Generation and molecular characteristics of a highly attenuated GPV strain through adaptation in GEF cells.

BACKGROUND: Goose parvoviruses (GPVs) spread globally and cause a huge economic loss to the poultry industry. Although the attenuated GPV vaccines play a key role in preventing the disease caused by GPV, the molecular basis for the attenuation of GPV is barely known. RESULTS: A highly attenuated GPV strain, GPV-CZM-142, was generated through blindly passaging of the highly pathogenic strain, GPV-CZM, in goose embryonic fibroblasts (GEF) for 142 generations. The GEF-adapted GPV strain's virulence was 10,000 times weaker than its wild type counterpart, GPV-CZM, based on the ELD50 (50% Embryo Lethal Dose). By comparing with the wild type strain, genome sequencing analysis identified adapted mutations either in ITR or in NS and VP1 of GPV-CZM-142. CONCLUSIONS: The highly attenuated GPV strain, GPV-CZM-142, provides a GPV vaccine candidate, and the identified virulence-related mutations give a novel insight into the molecular determinants of GPV virulence.

Construction and sequencing of an infectious clone of the goose embryo-adapted Muscovy duck parvovirus vaccine strain FZ91-30.

BACKGROUND: Muscovy duck parvovirus (MDPV) is the etiological agent of Muscovy duckling parvoviral disease, which is characterized by diarrhea, locomotive dysfunction, stunting, and death in young ducklings, and causes substantial economic losses in the Muscovy duck industry worldwide. FZ91-30 is an attenuated vaccine strain that is safe and immunogenic to ducklings, but the genomic information and molecular mechanism underlining the attenuation are not understood. METHODS: The FZ91-30 strain was propagated in 11-day-old embryonated goose eggs, and viral particles were purified from the pooled allantoic fluid by differential centrifugation and ultracentrifugation. Single-stranded genomic DNA was extracted and annealed to form double-stranded DNA. The dsDNA digested with NcoI resulted two sub-genomic fragments, which were then cloned into the modified plasmid pBluescript II SK, respectively, generating plasmid pBSKNL and pBSKNR. The sub-genomic plasmid clones were sequenced and further combined to construct the plasmid pFZ that contained the entire genome of strain FZ91-30. The complete genome sequences of strain FM and YY and partial genome sequences of other strains were retrieved from GenBank for sequence comparison. The plasmid pFZ containing the entire genome of FZ91-30 was transfected in 11-day-old embryonated goose eggs via the chorioallantoic membranes route to rescue infectious virus. A genetic marker was introduced into the rescued virus to discriminate from its parental virus. RESULTS: The genome of FZ91-30 consists of 5,131 nucleotides and has 98.9 % similarity to the FM strain. The inverted terminal repeats (ITR) are 456 nucleotides in length, 14 nucleotides longer than that of Goose parvovirus (GPV). The exterior 415 nucleotides of the ITR form a hairpin structure, and the interior 41 nucleotides constitute the D sequence, a reverse complement of the D' sequence at the 3' ITR. Amino acid sequence alignment of the VP1 proteins between FZ91-30 and five pathogenic MDPV strains revealed that FZ91-30 had five mutations; two in the unique region of the VP1 protein (VP1u) and three in VP3. Sequence alignment of the Rep1 proteins revealed two amino acid alterations for FZ91-30, both of which were conserved for two pathogenic strains YY and P. Transfection of the plasmid pFZ in 11-day-old embryonated goose eggs resulted in generation of infectious virus with similar biological properties as compared with the parental strain. CONCLUSIONS: The amino acid mutations identified in the VP1 and Rep1 protein may contribute to the attenuation of FZ91-30 in Muscovy ducklings. Plasmid transfection in embryonated goose eggs was suitable for rescue of infectious MDPV.

Parvovirus B19 in pregnancy: prenatal diagnosis and management of fetal complications.

PURPOSE OF REVIEW: Parvovirus B19 infection is often considered a mild and self-limiting disease of minor clinical importance. This review aims to raise awareness of recently discovered potentially devastating consequences of this infection in pregnancy, and provides updated guidelines on diagnosis and management. RECENT FINDINGS: In contrast to previous beliefs, parvovirus B19 infection during any stage of pregnancy may not only cause fetal death, but may also result in severe and irreversible neurological sequelae in survivors. Improved diagnostic techniques allow more reliable and earlier diagnosis of fetal disease. SUMMARY: Clinicians need to be aware of the risk of adverse outcome of parvovirus B19 infection in pregnancy, and sometimes the long interval between exposure and fetal symptoms. Accurate diagnosis using PCR and weekly ultrasound checks ups with Doppler measurement of middle cerebral artery flow velocity up to 20 weeks postexposure may improve detection of fetal disease. More timely treatment likely results in improved outcome.

Management and outcome of pregnancies with parvovirus B19 infection over seven years in a tertiary fetal medicine unit.

OBJECTIVES: To determine rates of fetal anaemia and pregnancy outcome in susceptible pregnant women infected with human parvovirus B19 infection in a tertiary fetal medicine department over a 7-year period. Additional features enabling identification of fetuses that progress to severe anaemia were also investigated. METHODS: Forty-seven susceptible, pregnant women with confirmed parvovirus infection referred to a regional fetal medicine unit, over a 7-year period (1999-2006), were identified. Where possible maternal serum AFP measurements were obtained from second-trimester serum screening and the presence or absence of echogenic bowel noted. RESULTS: Of the 47 cases, one was excluded. Of the remaining 46 cases, 34 (74%) showed no signs of fetal anaemia and delivered at term. The remaining 12 (26%) showed signs of fetal anaemia. Eight of the 12 developed hydrops and underwent fetal blood sampling and transfusion (median pretransfusion Hb 3.6 g/dl). Seven of the 8 transfused fetuses were thrombocytopenic with a platelet count <150 x 10(9)/l, with 2 fetuses having platelet counts <50 x 10(9)/l. The median gestation age at transfusion was 22 weeks (range 18-27 weeks). The median number of weeks between seroconversion and transfusion was 6 (range 3-12). The signs of anaemia resolved after one transfusion in 5 of the 8 transfused fetuses and they subsequently delivered at term. There were 2 fetal deaths during or shortly after transfusion and one neonatal death following delivery at 28 weeks gestation due to severe pre-eclampsia, 5 days after successful transfusion. CONCLUSIONS: Following parvovirus seroconversion, the incidence of significant fetal anaemia requiring transfusion was 17%. Seroconversion after 21 weeks did not result in severe fetal anaemia. Significant anaemia requiring intervention did not occur 12 weeks after maternal seroconversion. We did not demonstrate a correlation with either maternal serum AFP or the presence of fetal echogenic bowel and the development of severe fetal anaemia. Because of the association between fetal anaemia and severe thrombocytopenia, it may be prudent to have compatible platelets available at the time of fetal blood sampling.

Fetal peritonitis due to appendiceal rupture: a rare complication of hydrops.

A rare complication (appendiceal perforation with meconium peritonitis) was observed in a second trimester fetus affected by nonimmune fetal hydrops due to parvovirus B-19 infection. The complication is not considered specific to this or any other etiology for hydrops, which is highly heterogeneous; rather it is an expression of the fragility and friability of edematous tissues.

Fetal parvovirus B19 infection in a twin pregnancy with 1 twin presenting with hydrops fetalis and the other asymptomatic: a case report.

BACKGROUND: Parvovirus B19 infection during pregnancy is a known cause of nonimmune hydrops, while about one third of maternal infection is asymptomatic. Spontaneous abortion, hydrops fetalis, intrauterine growth retardation and meconium peritonitis are possible manifestations of fetal infection, but the infection can also be asymptomatic. CASE: Both fetuses in a dichorionic, diamniotic twin pregnancy were infected with parvovirus B19 infection, but only 1 fetus presented with hydrops fetalis. That fetus died, while the other remained asymptomatic throughout the pregnancy. The mother had a normal delivery at 39 weeks of gestation, and the neonatal outcome of the surviving twin was normal apart from early-onset neonatal jaundice due to ABO incompatibility. CONCLUSION: This case, together with others, illustrates that parvovirus B19 infection in twin fetuses can present differently despite the fact that the fetuses are nurtured in rather similar intrauterine environments. A severe complication in 1 twin does not preclude a normal outcome in the other. The possibility of different manifestations should be borne in mind so that parvovirus B19 infection remains high on the list of differential diagnoses of nonimmune hydrops affecting only 1 fetus in a twin pregnancy.

Detection of parvovirus B19 infection in formalin-fixed and paraffin-embedded placenta and fetal tissues.

Parvovirus B19 infection was first discovered in 1975 and it is implicated in fetal death from hydrops fetalis the world over. Diagnosis is usually made through histological identification of the intranuclear inclusion in placenta and fetal organs. However, these cells may be scarce or uncharacteristic, making definitive diagnosis difficult. We analyzed histologically placentas and fetal organs from 34 cases of non-immune hydrops fetalis, stained with Hematoxylin and Eosin (HE) and submitted to immunohistochemistry and polymerase chain reaction (PCR). Of 34 tissue samples, two (5.9%) presented typical intranuclear inclusion in circulating normoblasts seen in Hematoxylin and Eosin stained sections, confirmed by immunohistochemistry and PCR. However, PCR of fetal organs was negative in one case in which the placenta PCR was positive. We concluded that parvovirus B19 infection frequency is similar to the literature and that immunohistochemistry was the best detection method. It is highly specific and sensitive, preserves the morphology and reveals a larger number of positive cells than does HE with the advantage of showing cytoplasmic and nuclear positivity, making it more reliable. Although PCR is more specific and sensitive in fresh or ideally fixed material it is not so in formalin-fixed paraffin-embedded tissues, frequently the only one available in such cases.

Detection of parvovirus B19 infection in formalin-fixed and paraffin-embedded placenta and fetal tissues

Parvovirus B19 infection was first discovered in 1975 and it is implicated in fetal death from hydrops fetalis the world over. Diagnosis is usually made through histological identification of the intranuclear inclusion in placenta and fetal organs. However, these cells may be scarce or uncharacteristic, making definitive diagnosis difficult. We analyzed histologically placentas and fetal organs from 34 cases of non-immune hydrops fetalis, stained with Hematoxylin and Eosin (HE) and submitted to immunohistochemistry and polymerase chain reaction (PCR). Of 34 tissue samples, two (5.9 percent) presented typical intranuclear inclusion in circulating normoblasts seen in Hematoxylin and Eosin stained sections, confirmed by immunohistochemistry and PCR. However, PCR of fetal organs was negative in one case in which the placenta PCR was positive. We concluded that parvovirus B19 infection frequency is similar to the literature and that immunohistochemistry was the best detection method. It is highly specific and sensitive, preserves the morphology and reveals a larger number of positive cells than does HE with the advantage of showing cytoplasmic and nuclear positivity, making it more reliable. Although PCR is more specific and sensitive in fresh or ideally fixed material it is not so in formalin-fixed paraffin-embedded tissues, frequently the only one available in such cases.

O parvovírus B19 foi detectado em 1975 e desde sua descoberta tem se mostrado um agente infeccioso importante em seres humanos, cujo diagnóstico pode ser feito pelo exame histológico através do encontro de inclusão nuclear em tecidos fetais ou placentários. No entanto, estas células podem ser escassas ou não apresentarem características típicas, dificultando o diagnóstico. Analisamos placentas e órgãos fetais de 34 casos de hidropisia fetal não-imune corados com Hematoxilina e Eosina (HE) e submetidos à reação em cadeia da polimerase (PCR) e imuno-histoquímica (IH). Em dois casos (5,9 por cento) houve positividade na placenta pelo HE, IH e PCR. No entanto, PCR dos órgãos fetais foi negativa em um destes casos enquanto que a identificação pôde ser feita por IH e histologia. Concluímos que a freqüência do parvovírus B19 é similar à literatura e a reação IH foi o melhor método de detecção, com identificação mais específica e segura, permitindo identificação citoplasmática, o que não é possível pelo exame histopatológico. A PCR pode apresentar falsa negatividade, provavelmente pela fixação, não identifica as células e é mais dispendiosa. Embora mais específica e sensível em material a fresco ou idealmente fixado isto não ocorre com tecidos fixados em formalina e embebidos em parafina, freqüentemente os únicos disponíveis.

Hydrops fetalis and neonatal death from human parvovirus B19: an unusual complication.

A case of prenatally diagnosed human parvovirus B19 (HPVB19) infection is reported. The neonate died after intrauterine therapy and premature delivery. The fetus was diagnosed with oedema, cardiomegaly, poor myocardial contractility and a pericardial effusion at 24/40 weeks' gestation. Ultrasound using colour flow Doppler showed a midcerebral artery peak systolic velocity (MCA PSV) raised at 45 cm/s, suggesting fetal anaemia. This was confirmed on fetal blood sampling, but recovery was suggested with a reticulocyte count of 16.8%. The fetal karyotype was normal, 46,XY. Fetal IgM was positive for Parvovirus. A week later, severe fetal anaemia was suspected and intrauterine transfusion carried out. Altogether three transfusions were given. At 31/40 weeks, the mother presented to her local hospital with suspected preterm labour, a caesarean section was carried out because of fetal compromise on cardiotocography. The baby was in poor condition at birth and resuscitation was stopped at 45 min of age. The post-mortem examination confirmed the hydrops and proved persistent Parvovirus infection, cardiac involvement and severe liver fibrosis.HPVB19 generally follows a benign course with intrauterine therapy; however, in this case, the fetus died despite successful transfusions. The reasons for this are discussed.

[Parvovirus B19 infection--dangerous for the fetus]. / Parvovirus B19-infeksjon--en livstruende fostersykdom.

BACKGROUND: Human parvovirus B19 infection is a common and usually benign disease in children, but may cause severe complications in pregnancy. We determined the proportion of the pregnant population at risk of infection and present a recent cluster of cases with fetal complications, hoping to raise the general awareness. MATERIAL AND METHODS: Five cases of fetal parvovirus B19 infection during 2001--2 illustrate the delay in diagnosis, treatment and outcome. During the same period, the number of positive and negative serological tests was noted for the catchment area and the entire country. Additionally, 206 consecutive pregnant women were tested to determine susceptibility. RESULTS: Out of the five cases of fetal complications, all had cardiac involvement, three had anaemia, and two fetuses died. Lag-time to fetal diagnosis was six, eight, and 13 weeks for those with known maternal disease. In Norway, seroconversion is similar to that found in other Nordic countries; 36 % (74/206) of the pregnant population is seronegative. In Norway, parvovirus B19 infection is more frequently reported in the January-June period. A third of the pregnant population is susceptible to infection. Early testing in cases of exposure or suspected infection in pregnancy is recommended in order to ensure appropriate follow-up and treatment.

Fetal morbidity and mortality after acute human parvovirus B19 infection in pregnancy: prospective evaluation of 1018 cases.

OBJECTIVE: To determine more precisely the incidence of fetal complications following maternal parvovirus B19 infection at various gestational ages. METHODS: An observational prospective study of 1018 pregnant women whose acute B19 infection was serologically confirmed in our laboratory. RESULTS: The observed rate of fetal death throughout pregnancy was 6.3% (64/1018) (95% confidence interval [CI]: 4.9, 8.0). The fetal death rate for those infected within the first 20 weeks of gestation (WG) was 64/579 (11.0%). Fetal death was only observed when maternal B19 infection occurred before the completed 20 WG. The observed stillbirth proportion was 0.6% (6/960). Three of six stillbirth cases presented with fetal hydrops. The overall risk of hydrops fetalis was 3.9% (40/1018) (95% CI: 2.8, 5.3). Three of 17 cases with non-severe hydrops and 13 of 23 cases with severe hydrops received intrauterine transfusion(s). The proportion of fetuses with severe hydrops that survived following fetal transfusions was 11/13 (84.6%). All of the non-transfused fetuses with severe hydrops died. CONCLUSION: Our data demonstrate a relevant B19-associated risk of fetal death, which is largely confined to maternal B19 infection in the first 20 WG. Timely intrauterine transfusion of fetuses with severe hydrops fetalis reduces the risk of fetal death. Parvovirus B19-associated stillbirth without hydropic presentation is not a common finding.

Amniotic band syndrome with significant orofacial clefts and disruptions and distortions of craniofacial structures.

Amniotic band syndrome (ABS) is a rare disorder in which bands of mesoderm that emanate from the chorionic side of the amnion and insert on the fetal body can generate a wide variety of disfiguring and disabling malformations. It usually is sporadic, and the incidence is approximately 1 in 15,000 live births, and affected children typically require involvement of several pediatric surgical subspecialties. The authors describe a case of ABS with extensive craniofacial anomalies.

Long term follow up of serostatus after maternofetal parvovirus B19 infection.

BACKGROUND: Maternofetal parvovirus B19 infection may result in fetal hydrops or abortion. Chronic infection has been associated with long term complications (polyarthritis, persistent aplastic anaemia, hepatitis). In pregnancy maternal immunosuppression caused by a TH2 dominant response to viral antigens has been observed. There is little information on long term reactivity to intrauterine infection. AIMS: To assess the serological status in children and their mothers after maternofetal parvovirus B19 infection and development of fetal hydrops. METHODS: A total of 18 children and their mothers, and 54 age matched control infants were studied. Main outcome measures were parvovirus B19 DNA, specific IgM and IgG against the virus proteins VP1/VP2, and NS-1 in venous blood. RESULTS: Parvovirus B19 DNA and antiparvovirus B19 (IgM) were undetectable in all sera. A significant larger proportion of maternal sera compared to study children's sera contained IgG against the non-structural protein NS-1. Mean levels of VP1/VP2 IgG antibodies were significantly lower in the children than in their mothers (48 (36) v 197 (95) IU/ml). There was no history of chronic arthritis in mothers and children. Five women had subsequent acute but transient arthritis postpartum, which was not correlated with antibodies against NS-1. CONCLUSIONS: Serological evidence of persistent infection after maternofetal parvovirus B19 disease could not be detected. Increased maternal prevalence of anti NS-1 (IgG) and increased levels of antiparvovirus B19 (IgG) may reflect prolonged viraemia compared to fetal disease.

Prenatal diagnosis of parvovirus B19-induced hydrops fetalis by chemiluminescence in situ hybridization.

Parvovirus B19 can be transmitted transplacentally from the infected mother to the fetus during pregnancy, and hydrops fetalis, abortion, or stillbirth can result. In our study we explored the use of chemiluminescence in situ hybridization to detect B19 DNA on cord blood cells, amniotic fluid cells, and pleuric fluid cells from several cases of hydrops fetalis. B19 DNA was detected by using digoxigenin-labeled probes immunoenzymatically visualized with the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate for alkaline phosphatase. The luminescent signal emitted from the hybridized probes was detected, analyzed, and measured with a high-performance, low-light-level imaging luminograph connected to an optical microscope and to a personal computer for the quantification and localization of the chemiluminescent emission inside individual cells.

Diagnosis and incidence of fetal parvovirus infection in an autopsy series: II. DNA amplification.

This study evaluates the practical utility of the polymerase chain reaction (PCR) as a diagnostic method for intrauterine fetal parvovirus infection in cases of hydrops fetalis. Paraffin-embedded, formalin-fixed fetal tissues from cases of hydrops fetalis were assessed for parvovirus B19 by histology and PCR in conjunction with 32P hybridization. Of 673 fetal and neonatal autopsies performed at Women and Infants' Hospital for the years 1985 through 1990, 32 cases were determined to have hydrops fetalis, of which five were positive for parvovirus infection by both histology and the PCR. PCR was not used in seven (22%) of the 32 hydrops cases because 1 microgram of DNA was not available for study. Histology was as sensitive as PCR in detecting parvovirus B19 in fetal autopsy tissues from cases of hydrops fetalis, and could be used reliably in each case to diagnose parvovirus infection. In our hands, histology is as sensitive as PCR and less labor-intensive. We would reserve PCR for cases without inclusions and with a strong suspicion of parvovirus infection, or for fluids in which histological analysis is not available.

Diagnosis and incidence of fetal parvovirus infection in an autopsy series: I. Histology.

This study was undertaken to obtain additional information about the incidence and characteristics of fetal parvovirus B19 infection in an unselected autopsy series, and to assess the distribution and quantitation of inclusions in various organs. Autopsy records from 673 fetal and neonatal autopsies performed at Women and Infants' Hospital during 1985 through 1990 were reviewed. Thirty-two cases of hydrops fetalis were identified, and, of these, 5 had parvovirus infection. This gives an incidence of fetal parvovirus infection resulting in hydrops fetalis of 0.7% among all autopsies, and a 16% incidence among cases of hydrops. Thirty-five percent of the cases of hydrops had malformations; a muscular ventricular septal defect was noted in one of the 5 cases of parvovirus infection. All 5 parvovirus cases had characteristic erythroid nuclear inclusions, and these inclusions were resistant to tissue degenerative changes. The most reliable tissue for histologic diagnosis was the liver, followed by heart and lung. Only 2 of 5 placentas had diagnostic inclusions, making examination of the placenta alone insufficient for ruling out fetal parvovirus infection.

[Molecular biological demonstration of parvovirus infection in fetal tissue]. / Molekularbiologischer Nachweis einer Parvovirusinfektion in fetalem Gewebe.

We report on the morphological findings in 16 fetuses with serologically confirmed maternal parvovirus B19 infection. Typical routine morphological findings of the hydropic fetuses were the presence of abnormal erythroblasts with typical nuclear inclusions. These infected cells positively stained immunohistochemically with antibodies against a recombinant virus protein, as well as they ultrastructurally contained virus particles. Using in-situ hybridization techniques, we were able to demonstrate the presence of parvovirus B19 genome in the infected cells, while no other cell type was shown to contain virus genome. According to our results the differential diagnosis of fetal parvovirus B19 infection should be considered in each case with hydrops fetalis of unknown origin. The careful routine microscopic examination of fetal tissue may provide evidence for parvovirus infection which should be confirmed by in-situ hybridization analysis.