Coding-complete sequence of a vaccine-derived recombinant porcine reproductive and respiratory syndrome virus strain isolated in Hungary.

Porcine reproductive and respiratory syndrome virus 1 is a major cause of swine morbidity and mortality in various parts of the world, including Hungary. A national elimination programme to reduce the associated economic burden was initiated in Hungary in 2012. Using extensive laboratory surveillance, we identified and isolated an unusual PRRSV strain. The complete coding sequence of this isolate was determined and analyzed. The genome of this Hungarian PRRSV1 strain, HUN60077/16, is 15,081 nucleotides in length. Phylogenetic and recombination analysis showed a mosaic structure of the genome where a large fragment of ORF1b and the genomic region coding for ORF3 to ORF7 showed a very close genetic relationship to the vaccine virus Unistrain, while the ORF1a region, the 3' end of ORF1b, and the whole ORF2 were only distantly related to this or any other PRRSV1 strain whose genome sequence is available in the GenBank database. Genomic characterization of PRRSV strains is crucial when possible vaccine-associated cases are identified. This approach not only helps to identify genetic interactions between vaccine and wild-type PRRSV1 strains but may also be needed to prevent trust in commercial vaccines from being undermined.

Correction to: Investigation of brain structure in the 1-month infant.

The authors regret that, in this article, there was an error in the analyses comparing infant male and female regional brain volume differences.

Investigation of brain structure in the 1-month infant.

The developing brain undergoes systematic changes that occur at successive stages of maturation. Deviations from the typical neurodevelopmental trajectory are hypothesized to underlie many early childhood disorders; thus, characterizing the earliest patterns of normative brain development is essential. Recent neuroimaging research provides insight into brain structure during late childhood and adolescence; however, few studies have examined the infant brain, particularly in infants under 3 months of age. Using high-resolution structural MRI, we measured subcortical gray and white matter brain volumes in a cohort (N = 143) of 1-month infants and examined characteristics of these volumetric measures throughout this early period of neurodevelopment. We show that brain volumes undergo age-related changes during the first month of life, with the corresponding patterns of regional asymmetry and sexual dimorphism. Specifically, males have larger total brain volume and volumes differ by sex in regionally specific brain regions, after correcting for total brain volume. Consistent with findings from studies of later childhood and adolescence, subcortical regions appear more rightward asymmetric. Neither sex differences nor regional asymmetries changed with gestation-corrected age. Our results complement a growing body of work investigating the earliest neurobiological changes associated with development and suggest that asymmetry and sexual dimorphism are present at birth.

Resurgence of rabies in Hungary during 2013-2014: An attempt to track the origin of identified strains.

In 2013-2014, accumulation of rabies episodes raised concerns regarding ongoing elimination programme in Hungary. Nearly four dozen cases were identified over a 13-month period in the central region of the country far behind the immunization zones. Although the outbreak was successfully controlled, the origin of disease remained unknown. In this study, we sequenced the partial N and G genes from 47 Hungarian rabies virus (RV) strains isolated from the 2013-2014 outbreak. Sequencing and phylogenetic analysis of the N and G genes showed that the Hungarian RV isolates share high nucleotide similarity among each other (up to 100%). When analysing the N gene, comparable sequence similarity was seen between the outbreak strains and some historic Romanian RV strains. Unfortunately, in the lack of available sequence data from the Romanian RV strains, the genetic relationship within the G gene could not be determined. Phylogenetic analysis of Hungarian RV isolates detected in the past revealed that multiple independent RV lineages circulated in our country over the past 25 years. The parental strain of the 2013-2014 outbreak may have been imported independently perhaps from east through transborder movement of a reservoir animal. Next to the introduction, this imported RV strain seems to have spread clonally in the affected area. Our findings indicate that despite effective control measures that, overall, minimized the incidence of rabies over the past decade, field and laboratory monitoring needs to be continued to make rabies elimination programme in Hungary successful.

Mapping White Matter Microstructure in the One Month Human Brain.

White matter microstructure, essential for efficient and coordinated transmission of neural communications, undergoes pronounced development during the first years of life, while deviations to this neurodevelopmental trajectory likely result in alterations of brain connectivity relevant to behavior. Hence, systematic evaluation of white matter microstructure in the normative brain is critical for a neuroscientific approach to both typical and atypical early behavioral development. However, few studies have examined the infant brain in detail, particularly in infants under 3 months of age. Here, we utilize quantitative techniques of diffusion tensor imaging and neurite orientation dispersion and density imaging to investigate neonatal white matter microstructure in 104 infants. An optimized multiple b-value diffusion protocol was developed to allow for successful acquisition during non-sedated sleep. Associations between white matter microstructure measures and gestation corrected age, regional asymmetries, infant sex, as well as newborn growth measures were assessed. Results highlight changes of white matter microstructure during the earliest periods of development and demonstrate differential timing of developing regions and regional asymmetries. Our results contribute to a growing body of research investigating the neurobiological changes associated with neurodevelopment and suggest that characteristics of white matter microstructure are already underway in the weeks immediately following birth.

Hemodynamic changes in patients with arteriovenous malformations assessed using high-resolution 3D radial phase-contrast MR angiography.

BACKGROUND AND PURPOSE: Arteriovenous malformations have a high lifetime risk of hemorrhage; however, treatment carries a significant risk of morbidity and mortality, including permanent neurologic sequelae. WSS and other hemodynamic parameters are altered in patients with symptomatic AVMs, and analysis of hemodynamics may have value in stratifying patients into different risk groups. In this study, we examined hemodynamic data from patients with stable symptoms and those who presented with acute symptoms to identify trends which may help in risk stratification. MATERIALS AND METHODS: Phase-contrast MRA using a radial readout (PC-VIPR) is a fast, high-resolution technique that can acquire whole-brain velocity-encoded angiograms with scan times of approximately 5 minutes. Ten patients with AVMs were scanned using PC-VIPR; velocity, area, flow, and WSS in vessels feeding the AVMs and normal contralateral vessels were calculated using velocity data from the phase-contrast acquisition. RESULTS: Patients with an asymptomatic presentation or mild symptoms (n = 4) had no significant difference in WSS in feeding vessels compared with normal contralateral vessels, whereas patients presenting with hemorrhage, severe headaches/seizures, or focal neurologic deficits (n = 6) had significantly higher WSS in feeding vessels compared with contralateral vessels. CONCLUSIONS: In this study, we demonstrate that estimates of WSS and other hemodynamic parameters can be obtained noninvasively in patients with AVMs in clinically useful imaging times. Variation in WSS between feeders and normal vessels appears to relate to the clinical presentation of the patient. Further analysis of hemodynamic changes may improve characterization and staging of AVM patients, when combined with existing risk factors.

Velocity measurements in the middle cerebral arteries of healthy volunteers using 3D radial phase-contrast HYPRFlow: comparison with transcranial Doppler sonography and 2D phase-contrast MR imaging.

BACKGROUND AND PURPOSE: We have developed PC HYPRFlow, a comprehensive MRA technique that includes a whole-brain CE dynamic series followed by PC velocity-encoding, yielding a time series of high-resolution morphologic angiograms with associated velocity information. In this study, we present velocity data acquired by using the PC component of PC HYPRFlow (PC-VIPR). MATERIALS AND METHODS: Ten healthy volunteers (6 women, 4 men) were scanned by using PC HYPRFlow and 2D-PC imaging, immediately followed by velocity measurements by using TCD. Velocity measurements were made in the M1 segments of the MCAs from the PC-VIPR, 2D-PC, and TCD examinations. RESULTS: PC-VIPR showed approximately 30% lower mean velocity compared with TCD, consistent with other comparisons of TCD with PC-MRA. The correlation with TCD was r = 0.793, and the correlation of PC-VIPR with 2D-PC was r = 0.723. CONCLUSIONS: PC-VIPR is a technique capable of acquiring high-resolution MRA of diagnostic quality with velocity data comparable with TCD and 2D-PC. The combination of velocity information and fast high-resolution whole-brain morphologic angiograms makes PC HYPRFlow an attractive alternative to current MRA methods.

The effect of spatial resolution on wall shear stress measurements acquired using radial phase contrast magnetic resonance angiography in the middle cerebral arteries of healthy volunteers. Preliminary results.

We have recently implemented radial phase-contrast techniques that allow high resolution angiograms with velocity information to be acquired within clinically-useful imaging times. 10 healthy volunteers were scanned using PC-VIPR and PC-SOS, two high resolution phase-contrast techniques at spatial resolutions of 0.67×0.67×0.67 mm(3) and 0.4×0.4×1 mm(3) respectively. The velocity measurements from the two acquisitions were imported into a custom Matlab runtime environment that automatically calculated WSS values using Green's Theorem and B-spline interpolation. Time average axial WSS was 1.069 N/m(2) (95% confidence interval: 0.8628< x < 1.276) in the left and right middle cerebral arteries of the 10 healthy volunteers (n=20) when scanned by PC-VIPR, and 1.670 N/m(2) when scanned by PC-SOS (95% confidence interval: 1.395 < x < 1.946). This difference in means was statistically significant (p < 0.002). Previous investigators have found that higher spatial resolution results in higher WSS measurements because smaller voxel size results in fewer partial volume effects. This was true in our study as well. In this study, we found that PC-SOS has significantly higher spatial resolution than PC-VIPR and this followed in the WSS measurements. Higher in-plane spatial resolution allows WSS calculations to be performed more accurately because of increased precision near the vessel boundary.

Simulation of relative temporal resolution of time-resolved MRA sequences.

Time-resolved MRA sequences are typically characterized by the display frame rate. However, true temporal resolution should be defined in a manner analogous to spatial resolution; it is not the ability of a sequence to update rapidly but rather the ability to discern changes that occur within a small time that should characterize temporal resolution. For view-shared methods like Keyhole and time-resolved imaging of contrast kinetics (TRICKS), regions of k-space from multiple time frames are combined to form a single reconstructed time frame. This often causes the time needed to acquire all k-space data points to be significantly longer than the displayed frame time, resulting in a poor frequency response. Simulated here are the temporal impulse response and temporal frequency response (TFR) curves of three time-resolved MRA methods, including the recently introduced highly-constrained backprojection local reconstruction (HYPR LR) method. It is found that the HYPR LR reconstruction method exhibits a better TFR for a larger spectrum of temporal and spatial frequencies than the Keyhole and TRICKS methods.

Pacheco's disease in a Hungarian zoo bird population: a case report.

An epizootic of Pacheco's disease is reported from a zoo bird population. The infection was introduced by wild-captured Patagonian conures (Cyanoliseus patagonus) despite 61 days of quarantine. The disease affected several parrot species and, interestingly, three out of seven bearded barbets (Lybius dubius). The mortality rate was 30.93%. Autopsy revealed abdominal hyperaemia with liver haemorrhages and, in less rapid cases, yellowish discoloration and fragility of the liver. Death was caused by the collapse of circulation. Histopathology demonstrated liver cell necrosis, disintegration of the lobular structure, and a few intranuclear inclusion bodies. Icosahedral virions were detected by electron microscopy. The virus was isolated in the allantoic cavity of embryonated chicken eggs as well as in chicken embryo fibroblast cell culture. A 281-bp-long fragment of psittacid herpesvirus DNA was detected by PCR in cell culture material and liver samples of the affected birds. To our knowledge this is the first report of Pacheco's disease in bearded barbets as well as the first occurrence of Pacheco's disease in Hungary.

Simultaneous detection of three porcine viruses by multiplex PCR.

Specific oligonucleotide primers were selected and combined in a multiplex arrangement, in order to detect simultaneously three economically important porcine viruses by polymerase chain reaction (PCR). The pathogen panel was comprised of viruses that cause reproductive failure in infected herds: Aujeszky's disease virus (ADV), porcine parvovirus (PPV) and porcine respiratory and reproductive syndrome virus (PRRSV). In order to reduce the time required for the detection of the pathogens, the assay was optimised to a RapidCycler PCR instrument. The multiplex PCR assay was shown to be specific, sensitive and rapid, because the results were read in less than 60 min after sample preparation. Due to its speed, efficiency and sensitivity, the described rapid multiplex PCR assay serves as a useful novel tool in the veterinary diagnostic laboratories for the quick and complex detection of these important porcine pathogens.

Rapid and simultaneous detection of avian influenza and newcastle disease viruses by duplex polymerase chain reaction assay.

A duplex reverse transcription-polymerase chain reaction (dRT-PCR) assay has been developed for the simultaneous, rapid and specific detection/discrimination of avian influenza virus (AIV) and Newcastle disease virus (NDV). Primers targeting the matrix protein gene (M) of AIV and the fusion protein gene (F) of NDV were evaluated experimentally with 13 AIV and 19 NDV strains. PCR products of the expected size of 144 bp and 316 bp were amplified from AIV/NDV samples, respectively, while no cross-reaction was observed with negative controls or with 16 other avian pathogens. The endpoint of detection was defined as approximately 10(+0.5) 50% egg infectious dose (EID(50))/0.2 ml for AIV and 10(+2.2) EID(50)/0.2 ml for NDV. The assay was able to detect AIV/NDV with similar sensitivity in spiked stool samples and in specimens from vaccinated birds. The developed dRT-PCR assay is a rapid, cost-effective tool, which provides powerful novel means for the early diagnosis of avian influenza and Newcastle disease.

Application of polymerase chain reaction and virus isolation techniques for the detection of viruses in aborted and newborn foals.

The occurrence of two important pathogens, equine herpesvirus 1 (EHV1) and equine arteritis virus (EAV) causing abortions, perinatal foal mortality and respiratory disease, was investigated by polymerase chain reaction (PCR) and virus isolation to demonstrate the presence of abortigenic viruses in samples from 248 horse fetuses in Hungary. We found 26 EHV1- and 4 EAV-positive aborted or prematurely born foals from 16 and 4 outbreaks, respectively, proving that despite the widely applied vaccination, EHV1 is a far more important cause of abortions in the studs than EAV. We compared the virus content of different organs of the fetuses by PCR and isolation to identify the organ most suitable for virus demonstration. Our investigations indicate that the quantity of both viruses is highest in the lungs; therefore, according to our observations, in positive cases the probability of detection is highest from lung samples of aborted or newborn foals. Both the PCR and the virus isolation results revealed that the liver, though widely used, is not the best organ to sample either for EHV1 or for EAV detection. From the analysis of the epidemiological data, we tried to estimate the importance of the two viruses in the Hungarian horse population.

Genetic variation of the prevailing porcine respiratory and reproductive syndrome viruses occurring on a pig farm upon vaccination.

Recurrence of porcine respiratory and reproductive syndrome (PRRS) was observed on a pig farm after introducing two PRRS live virus vaccines to combat preceding outbreaks. The phylogenetic analysis of the nucleotide sequence encoding the GP5 envelope glycoprotein and the nucleocapsid protein coding sequences (ORF5 and ORF7, respectively) showed a close genetic relationship between the new outbreak-related and one of the vaccine viruses, while the prevailing PRRS virus genetic variants disappeared from the farm. These findings, supported by the epidemiological data, indicate that the new variant PRRS viruses might originate from a vaccine virus and demonstrate the limited efficacy of modified live vaccines against heterologous PRRS virus strains.

Phylogenetic analysis of rabbit haemorrhagic disease virus (RHDV) strains isolated between 1988 and 2003 in eastern Hungary.

To define the genetic variability of RHDV strains collected in eastern Hungary, liver samples from rabbits that had died of RHD were collected between 1988 and 2003. The phylogenetic analysis of a 528-nucleotide-long portion of the gene encoding the VP60 capsid protein assigned the strains into three genogroups. The first group contained viruses from 1988-1993, and a second group comprised isolates from 1994-2002. A third group comprised all of the tested representatives of the RHDVa subtype and a Hungarian isolate from 2003. These findings were supported by the alignments of the deduced amino acid sequences of the VP60 gene and strongly suggest the presence of the RHDVa subtype in Hungary.

Genetic characterization of type 2 porcine circoviruses detected in Hungarian wild boars.

Porcine circoviruses (PCV) are present in pigs worldwide; they are grouped into two types: PCV1 comprising non-pathogenic viruses and PCV2 responsible for several clinical manifestations. Both types are frequently detected in domestic pigs, the prevalence and role of PCV in wild boars however, is not well studied. During the years 2002-2003 over 2000 organ samples of Hungarian wild boars were collected, grouped and samples from 307 different animals were tested by polymerase chain reaction for the presence of PCV. 35.5% of the wild boars were positive for one or both PCV types and PCV2 was detected in 20.5% of the animals. The PCV2 viruses were divided into 7 groups (WB-H1-7) based on sequencing data and genomes representing these groups were sequenced completely. The wild boar PCV2 groups were distributed evenly in the geographical region, regardless of the time and place of collection. The phylogenetic analysis of the PCV2 sequences of wild boar and domestic pig origin showed the possibility of an epidemiological link between wild boar and domestic pig infections. Interestingly, the complete nucleotide sequence of the viruses and the predicted amino acid sequence of the replication associated protein (ORF1) grouped the viruses similarly, whereas the capsid protein (ORF2) comparisons revealed different relations among the groups, suggesting the possibility of genomic recombination in PCV2.

Application of real-time RT-PCR utilising lux (light upon extension) fluorogenic primer for the rapid detection of avian influenza viruses.

A real-time RT-PCR assay utilising light upon extension fluorogenic primer (LUX RT-PCR) was developed for the rapid and efficient detection of avian influenza viruses (AIV). The assay detected each of the AIV isolates tested (16/16) and gave negative results with heterologous pathogens (17/17). The detection limit of the assay proved to be 10(-0.5) EID50/0.2 ml and 10(1.5) EID50/0.2 ml in allantoic fluid of virus-infected embryonated chicken eggs and in spiked chicken faeces samples, respectively. Based on its specificity, sensitivity and relative simplicity, the LUX RT-PCR assay provides a novel, rapid and cost-effective diagnostic tool for avian influenza surveillance and monitoring programs.

Cytopathogenicity markers in the genome of Hungarian cytopathic isolates of bovine viral diarrhoea virus.

Since genetic recombination is a major factor in the evolution of the cytopathogenic (cp) bovine viral diarrhoea virus (BVDV) biotypes, in this study the cytopathogenicity markers were investigated in the genomes of two cp BVDV strains recently isolated from mucosal disease (MD) cases in Hungary. In the genome of strain H4956, a Jiv-like insertion was found similar to those described in reference strain NADL and in other BVDV 1, BVDV 2 and border disease virus (BDV) strains. The 133 amino acid Jiv-like sequence is inserted at nucleotide position 4984 (amino acid position 1533), 9 nucleotides upstream of that of strain NADL. The insertion showed 96% amino acid sequence identity with the cellular Jiv protein. In the genome of cp BVDV strain H115/PCR, an ubiquitin-containing duplication was found. The duplicated sequence started at nucleotide position 7978 (amino acid 2531) in the NS4B gene. The duplication contained a complete ubiquitin monomer of 76 amino acids and the complete NS3 gene starting at nucleotide position 5153 (amino acid 1589), which corresponds to the first N-terminal amino acid of NS3. The duplication was located further downstream of the known ubiquitin-containing genomic regions of cp BVDV strains, and it consisted of the shortest inserted nucleotide sequence. The insertions and duplication of strains H4956 and H115/PCR further confirmed that recombinations occurring at positions A and B are the most common mechanisms leading to the development of BVDV cytopathogenicity.