Renal Syndrome Virus Research Articles

MIXED INFECTION - HANTAVIRUS HAEMORRHAGIC FEVER WITH RENAL SYNDROME AND HERPES SIMPLEX VIRUS ENCEPHALITIS

Introduction: This case report describes a patient treated at the Clinic of Infectious Diseases of University Hospital “St. George” Plovdiv, with two infections occurring at the same time – haemorrhagic fever with renal syndrome (HFRS) and herpes simplex virus (HSV) encephalitis. Results: A 53-year-old male presented with fever, flu-like syndrome and renal impairment with mild nitrogen elevation. The patient was treated at the Clinic of Nephrology for one week. Several hours after discharge he had three generalised tonic-clonic seizures and was admitted to the intensive care unit with status epilepticus and acute respiratory failure requiring intubation and mechanical ventilation for 4 days. Lumbar puncture results showed: white blood cells – 1.10 6 /l, glucose – 5.3 mmol/l, total protein – 1.6 g/l, increased immunoglobulin levels. The patient was transferred to the Clinic of Infectious Diseases with suspicion of viral encephalitis.
 HSV type 1 was detected in the cerebrospinal fluid by PCR. Due to suspected hantavirus infection, serum was examined and IgM and IgG antibodies against the HFRS virus were detected by ELISA. Specific treatment with acyclovir was started and there was a rapid improvement in the clinical condition. Convulsions did not recur. Nitrogen levels normalised and there was a short period of polyuria. Thrombocytopenia quickly resolved without occurrence of haemorrhagic syndrome. In the course of the disease, the patient developed mild diarrhoeal syndrome and left thrombophlebitis. He was discharged clinically well with negative PCR results for HSV.
 In conclusion, we can assume that as a result of immunosuppression due to HFRS, HSV type 1 reactivated and led to herpes encephalitis.

Clinical and virological characteristics of hantavirus infections in a 2014 Croatian outbreak.

Croatia is endemic for hemorrhagic fever with renal syndrome (HFRS), with both Puumala (PUUV) and Dobrava virus (DOBV) documented. Several large outbreaks were recorded in 1995, 2002, and 2012. We analyzed demographic, clinical, laboratory, and virological characteristics of HFRS cases detected in three geographically close natural foci (Ogulin, Slunj, and the Plitvice Lakes surroundings) during the 2014 outbreak. From January to December 2014, 122 patients with suspected HFRS were tested for hantavirus IgM/IgG antibodies using an indirect immunofluorescence assay (IFA). Cross-reactive samples were further tested using a western blot (WB). For hospitalized patients from Ogulin area, clinical and laboratory data were analyzed. Acute infection was documented in 57 (46.7%) patients, of whom 75.4% were hospitalized. Ten (8.2%) patients were found to be IgG seropositive. Patients were 15-69 years of age and predominantly male (74.5%). The outbreak started in winter months, with most cases recorded from May to July (80.7%). The most frequently reported symptoms were fever (96.3%), chills/shivering (62.9%), and lumbar pain (48.1%). Mild clinical form was found in 66.7% patients, moderate in 18.5%, and severe in 14.8% patients (all but one infected with PUUV). One patient died. Using IFA, 48.8% patients showed monotypic antibody response, while in 51.2%, cross-reactive antibodies were found. PUUV was confirmed in 94.7% and DOBV in 5.3% HFRS cases by WB. Central mountainous Croatian regions are still highly endemic areas for HFRS. A higher percentage of severe PUUV infections could be at least partly associated with a patient's immune status.

Features of the replication of hantaviruses - pathogens of hemorrhagic fever with renal syndrome in cell cultures of different origin

There are presented results of the adaptation ofpathogens of hemorrhagic fever with renal syndrome (HFRS) virus Puumala and Dobrava strains, to the reproduction in cell cultures of the simian origin - Green monkey kidney (Vero, 4647), Green monkey spleen (455), calf kidney (PT-1) and sheep kidney (4184). Hantaviruses were shown to be able to adapt to the replication in the new cellular system differing in species origin from the culture of Vero E6 origin in which the strains were isolated. This fact demonstrates the heterogeneity of hantavirus population, with the existence of «quasispecies» which can recognize and bind to ligands (b3 integrins) of the other species specificity. The highest yield of the virus, which is comparable with the culture of Vero E6, culture Vero provides. In Vero cells after 3-4 passages Puumala and Dobrava-Belgrade viruses replicate also actively as in a permissive culture Vero E6, that proposes the use of Vero culture cell allowed for the production of immunobiological drugs administered to people as a substrate in the delivery of vaccines against HFRS.

Establishment of a novel one-step reverse transcription loop-mediated isothermal amplification assay for rapid identification of RNA from the severe fever with thrombocytopenia syndrome virus

As an emerging infectious disease, severe fever with thrombocytopenia syndrome virus (SFTSV) infection has been found in many areas of China. Suitable laboratory diagnostic method is urgently needed in clinical detections and epidemiological investigations. In this study, a modified, low-cost and rapid visualized one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of RNA from the SFTSV has been established. In order to avoid the risk of aerosol contamination and facilitate the naked eye to observe, a microcrystalline wax–dye capsule wrapping the highly sensitive DNA fluorescence dye SYBR Green I was added to the RT-LAMP reaction tube before the initiation of the assay. The detection limit of the established RT-LAMP assay was 10 fg template RNA per reaction mixture. The RT-LAMP assay was confirmed to be high specific to SFTSV, and no cross-reaction was found with the detection of the Chikungunya fever virus, Hemorrhagic Fever with Renal Syndrome virus (HFRSV), and Dengue fever virus. The assay was then applied for the detection of SFTSV RNA in 32 clinical serum samples and showed 94.4% consistence with the detection results of the real-time RT-PCR. The whole process, from sample preparation to result reporting, can be completed within 2h. This adapted, cost efficient and quick visualized RT-LAMP method is feasible for SFTSV field diagnosis in resource-limited field settings.

PXII-22 Investigation of enterovirus RNA, enterovirus antibodies, GAD antibodies and HLA genotyping in pediatric patients with type 1 diabetes mellitus

In this chapter, we describe 73 zoonotic viruses that were isolated in Northern Eurasia and that belong to the different families of viruses with a single-stranded RNA (ssRNA) genome. The family includes viruses with a segmented negative-sense ssRNA genome (families Bunyaviridae and Orthomyxoviridae) and viruses with a positive-sense ssRNA genome (families Togaviridae and Flaviviridae). Among them are viruses associated with sporadic cases or outbreaks of human disease, such as hemorrhagic fever with renal syndrome (viruses of the genus Hantavirus), Crimean–Congo hemorrhagic fever (CCHFV, Nairovirus), California encephalitis (INKV, TAHV, and KHATV; Orthobunyavirus), sandfly fever (SFCV and SFNV, Phlebovirus), Tick-borne encephalitis (TBEV, Flavivirus), Omsk hemorrhagic fever (OHFV, Flavivirus), West Nile fever (WNV, Flavivirus), Sindbis fever (SINV, Alphavirus) Chikungunya fever (CHIKV, Alphavirus) and others. Other viruses described in the chapter can cause epizootics in wild or domestic animals: Geta virus (GETV, Alphavirus), Influenza A virus (Influenzavirus A), Bhanja virus (BHAV, Phlebovirus) and more. The chapter also discusses both ecological peculiarities that promote the circulation of these viruses in natural foci and factors influencing the occurrence of epidemic and epizootic outbreaks

Safety and immunogenicity of bivalent inactivated vaccine against haemorrhagic fever with renal syndrome in a phase II trial on healthy Chinese volunteers

We have developed a cell culture-derived, inactivated bivalent vaccine against both Haemorrhagic Fever with Renal Syndrome (HFRS) viruses 84 Fli (type I) and L99 (type II). This purified vaccine was tested in the clinical phase I/II trial in healthy non-infected adults in China. About 518 volunteers in Southern region and 650 volunteers from the Northern region of China were vaccinated. Volunteers then received one or two boosts at two weeks post-vaccination. No obvious side effects were observed among vaccinees after each inoculation. More than 87% of vaccinees have exhibited high neutralising antibody titers by Plaque Reducing Neutralising Test (PRNT) against both types I and II viruses and 100% positive seroconversion of IgG antibody by ELISA. These results indicate that our purified cell culture-derived bivalent vaccine is safe, well-tolerated and capable of inducing a protective immunity.

Experimental study on enzyme dot assay for detection of hemorrhagic fever with renal syndrome antigen

To establish a new and efficient method(IEDA) for detection of hemorrhagic fever with renal syndrome virus (HFRSV) antigen. An immune enzyme dot assay (IEDA) with mixture of three sorts anti-HFRSV-IgG, which was obtained from rabbit vaccinated with EHFV R22, Chen and Hubei strain was employed to detect HFRSV antigen in serum and urine from epidemic hemorrhagic fever (EHF) patients, and compared with indirect immune fluorescence assay (I-IFA), 76 serum samples and 40 urine samples were detected in this study. The results showed that the total positive rate of HFRSV antigen detected by IEDA was 73.68% in serum and 65.00% in urine, while that detected by I-IFA was 75.00% and 70.00%, respectively. The positive rate in primary phase (within 5 days) of HFRSV antigen detected by IEDA was 94.34% in serum and 83.33% in urine, while that detected by I-IFA was 64.42% and 55.56%, respectively, there was significant difference in both serum and urine detections. Correlation study showed a high correlation in the result of IEDA and I-IFA. The results of this study suggested that the IEDA, as compared with I-IFA, was a more specific, sensitive, rapid and simple method with higher positive rate in primary phase. IEDA could be widely used for early diagnosis of HFRS in hospital at grassroots level.

The effects of hybridoma growth factor in conditioned media upon the growth, cloning, and antibody production of heterohybridoma cell lines

Interleukin 6 (IL-6)/hybridoma growth factor (HGF) has been shown to be the requirement for growth of murine hybridomas in vivo or in vitro. In this paper, two kinds of conditioned media (CM) from the culture supernatants of a human fibroblast cell line CRL1506 and a cloned Epstein-Barr virus (EBV) transformed human lymphoblastoid cell line (LCL) N23 were found to have IL-6 activity by strongly promoting the growth, antibody secretion (increase of one- to three-fold), and cloning efficiencies of heterohybridomas secreting human monoclonal anti-hemorrhagic fever with renal syndrome virus antibodies and LCL. Since these CM contained no detectable IL-2, and IL-4 had no effects on the growth of the cell lines, IL-6 was considered to be the main active component of the CM responsible for promoting hybridoma growth. This effect was further confirmed by IL-6-dependent cell line 7TD1 bioassay (IL-6 activity in the CM ranging from 1,000 to 10,000 units/ml). Moreover, we successfully established four EBV-transformed lymphoblastoid cell lines at single-cell level by adding an equal volume of CRL1506-CM to 10% FCS-RPMI1640 in limiting dilution. Finally, it is worth noting that the sensitivity of the heterohybridomas to the two kinds of CM was not the same and was not consistent with that of their parental myeloma cell lines. Thus, it suggests that the CM might contain more than one factor, and the choice of proper conditioned media should be very useful for human monoclonal antibody production.

Antigenic variation of European haemorrhagic fever with renal syndrome virus strains characterized using bank vole monoclonal antibodies

Monoclonal antibodies (MAbs) against Puumala (PUU) virus, the aetiological agent of nephropathia epidemica, were produced by fusing activated spleen cells from a bank vole (Clethrionomys glareolus) with the mouse myeloma cell line SP2/0. This novel approach, utilizing the natural vector of PUU virus for hybridoma production, proved to be highly efficient, and eight stable PUU virus-specific heterohybridomas were isolated and characterized. The bank vole MAbs were all specific for the nucleocapsid protein (N) of PUU virus, as determined by immunoprecipitation. When evaluated by additivity immunoassays, the MAbs were found to recognize several different, distinct or overlapping, epitopes on N. The MAbs were used in immunofluorescence assays to compare eight PUU-related virus isolates, and the prototype Hantaan, Urban rat and Prospect Hill viruses. The reactivity varied among the different MAbs and could be classified into five groups. One MAb reacted exclusively with PUU-related viruses; two MAbs reacted with all PUU-related virus strains tested, as well as Prospect Hill virus, but did not react with Urban rat virus and Hantaan virus; one MAb reacted with all PUU-related virus strains tested and weakly with Hantaan virus, but not with Urban rat and Prospect Hill viruses; two MAbs reacted with all the virus strains tested. Two virus strains, K-27 and CG-1820, isolated in the western U.S.S.R., were distinguished from the other PUU-related virus strains by two MAbs, suggesting that the large group of independently isolated PUU-related viruses may be more heterogeneous than previously believed.

Renal Lesions in Rats Infected with Rattus Serotype Hantavirus (SR‐11 strain)

Hemorrhagic fever with renal syndrome (HFRS) virus, strain SR-11 (SR) was inoculated intraperitoneally into specific-pathogen-free (SPF) newborn rats, from which the kidney lesions were examined pathologically. The infected rats revealed proteinuria on and after 16 days postinoculation (PI). Histologically, the epithelial cells of the renal tubules showed mild vacuolar and granular degeneration with cytoplasmic inclusion bodies (CIB) on and after 16 days PI. Ultrastructurally, a decrease in number of mitochondria and endocytic vesicles was recognized in the epithelial cells of the proximal renal tubules. Occasionally, both the proximal and distal renal tubular cells had CIB near well-developed Golgi apparatus on and after 13 days PI. Immunohistochemically, CIB were positive for anti-SR nucleocapsid antibody, but negative for anti-SR envelope protein antibody. From the results obtained here, it was concluded that the proteinuria in rats infected with HFRS virus resulted from an insufficiency of reabsorption in the proximal renal tubules, and that CIB consisted of the viral nucleocapsid protein.

INCIDENCE OF ANTIBODIES AGAINST HUMAN IMMUNODEFICIENCY VIRUS, HUMAN T-CELL LYMPHOTROPIC VIRUS TYPE 1, HEPATITIS B VIRUS, HEMORRHAGIC FEVER WITH RENAL SYNDROME VIRUS AND CHLAMYDIA IN TONGA AND WESTERN SAMOA

Among the populations of Tonga and Western Samoa, serum antibodies against human immunodeficiency virus or hemorrhagic fever with renal syndrome virus were not detected (0/904 and 0/192). No serum samples were considered to be positive for antibody against human T-cell lymphotropic virus type 1 (0/527). Hepatitis B antigen and antibody were found in 4% (8/192) and 47% (90/192), respectively. Chlamydia trachomatis IgG and C. psittaci IgG antibodies were detected in 39% (75/192) and 47% (91/192), respectively. The possibilities of the spread of human immunodeficiency virus and hemorrhagic fever with renal syndrome virus on the islands when the viruses invade from abroad were discussed.

Nucleotide sequence of the M genome segment of hemorrhagic fever with renal syndrome virus strain B-1

Nucleotide sequence of the M genome segment of hemorrhagic fever with renal syndrome virus strain B-1

Comparison of Virulence between Two Strains of Rattus Serotype Hemorrhagic Fever with Renal Syndrome (HFRS) Virus in Newborn Rats

Two strains of hemorrhagic fever with renal syndrome (HFRS) virus from Rattus, SR-11 and KI-262, showed virtually identical antigenicity but differed from prototype strain Hantaan 76-118 (Apodemus origin) in a neutralization test. Wistar newborn rats inoculated intraperitoneally (i.p.) with SR-11, which was isolated from a laboratory rat associated with an outbreak of HFRS, developed clinical signs such as ataxia and limb paralysis and died at about 18 days after inoculation. The LD50 of SR-11 in 1-day-old rats was 10(1.2) focus-forming units (FFU). In contrast, the animals inoculated i.p. or intracerebrally with 10(4) FFU of KI-262, which was from a wild rat in a dumping-ground area--an enzootic focus where no human cases have been recorded--did not show any significant clinical signs. The susceptibility of rats to SR-11 fatal infection was age-dependent. Virus titers in brains, lungs, kidneys, and livers of the rats inoculated with SR-11 were significantly higher than those in the same organs of the animals infected with KI-262. Necrosis of neurons in the brain tissue occurred in the rats infected with SR-11, while it was mild in the animals infected with KI-262.

Virion proteins of viruses causing hemorrhagic fever with renal syndrome; monoclonal antibody analysis

SDS-polyacrylamide gel electrophoresis and Western blotting analysis by using 9 clones of monoclonal antibodies specific for hemorrhagic fever with renal syndrome (HFRS) viruses were carried out on the virion proteins of 17 strains of HFRS viruses isolated from different areas in Asia and different hosts such as Apodemus agrarius, Rattus norvegicus, domestic cat and HFRS patients. Polypeptide with apparent molecular weight about 50 kitodalton (Kd) could be detected in all 17 strains of HFRS viruses. On this polypeptide there are two different antigenic determinants and one of them might be genus specific.

Experimental Labyrinthitis in Rats Caused by Infecting with Virus Causing Hemorrhagic Fever with Renal Syndrome (HFRS)

Acute and persistent viral infection of the inner ear of rats was induced with hemorrhagic fever with renal syndrome (HFRS) virus following intraperitoneal inoculation. Newborn (within 24 h after birth) rats were inoculated intraperitoneally (i.p.) with HFRS virus (B-1 strain), and the cochlea was examined using an immunohistochemical technique for the localization of viral antigens 1 week (acute infection) and 1 year (persistent infection) after infection. Virus-specific antigens were detected in the inner ear structures of the rats suffering from both acute and persistent infection. Morphological changes in the inner ear structures of these rats were observed, with hemorrhage and infiltration of lymphocytes and macrophages. Moreover virus could be isolated from the blood of all infected rats. In the present study, we have proved that HFRS virus can infect the inner ear via the viremia.

Role of maternal antibody in protection from hemorrhagic fever with renal syndrome virus infection in rats

The effect of maternal antibody on the protection of newborn rats from infection of HFRS virus strain SR-11 was examined. Antibody to HFRS virus was transferred from immune dams to their offspring prenatally as well as postnatally. IgG antibody was detected in the sera of fetuses by IFA test (titers from 1:64 to 1:256) and in fetal fluids (1:32) obtained from the uteri of immune dams at 20 days after mating. In the sera of the newborn, IgG titers of maternal antibody ranged from 1:64 to 1:256 just after birth, reached a peak titer around 1:2,048 at 2 weeks after birth, then declined and disappeared at about 8 weeks of age. No IgA and IgM antibodies were detected in the sera of fetuses and newborns. After intraperitoneal challenge by strain SR-11 (10(2.2) LD50), death and infection of 2-day-old rats from immune dams were prevented by the presence of maternal antibody. The protective effect of maternal antibody remained in 8-week-old rats having an IFA titer of maternal antibody as low as 1:16, and even in some 10-week-old rats with negative tests for maternal antibody.

Virus-specific proteins and RNAs from cells infected with hemorrhagic fever with renal syndrome (HFRS) viruses derived from endemic areas of the U.S.S.R.

Cell-associated proteins of rodent (CG-1820 strain) and human (K-27 and 360 strains) hantaviruses isolated in the European endemic areas of the U.S.S.R. are antigenically similar as revealed by immunoprecipitation and immunoblotting assays. Nucleocapsid-associated RNAs of representative hantaviruses of four antigenic groups [Sugiyama et al. (1987) J Gen Virol 68: 979-987] have unique PAGE patterns. "Electropherotyping" of the RNAs isolated from infected cells might be used for identifying and distinguishing hantaviruses.

Effects of human and murine interferons against hemorrhagic fever with renal syndrome (HFRS) virus (Hantaan virus)

The effects of human interferons (IFN-α, IFN-β, IFN-γ) on the replication of Hantaan virus (HV) in Vero E6 cells were examined. Pretreatment of cells with human IFNs resulted in dose-dependent inhibition of HV plaque formation. Of the 3 human IFNs, IFN-β inhibited virus replication most effectively. Pretreatment of murine macrophage cells with mouse IFN-β also resulted in an inhibition of viral growth and then the effect of murine IFN-β in newborn ICR mice infected with HV was also examined. When newborn mice were inoculated intraperitoneally with HV, their survival rate was approximately 20%. When they were treated with interferon 6 h before infection with virus, their survival rate was 85–90%. When IFN and virus were injected simultaneously into the intraperitoneal cavity, the survival rate of the mice was also higher than that of untreated mice. When the mice were treated with IFN for 2 or 7 consecutive days after infection, their survival rate was 70%. These results suggest that IFN may be effective for both prophylactic and therapeutic purposes in Hantaan virus infection.

Four serotypes of haemorrhagic fever with renal syndrome viruses identified by polyclonal and monoclonal antibodies.

Antigenic relationships among 20 strains of haemorrhagic fever with renal syndrome (HFRS) viruses isolated in Korea, China, U.S.S.R., Finland, Japan and U.S.A. were examined with rat immune sera, patient sera, eight monoclonal antibodies against the SR-11 strain and 10 monoclonal antibodies against the 76-118 strain. Antigen analyses by indirect immunofluorescent antibody and immune adherence haemagglutination tests using polyclonal and monoclonal antibodies demonstrated that HRFS viruses may be divided into four serotypes, i.e. Apodemus (Type 1), Rattus (Type 2), Clethrionomys (Type 3) and Microtus (Type 4). Further, it was demonstrated that Type 1 could be divided into three subtypes and Type 3 into two subtypes. The two sets of monoclonal antibodies were useful for identification of the antigenic types of viruses isolated from patients in endemic areas.

Persistent infection of rats with haemorrhagic fever with renal syndrome virus and their antibody responses.

Newborn (within 24 h after birth), 1-week-old and 6-week-old (adult) rats were inoculated with a Hantaan-related virus (B-1) and attempts were made to isolate the virus from various organs. Virus-specific antigens were detected in various organs of newborn rats. Moreover virus could be isolated from almost all their organs even 25 weeks after infection. In contrast, in rats infected at 6 weeks of age the virus could be isolated from various organs but its concentration decreased progressively with time. The levels of haemagglutination-inhibiting (HI) and neutralizing antibodies to B-1 virus in the sera were measured. In adult rats, HI antibodies were first detected 2 weeks after infection and their titre rose to a maximum after 5 weeks. On the other hand, in newborn rats the levels of antibodies remained low until 5 or 6 weeks after infection and started to increase to a high level more than 9 weeks after infection. Furthermore, in rats infected soon after birth IgM antibodies predominated for a long time and these antibodies also neutralized infectivity at a high level. These data suggest that the induction of a high titre of neutralizing antibodies mainly of the IgM type does not result in virus clearance in newborn rats and that cellular immunity may be important for virus clearance in vivo.