Generation and characterization of genetic reassortants between Puumala and Prospect Hill hantavirus in vitro

Abstract

Hantaviruses belong to the family Bunyaviridae characterized by tri-segmented RNA genomes. Depending on the hantavirus species, infection can lead to hantavirus cardiopulmonary or haemorrhagic fever with renal syndrome. In vitro studies suggest that pathogenic hantaviruses evade induction of innate antiviral responses, and this ability might determine the virulence in humans. Since reverse genetic systems are not available, in vitro reassortment is currently the only way to culture defined hantavirus variants. Here, we demonstrate for the first time the generation of a reassortant between a pathogenic Old World and a non-pathogenic New World hantavirus in vitro. The reassortant contained the glycoprotein coding M-segment derived from the pathogenic Puumala virus (PUUV) and the other genomic segments coding for the nucleocapsid protein and RNA-dependent RNA-polymerase from Prospect Hill virus (PHV), which is taken as nonpathogenic in humans. Exchange of the M-segment was confirmed by sequencing and virus neutralization test with PUUV-specific sera. Functional analysis of the reassortant and parental viruses revealed characteristic growth kinetics and innate immune responses as determined by expression analyses for lambda interferon and MxA, and by interferon-stimulated response element reporter gene studies. Consistent with previous studies with other pathogenic hantaviruses, PUUV elicited reduced innate responses if compared with PHV. In all these functional assays the reassortant revealed PHV-like phenotypes. Thus, neither the PUUV Msegment nor entry via specific M-segment directed pathways modulated the virus type-specific innate responses. Moreover, the data imply that this approach might be an option for production of attenuated viruses that could be used as vaccines against pathogenic hantaviruses.

Genetic reassortment between high-virulent and low-virulent Dobrava-Belgrade virus strains

“…In contrast, Slo/ Af elicited an early and strong MxA protein expression in HuH7 cells, which decreased steadily with establishment of the infection. Interestingly, we found the same transient MxA expression pattern in HuH7 cells infected with PUUV [20]. This result might suggest that at later time points during infection DOBV Slo/Af (and also PUUV) develops an antagonistic activity which interferes with activation of the IFN response or directly targets MxA for degradation.…”

“…As a consequence, the absence of natural genetic reassortment has been taken as one of the criteria in the hantavirus species definition [16]. In cell culture, genetic reassortment has been shown for American hantaviruses of the same or of different species [17][18][19] as well as between American Prospect Hill virus and European Puumalavirus [20]. In vitro reassortment has not been studied for European Dobravavirus.…”

Characterization of Two Substrains of Puumala Virus That Show Phenotypes That Are Different from Each Other and from the Original Strain

“…Reassortments are frequently observed for PUUV in the natural hosts, and it seems that there is a nonrandom distribution of genomic segments, with S and L preferentially originating from the same isolate found in the reassortments (45). Cocultivation of Andes and Sin Nombre hantaviruses also resulted in reassortments with homologous S and L segments (46), as did a reassortment between PUUV and Prospect Hill virus containing a PUUV M segment and Prospect Hill virus S and L segments (18). However, our data do not exclude the possibility that the M segment found in PUUV-La and PUUV-Sm is well conserved within or might even represent the only M-segment variant in the different substrains that constitute the PUUV-Pa strain.…”

Hantavirus Emergence in Rodents, Insectivores and Bats

“…The genus Tospovirus contains only plant pathogens transmitted by arthropods whereas the genera Orthobunyavirus, Nairovirus and Phlebovirus comprise arthropod-borne bunyaviruses including zoonotic representatives causing disease in human and animals. In fact, reassortment has been reported for closely related hantaviruses in vitro, but also in nature (Li et al, 1995;Rodriguez et al, 1998;Kirsanovs et al, 2010;Klempa et al, 2003;Razzauti et al, 2008;Zou et al, 2008;Razzauti et al, 2009;Handke et al, 2010). Thus, the criteria for the definition of a virus species within the different genera of the family differ .…”