mean HA titre 825.6 ± 261.1 to mean HA, 270.8 ± 132.1 [p < 0.05]. Also treating parvovirus infected dogs with a 12% AMS drug formulation reduced mortality due to the virus from 100% to zero [p < 0.01].

Human parvovirus B19-associated early postoperative acquired pure red cell aplasia in simultaneous pancreas-kidney transplantation:A case report

BACKGROUND Acquired pure red cell aplasia(aPRCA)related to human parvovirus B19(HPV B19)is rarely reported in simultaneous pancreas-kidney transplantation(SPKT)recipients;there has yet to be a case report of early postoperative infection.In this current study,we report the case of a Chinese patient who experienced the disease in the early postoperative period.CASE SUMMARY A 63-year-old man,with type 2 diabetes and end-stage renal disease,received a brain dead donor-derived SPKT.Immunosuppression treatment consisted of tacrolimus,prednisone,enteric-coated mycophenolate sodium(EC-MPS),and thymoglobulin combined with methylprednisolone as induction.The hemoglobin(Hb)level declined due to melena at postoperative day(POD)3,erythropoietinresistant anemia persisted,and reticulocytopenia was diagnosed at POD 20.The bone marrow aspirate showed decreased erythropoiesis and the presence of giant pronormoblasts at POD 43.Metagenomic next-generation sequencing(mNGS)of a blood sample identified HPV B19 infection at POD 66.EC-MPS was withdrawn;three cycles of intravenous immunoglobulin(IVIG)infusion therapy were administered;and tacrolimus was switched to cyclosporine.The HPV B19-associated aPRCA resolved completely and did not relapse within the 1-year follow-up period.The diminution in mNGS reads was correlated with Hb and reticulocyte count improvements.CONCLUSION HPV B19-associated aPRCA can occur at an early period after SPKT.An effective therapy regimen includes IVIG infusion and adjustment of the immunosuppressive regimen.Moreover,mNGS can be used for the diagnosis and to reflect disease progression.

Generation of a Canine-origin Neutralizing scFv Against Canine Parvovirus

Canine parvovirus(CPV)is a high morbidity and lethality virus which causes severe enteric disease in dogs.In an attempt to gain canine-origin neutralizing antibodies against CPV,single chain variable fragment(scFv)bacteria display libraries against the protective antigen VP2 were constructed and screened.VP2 specific scFvs were selected following three rounds of screening procedures.Selected scFvs were characterized by FCM and ELISA.Seven scFvs showed high affinity and specific binding to CPV.Moreover,the neutralizing activity of the antibody was preliminarily identified by CPV(100 TCID_(50))in vitro and scFv-2 showed the neutralizing ability with a titer of 32768.This study generated the first canine-origin neutralizing scFv against CPV.The neutralizing scFv would be constructed into full-length antibody in the future.This study laid the foundation for the generation of an effective therapeutic reagent with long half-life and no immunological rejection for the prevention and treatment of CPV infection.

Human parvovirus B19-associated hematopathy in HIV disease:need for clinicopathological revisit

Persons living with HIV infection occasionally suffer from anemia due to varying causes.These include the use of zidovudine,malnutrition especially vitamin B12and iron deficiency,opportunistic infections by Mycobacterium tuberculosis,Pneumocystis jiroveci,and direct hematological effects of HIV infection itself within the marrow microenvironment.Persistent Parvovirus B19（B19V）infection is a clinically important and treatable etiology of anemia in HIV-infected persons.

Pure red cell aplasia due to parvovirus B19 infection after liver transplantation:A case report and review of the literature

Pure red cell aplasia (PRCA) due to parvovirus B19 (PVB19) infection after solid organ transplantation has been rarely reported and most of the cases were renal transplant recipients. Few have been described after liver transplantation. Moreover, little information on the management of this easily recurring disease is available at present. We describe the first case of a Chinese liver transplant recipient with PVB19-induced PRCA during immunosuppressive therapy. The patient suffered from progressive anemia with the lowest hemoglobin level of 21 g/L. Bone marrow biopsy showed selectively inhibited erythropoiesis with giant pronormoblasts. Detection of PVB19-DNA in serum with quantitative polymerase chain reaction (PCR) revealed a high level of viral load. After 2 courses of intravenous immunoglobulin (IVIG) therapy, bone marrow erythropoiesis recovered with his hemoglobin level increased to 123 g/L. He had a low- level PVB19 load for a 5-mo follow-up period without recurrence of PRCA, and finally the virus was cleared. Our case indicates that clearance of PVB19 by IVIG in transplant recipients might be delayed after recovery of anemia.

Parvovirus B19 induced hepatic failure in an adult requiring liver transplantation

Parvovirus B19 induced acute hepatitis and hepatic failure have been previously reported, mainly in children. Very few cases of parvovirus induced hepatic failure have been reported in adults and fewer still have required liver transplantation. We report the case of a 55-year-old immunocompetent woman who developed fulminant hepatic failure after acute infection with Parvovirus B19 who subsequently underwent orthotopic liver transplantation. This is believed to be the first reported case in the literature in which an adult patient with fulminant hepatic failure associated with acute parvovirus B19 infection and without hematologic abnormalities has been identified prior to undergoing liver transplantation. This case suggests that Parvovirus B19 induced liver disease can affect adults, can occur in the absence of hematologic abnormalities and can be severe enough to require liver transplantation.

Development of a Polyclonal Antibody-based AC-ELISA and Its Comparison with PCR for Diagnosis of Canine Parvovirus Infection

A polyclonal antibody-based antigen-capture ELISA (AC-ELISA) has been developed for detection of Canine parvovirus (CPV) antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as the capture antibody, guinea pig anti-CPV polyclonal antibody as tracing antibody and anti-guinea pig HRPO conjugate as the detection system. The optimum dilution of the capture antibody and the tracing antibody capable of detecting the CPV-2 antigens was found to be 1:1 600 and 1:400, respectively, in the check-board titration. In this study, a total of 152 samples (129 faecal samples and 23 cell culture supernatant) were tested both by AC-ELISA and by polymerase chain reaction (PCR). Of the samples tested, 69 and 78 samples were found positive by AC-ELISA and PCR, respectively. The AC-ELISA had relative sensitivity, relative specificity and accuracy of 88.4%, 100.0% and 91.4% respectively. The analytical sensitivity of AC-ELISA was estimated to be 102.8 TCID50/mL whereas PCR sensitivity was 100.8 TCID50/mL. The AC-ELISA is a simple, quick and reliable method for screening large numbers of faecal samples of dogs suspected of CPV infection.

Killing of p53-deficient hepatoma cells by parvovirus H-1 and chemotherapeutics requires promyelocytic leukemia protein

AIM: To evaluate the synergistic targeting and killing of human hepatocellular carcinoma (HCC) cells lacking p53 by the oncolytic autonomous parvovirus (PV) H-1 and chemotherapeutic agents and its dependence on functional promyelocytic leukemia protein (PML). METHODS: The role of p53 and PML in regulating cy-totoxicity and gene transfer mediated by wild-type (wt) PV H-1 were explored in two pairs of isogenic human hepatoma cell lines with different p53 status. Further-more,H-1 PV infection was combined with cytostatic drug treatment. RESULTS: While the HCC cells with different p53 status studied were all susceptible to H-1 PV-induced apoptosis,the cytotoxicity of H-1 PV was morepronounced in p53-negative than in p53-positive cells. Apoptosis rates in p53-negative cell lines treated by genotoxic drugs were further enhanced by a treatment with H-1 PV. In flow cytometric analyses,H-1 PV infection resulted in a reduction of the mitochondrial transmembrane potential. In addition,H-1 PV cells showed a significant increase in PML expression. Knocking down PML expression resulted in a striking reduction of the level of H-1 PV infected tumor cell death. CONCLUSION: H-1 PV is a suitable agent to circumvent the resistance of p53-negative HCC cells to genotoxic agents,and it enhances the apoptotic process which is dependent on functional PML. Thus,H-1 PV and its oncolytic vector derivatives may be considered as therapeutic options for HCC,particularly for p53-negative tumors.

RELATIONSHIP BETWEEN HUMAN PARVOVIRUS B19 INFECTION AND APLASTIC ANEMIA

Objective. To explore the relationship between human parvovirus B19 (HPV B19) infection and aplastic anemia (AA) and to investigate the role of HPV B19 in the occurrence of AA. Methods. The presence of HPV B19 DNA was detected in the peripheral blood samples of 60 patients with AA (children 38 and adults 22) by nested polymerase chain reaction (PCR) assay, and 30 healthy persons were selected as control. Results. Sixteen (26.7％ ) of 60 AA cases were HPV B19 DNA positive, while all the samples in the control group were negative for HPV B19 (P = 0.000914). Among the case group, the positive rates of HPV B19 DNA were 21.4％ (6 / 28), 30.0％ (3 / 10), 20.0％ (1 / 5) and 35.3％ (6 / 17) in children acute AA (AAA), children chronic AA (CAA), adults AAA and adults CAA patients respectively, which were significantly higher than that in the control group. Furthermore, there was no remarkable difference between children AA and adults AA in the 16 HPV B19 DNA positive patients; neither was there between AAA and CAA. Conclusions. HPV B19 infection is not only correlated with the occurrence of children AAA and CAA, but also with adults AAA and CAA, and might be an important viral cause for AA in humans.

Molecular Cloning and Prokaryotic Expression of Non-Structural Protein NS1 Gene of Porcine Parvovirus

Porcine parvovirus (PPV) is one of the major agents causing swine reproductive failure. NS1 protein is a non-structural protein of PPV and can be used as a reagent for differentiation of vaccinated animals and infected ones. In present study, a recombinant plasmid pET28a/NS1 was constructed by cloning the coding sequence for NS1 of PPV into pET28a, a bacterial expression vector. The NS1 protein was expressed in E. coli BL21(DE3) after induced by IPTG and the recombinant fusion protein was purified with affinity chromatography. Expression amount of NS1 protein was improved by optimizing the inducing parameters. The recombinant NS1 protein is reactive to PPV positive sera in Western blot and ELISA test and therefore can be applicable in differential diagnosis of PPV infections.

RESEARCH AND DEVELOPMENT OF CANCER-TARGETING VECTORS: AN EXPLORATION ON ONCOTROPISM AND ONCOSUPPRESSION OF AUTONOMOUS PARVOVIRUSES

Autonomous parvoviruses are single-stranded DNA viruses with a genome size of about 5 kb. They are characterized by their oncotropism and oncosuppression to the hosts. After infection the viral genes are not integrated into the chromosome of host cells. Autonomous parvoviruses could be used as potential vectors carrying heterologous therapeutic genes for the construction of recombinant autonomous parvoviruses. The specific advantages of these viral vectors to cancer gene therapy are discussed.

Detection of Human Parvovirus B19 Nonstrutural Protein DNA by Nested-Polymerase Chain Reaction in Gravida Serum and Pregnant Tissues

A new nested-polymerase chain reaction （nested-PCR） assay was developed to detect human parvovirus B19 DNA corresponding to the nonstructural protein in clinical specimens in a routine diagnostic laboratory. The sensitivity of this highly specific assay was up to 0. 005 fg of B19 DNA. Parvovirus B19 was identified in sera of 20 pregnant women with abnormal pregnant outcome. Among these 20 cases, intrauterine parvovirus infection did exist in 7 pregnant women because parvovirus B19 DNA was detected in the pregnant tissues of them such as placenta tissues, chorionic villi, amniotic fluid, fetal spleen, liver and abdominal fluids.

A preliminary analysis of antineoplastic activity ofparvovirus MVMp NS-1 proteins

Human gastric cancer MKN-45 cells were transfectedwith pULB 3238, a plasmid carrying MVMp NS-1 genewith its original P4 promoter replaced by the glucocorticoid inducible promoter MMTV-LTR. After the integration and expression of NS-1 gene, some of the transfectantsdied, while others remained alive, but the growth featuresof survived cells were changed. For further study on theantineoplastic function of parvoviral NS-1 protein in vivo,transgenic mice carrying NS-1 genes were established byconventional method. Among 4 founders, one of them wasfound to be able to transmit the transgene to around 50%of their offsprings. RT-PCR was performed to indicate theexpression of NS-1 gene in transgenic mice and its mRNAappeared in a variety of tissues. The expression of integrated NS-1 gene may correlate with the decreased incidence of tumor induced in vivo by chemical carcinogens.

Epidemiology Survey of Porcine Parvovirus and Porcine Circovirus Type 2 in Sichuan Province

[ Objective] The study aimed to analyze the prevalence of Porcine Parvovirus （PPV） and Porcine Circovirus Type 2 （ PCV2 ） and the mixed infection in Sichuan Province to lay the foundation for further predicting the tendency of the plague and formulating appropriate prevention and control strategies. [ Method] Two hundred and seventy -three samples were collected from 21 large pig farms in Sichuan province, and epidemiology of PPV and PCV2 were investigated by PCR detecting. [ Result] The positive rate of PPV was 17.22% （47/273）, and positive rate of pig farms was 38.1% （8/21）, meanwhile it also displayed the feature that infection rate of boar was higher than that of piglets; The positive rate of PCV2 was 52.38% （143/273）, and positive rate of pig farms was 85.7% （18/21）, and it showed the trend that the infection rate of PCV2 was rising with the growth of the age. The co-infection rate of PPV and PCV2 was 10.62% （29/273）, and co-infection rate of pig farms was 28.7% （6/21）, which mainly concentrated in the sow and nursery piglets. Only 14.3% （3/21） pig farms was epidemiologically negative of PPV and PCV2. [ Conclusion] PPV and PCV2 and co-infection was widely popular in Sichuan province, and it did more serious harm to the pig-raising industry.

Epidemiological Survey on Canine Parvovirus Disease in Taizhou Region,Jiangsu Province,China

The canine parvovirus disease is an acute infectious disease caused by canine parvovirus(CPV). It is clinically characterized by severe vomiting,hemorrhagic enteritis,significant reduction in white blood cells and myocarditis. The disease with high incidence,highly infectious and high mortality has become one of the serious infectious diseases threatening dog raising industry in China. In this research,260 cases of canine parvovirus case from an Aite Pet Clinic in Taizhou City during January 2010 and March 2011 were analyzed. This study discloses the epidemiology of CPV in Taizhou region of Jiangsu Province,i. e.,the incidence of CPV and canine motility are closely correlated with age,breed,immune inoculation and season. This study provides useful guide for the clinical treatment of CPV in the future.