Nucleotide sequence characterization and phylogenetic analysis of hantaviruses isolated in Shandong Province,China

Background China is the most severe endemic area of hemorrhagic fever with renal syndrome （HFRS） in the world with 30 000-50 000 cases reported annually, which accounts for more than 90% of total number of cases worldwide. The incidence rate of the syndrome in Shandong Province is one of the highest in China, which has ever reached 50 per 100 000 persons per year. However, the molecular characteristics of hantaviruses （HV） epidemic in Shandong Province remain unclear. Therefore it is useful to clarify nucleotide sequence and phylogenetic characteristics of HV isolated in Shandong Province in order to provide better advices to control and prevent HFRS. Methods RNAs were extracted from sera of clinically diagnosed patients and positive rodent lungs that were detected by indirect immunofluorescent assay （IFA）. Partial M segments of HV were amplified from the RNAs with reverse transcription nested polymerase chain reactions （nested PCR） using hantavirus genotype specific primers. The nested PCR products were sequenced and compared with those from previously epidemic isolates in Shandong and with other representative HV sequences from GenBank. Phylogenetic tree analyses were performed based on the sequences of the M genes. Results Thirty-four HV isolates in Shandong showed 67.1%-100% nucleotide identities. The nucleotide homologies among 6 Hantaan viruses （HTNV） isolates in Shandong were 78.1%-98.7%, while the homologies among 28 Seoul virus （SEOV） isolates in Shandong were 93.7%-100%. There were at least 3 subtypes HTNV （H2, H5, H9） and 2 subtypes SEOV （S2, S3） in Shandong Province. Conclusions In Shandong Province, the homologies of HTNV were lower and there were no predominant subtypes, while the homologies of SEOV were higher and S3 was the predominant subtype. The homologies of SEOV from rodents were higher than those from patients. The distribution of subtypes in Shandong was similar to that of the adjoining provinces. Phylogenetic analyses of the sequences showed geographic clustering of HV in Shandong.

展望流行性出血热病原学研究

1976年在南朝鲜、1981年在我国相继成功地分离到流行性出血热病毒。这一突破性进展使我国也使全世界对这种疾病的研究和防治跨进一个新的阶段。

Comparison of indirect immunofluorescence and western blot method in the diagnosis of hantavirus infections

BACKGROUND Serologic cross-reactivity between hantaviruses often complicates the interpretation of the results.AIM To analyze the diagnostic value of indirect immunofluorescence assay(IFA)and western blot(WB)in the diagnosis of hantavirus infections.METHODS One hundred eighty-eight serum samples from Puumala(PUUV)and Dobrava(DOBV)orthohantavirus infected patients were analyzed.Serology was performed using commercial tests(Euroimmun,Lübeck,Germany).RESULTS Using IFA,49.5%of acute-phase samples showed a monotypic response to PUUV,while 50.5% cross-reacted with other hantaviruses.The overall cross-reactivity was higher for immunoglobulin G(IgG)(50.0%)than for immunoglobulin M(IgM)(25.5%).PUUV IgM/IgG antibodies showed low/moderate reactivity with orthohantaviruses Hantaan(12.3%/31.5%),Seoul(7.5%/17.8%),DOBV(5.4%/28.1%),and Saaremaa(4.8%/15.7%).Both DOBV IgM and IgG antibodies were broadly reactive with Hantaan(76.2%/95.2%),Saaremaa(80.9%/83.3%),and Seoul(78.6%/85.7%)and moderate with PUUV(28.5%/38.1%).Using a WB,serotyping was successful in most cross-reactive samples(89.5%).CONCLUSION The presented results indicate that WB is more specific than IFA in the diagnosis of hantavirus infections,confirming serotype in most IFA cross-reactive samples.

Diversity and Distribution of Host Animal Species of Hantavirus and Risk to Human Health in Jiuhua Mountain Area,China

Objective To investigate the diversity and the distribution of host animal species of hantavirus and the effect on human health in Jiuhua Mountain area,China.Methods The host animal species of hantavirus was surveyed by using the trap method and the species diversity was evaluated by using the Simpson,Shannon-Weaner,and Pielou indices.Hantavirus antigens or antibodies in lung and blood samples of all the captured host animals were detected by direct or indirect immunofluorescence.Results Nine animal species of hantavirus were distributed in the forest ecosystem of Jiuhua Mountain.Of these,Niviventer confucianus and Apodemus agrarius were predominant,and N.confucianus,Rattus norvegicus,and Mus musculus had relatively large niche breadth index values.The host animals in the eastern and western mountain regions shared similar biodiversity index characteristics,predominant species,and species structures.Hantavirus was detected in 5 host animal species in Jiuhua Mountain area,the carriage rate of hantavirus was 6.03%.The average density of host animals in forest areas of the mountainous area was only 2.20%,and the virus infection rate in the healthy population was 2.33%.Conclusion The circulation of hantavirus was low in the forest areas of Jiuhua Mountain and did not pose a threat to human health.

Stable Expression of Hantavirus H8205 Strain G1/IL-2 Gene and Immune Protection of the Fusion Gene

To explore the feasibility of stable expression of Hantavirus H8205 strain G1 segment and human IL-2 fusion gene in Vero cells, and to examine the immune protection effects on mice vaccinated with this recombinant eukaryotic expression vector containing Hantavirus G1 gene and IL-2 gene. With the help of lipofectamine, the Vero cells were transfected with pcDNA3.1/HisB-IL-2-G1 and the positive cells were selected by G418. IFAT and SDS-PAGE elec- trophoresis were used to determine the stable transfection and expression of recombinant protein. Each mouse was inoculated with plasmids intramuscularly （i.m.） three times, 2 boosts were given at 2-week intervals, serum anti-hantavirus antibodies were detected by ELISA and neutralizing antibodies （NAb） were detected by Plaque Reduction Neutralization Test. The fusion protein expressed in Vero cells was 78 kD, corresponding to the estimated molecular size. The neutralizing antibody titers of mice with pcDNA3.1/HisB-IL-2-G1 were 1：20-1：80. IL-2/G1 fusion gene could be transferred in Vero cells and stably express the fusion protein. Specific humeral immune responses in mice can be induced with the recombinant eukaryotic expression vector containing the fusion gene, which lays the foundation for further development of therapeutic HTNV vaccine.

Hemorrhagic fever with renal syndrome complicated with aortic dissection:A case report

BACKGROUND Hemorrhagic fever with renal syndrome is caused by hantaviruses presenting with high fever,hemorrhage,and acute kidney injury.Microvascular injury and hemorrhage in mucus were often observed in patients with hantavirus infection.Infection with bacterial and virus related aortic aneurysm or dissection occurs sporadically.Here,we report a previously unreported case of hemorrhagic fever with concurrent aortic dissection.CASE SUMMARY A 56-year-old man complained of high fever and generalized body ache,with decreased platelet counts of 10×10^9/L and acute kidney injury.The enzymelinked immunosorbent assays test for immunoglobulin M and immunoglobulin G hantavirus-specific antibodies were both positive.During the convalescent period,he complained sudden onset acute chest pain radiating to the back,and the computed tomography angiography revealed an aortic dissection of the descending aorta extending to iliac artery.He was diagnosed with hemorrhagic fever with renal syndrome and Stanford B aortic dissection.The patient recovered completely after surgery with other support treatments.CONCLUSION Hemorrhagic fever with renal syndrome complicated with aortic dissection is rare and a difficult clinical condition.Hantavirus infection not only causes microvascular damage presenting with hemorrhage but may be risk factor for acute macrovascular detriment.A causal relationship has yet to be confirmed.

Preparation and antigenic characterization of rat monoclonal antibodies againts Hantavirus

Monoclonal antibodies (McAb) to Hantavirus (HTV)were derived from the hybridoma fused with Lou/c plasmocytoma rat cells (IR983F) and spleen cells from lou/c inbred rat immunized with HTV Chen strain. Eleven cell lines producing monoclonal antibodies direc

STUDIES ON DYNAMIC CHANGES AND COMPARISONS OF SERUM SPECIFIC IGM AND IGG ANTIBODIES IN PATIENTS WITH HEMORRHAGIC FEVER WITH RENAL SYNDROME

The speciric IgM and IgG antibodies in serum samples from patients with hemorrhagic fever with renal syndrome (HFRS) were sequentially and simultaneously determined during the course of the disease. The results showed that the detectable days and the days reaching 100% positive rate after onset of illness in specific IgM antibodies were earlier than those in specific IgG antibodies. No significant differences were observed between the titers of specific IgM antibodies on different days after onset of the disease while the titers of specific IgG antibodies differed, significantly on different days after onset of illness (F'= 11. 72,P< 0. 01 ). The titers of specific IgM antibodies in different clinical types were not significantly different at the same illness day but the titers of specific IgG antibodies in various clinical types were significantly different from day 7 to day 8 after onset of disease (F' = 4. 004, P< 0. 05). The levels of specific IgM and IgG antibodies were more stable and the crossings of the IgM and IgG antibody curves presented later in patients with mild and middle clinical types than those in patients with severe and gravis types. These results suggest that the detection of specific IgM antibodies may be useful for the early laboratory diagnosis of HFRS,the excess Production of specific IgG antibodies may play a role in the formation of immune complexes and the exacerbation of pathologic damage in patients with severe and gravis types and the observations of the dynamic changes of specific IgM and IgG antibody curves may have some implications in evaluating the seventies of the disease and the prognosis of the patients with HFRS.

First Evidence of Hantavirus in Central Iran as an Emerging Viral Disease

Hantavirus is a zoonosis transmitted from rodents to humans. Asymptomatic infected rodents can secrete hantaviruses in the urine, feces, and saliva. The main route of infection transmission to human is aerosols contaminated with the virus. This study was designed to evaluate the serological and molecular prevalence of hantavirus as an emerging zoonoses disease among street sweepers in Isfahan province, central Iran. Serum samples from 200 street sweepers in healthy condition and those with recent renal failure were tested by ELISA (IgM and IgG). Molecular analysis was subsequently applied for IgM positive cases. From these samples, 9 (4.5%) were positive, of which 2 (22.22%) were positive for both IgM and RT-PCR, while 7 (77.77%) were positive for IgG. The mean age and work experience of the positive cases were 39.7 and 11.5 respectively. According to our observations, all positive cases reported prevalence of rodents in their work place. The logistic regression test showed that the age and work experience were not risk factors for being positive, but prevalence of rodents in work place was a risk factor for being positive, when compared with negative cases. This is the first comprehensive study on the prevalence of hantavirus with positive results coming from Iranian population, which can raise the public awareness for the hantavirus infections as a public health threat.

Viral disease

930440 Isolation and identification of epidemichemorrhagic fever virus from cerebrospinal flu-id.XU Wei(徐伟),WANG Lin(王麟).Ji-nan Infect Dis Hosp,Jinan,250021.Chin J MedLab Technol 1993;16(3):138—139.Cerebrospinal fluid samples of four patientswith epidemic hemorrhagic fever(EHF)werestudied virologically.From one patient withcomplication of encephalopathy,a strain of viruswas isolated by Veto—E6 cell culture.After aseries of specific serologic tests,including IFA,ELISA and NT,and animal inoculation were per-formed,the virus was finally identified to be Han-taan strain of the genus Hantavirus.This

新出现的疾病

新出现的疾病ＲｉｃｈａｒｄＬｅｖｉｕｓ等著葛建一，江世亮译从新病的出现和旧病复发中吸取教训，有助于我们对未来的流行病作好准备。和２５年前一样，近来瘟疫的威胁似乎已过时，甚至是中世纪的事了。传染病引起的死亡，被认为是卫生条件差和缺乏有效的抗菌素及疫苗的...

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, 200d--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 type Ⅰ （Hantaan virus, HTNV） and 3 of type Ⅱ （Seoul virus, SEOV）. Among 17 samples in spring and summer, 14 were sero-positive, of which 5 cases were of type Ⅰ and 9 of type Ⅱ . So it concludes that both of the two types of Hantavirus exist in Heilongjiang. The type Ⅰ is the main pathogen of HFRS in winter, and typeⅡ is the main in spring and summer.

Genotyping and preparation of the recombinant nucleocapsid protein antigen of hantavirus

Objective To identify new recombinant antigens with potential for diagnosis of hemorrhagic fever with renal syndrom (HFRS) and establish reverse transcription-polymerase chain reaction-restriction fragments length polymorphism (RT-PCR-RFLP) for genotyping of hantavirus.Methods One group of primers was used to clone the full-length S genome segment and the partial S genorme segment of the N-terminal. The two cloned genes were both fusionally expressed and nonfusionally expressed in the T7 system. The other group of primers was used to establish a RT-PCR method to detect RNAs in 37 virus isolates, 2 positive standard virus strains of hantavirus and 5 negative controls.The method for typing RFLP was set up by digesting the PCR products of 20 virus isolates with Ras Ⅰ and Hind Ⅲ.Results The non-fusionally expressed products with a working concentration of 1:10 000 by chapping enzyme-linked immunosorbent assay (cELISA), presented good biological activity though yields were lower than that of the fusionally expressed products.The specific component of the hantavirus genome (299 bp or 577 bp) wes seen in all viral samples. The genotyping of hantavirus showed that 9 out of the total were hantann (HTN) viruses, 8 were seol (SEO) viruses and 3 were not determined.Conclusions The good working titrer of expressed recombinant antigen showed that it has the potential to replace the natural antigen for detecting hantavirus antibodies. On comparison with cELISA, the detection rates for these two methods were 100% and 84.6%, and the coincidence rate was 84.6%. The former had a 15.4% higher sensitivity than the latter. The typing efficiency of RT-PCR-RFLP and sero-typing method was 85% (17/20) and 55% (11/20), respectively, showing that the former was 30% higher than the latter, while their results were highly consistant.

Hantavirus infection:a global zoonotic challenge

Hantaviruses are comprised of tri-segmented negative sense single-stranded RNA, and are members of the Bunyaviridae family. Hantaviruses are distributed worldwide and are important zoonotic pathogens that can have severe adverse effects in humans. They are naturally maintained in specific reservoir hosts without inducing symptomatic infection. In humans, however,hantaviruses often cause two acute febrile diseases, hemorrhagic fever with renal syndrome(HFRS) and hantavirus cardiopulmonary syndrome(HCPS). In this paper, we review the epidemiology and epizootiology of hantavirus infections worldwide.

Detection and characterization of three zoonotic viruses in wild rodents and shrews from Shenzhen city,China

Diverse species of rodents and shrews, which are abundant worldwide, harbor a variety of viruses;some of these are closely related to human viruses and possess zoonotic potential. Previously studies have demonstrated that the mammarenavirus and hantavirus carried by rodents or shrews could cause diseases in human population. To determine the distribution of zoonotic viruses in Shenzhen city, the major city in southern China with a high population density, we analyzed 225 rodents(Rattus norvegicus and Rattus flavipectus) and 196 shrews(Suncus murinus) from urban and rural districts for the presence of mammarenavirus, hantavirus, and hepatitis E virus(HEV) by RT-PCR targeting the conserved regions. The infection rates for mammarenavirus, hantaviruses,and HEV in rodents and shrews were 3.56%, 6.89%, and 1.66%, respectively. Partial genome fragment analysis indicated that mammarenavirus and hantavirus strains had more than 90% and 99% nucleic acid identity with Cardamones virus and Seoul virus, respectively, which cause diseases in humans. Although the present HEV strains identified are typically found worldwide,phylogenetic analysis demonstrated a divergence of 16%. To our knowledge, the present work is the first report of the prevalence of mammarenavirus, hantaviruses, and rat HEV strains in rodents and shrews from Shenzhen city, China. Our findings highlight the zoonotic potential of rodent-and shrew-borne mammarenavirus and hantavirus, and the biodiversity of rat HEV isolates in Shenzhen city. The present work suggests that utilization of good hygiene habits is important to minimize the risk of zoonosis.

Hantavirus Infection during Pregnancy

Hantavirus infection is a global health challenge,causing widespread public concern.In recent years,cases of hantavirus infection in pregnant women have been reported in many countries.The infected pregnant women and their fetuses appear to have more severe clinical symptoms and worse clinical outcomes.Hence,to study the prevalence of hantavirus infection in pregnant women,this study will focus on the epidemiological distribution of the virus,different virus species penetrating the placental barrier,and factors affecting the incidence and clinical outcome of the infection in pregnant women and their fetuses.In addition,this review will also discuss the diagnostic tools and treatments for pregnant patients and provide an overview of the relevant future research.

Enhancing DNA vaccine potency against hantavirus by co-administration of interleukin-12 expression vector as a genetic adjuvant

Background The heavy incidence and mortality of hemorrhagic fever with renal syndrome, as well as no specific drugs in curing the disease, clearly indicate the need for development Of the more effective hantavirus vaccine. Refining the DNA vaccination strategy to elicit more clinically efficacious immune responses is now under intensive investigation. In the present study, we examined the effects of using an interleukin-12 expression plasmid as a genetic adjuvant to enhance the immune responses induced by a DNA vaccine based on the S gene encoding nucleocapsid protein against hantavirus. Methods BALB/c mice were immunized three times by intramuscular inoculations of DNA vaccine encoding of hantanvirus nucleoeapsid protein alone or in combination with a plasmid expressing murine interleukin-12 (pcIL-12). Booster immunizations were employed 2 times at 2-week interval. To evaluate the Immoral and cellular immune responses, antigen-specific lymphocyte proliferation and antibody production were assayed by MTT method and ELISA respectively. The level of interleukin-4 and interferon-gamma in the splenic lymphocytic cultured supernatant were detected with ELISA kit at day 5, 10, 17, 35 and 42 after primary immunization. Results Antigen-specific IgG antibodies was increased markedly at day 17 in the experiment groups and reached a plateau after day 35. As pcIL-12 co-injected, a significant inhibition of antigen-specific IgG levels was displayed over the period and the antibody mean titre was decreased to only about 1: 50 at day 42 after primary immunization, significantly lower than the group immunized with pcDNA3.1 + S alone, in which the mean titre was about 1:70. Interferon-gamma was increased remarkably by the co-injection of pcIL-12 compared with the injection of pcDNA3.1 + S alone. However, the production of interleukin-4 was inhibited by pcIL-12 coinjection. Furthermore, pcIL-12 co-injection efficiently enhanced antigen-specific lymphocyte proliferation. Conclusion Humoral and cytokine responses elicited by pcDNA3.1 + S inoculation can be modulated by coinoculation with pcIL-12 and efficiently induced Th1-dominant immune responses.

Lack of association between integrin αvβ3 gene polymorphisms and hemorrhagic fever with renal syndrome in Han Chinese from Hubei, China

Hantaviruses belong to the family Bunyaviridae and cause hemorrhagic fever with renal syndrome （HFRS） in humans. 133 integrins, including αvβ3 and αⅡbβ3 integrins, act as receptors on endothelial cells and play key roles in cellular entry during the pathogenesis of hantaviruses. Previous study demonstrated that the polymorphisms of integrin αⅡbβ3 are associated with susceptibility to hantavirus infection and the disease severity of HFRS in Shaanxi Province of China, rather than in Finland. However, the polymorphisms of integrin av133 in patients with HFRS was incompletely understood. Here, we aimed to investigate the associations between polymorphisms in human integrin αvβ3 and HFRS in Han Chinese individuals. Ninety patients with HFRS and 101 healthy controls were enrolled in this study. Analysis of five single nucleotide polymorphism （SNP） sites （rs3768777 and rs3738919 on ITGAV; rs13306487, rs5921, and rs5918 on ITGB3） was performed by TaqMan SNP genotyping assays and bi-directional PCR allele-specific amplification method. No significant differences were observed between the HFRS group and controls regarding the genotype and allele frequency distributions of any of the five SNP sites, and no associations were found between ITGAV polymorphisms/genotypes and disease severity. In conclusion, our results implied that these five SNPs in the integrin αvβ3 gene were not associated with HFRS susceptibility or severity in Han Chinese individuals in Hubei Province.

Home range variability,spatial aggregation,and excursions of Akodon azarae and Oligoryzomys flavescens in Pampean agroecosystems

Rodents are reservoirs of various types of hantavirus,some of which are agents of hantavirus pulmonary syndrome in humans.Each hantavirus is associated with a single rodent host species but successive spill-over events may eventually lead to host-switching and new species’becoming host of a given pathogen.This study aims to gain an understanding of the spatial ecology of two hantavirus-host species,Akodon azarae,and Oligoryzomys flavescens,by identifying factors modulating their home range sizes and stability,and by evaluating intra-and interspecific spatial aggregation for these species and a third one—Oxymycterus rufus—living in sympatry.For this,eleven capture-mark-recapture surveys were carried out,spanning 22 months.We found that A.azarae males have larger and more mobile home ranges than females,independently of the season.Consequently,males could likely have a more relevant role in the transmission of hantavirus because of their greater exposure both to a higher number of contacts between individuals and viral contamination of the environment.Contrasting,O.flavescens individuals showed negligible displacements of their home range through time,which could limit the range of hantavirus spread in host populations.Since O.flavescens is host to Lechiguanas hantavirus(pathogenic to humans)this result encompasses epidemiological relevance,for it may imply the existence of local foci of infection.Additionally,individuals of both species performed excursions outside their home ranges.These events could enable hantavirus spread over distances beyond the normal range of movements and lead to new hantavirus outbreaks in formerly non-infected rodent populations,favoring the persistence of the virus in nature.

Spatial structure of rodent populations and infection patterns of hantavirus in seven villages of Shandong Province from February 2006 to January 2007

Background Hemorrhagic fever with renal syndrome （HFRS） is endemic in Junan county, Shandong Province, China. We conducted geographic information system （GIS）-based spatial analysis with the objective of estimating the spatial distribution of rodent populations and their hantavirus infection patterns, to describe the spatial relationships of hantavirus strains in small ecological areas and to identify key areas in endemic areas of HFRS for future public health planning and resource allocation.