Virus- and cell type-specific effects in orthohantavirus infection.

Orthohantaviruses Hantaan (HTNV) and Puumala (PUUV) virus cause hemorrhagic fever with renal syndrome (HFRS), that is characterized by acute renal failure with often massive proteinuria and by morphological changes of the tubular and glomerular apparatus. Orthohantaviral N protein is found in renal cells and plays a key role in replication. However, the replication in human renal cells is not well characterized. Therefore, we examined the orthohantaviral infection in different human renal cells. Differences in localization of N protein, release of particles, and modulation of the actin cytoskeleton between both virus species are observed in human renal cells. A substantial portion of HTNV N protein demonstrates a filamentous pattern in addition to the typical punctate pattern. Release of HTNV depends on an intact actin and microtubule cytoskeleton. In contrast, PUUV N protein is generally localized in a punctate pattern and release of PUUV does not require an intact actin cytoskeleton. Infection of podocytes results in cytoskeletal rearrangements that are more pronounced for HTNV. Analyzing Vero E6 cells revealed differences compared to human renal cells. The pattern of N proteins is strictly punctate, release does not depend on an intact actin cytoskeleton and cytoskeletal rearrangements are not present. No virus-specific variations between HTNV and PUUV are observed in Vero E6 cells. Using human renal cells as cell culture model for orthohantavirus infection demonstrates virus-specific differences and orthohantavirus-induced cytoskeletal rearrangements that are not observed in Vero E6 cells. Therefore, the choice of an appropriate cell culture system is a prerequisite to study orthohantavirus pathogenicity.

The Long Noncoding RNA NEAT1 Exerts Antihantaviral Effects by Acting as Positive Feedback for RIG-I Signaling.

Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs (lncRNAs) play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus (HTNV) infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon (IFN-ß) production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I-IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein (SFPQ) by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections.IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs (lncRNAs) as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon (IFN) responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I-IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.

Hantavirus-like particles generated in CHO cells induce specific immune responses in C57BL/6 mice.

A safe and effective hantavirus vaccine is highly desirable since hantaviruses are distributed worldwide and cause an acute and often fatal disease (hemorrhagic fever with renal syndrome, HFRS). Virus-like particles (VLPs) displaying functional viral proteins could provide effective vaccines against a few viruses, but their ability to induce hantavirus-specific immune response has not been adequately investigated. To measure the immunogenicity of Hantaan virus-like particles (HTN-VLPs) vaccine, we generated recombinant HTN-VLPs by co-expressing Hantaan virus nucleocapsid (N) protein and glycoproteins (Gn and Gc) in Chinese hamster ovary (CHO) cells. We compared intramuscular versus subcutaneous administration of HTN-VLPs for the ability to induce specific immune response against Hantaan virus infection. Mice that received both intramuscular and subcutaneous immunizations of HTN-VLPs were sufficiently stimulated specific antibody response against Hantaan virus N protein and glycoproteins, which was comparable to Chinese commercial inactivated bivalent hantaviruses vaccine. Moreover, vaccination with HTN-VLPs also resulted in the induction of higher levels of specific cellular response to N protein than that of inactivated vaccine. Our results provide an important insight towards the development of hantaviruses-like particles as a potential candidate vaccine for the control and prevention of hantaviruses infection.

[The in vitro antiviral activity against the causative agent of hemorrhagic fever with renal syndrome].

It is shown that primary screening of drugs against the pathogens of hemorrhagic fever with renal syndrome (HFV) may be performed, by using two approaches in estimating the suppression of plague formation and that of viral reproduction in the cultured cells. It is expedient to make a primary screening of interferon and its inductors to estimate the suppression of viral reproduction in the cultured Vero E6, PSEV, and CL-17 cells, the infection multiplicity should be hundredth parts of BOE/cell. Among the test agents, there are virazole and realdiron that are the most active drugs against Hantaan virus, 76-118 strain, which virually completely suppress the reproduction of the study causative agent, when used even at concentrations of 1-5 microg/ml and 100 U/ml, respectively.

Dynein-dependent transport of the hantaan virus nucleocapsid protein to the endoplasmic reticulum-Golgi intermediate compartment.

In contrast to most negative-stranded RNA viruses, hantaviruses and other viruses in the family Bunyaviridae mature intracellularly, deriving the virion envelope from the endoplasmic reticulum (ER) or Golgi compartment. While it is generally accepted that Old World hantaviruses assemble and bud into the Golgi compartment, some studies with New World hantaviruses have raised the possibility of maturation at the plasma membrane as well. Overall, the steps leading to virion assembly remain largely undetermined for hantaviruses. Because hantaviruses do not have matrix proteins, the nucleocapsid protein (N) has been proposed to play a key role in assembly. Herein, we examine the intracellular trafficking and morphogenesis of the prototype Old World hantavirus, Hantaan virus (HTNV). Using confocal microscopy, we show that N colocalized with the ER-Golgi intermediate compartment (ERGIC) in HTNV-infected Vero E6 cells, not with the ER, Golgi compartment, or early endosomes. Brefeldin A, which effectively disperses the ER, the ERGIC, and Golgi membranes, redistributed N with the ERGIC, implicating membrane association; however, subcellular fractionation experiments showed the majority of N in particulate fractions. Confocal microscopy revealed that N was juxtaposed to and distributed along microtubules and, over time, became surrounded by vimentin cages. To probe cytoskeletal association further, we probed trafficking of N in cells treated with nocodazole and cytochalasin D, which depolymerize microtubules and actin, respectively. We show that nocodazole, but not cytochalasin D, affected the distribution of N and reduced levels of intracellular viral RNA. These results suggested the involvement of microtubules in trafficking of N, whose movement could occur via molecular motors such as dynein. Overexpression of dynamitin, which is associated with dynein-mediated transport, creates a dominant-negative phenotype blocking transport on microtubules. Overexpression of dynamitin reduced N accumulation in the perinuclear region, which further supports microtubule components in N trafficking. The combined results of these experiments support targeting of N to the ERGIC prior to its movement to the Golgi compartment and the requirement of an intact ERGIC for viral replication and, thus, the possibility of virus factories in this region.

Hypopituitarism as a late complication of hemorrhagic fever.

We report three patients who developed hypopituitarism as a late complication of hemorrhagic fever with renal syndrome (HFRS). Their past history, physical examination, and endocrine investigation confirmed hypopituitarism. Magnetic resonance imaging of the pituitary revealed atrophic pituitary gland with an empty sella. Hemorrhagic fever is endemic in certain regions of the Balkans, and this preliminary report suggests the importance of investigating the endocrine status in every patient who survived HFRS.

Bovine aortic endothelial cells are susceptible to Hantaan virus infection.

Hantavirus serotype Hantaan (HTN) is one of the causative agents of hemorrhagic fever with renal syndrome (HFRS, lethality up to 10%). The natural host of HTN is Apodemus agrarius. Recent studies have shown that domestic animals like cattle are sporadically seropositive for hantaviruses. In the present study, the susceptibility of bovine aortic endothelial cells (BAEC) expressing alpha(V)beta(3)-integrin to a HTN infection was investigated. Viral nucleocapsid protein and genomic RNA segments were detected in infected BAEC by indirect immunofluorescence assay, Western blot analysis, and reverse transcription-polymerase chain reaction (RT-PCR), respectively. The results of this study strongly support our previous observation on Puumala virus (PUU) that has been propagated efficiently in BAEC. These findings open a new window to contemplate the ecology of hantavirus infection and transmission route from animal to man.

[Detection of hantaan virus from gamasid mite and chigger mite by molecular biological methods].

OBJECTIVE: To study the proliferation and location of hantaan virus (HV) in gamasid mites and chigger mites. METHODS: HV RNA in gamasid mites and chigger mites were detected by reverse transcription, polymerase chain reaction (RT- PCR) and in situ hybridization. RESULTS: The smallest quantity of mite from which HV RNA could be detected was 5 mites group. The titers of -and proliferated in mites HV RNA could be found in ovary cells and dug cells of gamasid mites and chigger mites by in situ hybridization. CONCLUSIONS: The results showed that HV could be trans-stadially transmitted and proliferated in mites, and HV always located in ovary and dug organs of mites. These results provide direct evidence at molecular level for the role of gamasid mites and chigger mites as vectors in transmission of HV.

[Passage adaptability of candidate strains for hemorrhagic fever with renal syndrome purified vaccine in Vero cells and their immunogenicity].

OBJECTIVE: To adapt the candidate strains of hemorrhagic fever with renal syndrome (HFRS) purified vaccine to Vero cells and to study their antigenicity and immunogenicity. METHODS: The viral strains H8207 (Hantaan virus, HTN) and Y86013 (Seoul virus, SEO) were continuously propagated in Vero cell by the terminal dilution method and studied the characteristics of virus multiplication, viral titers and the amounts of virus antigen after serial passages. Three batches of crude monovalent inactivated vaccine were developed using the different passages of these 2 viral strains. RESULTS: The strains H8207 and Y86013 adapted to Vero cells and stably grew on the cells with high titers. Rabbits immunized with the crude vaccines of H8207 and Y86013 showed 100% sero-conversion and the neutralizing antibody titers of the rabbit immune sera reached 1?10 at 4 weeks after 2 times of immunization. CONCLUSIONS: The results suggest that these 2 candidate strains had adapted to Vero cells, possessed high titers and good immunogenicity and be feasible to prepare the HFRS purified vaccine in Vero cells.

[Distribution of hemorrhagic fever with renal syndrome virus in gamasid mites and chigger mites].

OBJECTIVE: To study the distribution of hemorrhagic fever with renal syndrome virus (HFRSV) in mites. METHODS: In situ reverse transcription-polymerase chain reaction (IS RT-PCR) was used for detecting the distribution of HFRSV in mites. RESULTS: HFRSV RNA was mainly located in ovary and mid-gut tissues of gamasid mites and chigger mites. The positive signal intensity in the third and fourth generations of gamasid mite was stronger than that in the first and second generations, and that in nymph of chigger mite more than larva. CONCLUSION: Both chigger mite and gamasid mite could play an important role in the transmission of HFRSV.

Application of cDNA microarray technique to detection of gene expression in host cells infected with viruses.

cDNA microarray technique was used to monitor changes in mRNA levels in cells after Hantaan virus (HTNV) infection. The values of the ratio of medians for HTNV and Japanese encephalitis virus (JEV) at the early stage of infection were compared and found similar, suggesting that the same or similar genes are associated with the early events of infection with either virus. The reproducibility of values of the "ratio of medians" for HTNV was examined. We found that applying cluster analysis to the gene expression data groups efficiently together genes with the same function. Therefore, in analyzing the effects of viral infection on host cells by the cDNA microarray technique, clustering data appear to be necessary for gaining biological meaning from a dump of gene expression profiles obtained from virus-infected cells.

[Proliferation and location of Hantaan virus in gamasid mites and chigger mites, a molecular biological study].

OBJECTIVE: To study the proliferation and location of Hantaan virus (HV) in gamasid mites and chigger mites and the significance of gamasid mites and chigger mites as vectors of transmission of hemorrhagic fever with renal syndrome (HFRS). METHODS: Gamasid mites collected from the nests of wild rodents and chigger mites collected from the bodies of wild rodents and thick growth of grass in the field were raised. Two oligonucleotide primers were developed based on the gene fragments of cDNA of HV 76 - 118 strain to be used in RT-PCR. RNA was extracted from the suspension of the gamasid mites from the nests where rodents no HV had been found in whose lungs lived and from the unfed larvae of chigger mites, being formed groups of 5, 10, 30, or 50 individuals. RT-PCR was conducted to detect the HV-RNA in such suspension. The larvae, nymphs, and imagines of both kinds of mites were ground to make suspension at an interval of 20 days. Vero-E(6) cells were inoculated to measure the titer of 50% tissue culture infective dose (TCID(50))/ml of HV. Frozen sections of larvae, nymphs, and imagines of both kinds of mites were made. RT-PCR and in situ hybridization were conducted to detect the distribution of HV-positive particles. Monoclonal antigen technique was used to compare the antigenicity of the HV-RNA from the rodents, mites, and patients from the same epidemic areas. RESULTS: HV-RNA was detected in gamasid mites and chigger mites. Except in the larvae of chigger mites 60 days after collection, titers of HV were detected and increased gradually in mites at different stages of life cycle. HV-RNA positive particles were detected in the epithelial cells of midgut and ovary, with the signal denser and more numerous in nymphs than in larvae. The genotypes of HV from rodents, mites and patients in the same endemic areas were identical: HTN type virus. CONCLUSION: HV can be transmitted transstadially and proliferated in mites, gamasid mites and chigger mites play the role of vectors of transmission for HFRS.

[Detection and proliferation of hemorrhagic fever virus in chigger mites].

OBJECTIVE: To study the impact of Leptotromhidium scutellaris as a vector in transmission of hemorrhagic fever. METHODS: Reverse transcription-polymerase chain reaction (PCR) and in situ hybridization technique were used to detect RNA of hemorrhagic fever with renal syndrome virus (HFRSV). TCID(50)/ml of HFRSV were titrated for larva and nymph of chigger mites periodically. RESULTS: RNA of HFRSV was detected in the tissues of chigger mites, such as ovary cells, etc. by PCR and in situ hybridization. Titration of TCID(50)/ml showed that HFRSV could be transmitted transstadially and proliferated in chigger mites. CONCLUSION: It provides direct evidence at molecular level for the role of chigger mite as a vector in transmission of HFRSV with theoretical and practical importance in prevention of HFRS.

Characterization of the mode of Hantaan virus infection in adult mice using a nested reverse transcriptase polymerase chain reaction: transient virus replication in adult mice.

A polymerase chain reaction (PCR) for the detection of hantavirus genome was established and applied to analyze the mode of infection of Hantaan virus in adult ICR mice. The cDNA for the S genome segment of Hantaan virus was reverse-transcribed from the total RNA of organs of the infected mice. The sequence in the S genome segment of Hantaan virus was successfully amplified by reverse transcriptase (RT)-PCR followed by nested PCR. In 5-week-old ICR mice inoculated intraperitoneally with Hantaan virus, strain 76-118 (1.3 x 10(5) FFU/mouse), the virus was detected in clots and lungs from 3 to 10 days post-inoculation (p.i.) by nested PCR and virus-isolation techniques. No virus was detected in any specimens collected on 1 day and after 28 days p.i., and in spleens and brains through the observation period by both methods. The antibody which was measured by indirect immunofluorescence antibody assay (IFA) appeared at 7 days p.i. and the geometric mean titer was elevated to its maximum level of 1:203 at 10 days p.i., maintaining the same level until 35 days p.i. These results suggest that adult mice are transiently infected with Hantaan virus.

[Propagation of the HTV in primary human embryonic kidney and lung cell culture].

2 strains of Hantaan virus (HTV, 76-118, Hubei-114) have been propagated successfully in cultured primary human embryonic kidney (HEK) and lung (HEL) cells. Cytopathic effect (CPE) was observed in the two kind of cells on day 5 to 7 postinoculation which showed the cell became round and clustered, then detached. The replicating peak of the Hubei-114 in two kinds of cell cultures appeared on the 11th day and another strain on the 14th or 17th day after infection. The ultrastructure changes were observed with EM and IEM, which stained by ICGT before embedding. It was discovered that the mitochondia atrophied and decreased, and inclusion bodies in the cytoplasma of HEK and KEL cells. A large amount of gold granulae were found in the inclusion bodies and the virions were seen occasionally. Contamination with other agents have been ruled out. Our data suggest that the replicating characters of HTV in these cell systems might be possible for the pathogenicity of HFRS for human.

Comparison of virulence between Seoul virus strain SR-11 and Hantaan virus strain 76-118 of hantaviruses in newborn mice.

Virulence of hantavirus strain of SR-11 Seoul virus and Hantaan 76-118 (HTN) of Hantaan virus were compared. Infections of both strains were lethal in newborn mice. However, inoculum required to cause lethal infection was about 4,000 times higher for strain HTN (1.65 x 10(3) PFU/mouse/LD50) than for strain SR-11 (0.36 PFU). Thus, both strains were considered pathogenic to newborn mice but they possessed different levels of virulence. The assay system used for these strains in newborn mice proved to be useful in the study of hantavirus virulence. Growth curves of the two strains in CV-7 cell cultures were compared. Strain SR-11 was shown to have higher activity of virus replication and virus release into the culture fluids than strain HTN. The possibility of a relationship between replication activity and high levels of virulence in mice was suggested.