Korean hemorrhagic fever: propagation of the etiologic agent in a cell line of human origin.

The etiologic agent of Korean hemorrhagic fever has been propagated in a human cultured cell line derived from a carcinoma of the lung. The cells, described as type II, alveolar epithelial, support replication of the agent and successive passages. Antigen of the Korean hemorrhagic fever agent is readily detected in infected cells by means of direct or indirect fluorescent antibody techniques. Previous attempts to propagate this agent in vitro had been unsuccessful.

Antigenic and genetic properties of viruses linked to hemorrhagic fever with renal syndrome.

Hemorrhagic fever with renal syndrome (HFRS) comprises a variety of clinically similar diseases of viral etiology that are endemic to and sporadically epidemic throughout the Eurasian continent and Japan. Although HFRS has not been reported in North America, viruses that are antigenically similar to HFRS agents were recently isolated from rodents in the United States. Examination and comparison of eight representative isolates from endemic disease areas and from regions with no known associated HFRS indicate that these viruses represent a new and unique group that constitutes a separate genus in the Bunyaviridae family of animal viruses.

Epidemic hemorrhagic fever with renal syndrome. A broadening horizon.

A virus identical to or closely related to the viruses of the epidemic hemorrhagic fevers and/or nephropathia epidemica has been demonstrated serologically in rats from geographically separate areas of this country by three groups of investigators. This information suggests a previously unrecognized health threat that should be considered in the differential diagnosis of patients initially seen with febrile illnesses and acute renal failure. The salient features of these viral diseases and the clues that should alert the clinician to seek serologic confirmation of Hantaan or an immunologically related virus are discussed herein.

Antigenic and molecular characterization of hantavirus isolates from China.

Hemorrhagic fever with renal syndrome (HFRS) is caused by certain viruses in the genus Hantavirus, family Bunyaviridae, and is a major public health problem in China. By using molecular and serological tests, we characterized 15 hantaviruses isolated either from patients with HFRS or from rodents captured in endemic areas of China. By cross plaque-reduction neutralization tests performed with rabbit immune sera, we identified two serologically distinct groups of viruses, comprised of those related to Hantaan virus, and those related to Seoul virus. To study the genetic relationships among these viruses, we amplified a 330 base pair region of the medium (M) genome segment of each isolate by reverse transcription and polymerase chain reaction (PCR) and compared the nucleotide sequences to those of other, well-characterized hantaviruses. In addition, we PCR-amplified and analyzed the entire coding region of the small (S) genome segment of each isolate by restriction enzyme digestion with a battery of enzymes. The results of our genetic analyses of both the M and S segments of these isolates confirmed our serological data, indicating that Hantaan and Seoul viruses co-circulate in endemic disease regions of China. We constructed a phylogenetic tree based on multiple alignment of the partial M segment sequences. The resulting dendrogram distinguished three genetic subtypes of Hantaan viruses and one type of Seoul virus.

Hantavirus: an old bug learns new tricks.

Hantaviruses are a diverse group of RNA arboviruses in the Bunyaviridae family. Although their role as the causative agents of HFRS has been well established, the recent outbreak of a new disease in the Southwest clearly demonstrates the protein clinical manifestations that this pathogen can produce. Furthermore, whereas hantaviruses have been characterized largely as focal agents in the production of geographically delimited diseases, recent trends indicate that endemic areas for the virus are expanding. Outbreaks often occur in clusters as a result of the epizoology of rodent hosts, but isolated cases of hantavirus-related disease also may be observed. Although hantaviruses have proven their pathogenic capability in other areas of the world, it was perceived widely that they were of little consequence to public health in the United States. However, as more is learned about the nature of this truly global infectious agent, its potential danger to mankind becomes increasingly apparent. It is hoped that continued research will elucidate all the facets of hantavirus-induced disease.

Comparison of European isolates of viruses causing hemorrhagic fever with renal syndrome by a neutralization test.

Different virus isolates causing hemorrhagic fever with renal syndrome (HFRS) were compared using a neutralization test. Patient convalescent sera and antisera prepared in rabbits were used to compare Puumala-related Hantavirus isolates from Finland, Sweden, Belgium, and the USSR. The majority of European isolates were indistinguishable from each other using both homologous rabbit antisera and patient convalescent sera. The European isolates of HFRS were also compared with prototype Hantaan (the etiologic agent of Korean hemorrhagic fever). The one-way cross-reaction between the Hantaan and Puumala viruses, previously described using human convalescent sera tested by indirect immunofluorescence and immunoprecipitation, was also seen by the neutralization test.

Intraspecific transmission of Hantaan virus, etiologic agent of Korean hemorrhagic fever, in the rodent Apodemus agrarius.

Experimental parameters of infection and intraspecific transmission of Hantaan virus, the etiologic agent of Korean hemorrhagic fever, in Apodemus agrarius rodents were determined. Mice inoculated by the intramuscular route experienced viremia for about 1 week beginning on day 7. After 3 weeks, immunofluorescent and neutralizing antibodies were present and no mouse ever developed signs of acute illness. Virus was recovered from lung, kidney, salivary gland, and liver, and virus excretion in urine, saliva, and feces occurred from about day 10 through day 360 (urine) post-inoculation. Antigen, but not infectious virus, was persistent in lung tissue for as long as 1 year. Horizontal contact infection occurred among cage-mates regardless of sexual pairing up to 360 days after infection and no evidence for participation of ectoparasitic arthropods in such transmission was obtained.

Pathogenesis of Hantaan virus infection in suckling mice: clinical, virologic, and serologic observations.

Hemorrhagic fever with renal syndrome (HFRS) is a debilitating disease of humans caused by Hantaan virus (HV), the prototype member of a newly proposed genus of Bunyaviridae. Studies of HV pathogenesis have been limited by the absence of a well defined model for a virus-induced disease state. In an attempt to devise a model for HV pathogenesis in laboratory rodents, newborn outbred suckling ICR mice were shown to be uniformly susceptible to lethal infection with non-mouse adapted HV by intracerebral (IC), intraperitoneal (IP), intramuscular (IM), and subcutaneous (SC) inoculation routes. Clinical course, mean time to death, and fatal outcome were age-dependent. With an inoculum of 10 LD50, mortality was 100% in mice infected within 72 hr of birth, but declined to 50% by 7 days. By 2-2.5 weeks, animals developed complete resistance to clinical disease. Virus was consistently detected in serum by day 6 post-infection in IC- and IP-inoculated animals, and reached peak levels of congruent to 10(5) PFU/ml by day 8. Mice infected IM and SC showed delays in onset of viremia, but achieved similar titers. Immunofluorescent antibody appeared by 17-18 days, and neutralizing antibody by 15 days, in all experimental groups. Two of 8 inbred mouse strains were identified as resistant to clinical disease: SJL/J and A/J.

Epidemiological investigations following an outbreak of hemorrhagic fever with renal syndrome in Greece.

Epidemiological investigations were conducted following an outbreak of hemorrhagic fever with renal syndrome (HFRS) which occurred in the state of Epirus, northwestern Greece, in July and August 1983. A total of 8 patients were hospitalized during the outbreak; 3 were severely ill and 1 died. A serosurvey made in May 1984 sampled 184 of the approximately 400 residents of the village of Tsepelovo, where 4 patients resided, and found 12 (6.5%) persons, including convalescent sera from 4 patients, with antihantaviral antibody by immunofluorescent antibody (IFA) tests. Small mammal collections found house rats common in the village, but none exhibited anti-hantaviral antibody. Collections in nearby fields and mountains found Apodemus flavicollis rodents common, and 2 (6%) of 33 captured had high IFA anti-hantavirus antibody. Virus isolation attempts from rodent tissues were unsuccessful. Testing of convalescent patients' sera by IFA and plaque reduction neutralization tests indicated that the etiological agent was neither Puumala virus nor Seoul virus, but appears to be a strain of Hantaan virus or perhaps a new virus. The rodent host of this virus may be A. flavicollis, and the distribution of this species corresponds with previously reported cases of severe HFRS described elsewhere in central Europe.

Grouping of hantaviruses by small (S) genome segment polymerase chain reaction and amplification of viral RNA from wild-caught rats.

A single pair of consensus primers in the polymerase chain reaction (PCR) amplified a conserved region of the small genome segment of twenty hantavirus isolates. Isolates tested included representatives of the four recognized hantaviruses, Hantaan, Seoul, Puumala and Prospect Hill, as well as isolates from Mus musculus (Leakey), Bandicota indica (Thailand 749), and Suncus murinus (Thottapalayam). Viruses from the Nairovirus and Phlebovirus genera yielded negative results. The amplification products were 281-nucleotide pairs (np) in length, with the exception of Thottapalayam, which had an amplification product of approximately 320 np. Products of all isolates were detected by Southern hybridization with a 32P-labeled Hantaan 76-118 amplicon, while an oligonucleotide probe to a conserved region of the amplified fragment failed to detect some isolates of Seoul and Puumala viruses. Restriction endonuclease analysis allowed three groupings: Hantaan-like viruses, Seoul-like viruses, and a diverse group of patterns for the other viruses. Differences were found within the Seoul-like virus group by this method, whereas the Hantaan-like viruses were shown to be similar. RNA extracted from tissues of seropositive and seronegative rats trapped in Baltimore showed the practical application of the test. Hantavirus-specific RNA was detected in 12 (92%) of 13 seropositive rats, but not in seronegative rats. This simple method for detecting and characterizing hantaviruses has potential for epidemiologic studies and for diagnosing human hantavirus infections.

Isolation of a Hantaan-related virus from Brazilian rats and serologic evidence of its widespread distribution in South America.

A serosurvey of domestic rats was conducted in several South American cities between September 1982 and March 1983 for evidence of hantavirus infection. Antibody-positive rats were found in Belem, São Paulo and Recife-Olinda, Brazil and in Buenos Aires, Argentina, with the highest antibody prevalence rate detected in Belem (30 positive of 54 tested, 56%). A virus isolated from tissues of a Rattus norvegicus captured in Belem, was shown to be antigenically similar to Girard Point viruses isolated from domestic rats captured in the United States and clearly distinct from prototype Hantaan virus, causative agent of Korean hemorrhagic fever in Asia. This represents the first isolation of a virus of the genus Hantavirus from South America and supports previous observations that indicate a widespread distribution of urban rat-associated hantaviruses. The abundance of domestic rats and their regionally high antibody rates suggest that risk of human hantavirus infection in some locations of South America may be significant.

Serological survey of Prospect Hill virus infection in indigenous wild rodents in the USA.

We found serological evidence of infection with Prospect Hill virus, a Hantaan-like virus isolated from meadow voles (Microtus pennsylvanicus), in microtine and cricetid rodents trapped in Maryland, West Virginia, Minnesota and California, USA. Fluorescent antibodies were detected in sera from M. pennsylvanicus (74/277), M. californicus (39/185), Clethrionomys gapperi (5/51), Peromyscus maniculatus (4/22) and P. truei (1/11). Sera from seropositive P. maniculatus contained neutralizing antibodies against Prospect Hill virus, confirming that infection with Prospect Hill virus or antigenically related viruses is not restricted to microtine rodents in the USA. Despite the widespread distribution of Prospect Hill virus in indigenous rodents, the recent demonstration that American mammalogists are only rarely infected supports the view that the overall risk of Prospect Hill virus infection in man is low.

Infection of laboratory workers with hantavirus acquired from immunocytomas propagated in laboratory rats.

Hantavirus has been isolated in cell culture from rat immunocytomas used and stored at a research laboratory in the U.K. where there was evidence of a laboratory-acquired infection leading to haemorrhagic fever with renal syndrome. Both transplantation into LOU/M/Wsl rats and storage of passaged immunocytomas at -70 degrees C over a period of 8-10 years had not eliminated the virus. The isolates were identified as Hantavirus by means of serum obtained from patients with hantavirus infection as well as polyclonal serum derived from laboratory animals. This paper identifies a potential source of hantavirus infection in laboratories. The importing of rats, rat immunocytomas and anti-immunocytoma serum in relation to the potential risks of laboratory-acquired hantavirus infection is discussed.

Different pathohistological presentations of acute renal involvement in Hantaan virus infection: report of two cases.

We present two patients with Hantaan virus infection, admitted to the Department of Nephrology, Skopje, at the same time, with the same clinical presentation (chills, fever, abdominal pain, hemorrhages, nausea, headache, proteinuria, hematuria, oliguria, acute renal failure) but with different pathohistological findings and different disease courses. In the first case diffuse proliferative glomerulonephritis was found, with a complete recovery of renal function after a month, with a mild proteinuria and erythruria during the second and the third month. In the second case, glomeruli were normal in general, with slight mesangial proliferation found in two out of twenty, but interstitial edema, lymphocyte infiltrations and tubular changes were noted. Complete recovery was not noted after 3 months of follow-up. The patient is now without hemodialysis treatment, with polyuria, in the stable phase of chronic renal failure which is not improving.

Severe hemorrhagic complications from infection with nephropathia epidemica strain of Hantavirus.

In the following we describe a case of severe hemorrhagic fever with renal syndrome (HFRS) caused by Puumala infection. The diagnosis was made by immunofluorescence technique and by solid phase enzyme immunoassay using recombinant nucleocapsid antigen of a Puumala serotype strain. Such a clinical course with severe bleeding complications is considered untypical for Puumala induced HFRS.

Effects of Hantaan virus on human endothelial cells and their significance in pathogenesis of hemorrhagic fever with renal syndrome.

The effects of Hantaan virus (HTNV) on human endothelial cells (HECs) were investigated both in vivo and in vitro. The 76-118 strain or SR-11 strain of HTNV were inoculated into HECs monolayers respectively, and the virus antigens could be detected on the seventh day of the first passage after inoculation by immunofluorescent technique. The HTNV could also be isolated through cultures of Vero E-6 cells. HTNV particles and inclusion bodies together with various changes in the organelles were observed in the infected cells by transmission electronic microscopy (TEM), and the immunoenzyme positive virus particles were seen by immunoelectronic microscopy. Samples of skin biopsy were individually obtained from 14 cases of hemorrhagic fever with renal syndrome on the second to the fifth day after the onset of the illness. It was found that HTNV antigens were widely distributed in the cytoplasm of endothelial cells of the samples from 5 out of 14 cases by avidin-biotin-peroxidase complex staining, and morphologic changes of the endothelial cells similar to those observed in vitro, were also seen by TEM. The results indicated that HEC is one of the target cells susceptible to HTNV. The virus could invade and propagate in HECs, and could induce damage to the latter.

Isolation of a strain of Hantaan virus from peritoneal exudate cells of a patient with hemorrhagic fever with renal syndrome.

A new strain of Hantaan virus (HTNV), GH716, was isolated from the peritoneal exudate cells (PEC) of a patient with severe hemorrhagic fever with renal syndrome (HFRS). The isolate was propagated in Vero E6 cells. At each passage the virus-infected cells were examined for HTNV with immunofluorescence technique using monoclonal antibodies (McAb) 25-1 and 84-1 against HTNV. From passage 5 on, fluorescent intensity tended to stabilize at (+++) and infectivity titer reached 10(5) TCID50/ml. Using 14 McAb to 76-118 and BI strains of HTNV, we found that strain GH716 was antigenically similar to strain 76-118 (Apodemus type) and different from strain R22 (Rattus type), suggesting that GH716 may fall into the Apodemus type of HTNV. This is the first isolation of an HTNV from human PEC collected on the tenth day of illness. The successful isolation of strain GH716 may provide an alternative source of obtaining HTNV during the later stages of HFRS.

The duration of viremia patients with epidemic hemorrhagic fever.

The duration of viremia in patients with epidemic hemorrhagic fever (EHF) was studied using immunofluorescence technique and cell culture assays. The duration of virus in plasma of EHF patients was about 1 week, which was concomitant with the febrile phase of the disease. Comparatively, the separation rate of viruses in peripheral blood mononuclear cells (PBMC) was greater (1.8 times) than in plasma, while the detected peak of EHF (in 4-7 days after onset of the disease) was found to be 2 or 3 days less than that of plasma, thus the detectable viremia was prolonged until the 8th to 11th day of the disease. High titre EHF antibody of IgG, TgM was unable to neutralize the virus in human blood. The results revealed that a blood-cell-associated prolonged viremia is one of the characteristics of EHF infection.