The intracellular replication and molecular virulence mechanisms of Rabbit haemorrhagic disease virus (RHDV) are poorly understood, mainly due to the lack of an effective cell culture system for this virus. To increase our understanding of RHDV molecular biology, the subcellular localisation of recombinant non-structural RHDV proteins was investigated in transiently transfected rabbit kidney (RK-13) cells. We provide evidence for oligomerisation of p23, and an ability of the viral protease to cleave the p16:p23 junction in trans, outside the context of the nascent polyprotein chain. Notably, expression of the viral polymerase alone and in the context of the entire RHDV polyprotein resulted in a redistribution of the Golgi network. This suggests that, similar to other positive-strand RNA viruses, RHDV may recruit membranes of the secretory pathway during replication, and that the viral polymerase may play a critical role during this process.

Hantaan virus (HTNV) causes severe human disease. The HTNV genome consists of three ssRNA segments of negative polarity that are complexed with viral nucleocapsid (N) protein. How the human innate immune system detects HTNV is unclear. RNA helicase retinoic acid-inducible gene I (RIG-I) does not sense genomic HTNV RNA. So far it has not been analysed whether pathogenassociated molecular patterns generated during the HTNV replication trigger RIG-I-mediated innate responses. Indeed, we found that knock-down of RIG-I in A549 cells, an alveolar epithelial cell line, increases HTNV replication and prevents induction of 29,59-oligoadenylate synthetase, an interferon-stimulated gene. Moreover, overexpression of wild-type or constitutive active RIG-I in Huh7.5 cells lacking a functional RIG-I diminished HTNV virion production. Intriguingly, reporter assays revealed that in vitro-transcribed HTNV N RNA and expression of the HTNV N ORF triggers RIG-I signalling. This effect was completely blocked by the RNA-binding domain of vaccinia virus E3 protein, suggesting that dsRNA-like secondary structures of HTNV N RNA stimulate RIG-I. Finally, transfection of HTNV N RNA into A549 cells resulted in a 2 log-reduction of viral titres upon challenge with virus. Our study is the first demonstration that RIG-I mediates antiviral innate responses induced by HTNV N RNA during HTNV replication and interferes with HTNV growth. INTRODUCTIONHuman infections with hantaviruses are on the rise due to enhanced human contact with rodents, their main reservoir (Ludwig et al., 2003;Schmaljohn & Hjelle, 1997;Ulrich et al., 2002). Humans are infected after inhalation of aerosols from excreta shed by chronically infected rodents that do not show obvious symptoms. The outcome of human infection is variable and depends on the infecting hantavirus species. Pathogenic hantavirus species elicit highly lethal diseases, hantavirus cardiopulmonary syndrome (HCPS) or haemorrhagic fever with renal syndrome (HFRS) (Krüger et al., 2001;Muranyi et al., 2005). Hantaan virus (HTNV), the prototype member of the genus Hantavirus in the family Bunyaviridae, causes HFRS with a case fatality rate up to 15 %.Hantaviruses are enveloped and contain a tripartite ssRNA genome of negative polarity (Schmaljohn & Nichol, 2007). It consists of a small, medium and large segment that encode the nucleocapsid (N) protein, envelope glycoproteins (Gn and Gc) and RNA-dependent RNA polymerase (RdRp). The 59 and 39 termini of the hantaviral genome form a panhandle structure. HTNV replication starts with virion attachment to integrin b3 (CD61), a receptor for pathogenic hantaviruses (Gavrilovskaya et al., 1998(Gavrilovskaya et al., , 1999. Recently, decay-accelerating factor (CD55) and receptor for globular heads of C1q (gC1qR) have been defined as additional HTNV receptors (Choi et al., 2008; Krautkrämer & Zeier, 2008). After entry by endocytosis and acidification of endosomes fusion between endosomal membrane and viral envelope takes place. After release of the viral nucleocapsid int...

BackgroundOnly one strain (the Czech CAPM-v351) of rabbit haemorrhagic disease virus (RHDV) has been released in Australia and New Zealand to control pest populations of the European rabbit O. cuniculus. Antigenic variants of RHDV known as RHDVa strains are reportedly replacing RHDV strains in other parts of the world, and Australia is currently investigating the usefulness of RHDVa to complement rabbit biocontrol efforts in Australia and New Zealand. RHDV efficiently kills adult rabbits but not rabbit kittens, which are more resistant to RHD the younger they are and which may carry the virus without signs of disease for prolonged periods. These different infection patterns in young rabbits may significantly influence RHDV epidemiology in the field and hence attempts to control rabbit numbers.MethodsWe quantified RHDV replication and shedding in 4–5 week old rabbits using quantitative real time PCR to assess their potential to shape RHDV epidemiology by shedding and transmitting virus. We further compared RHDV-v351 with an antigenic variant strain of RHDVa in kittens that is currently being considered as a potential RHDV strain for future release to improve rabbit biocontrol in Australia.ResultsKittens were susceptible to infection with virus doses as low as 10 ID50. Virus growth, shedding and transmission after RHDVa infection was found to be comparable or non-significantly lower compared to RHDV. Virus replication and shedding was observed in all kittens infected, but was low in comparison to adult rabbits. Both viruses were shed and transmitted to bystander rabbits. While blood titres indicated that 4–5 week old kittens mostly clear the infection even in the absence of maternal antibodies, virus titres in liver, spleen and mesenteric lymph node were still high on day 5 post infection.ConclusionsRabbit kittens are susceptible to infection with very low doses of RHDV, and can transmit virus before they seroconvert. They may therefore play an important role in RHDV field epidemiology, in particular for virus transmission within social groups during virus outbreaks.