Case report: severe neurological manifestation of Dobrava hantavirus infection

In Europe infection with Puumala or Dobrava viruses causes hemorrhagic fever with renal syndrome (HFRS). In the course of HFRS, mild neurological symptoms such as headache, vertigo, and nausea are common. However, the data about the occurrence of severe, potentially life-threatening neurological manifestations are rather scarce. Here, we present a case of HFRS with serologically proven Dobrava virus infection complicated by epileptic seizures and hemiparesis due to focal encephalitis.     

Genetic detection of Dobrava/Belgrade virus in a Czech patient with Haemorrhagic fever with renal syndrome

In the summer of 2008, a 15-year-old boy was hospitalized in a paediatric intensive care unit in the Czech Republic. Laboratory diagnosis of hantavirus infection was established by serological and molecular methods. Sequence and phylogenetic analyses showed that the causative strain was Dobrava/Belgrade virus, which is genetically closer to strains associated with Apodemus flavicollis rodents.     

Puumala and Dobrava viruses cause hemorrhagic fever with renal syndrome in Bosnia-Herzegovina: Evidence of highly cross-neutralizing antibody responses in early patient sera

Hantavirus infection was diagnosed serologically by μ-capture IgM and IgG ELISAs in hemorrhagic fever with renal syndrome (HFRS) patients admitted to Tuzla Hospital, Bosnia-Herzegovina. The results indicated that more than one hantavirus caused the outbreak. To address the question of which hantavirus serotypes were involved, sequentially drawn sera were analyzed by focus reduction neutralization test (FRNT) for antibodies against Puumala, Hantaan, Dobrava, and Seoul hantaviruses. The data revealed that acute- or early convalescent-phase sera, even when drawn as late as 3 weeks after the onset of disease, could not be used for typing of the causative hantavirus; a significant number of these samples showed similar reactivity of neutralizing antibodies to several different hantavirus serotypes. Moreover, although several acute-phase sera showed the highest FRNT titer to Hantaan virus, convalescent sera from these patients in all cases showed high specificity for Puumala or Dobrava viruses. This phenomenon, interpreted as a cross-neutralizing primary antibody response, makes several earlier reports concerning causative agents of HFRS questionable. Serological examination of small rodents trapped in the endemic area identified Puumala- and Dobrava-like virus infections. RT-PCR and sequencing of rodent lung samples identified Dobrava virus in one yellow-necked field mouse (Apodemus flavicollis). Cross-FRNT data, using polyclonal rabbit antibodies, clearly confirmed Dobrava virus as a unique hantavirus serotype. In conclusion, the results revealed that both Puumala- and Dobrava-like viruses caused HFRS in Bosnia-Herzegovina, whereas no signs of Hantaan or Seoul virus involvement were found. J. Med. Virol. 53:51–59, 1997. © 1997 Wiley-Liss, Inc.     

Using the polymerase chain reaction to maintain DNA probe inventories in clinical and diagnostic laboratories

Clinical and diagnostic DNA laboratories must maintain a large inventory of DNA probes for use in hybridization studies. The preparation of plasmid DNA and isolation of DNA fragments for use as probes in both expensive and time consuming. We present here a rapid and relatively inexpensive method of producing large amounts of DNA fragments from stocks, using the polymerase chain reaction (PCR). Our experience over the past year using this technique has been very positive and we believe many laboratories could benefit by employing such a labor-saving approach to maintaining DNA probes. The technique uses the bacteriophage M13 DNA sequencing primers to amplify cloned inserts contained in commonly used plasmid vectors. As examples, we illustrate the use of DNA produced in this manner as probes for linkage analysis of the fragile X syndrome and for detection of deletions in the Duchenne muscular dystrophy gene. We have also found that at least two probes can be amplified in the same PCR reaction, allowing the detection of two different restriction fragment length polymorphisms (RFLP) simultaneously. It should be possible for laboratories to devise strategies particular to their individual needs using more than one DNA probe produced in the same PCR reaction to detect RFLP's. Such strategies would need only to consider that the predicted alleles of the multiple polymorphisms do not migrate to the same position during electrophoresis. Stocks of single or multiple probes produced by the PCR could then be maintained for more rapid Southern analyses.     

Identification and characterization of a Hantavirus strain of unknown origin by nucleotide sequence analysis of the cDNA derived from the viral S RNA segment

The genetic characterization of a serologically Hantaan-like virus but of unknown origin (termedDX) was carried out by molecular cloning and nucleotide sequencing of the corresponding cDNA of the viral S RNA segment. The S RNA was found to be 1765 nucleotides long with 3 and 5 termini being complementary for 24 bases. The virus messenger-sense RNA contains one major open reading frame (ORF) encoding 428 amino acids or a 50 kD polypeptide. A comparison of the DX S RNA segment to those of Sapporo rat, Hantaan, Puumala/Hällnäs B1, and Prospect Hill viruses reveals 95.4, 71.3, 55.3, and 60.9% homology at the nucleotide sequence level, and 94.7, 80.1, 58.4, and 59.8% at the deduced amino acid sequence level. Thus Hantavirus strain DX is very closely related to Sapporo rat virus. We also analyzed the S RNA segments of these Hantaviruses for the presence of a second ORF encoding a potential nonstructural NSs protein. All potential second ORFs detected in the different S RNA segments differ substantially in length and position among the viruses, despite the high conservation of the nucleotide sequences and the overall structure of the nucleocapsid proteins. This suggests that the nucleocapsid protein is the only polypeptide encoded by Hanta-virus S RNA segments, setting them apart from the other members of the Bunyaviridae family.     

汉坦病毒R36株的基因分型研究

采用汉坦病毒Ｍ片段特异性核苷酸分型引物，对Ｒ３６等７株汉坦病毒进行反转录和聚合酶链式反应扩增鉴定，结果Ｒ３６株仅能被Ⅰ型分型引物扩增出特异性片段，而不能被Ⅱ型引物扩增；ＰＣＲ产物的序列分析结果表明，Ｒ３６与ＨＴＮ型病毒７６－１１８株的同源性为７８．４％，与ＳＥＯ型病毒Ｒ２２株的同源性为６８．１％。文中对Ｒ３６与汉坦病毒其它毒株的核苷酸同源性进行了配对比较。该项研究为从核苷酸水平对Ｒ３６株的分型，提供了科学依据。     

我国疫区HFRS患者汉滩病毒核蛋白特异性CTL克隆的建立

目的：探讨汉滩病毒核衣壳蛋白（HTNV-NP）诱生的特异性细胞免疫的分子基础，阐明HFRS的发病机理。方法：分离HFRS患者的PBMC，建立HTNV-NP特异性的CTL克隆，同时将克隆有HINV S基因不同片段的真核表达载体转染患者自身的B淋巴母细胞系（BLCL），建立CTL靶细胞系，并进行细胞杀伤试验。结果：建立的特异性CTL克隆对表达完整NP、NP羧基和氨基端肽段的靶细胞均有比较明显的杀伤效应，平均杀伤率分别为50.2%、39.0%和25.4%。结论：NTNV-NP优势T细胞表位可能主要位于病毒核蛋白的羧基端。     

Identification of Medically Important Candida Species by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Analysis of the rDNA ITS1 and ITS2 Regions

Aim: We aimed to identify the distribution of species in candidal strains isolated from clinical samples and restriction fragment length polymorphism (RFLP) method based on Msp I and Bln I restrictive enzyme cuts of polymerase chain reaction (PCR) products after the amplification of ITS1 and ITS2 regions of rDNA genotypically. Materials and Methods: One hundred and fifty candidal strains isolated from various clinical samples were studies/included. Phenotypic species assessment was performed using automated VITEK-2 system and kit used with the biochemical tests. Common genomic region amplification peculiar to candidal strains was carried out using ITS1 and ITS2 primer pairs. After the amplification, PCR products were cut with Msp I and Bln I restriction enzymes for species identification. Results: The majority of Candida isolates were isolated from urine (78.6%) while other isolates were composed of strains isolated from swab, wound, blood and other samples by 11.3%, 3.3%, 2% and 4.7%, respectively. The result of RFLP analysis carried out with Msp I and Bln I restriction enzymes showed that candidal strains were Candida albicans by 45.3%, Candida glabrata by 19.3%, Candida tropicalis by 14.6%, Candida parapsilosis by 5.3%, Candida krusei by 5.3%, Candida lusitaniae by 0.6% and other candidal strains by 9.3%. Conclusion: When the ability to identify Candida to species level of phenotypic and PCR-RFLP methods was assessed, a great difference was found between these two methods. It may be argued that Msp I and Bln I restriction enzyme fragments can be used in the identification of medically important Candida species. Further studies are needed to develop this kind of restriction profile to be used in the identification of candidal strains.     

Restriction fragment length polymorphism in HLA class II genes of Latin-American caucasian celiac disease patients

In the present study Latin-American celiac disease patients were analyzed for the frequency of certain HLA class II restriction fragment length polymorphisms in order to investigate whether they exhibited the normal associated alleles or showed unusual class II variants. A DPB/RsaI 4.0-kb fragment that was shown to be significantly increased among North Americans celiac disease patients of the DR3,DQw2 haplotype was found with similar frequency in Latin-American control and celiac disease individuals. A DPA/BglII 3.7-kb fragment previously shown to be increased among British celiac disease patients was also present with similar frequency among Latin-American control and celiac disease individuals. These results show that the frequency of the HLA-DP region-derived restriction fragment length polymorphisms linked to celiac disease differs between Caucasian populations of different ethnic backgrounds (Anglo-Saxon and Latin-American). On the other hand, DNA samples from 13 patients and 14 controls bearing the DR5/DR7 phenotype (which is significantly associated with celiac disease in Latin populations) were investigated for the presence of particular restriction fragment length polymorphisms disproportionally present in celiac disease individuals. No significant differences were found between controls and patients when the DNA was analyzed with 10 different restriction enzymes and probes for DRB, DQA, DQB, and DPB HLA class II sequences.     

Determination of haemophilia A carrier status from hair samples using polymerase chain reaction technique

The usefulness of intragenic restriction fragment length polymorphisms (RFLPs) for Bc1I, HindIII and XbaI, adapted for polymerase chain reaction (PCR), was tested for the detection of haemophilia A carrier status in the consultand of a family in which only haematological information was available on the inheritance of the trait. Hair follicles were used as the non-invasive source of DNA. The mother was found to be homozygous for Bc1I and heterozygous for HindIII sites, whereas her status as regards informativeness could not be established for XbaI. On the basis of HindIII RFLP, the daughter was found to be a carrier of the haemophilia trait. This was confirmed by sequencing the amplified intron 19 of the mother and the daughter. The RFLP for XbaI did not appear to be suitable for carrier detection using PCR due to the difficulty of establishing homozygosity or heterozygosity from the results of digestion of the amplified product.     

黑龙江地区肾综合征出血热患者中病毒基因检测及流行病学意义

目的进一步了解黑龙江地区HFRS患者中汉坦病毒的基因类别,同时对该病近年来的流行病学特征进行初步分析。方法采用RT-PCR技术对31份肾综合征出血热(HFRS)患者早期血清中的病毒进行基因分型,并根据患者发病季节分成两个感染时段,即冬季(2003年11月—2004年2月),春夏季(2004年4月—2004年9月),再结合临床症状综合分析。结果31份血清中,共有22份血清检测阳性,其中冬季感染时段14份血清,有8例检出阳性(Ⅰ型5例,Ⅱ型3例);春夏季感染时段17份血清,有14例检出阳性(Ⅰ型5例,Ⅱ型9例)。结论黑龙江地区不仅存在汉坦病毒基因Ⅰ型,且同时存在Ⅱ型;冬季以Ⅰ型为主,春夏季以Ⅱ型为主。     

Detection of Hantavirus gene from peripheral blood of patients with HFRS in Heilongjiang province and the epidemiological significance

The aim of this study is to further understand the genotype of Hantavirus(HV) from peripheral blood of patients with hemorrhagic fever with renal syndrome (HFRS) and the epidemiological significance of this disease in Heilongjiang province in recent years. Thirty-one serum samples of clinically diagnosed patients with HFRS were examined by RT-PCR to decide the genetic subtype. On the basis of infection season, the serum samples were divided into two groups: winter (Nov, 2003—Feb, 2004) , spring and summer (April, 2004—Sep, 2004). Further analysis was performed in combination with clinical symptoms. It was found that among the total 31 samples, 22 were sero-positive. Among 14 serum samples in winter, 8 were sero-positive, of which 5 cases were of typeⅠ(Hantaan virus, HTNV) and 3 of typeⅡ(Seoul virus, SEOV). Among 17 samples in spring and summer, 14 were sero-positive, of which 5 cases were of typeⅠand 9 of typeⅡ. So it concludes that both of the two types of Hantavinis exist in Heilongjiang. The typeⅠis the main pathogen of HFRS in winter, and typeⅡis the main in spring and summer.     

中国汉族人群中肿瘤坏死因子-α-238位点基因多态性与血糖、血脂关系的研究

目的 :了解肿瘤坏死因子 α(tumornecrosisfactor alpha,TNF α)启动子上游 -2 3 8位点基因多态性在中国汉族正常人群中的分布及其与血糖、血脂间的关系。方法 :应用聚合酶链反应 (PCR)和限制性片段长度多态性 (RFLP)方法对 182例正常个体基因组DNA中TNF α -2 3 8位点基因多态性进行了观察 ,同时检测血糖、血脂 ,探讨其与基因型间的关系。结果 :正常汉族人群中TNF α -2 3 8位点基因型以GG型发生频率最高 ( 96.7% ) ,GA型较少 ( 3 .3 % )。这种分布特点在正常男女间差异不显著 ,且基因型间血糖、血脂水平比较差异无显著性 (P >0 .0 5 )。与其它种族相比较 ,TNF α -2 3 8位点基因型在中国正常人群中的分布与日本人群中的分布较为接近 ,而与西班牙、西非、荷兰、意大利等人群中的分布差异显著。结论 :TNF α -2 3 8位点基因多态性与血糖、血脂水平无相关性 ,但不同种族间分布存在着一定的差异 ,这种差异有可能是导致一些疾病在不同种族间发病率和临床表现存在不同的因素之一     

Hantaan virus infection in suckling mice: virologic and pathologic correlates

The Hantaan virus suckling mouse model was examined to delineate virologic and histopathologic characteristics of infection at the organ level. Viral antigen and infectious virus were detected in all organs examined, with highest titers achieved in brain, lung, and kidney. A constellation of histologic lesions was identified in brain (diffuse meningoencephalitis with bilaterally symmetrical thalamic necrosis), liver (pericholangiohepatitis), lung (pneumonitis), and spleen (lymphoid hyperplasia); this tetrad is apparently unique to this model system. The chronology of clinical, virologic, serologic, and pathologic findings in Hantaan-infected newborn mice suggests an immune-mediated mechanism in disease pathogenesis.     

Molecular analysis of HLA-DQ A alleles in coeliac disease lack of a unique disease-associated sequence.

Susceptibility to coeliac disease is strongly associated with some HLA class II antigens, encoded by the HLA-D region. Since the HLA-DQ locus seems to be primarily involved, we have analysed by polymerase chain reaction amplification and allele-specific oligonucleotide hybridization the most polymorphic region of the HLA-DQ A1 gene. No difference was observed between the 20 coeliac patients and 20 HLA-D-matched healthy controls who took part in the study. Furthermore, in patients and controls, the restriction fragment length polymorphism analysis of the HLA-DQ A gene using the restriction enzyme BglII did not disclose any specific disease-associated fragment. Our results are not consistent with a unique DQ A coeliac disease-associated sequence, but rather with the hypothesis that some polymorphic residues or allelic hypervariable regions, although found also in the normal population, can predispose to coeliac disease due to their higher frequency in this condition.     

Molecular characterisation of Cryptosporidium: an emerging parasite

Purpose: The aim of the present study was to determine the prevalence of Crytposporidium in local population and to understand its epidemiology by molecular methods. Methods: Faecal samples from 681 children and 804 adults, admitted to tertiary care hospitals in twin cities of Hyderabad and Secunderabad with complaints of diarrhoea; and six calves with diarrhoea, were screened for Cryptosporidium oocysts by microscopy and enzyme linked immunosorbent assay (ELISA). Polymerase chain reaction restriction fragment length polymorphism (PCR RFLP) based identification of Cryptosporidium species in positive specimens was done to elucidate epidemiology of Cryptosporidium. Results: Cryptosporidium was found in 52 (7.6%) children and 7(0.9%) adults and 1(16.6%) calf with diarrhoea. The prevalence of Cryptosporidium in children below five years of age was 8.2% and 14.3% in children in the age group of six months to one year. Of the 42 samples genotyped 29 (69%) were C. hominis and 8 (19%) were C. parvum and 5 (11.9%) were mixed infection with the two species. Conclusions: Children in the age group of six months to one year were found to be the most vulnerable. The occurrence of C. parvum, in nearly one third of cases in the present series indicates that the zoonotic transmission is of considerable significance in the epidemiology of Cryptosporidiosis in the study area.     

Spatiotemporal patterns of hemorrhagic fever with renal syndrome in Hebei Province,China, 2001-2016

Hemorrhagic fever with renal syndrome (HFRS) is highly endemic in China, where approximately 90% of the total human cases in the world are reported. The Hebei province, one of areas with the highest prevalence, has reported HFRS cases every year in the last two decades. This study describes the spatiotemporal patterns of HFRS in the Hebei province from 2001 to 2016, detects the high-risk spatiotemporal clusters of HFRS, and provides valuable information for planning and implementation of local preventive measures. For the purpose of the analysis, HFRS cases recorded during the sixteen years in the Hebei province were aggregated into three temporal periods (2001-2006, 2007-2012, and 2013-2016). Spatiotemporal analyses, including Global spatial autocorrelation analysis and Kulldorff's scan statistical analysis, were applied to analyze te spatiotemporal clusters of HFRS at the county level. The results revealed that the spatial extent of the HFRS epidemic in the Hebei province changed dynamically from 2001 to 2016, which indicated that a comprehensive preventative strategy should be implemented in the northeastern regions of the Hebei province in spring.