key: cord-292353-z86rjwle
authors: Hussein, Islam T.M.; Haseeb, Abdul; Haque, Absarul; Mir, Mohammad A.
title: Recent Advances in Hantavirus Molecular Biology and Disease
date: 2011-04-01
journal: Adv Appl Microbiol
DOI: 10.1016/b978-0-12-387022-3.00006-9
sha: 
doc_id: 292353
cord_uid: z86rjwle

Hantaviruses are emerging zoonotic pathogens that belong to the Bunyaviridae family. They have been classified as category A pathogens by CDC (centers for disease control and prevention). Hantaviruses pose a serious threat to human health because their infection causes two highly fatal diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). These pathogens are transmitted to humans through aerosolized excreta of their infected rodent hosts. Hantaviruses have a tripartite-segmented negative-sense RNA genome. The three genomic RNA segments, S, M, and L, encode a nucleocapsid protein (N), a precursor glycoprotein that is processed into two envelope glycoproteins (Gn and Gc) and the viral RNA-dependent RNA polymerase (RdRp), respectively. N protein is the major structural component of the virus, its main function is to protect and encapsidate the three genomic RNAs forming three viral ribonucleocapsids. Recent studies have proposed that N in conjunction with RdRp plays important roles in the transcription and replication of viral genome. In addition, N preferentially facilitates the translation of viral mRNA in cells. Glycoproteins, Gn and Gc, play major roles in viral attachment and entry to the host cells, virulence, and assembly and packaging of new virions in infected cells. RdRp functions as RNA replicase and transcriptase to replicate and transcribe the viral RNA and is also thought to have endonuclease activity. Currently, no antiviral therapy or vaccine is available for the treatment of hantavirus-associated diseases. Understanding the molecular details of hantavirus life cycle will help in the identification of targets for antiviral therapeutics and in the design of potential antiviral drug for the treatment of HFRS and HCPS. Due to the alarming fatality of hantavirus diseases, development of an effective vaccine against hantaviruses is a necessity.

renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). These pathogens are transmitted to humans through aerosolized excreta of their infected rodent hosts. Hantaviruses have a tripartite-segmented negative-sense RNA genome. The three genomic RNA segments, S, M, and L, encode a nucleocapsid protein (N), a precursor glycoprotein that is processed into two envelope glycoproteins (Gn and Gc) and the viral RNA-dependent RNA polymerase (RdRp), respectively. N protein is the major structural component of the virus, its main function is to protect and encapsidate the three genomic RNAs forming three viral ribonucleocapsids. Recent studies have proposed that N in conjunction with RdRp plays important roles in the transcription and replication of viral genome. In addition, N preferentially facilitates the translation of viral mRNA in cells. Glycoproteins, Gn and Gc, play major roles in viral attachment and entry to the host cells, virulence, and assembly and packaging of new virions in infected cells. RdRp functions as RNA replicase and transcriptase to replicate and transcribe the viral RNA and is also thought to have endonuclease activity. Currently, no antiviral therapy or vaccine is available for the treatment of hantavirus-associated diseases. Understanding the molecular details of hantavirus life cycle will help in the identification of targets for antiviral therapeutics and in the design of potential antiviral drug for the treatment of HFRS and HCPS. Due to the alarming fatality of hantavirus diseases, development of an effective vaccine against hantaviruses is a necessity.

Two major outbreaks that occurred in the past led to the discovery of hantaviruses and directed the attention of researchers toward the hantavirus-associated diseases. The first outbreak occurred during the Korean War (1950) (1951) (1952) (1953) , with more than 3000 cases of illness reported among the United Nations troops. The disease was initially named ''Korean hemorrhagic fever'' and is now commonly referred to as hemorrhagic fever with renal syndrome (HFRS). The second outbreak, which occurred in the Four Corners region of the United States in 1993, triggered the attention of the World Health Organization toward a new highly lethal disease. The disease was initially referred to as four-corner disease and is now called hantavirus pulmonary syndrome (HPS) or hantavirus cardiopulmonary syndrome (HCPS). Hantaviruses cause serious human illness with a mortality rate that ranges from 15% (for HFRS) to 50% (for HCPS). Twenty-five years after the Korean War, the etiologic agent of this disease, the Hantaan virus (HTNV), was identified in the striped field mouse (Apodemus agrarius) (Lee et al., 1981a) . Further studies revealed that the newly identified virus belonged to the Bunyaviridae family. In 1981, a new genus termed as ''hantavirus'' was introduced into the Bunyaviridae family, which included the viruses that cause HFRS (Fig. 2.1 ). Further investigations revealed that, unlike other members of the Bunyaviridae family, hantaviruses did not have an arthropod vector and exclusively establish a persistent infection in the population of their specific rodent hosts. The landmark discovery of HTNV encouraged more efforts to identify the etiologic agents of HFRS-related disease in Asia, Europe, and the United States. These efforts led to the discovery of Haantan-like viruses in other rodents from Far East Russia, China, and South Korea. Dobrava virus (DOBV), a distinct hantavirus, was isolated from Apodemus flavicollis, A. Agrarius, and Apodemus ponticus in Europe (Avsic-Zupanc et al., 1995 Golovljova et al., 2000; Jakab et al., 2007; Klempa et al., 2005 Klempa et al., , 2008 Klingstrom et al., 2006a) . In 1980s, a rat-born Seoul virus (SEOV) was found to cause HFRS in urban areas of Asia (Chan et al., 1987; Kim et al., 1995) . Around the same time period, Puumala virus (PUUV), the etiologic agent of nephropathia epidemica, a milder form of HFRS reported in 1930s in Europe, was identified in bank voles (Myodes glareolus) (Clement et al., 2003) . Despite these early efforts, the lack of advanced technology delayed the identification of etiologic agents of HFRS. However, the etiologic agent and rodent reservoir of HCPS, the Sin Nombre virus (SNV), were discovered within few weeks after the 1993s outbreak in the Four Corners region of the United States Nichol et al., 1993) . After this outbreak, other hantavirus strains causing HCPS were discovered and more than 2000 cases of HCPS have been reported throughout the Americas (Aquino et al., 2003; Barclay and Rubinstein, 1997; Bayard et al., 2004; Bohlman et al., 2002; Chu et al., 2006; Fulhorst et al., 1997; Hjelle et al., 1994 Hjelle et al., , 1996 Jiang et al., 1997; Johnson et al., 1997; Jones et al., 2008; Nichol et al., 1993; Vincent et al., 2000) . Currently, the hantavirus genus includes more than 21 species and more than 30 genotypes (Avsic-Zupanc et al., 2000; Chu et al., 2006) , although many more remain undiscovered due to the lack of advanced technology in other parts of the world including Africa, Middle East, and the Indian subcontinent. For example, recent discoveries of shrewborn hantavirus in several countries support the existence of other hantavirus species worldwide (Arai et al., 2007; Kang et al., 2009a) . Annually, 150,000-200,000 cases of hantavirus infection are reported worldwide. Due to high emergence and significant mortality of hantavirus infections, a serious attention from research scientists and world health organizations is required to promote public awareness and accelerate efforts for the treatment of hantavirus-associated diseases. Here, we summarize the findings about the microbiology of hantaviruses with an emphasis on nucleocapsid protein, glycoproteins Gn, Gc, and RNA-dependent RNA polymerase (RdRp). A brief synopsis about different aspects of hantavirus-associated disease is also presented in this chapter.

Hantavirus particles generally appear spherical or pleomorphic in electron micrographs ( Fig. 2. 2). They range in diameter from 80 to 120 nm Martin et al., 1985) . Hantaviruses have tripartite single-stranded negative-sense RNA genome that encodes an RdRp (large or L segment), two glycoproteins (medium or M segment), and a nucleocapsid protein (N) (small or S segment). The negative-sense genomes serve as templates for producing positive-sense complementary RNA (cRNA) and messenger RNA (mRNA). The total size of the RNA genomes ranges from 11,845 nucleotides for HTNV to 12,317 nucleotides for SNV (Hooper and Schmaljohn, 2001; Jonsson et al., 2010; Schmaljohn et al., 1985 Schmaljohn et al., , 1986 . The viral RNA segments are coated with the nucleocapsid protein forming three helical ribonucleoprotein (RNP) complexes ( Fig. 2.2) . The viral RdRp is associated with these nucleocapsids (Dahlberg et al., 1977; Obijeski et al., 1976) . The sequences at both the 3 0 and 5 0 termini of each RNA segment are complementary forming ''panhandle'' structures that are specifically recognized by the N protein and were shown to be important for viral transcription and replication. Viral mRNAs contain untranslated regions (UTRs) at both their 5 0 and 3 0 ends that flank the open reading frame (ORF) of the encoded viral protein. The 5 0 UTR of viral mRNA contains trinucleotide repeats (UAGUAGUAG), which were suggested to be involved in N-mediated translation initiation of viral mRNA. Similarly, a triplet repeat at the 3 0 UTR of vRNA has been proposed to play a role in the prime and realign mechanism of transcription initiation by the viral RdRp (Hewlett et al., 1977; Mir and Panganiban, 2004 , 2010 Pettersson and von Bonsdorff, 1975; Raju and Kolakofsky, 1989) . Hantaviruses are enveloped with an outer lipid bilayer derived from the Golgi membranes ( Fig. 2 .2). The envelope carries two viral-encoded glycoproteins, Gn and Gc, in the form of heterodimers which assemble into higher order oligomers and appear as projections or spikes on the outer surface of the virion (Hepojoki et al., 2010a; Huiskonen et al., 2009 Huiskonen et al., , 2010 Overby et al., 2008) . The virion consists of > 50% protein, 20-30% lipid, 7% carbohydrate, and 2% RNA (McCaughey and Hart, 2000; Obijeski and Murphy, 1977) . Hantaviruses can be easily inactivated by treatment with lipid solvents or nonionic detergents, which destroy the viral envelope (Schmaljohn and Nichol, 2007) . Mackow and Gavrilovskaya, 2001; Markotic et al., 2007; Raftery et al., 2002; Spiropoulou, 2001) . Several studies suggest that the attachment is mediated by the interaction of viral Gn protein with integrin receptors on the surface of host cells (Fig. 2.3) . The b1 and b3 integrins are considered to be the receptors for apathogenic and pathogenic hantaviruses, respectively (Gavrilovskaya et al., 1998 (Gavrilovskaya et al., , 1999 Larson et al., 2005) . However, it has been reported that hantaviruses can infect cells lacking integrin receptors, suggesting that integrins may not be the only receptors that hantaviruses employ to attach to host cells (Mou et al., 2006) . After attachment, cell entry is mediated via clatherin-coated pits, and virions are ultimately delivered to lysosomes ( Fig. 2.3 ). Within the endolysosomal compartment, virions are uncoated and three viral capsids are released into the cytoplasm ( Jin et al., 2002) . Viral RdRp initiates transcription and generates three mRNAs, one from each viral RNA segment S, M, and L. are translated on the rough endoplasmic reticulum (ER). The glycoprotein precursor is intrinsically cleaved at a highly conserved amino acid motif, WAASA, generating two glycoproteins, Gn and Gc, respectively (Lober et al., 2001; Ruusala et al., 1992; Spiropoulou, 2001) . After glycosylation in the ER, both Gn and Gc are transported to the Golgi complex, laying the foundations for final destinations where virions are maturated (Antic et al., 1992a; Plyusnin et al., 1997; Ravkov et al., 1998; Ruusala et al., 1992) . After initial rounds of transcription, viral RdRp switches to the replication mode and generates three viral genomic RNAs, which are encapsidated by the viral N protein to form three nucleocapsids. Mature viral nucleocapsids are transported to specific destinations on the Golgi membrane that are studded with the Gn and Gc proteins. However, the molecular mechanisms governing the specific recognition and encapsidation of viral genome by N, transport of capsids to Golgi, and budding of nascent virions into and out of Golgi complex are still unknown. Mechanisms of incorporation of viral RdRp into virus particles are fascinating and need more attention. There are reports suggesting that the assembly and maturation of New World hantaviruses take place on the plasma membrane (Ravkov and Compans, 2001) . The evidence supporting this pathway for assembly and maturation is based on observations that SNV and black Creek Canal virus (BCCV) particles were not observed in infected cells. However, localization of N protein from both old and new word hantaviruses on Golgi apparatus favors the possibility of their maturation on Golgi (Ramanathan and Jonsson, 2008; Ramanathan et al., 2007; Ravkov and Compans, 2001; Spiropoulou et al., 2003) .

Hantavirus nucleocapsid protein (N) is the major viral structural component; its main function is to protect and encapsidate the viral RNA forming viral RNP complex. It is encoded by the S segment vRNA and is abundantly expressed in the cytoplasm of infected cells. Therefore, it is considered as the most predominant viral antigen in the serologic response to infection. Hantaviral N protein has a molecular mass of approximately 50 kDa and contains 429-433 amino acid residues (de Carvalho Nicacio et al., 2001; Plyusnin et al., 1997; Van Epps et al., 1999) . It is highly conserved across hantaviruses and contains cross-reactive epitopes that encompass the first 100 amino acids at N-terminal (Elgh et al., 1996; Gott et al., 1997; Schmaljohn et al., 1986 Schmaljohn et al., , 1987 Yamada et al., 1995) . Further, serotype-specific conformational epitopes have been detected in about half of the C-termini of N proteins by serotype-specific monoclonal antibodies (MAbs) (Ruo et al., 1991; Yoshimatsu et al., 1996) .

N protein is a multifunctional molecule that interacts with host cellular proteins and plays several important roles to facilitate hantavirus replication in infected cells.

a. Viral RNA encapsidation and assembly Several studies showed that N binds specifically to a region near the 5 0 terminus of vRNA, which is indicative of its function during encapsidation (Gott et al., 1993; Jonsson et al., 2001; Mir and Panganiban, 2005; Osborne and Elliott, 2000; Severson et al., 1999) . It was also reported that N differentially interacts with minussense vRNA, plus-sense cRNA, and mRNA. Cell culture-based experiments have demonstrated that encapsidation requires full-length vRNA or cRNA molecules, since no mRNA molecules were detected in viral nucleocapsids ( Jin and Elliott, 1993) . Moreover, SNV N protein was shown to play the role of RNA chaperone that facilitates transient dissociation of misfolded RNA structures and creates opportunities for the folding of RNA into biologically functional and thermodynamically stable higher order structures (Mir and Panganiban, 2005) . A three-dimensional (3D) structural model of N protein is still lacking. However, the functionally important regions have been identified in several mutagenesis studies. The RNA-binding domain of N protein was mapped to the central conserved region that extends from amino acid 175 to 217 (Li et al., 2002) . In addition, the lysine residues dispersed between positions 175 and 429 and three more residues, including E192, Y206, and S217 located in the RNA-binding domain, were also shown to be important for RNA binding (Severson et al., 2005) . Hantavirus N protein undergoes protein-protein interaction and forms stable trimers both in vivo and in vitro. It has been shown that trimeric N recognizes the vRNA panhandle with specificity and high affinity (Mir et al., 2006) . N-panhandle interaction has been proposed to mediate the selective encapsidation and packaging of vRNA genome during virus assembly (Alfadhli et al., 2001 (Alfadhli et al., , 2002 Kaukinen et al., 2001 Kaukinen et al., , 2003 Kaukinen et al., , 2004 Mir and Panganiban, 2004) . Using the MultiCoil prediction algorithm (Wolf et al., 1997) , coiled-coils spanning amino acid residues (1-34 and 38-80) have been predicted for the N protein of several hantaviruses, including HTNV, SEOV, SNV, Prospect Hill virus (PHV), and Tula virus (TULV) (Alfadhli et al., 2001) . Both N-and C-terminal regions have been implicated in mediating the homotypic interaction between N molecules, and putative coiled-coil motifs in the N-terminal region of N protein have been proposed to facilitate trimerization. A ''head-to-head, tail-to-tail'' model for trimerization was proposed, where three monomers of N protein are brought together via interaction of their N-terminal coiledcoil domains. This initial contact is followed by the interaction between the C-terminal a-helices that form a shared hydrophobic space and thus consolidate the trimer formation (Alfadhli et al., 2001 (Alfadhli et al., , 2002 Kaukinen et al., 2001 Kaukinen et al., , 2003 Kaukinen et al., , 2004 b. Transcription and translation Apart from its role in packaging, N has been found to be involved in transcription and replication of viral genome in conjunction with viral RdRp or by interacting with template RNA during replication (Blakqori et al., 2003; Bridgen and Elliott, 1996; Ikegami et al., 2005; Kohl et al., 2004; Pinschewer et al., 2003) . Viral mRNAs are translated by the host cell translation machinery. Recent studies have shown that SNV N protein binds to mRNA 5 0 caps and 40S ribosomal subunit with high affinity and specificity ( Fig. 2.4) . These interesting findings have suggested that, in the host cell cytoplasm where cellular transcripts are competing for the same translation machinery, N protein facilitates the translation of hantaviral mRNAs by preferentially loading ribosomes onto capped viral transcripts (Klempa et al., 2008) . Further studies have shown that the sequence GUAGUAG, in the 5 0 UTR of hantavirus mRNA, is sufficient for preferential N-mediated translation initiation (Mir and Panganiban, 2010) . N-mediated translation initiation mechanism is different from the complex translation initiation strategy used by eukaryotic cells. It is likely that N initiates the translation of capped mRNA without the requirement of eIF4F complex, an amalgam of three initiation factors, eIF4E, eIF4G, and eIF4A (Panganiban and Mir, 2009) .

In host cells, after the completion of translation, the mRNAs are deadenylated and decapped, followed by degradation by exonucleolytic digestion from both 5 0 and 3 0 ends. In the 5 0 -3 0 degradation pathway, the decapping enzyme DCP2 in coordination with several other proteins removes the 5 0 mRNA cap, followed by degradation by exonuclease XRN1. This degradation mechanism takes place in discrete cytoplasmic foci called processing bodies (P-bodies) (Beckham and Parker, 2008) . Interestingly, a detailed analysis by confocal imaging revealed that N resides in cellular P-bodies. Further studies revealed that cellular 5 0capped mRNA oligoribonucleotides are rescued by N in virus-infected cells and stored in P-bodies for the later use as primers by the viral RdRp during transcription initiation . Moreover, it was reported that N protein has distinct cap and RNA-binding sites that independently interact with mRNA cap and viral genomic RNA, respectively. In addition, N can simultaneously bind to both mRNA cap and vRNA. N undergoes distinct conformational changes after binding to mRNA cap, vRNA, or both. The conformationally altered N with a capped primer loaded at the cap-binding site specifically binds the conserved 3 0 nine nucleotides of vRNA (3 0 AUCAUCAUC) and assists the bound primer to anneal at the 3 0 terminus. The annealed primer is later elongated by the RdRp and a nascent viral mRNA is synthesized. Therefore, it was suggested that the cap-binding site of N, in conjunction with RdRp, plays key roles during the transcription and replication initiation of vRNA genome . Taken together, these observations imply that N has a role in generating the capped RNA primer in P-bodies and also assists the capped primer to anneal to the 3 0 terminus of vRNA template during transcription initiation ( Fig. 2.4 ).

c. Interaction with cellular proteins N protein performs some ambassadorial functions by interacting with some cellular proteins to facilitate viral dissemination in infected cells. For example, interaction of N with actin filaments has been reported to mediate the transport of newly synthesized viral RNPs to the plasma membrane (Ravkov et al., 1998) . It was also shown that N protein interacts with the Fas-mediated apoptosis enhancer Daxx, a death-domain adaptor protein, which transduces death signals through the Jun N-terminal kinase (JNK) pathway (Yang et al., 1997) . In similar studies, interaction of N protein with Daxx and small ubiquitin-like modifiers (SUMO-1) pathway components in infected cells has been reported (Alfadhli et al., 2002; Cho et al., 2002; Kaukinen et al., 2003; Lee et al., 2003) .

The protein encoded by the largest ''L'' segment of hantaviruses is an RdRp. The RdRp is responsible for the replication and transcription of the viral genome. The negative-sense hantaviral RNAs (vRNAs) are copied by the L protein to produce positive-sense cRNA intermediates that serve as templates for genome replication and mRNA synthesis (Fig. 2.5 ).

Sequences of several hantavirus genomes are now available. From the available sequencing data, it is evident that the size of the L RNA of hantaviruses ($ 6.5 kb) is similar to those of other members of the Bunyaviridae family, except for the Tospovirus ($ 8.9 kb) and the Nairovirus ($ 12 kb) genera. The reverse complement of the L segment of all the members of the Bunyaviridae family has a single large ORF with flanking 5 0 and 3 0 UTRs. The predicted size of the protein encoded by this ORF is approximately 250 kDa, which has also been confirmed experimentally by few groups (Elliott et al., 1984; Kukkonen et al., 2004) .

There are a total of four classes of nucleotide polymerases: DNAdependent DNA polymerase (DdDp), DNA-dependent RNA polymerase (DdRp), RNA-dependent DNA polymerase (RdDp or reverse transcriptase), and RdRp. Amino acid sequence comparisons of all the nucleotide polymerases have revealed an interesting pattern of conserved motifs found in different classes of polymerases ( Fig. 2.6 ; Hansen et al., 1997; Joyce and Steitz, 1995) . Motifs A-D are shared by all polymerases. Motif E is found in those polymerases that recognize RNA as a template (reverse transcriptases and RdRPs). An extra motif F (also called as pre-motif A) is specifically found in RdRps (Bruenn, 2003; Hansen et al., 1997; Kamer and Argos, 1984; Muller et al., 1994; O'Reilly and Kao, 1998; Poch et al., 1989; Toh et al., 1985; Tordo et al., 1988) . The amino acid residues from these conserved motifs play critical roles in different aspects of the nucleotidyl transferase reaction of these enzymes, which include binding of metal ions, nucleoside triphosphates (NTPs), and the RNA/DNA template. Few additional conserved regions have been found in the amino acid sequences of RdRps of segmented negative strand RNA viruses (Aquino et al., 2003; de Haan et al., 1991; Kukkonen et al., 1998; Muller et al., 1994; Nemirov et al., 2003; Stohwasser et al., 1991) ; however, the functional importance of these motifs has not yet been validated.

Based on the organization of the conserved motifs in the amino acid sequence, the hantavirus RdRp is predicted to have a structure similar to other polymerases. The crystal structures of all polymerases show a common domain organization, which resembles a ''right hand,'' and it includes the subdomains denoted as ''finger,'' ''palm,'' and ''thumb'' (Kohlstaedt et al., 1992) . Among all known RdRps, an additional N-terminal domain is found that bridges the fingers and thumb domains to make a tunnel-like structure, which is otherwise open U-shaped in most of the other polymerases (Butcher et al., 2001; Lesburg et al., 1999; Ng et al., 2008) . The crystal structure of hepatitis C virus shows the palm subdomain to be constituted of b-strand of motifs A and C and a-helix of motif D. Motifs B and F, folded into an a-helix, form the fingers subdomain and the motif E, in form of an antiparallel b-sheet, is present between the palm and the thumb subdomains (Lesburg et al., 1999) . Conserved Arg and Lys residues in motif F and a highly conserved Asp in motif A play critical roles in binding of the RdRp with NTPs Bressanelli et al., 2002; Gohara et al., 2000 Gohara et al., , 2004 Huang et al., 1998; Joyce and Steitz, 1995) .

Conserved structural motifs among four classes of nucleotide polymerases: motifs A-D are found in all the polymerases. Motif E is found in polymerases that use RNA as a template. There is an extra motif F found only in RdRps.

Like other polymerases, the RdRps also show a dependence on binding of two divalent metal ions in the active site (Arnold et al., 1999; Doublie and Ellenberger, 1998; Rothwell and Waksman, 2005; Steitz et al., 1994) . The conserved Asp residues in motifs A and C play a crucial role in binding of the two metal ions. Mutation of these Asp residues has been shown to inactivate or alter the activity of several RdRps (Arnold et al., 1999; Jablonski and Morrow, 1995; Vazquez et al., 2000) . In a mutational analysis of Bunyamwera RdRp, replacement of the conserved aspartates of motifs A and C abolished the polymerase activity (Dunn et al., 1995) .

In a large-scale mutational study carried out on RdRp of Lassa virus (family Arenaviridae), an RdRp domain (between residues 1040 and 1540) was identified and a functional element within RdRp was found, which was important for transcription but not replication of the genome (Hass et al., 2008) .

RdRps of both positive-stranded RNA viruses such as poliovirus as well as negative-stranded RNA viruses including Sendai virus (Cevik et al., 2003; Smallwood et al., 2002) , human parainfluenza type 3 virus , measles virus (Cevik et al., 2004) a. Localization The site of RNA synthesis among the viruses of the Bunyaviridae family was believed to be the cytoplasm of the host cell. This was based on a study on La Cross virus in which the newly synthesized RNA was found to be in the cytoplasmic fraction where it was monitored by pulse-labeling followed by cell fractionation (Rossier et al., 1986) . But based on later studies on localization of the two viral proteins essential for RNA synthesis, namely, L and N, it is now thought that RNA synthesis among bunyaviruses is membrane associated. Kukkonen et al. (2004) studied the localization of L-GFP fusion protein and found it to localize in the perinuclear region (Kukkonen et al., 2004) . This is consistent with the fact that RNA synthesis of all positive-stranded viruses is membrane associated (Salonen et al., 2005) . Further studies on other viruses of this family will be required to confirm if that is a general mechanism employed by these viruses.

b. Mechanism of action Similar to all the other negative-sense RNA viruses, hantavirus genomic RNA segments contain UTRs on both the 5 0 and the 3 0 ends. The termini of the UTRs are partially complementary to each other due to which the segments fold to make a panhandle-like structure (Chizhikov et al., 1995; Kukkonen et al., 1998; Meyer and Schmaljohn, 2000; Padula et al., 2002) . Several minireplicon systems in which reporter genes (such as Renilla luciferase or chloramphenicol acetyltransferase) are flanked by the UTRs have been developed for members of the Bunyaviridae family (Blakqori et al., 2003; Dunn et al., 1995; Flick and Pettersson, 2001; Flick et al., 2003; Lopez et al., 1995) . Studies based on these minireplicon systems have shown that 5 0 and 3 0 UTRs serve as promoters for replication of the segments and transcription of the encoded reading frames. The UTRs are encapsidated by nucleocapsid protein and associate with RdRp both in the host cells and in the virion, and only these nucleocapsids are believed to be functional templates for mRNA synthesis and RNA replication by the viral RdRp.

c. mRNA synthesis The viral RdRp is responsible for the synthesis of viral mRNA. Several unique features involved in this process are detailed here. Sequence analysis of the 5 0 termini of mRNAs of several Bunyaviruses has revealed the presence of short (10-18 nucleotides) nontemplated 5 0 end sequences (Bishop et al., 1983; Garcin et al., 1995; Simons and Pettersson, 1991) . These sequences were later found to be derived from the 5 0 ends of host mRNAs by a mechanism similar to ''cap-snatching'' originally described for influenza virus. In this mechanism, a viral endonuclease cleaves the 5 0 termini of the host mRNAs and uses them as primers to initiate the transcription of viral mRNAs (Bouloy et al., 1978; Krug, 1981; Krug et al., 1979; Plotch et al., 1981) . The endonuclease activity required for cap-snatching is believed to reside in the viral polymerase itself (Patterson et al., 1984) .

Based on the observation that all the mRNAs contain a G residue at À 1 position, a prime and realign mechanism for mRNA synthesis in the Bunyaviridae family was proposed. In this mechanism, the G residue of the capped primer snatched from the host mRNA aligns with a C residue, upstream of the 3 0 terminus of the viral template. The primer is elongated few nucleotides by RdRp. The extended primer detaches and realigns to the 3 0 end of the terminus and then extends further to complete the mRNA synthesis (Garcin et al., 1995) .

Termination of mRNA synthesis and polyadenylation of the synthesized mRNA has been shown to vary among S, M, and L segments (Hutchinson et al., 1996) . Synthesis of S segment mRNA terminates downstream of a CCACCC motif found around 200 nucleotide downstream of the 5 0 end of the vRNA template. The S segment mRNA is not polyadenylated (Hutchinson et al., 1996) . The synthesis of the L mRNA extends to the 5 0 terminus of the L vRNA and is also not polyadenylated (Hutchinson et al., 1996) . However, the M segment of SNV was found to be polyadenylated and a potential polyadenylation-transcription termination signal was mapped and was found to be conserved among all hantaviruses (Hutchinson et al., 1996). d. RNA replication Replication of the vRNA genome is carried out by the viral RdRp through an antigenomic RNA (cRNA) intermediate. Unlike mRNA, the cRNA is exact complement of the viral genomic segments without any cap structure and host-derived sequences (Bishop et al., 1983; Eshita et al., 1985; Garcin et al., 1995; Obijeski et al., 1980; Raju and Kolakofsky, 1987) . The cRNA was shown to be encapsidated by the N protein (Patterson et al., 1984) . The 5 0 terminus of the vRNA in hantaviruses was found to contain uridine monophosohate (pU) instead of triphosphate (pppU; Chizhikov et al., 1995; Garcin et al., 1995) . This observation was explained by a prime and realign mechanism for cRNA and vRNA synthesis (Garcin et al., 1995) . Briefly, after elongation by few nucleotides, the capped RNA primer with a terminal ''G'' residue slides back on the 3 0 triplet repeat sequence of cRNA template, leaving a protruding GTP that is nonaligned with the 3 0 end. The protruding GTP is subsequently cleaved off by the polymerase leaving a pU residue at the 5 0 end (Garcin et al., 1995). e. Error, evolution, and editing The promiscuous nature of polymerization by RdRps due to lack of proof reading ability is thought to be the primary source of evolution in RNA viruses. This gives the virus an ability to replicate in different hosts and become pathogenic to humans. But the virus has to maintain a close window of variation to avoid the ''error catastrophe'' as too much of these errors may lead to generation of nonviable genome and reduction of the overall viral fitness (Crotty and Andino, 2002) . Drake et al. have estimated an error rate of approximately 1 mutation/replication/genome for hantaviruses (Drake, 1999) . The mean rate of evolutionary change in hantaviruses has been approximated to be within the range of 10 2 -10 4 substitutions/site/year (Ramsden et al., 2008) . RNA viruses employ several mechanisms to generate useful variations and to edit deleterious ones, which include reassortment and recombination (Barr and Fearns, 2010) . Natural reassortment has been detected among hantaviruses (Henderson et al., 1995; Li et al., 1995; Plyusnin et al., 1997) , and several experimental reassortants have been generated in the laboratory by infecting different strains of the same hantavirus (Ebihara et al., 2000; Rodriguez et al., 1998) . Recently, Handke et al. were able to generate in vitro reassortants between pathogenic PUUV and nonpathogenic PHV (Handke et al., 2010) .

RNA recombination in viruses usually occurs by a ''copy choice'' mechanism, in which a replicating polymerase stops copying one RNA molecule and switches to another (Copper et al., 1974; Worobey and Holmes, 1999) . A recombinant TULV was successfully generated through homologous recombination by infecting Vero E6 cells with one strain and by providing S cRNA of another TULV strain via plasmid-driven expression . A recent study in Bunyamwera virus (BUNV) has shown that recombination may also occur near the end of the templates to keep the ends intact (Walter and Barr, 2010 ).

The envelope glycoproteins are encoded by the viral M segment RNA. They are expressed as a glycoprotein precursor (GPC) polypeptide, which is cleaved by host signal peptidase in the ER lumen to generate N-terminal (G1 or Gn) and C-terminal (G2 or Gc) fragments (Fig. 2.7 ; Fazakerley and Ross, 1989; Spiropoulou et al., 2003) . Cleavage occurs after a highly conserved pentapeptide WAASA motif (Lober et al., 2001) . The GPC has neither been detected in infected cells nor in cells transfected with a plasmid encoding M segment mRNA, but was only detectable by in vitro translation of RNA transcripts. Therefore, it was suggested that the proteolytic processing of the GPC occurs during translation (Pensiero and Hay, 1992; Schmaljohn et al., 1987; Ulmanen et al., 1981) .

Gn and Gc are type I transmembrane proteins modified by N-linked glycosylation, with the N-terminus exposed on the surface of the virion and the C-terminus anchored in the membrane . They have high tendency to form disulfide bridges due to their high content of cysteine residues (4-7%) (Antic et al., 1992a) . SNV GPC is 1140 amino acids in length. The Gn of SNV consists of 652 residues (MW % 75 kDa), with a predicted transmembrane domain and a cytoplasmic tail (CT) domain of 142 amino acids. Gc is 488 residues in length (MW % 55 kDa) and has a predicted shorter CT of eight amino acids (Spiropoulou et al., 2003) . HTNV GPC has four hydrophobic domains. Domain I extends from position 1 to 17. Domains II (position 441-515) and IV (position 1097-1127) are the transmembrane domains of Gn and Gc, respectively (Spiropoulou et al., 1994) . Domain II is very long and the actual membrane spanning portion is not well defined. Domain III extends from position 627 to 648 and ends with the highly conserved pentapeptide motif WAASA before the N-terminus of Gc (Spiropoulou, 2001) . Most enveloped viruses bud from the plasma membrane into the extracellular space. However, in the case of family Bunyaviridae, many members of this family mature by budding into the lumen of the Golgi complex. Progeny virions are then released by fusion of secretory vesicles with the plasma membrane (Kuismanen et al., 1982; Schmaljohn and Nichol, 2007) . Additional evidence suggests that the assembly of New World hantaviruses (e.g., SNV and BCCV) may occur at the plasma membrane (Ravkov et al., 1997) . The reason why some viruses have evolved to bud from the Golgi membranes is poorly understood, but it might be due to the presence of Golgi retention signals in viral glycoproteins, which allow them to accumulate in the Golgi complex after their maturation in the ER (Antic et al., 1992b; Shi and Elliott, 2002; Spiropoulou et al., 2003) . There is no clear consensus on the exact location of these Golgi targeting signals in different members of family Bunyaviridae, and it appears that different viruses, even within a single genus, have their own strategies for Golgi targeting . For members of the Bunyavirus genus, this signal was mapped to the N-terminus of Gc (Lappin et al., 1994) . The same signal was reported to be located in the transmembrane domain and CT of Gn for members of genus Phlebovirus (Andersson et al., 1997; Gerrard and Nichol, 2002; Matsuoka et al., 1994) . It was also suggested that translocation of Gn and Gc from the ER to the Golgi complex depends on their interaction and heterodimerization because neither of them was able to leave the ER when expressed individually (Deyde et al., 2005; Ruusala et al., 1992; Shi and Elliott, 2002; Spiropoulou et al., 2003) .

Glycoproteins, Gn and Gc, play a significant role in the biology of hantaviruses, including virus entry to host cells, virulence, and assembly and packaging of new virions in infected cells.

a. Attachment and entry Hantaviruses enter the cells via receptormediated endocytosis. The acidic environment of the endosome facilitates fusion between the viral and cellular membranes, with a consequent release of the viral nucleocapsids into the cytoplasm ( Jin et al., 2002) . Viral glycoproteins mediate attachment of virus to endothelial cells via interaction with surface b3 integrin receptors (Arikawa et al., 1989; Gavrilovskaya et al., 1998 Gavrilovskaya et al., , 1999 b. Assembly and packaging Gn has a 142-residue C-terminal CT, which plays important roles in viral assembly and host-pathogen interactions. The lack of a matrix protein in hantaviruses, which plays an important role in the assembly and budding of many enveloped viruses, is thought to be substituted by a direct interaction between the nucleocapsid and envelope glycoproteins. Several lines of evidence seem to substantiate this model. The CT of Gn of Uukuniemi virus was shown by alanine scanning mutagenesis to mediate the packaging of a minigenome encoding a reporter gene into virus-like particles (VLPs; Overby et al., 2007) . Also, the CT domain of both Gn and Gc was shown to be indispensible for the replication of BUNV (Shi et al., 2007) . A highly conserved cysteine/ histidine-rich region of the Gn tail was recently shown by NMR spectroscopy to form two classical zinc fingers (Estrada et al., 2009 ). This suggested an involvement of this domain in nucleic acid binding or protein-protein interactions. However, it is not yet clear whether the CT interacts with the RNA genome or the protein component of the RNP during assembly. More recently, the N protein of PUUV was shown to coimmunoprecipitate with the glycoprotein complex. Mapping of the interaction sites revealed that the N protein has multiple binding sites in the CT of Gn and was also able to bind to the predicted CT of Gc (Hepojoki et al., 2010b). c. Virulence A clear association between viral glycoproteins and virulence was established by comparing the pathogenicity of virulent and attenuated or genetically reassorted strains of HTNV in an experimental newborn mouse model. Sequence comparison identified single amino acid changes in Gn and Gc as the genetic determinants responsible for the observed difference in viral virulence (Ebihara et al., 2000; Isegawa et al., 1994) . Gn consists of an external domain, a transmembrane domain and a C-termianl CT (Spiropoulou, 2001) . The CT of Gn appears to be a multifunctional virulence determinant that helps the virus to evade host innate responses and ensures productive infection in human endothelial cells. It contains immunoreceptor tyrosine-based activation motifs (ITAMs). These are cell-signaling elements involved in regulating the functions of immune and endothelial cells and their presence in Gn CT suggests a direct role in hantavirus pathogenesis (Geimonen et al., 2003b) . RIG-I (retinoic acid-inducible gene I) is an RNA helicase that triggers the cellular innate interferon (IFN) immune response upon detection of viral double-stranded RNA (Yoneyama et al., 2004) . The Gn CT of the pathogenic New York-1 virus (NY-1 V) was shown to inhibit the RIG-I-directed IFN responses (Alff et al., 2006 (Alff et al., , 2008 . To counteract this effect, it seems that cells have evolved a mechanism for downregulating Gn expression by targeting this protein for proteosomal degradation via polyubiquitination of the CT (Geimonen et al., 2003a) . These findings have created some controversy in the literature. It was initially reported that the Gn CTs of pathogenic (NY-1 V, HTNV, and Andes virus ANDV), but not the nonpathogenic hantavirus PHV, are exclusively degraded via the ubiquitinproteasome pathway in COS7 cells (Sen et al., 2007) . However, this view was challenged by Wang et al., who reported that the Gn CTs of the nonpathogenic TULV and PHV were proteosomally degraded in HEK-293 and Vero E6 cells. Therefore, it was concluded that this degradation is not related to viral pathogenesis (Wang et al., 2009 ).

Unlike other members of the Bunyaviridae family, hantaviruses are not transmitted by biting insects. Human infection occurs accidentally via inhalation of contaminated rodent excreta (Hart and Bennett, 1999) . Rodents are the main natural reservoir, where the virus can establish asymptomatic persistent infection. Within rodents, the disease is transmitted horizontally, mainly through aggressive behavior such as biting and scratching (Lee et al., 1981a) . It is mainly a rural disease associated with the risk factors of farming, hunting, and camping because those activities bring humans into close contact with the rodent reservoirs (Simpson et al., 2010) . The only hantavirus that causes disease in urban areas is SEOV because its host is the domestic rat (Mir, 2010) . Human-tohuman transmission is very rare and was reported only for ANDV in Argentina (Padula et al., 1998) .

Approximately 150,000-200,000 cases of hantavirus infection are reported annually worldwide (Hart and Bennett, 1999) . The geographic distribution of the disease reflects the distribution of the rodent host, hence the classification of hantaviruses into two main groups: Old World and New World ( Jonsson et al., 2010) . Occurrence of HFRS and HCPS depends on the type of virus causing the infection (Borges et al., 2006) . HFRS is primarily considered a Eurasian disease, which was first reported in Korea in 1951 and is caused by Old World hantaviruses, for example, HTNV, SEOV, DOBV, and PUUV. More than 150,000 cases of HFRS are reported annually, half of which occur in China only . HCPS appears to be confined to the Americas and is associated with New World hantaviruses, for example, SNV, NY-1 V, and ANDV. It was first discovered in 1993 in the Four Corners region of the southwestern United States (Nichol et al., 1993) . Approximately 300 cases of HCPS are reported each year in North and South America . SNV, which infects deer mouse, is the major cause of HCPS in North America (Fabbri and Maslow, 2001 ).

Rodents of the subfamily Murinae within family Muridae and the subfamilies Arvicolinae, Neotominae, and Sigmodontinae within family Cricetidae are considered to be the main natural reservoir hosts of hantaviruses. Old World hantaviruses, which cause HFRS, are carried by rodents of Myodes, Rattus, and Apodemus genera and New World hantaviruses, which cause HCPS are carried by the rodents of the subfamily Sigmodontinae. Recently, numerous novel hantaviruses from various sorcid and talpid insectivores have been discovered (Arai et al., 2007; Kang et al., 2009a,b; Song et al., 2007a,b,c) , but none of these viruses have been associated with any human disease.

An apparent coevolution of hantaviruses with their reservoir hosts is evident by the commonly observed close association of each hantavirus species with a certain rodent species. Moreover, phylogenetic analyses of hantavirus sequences and mitochondrial sequences from their rodent hosts suggest a long-standing coevolutionary history of hantaviruses with their rodent carriers ( Jackson and Charleston, 2004; Plyusnin, 2002; Plyusnin and Morzunov, 2001) . Depending on the population density, up to 50% of any given population of rodents are seropostive and considered as silent carriers for hantaviruses. Through experimental infections and field surveys, it has been shown that the transmission among rodents is exclusively horizontal and it occurs via inhalation of infected aerosols, through saliva or excreta, biting, and other aggressive behavioral interactions Glass et al., 1988; Hinson et al., 2004; Hutchinson et al., 2000; Kariwa et al., 1998; Lee et al., 1981a,b; Root et al., 2004) . Unlike other rodent-borne viruses such as arenaviruses, there is no vertical transmission from the dam to its offspring, and maternal antibodies can protect offspring from infection for several months (Kallio et al., 2006; Taruishi et al., 2008) . This view has been recently disputed by Calisher et al. (2009) , and several scenarios have been presented for the transmission of hantaviruses from infected to uninfected hosts (Calisher et al., 2009) .

In general, infection of the rodent hosts by their respective hantaviruses is thought to be asymptomatic and no overt disease is produced. However, several recent studies have reported development of some disease symptoms in the reservoir hosts such as pulmonary edema and periportal hepatitis in white-footed mouse Peromyscus leucopus infected with NY-1 V (Lyubsky et al., 1996) and SNV-infected deer mouse Peromyscus maniculatus (Netski et al., 1999) . Infection of hantavirus in the rodent host may also cause growth retardation as observed in Rattus norvegicus infected with SEOV (Childs et al., 1989) and P. maniculatus infected with SNV (Kanerva et al., 1998) . Nevertheless, the general absence of an overt illness in rodent hosts despite a persistent and lifelong infection is believed to be a smart way of survival for hantaviruses, by which they avoid killing their own hosts. It also highlights an amicable relationship between the virus and its host that was developed over hundreds of thousands of years of mutual interaction. The survival of rodent hosts in spite of the chronic viral infection is mediated by a combination of host's unique immunity against the virus and the viral genetic variability, which helps it evade the host's defense mechanisms (Schonrich et al., 2008) .

Persistently infected rodents shed the virus in urine, feces, and saliva. Inhalation of virus-contaminated aerosols is the major route of transmission to humans. However, the general view that rodents are the only infection source for humans has been disputed by Zeier et al. (2005) . It has been suggested that the close proximity of domestic animals such as cats, dogs, pigs, and cattle with rodents may cause some transmission events of hantaviruses to these animals. This species jump may cause the virus to evolve differently and result in more dangerous forms of the virus, which could be more pathogenic to humans .

In contrast to the asymptomatic infection of the rodent reservoir, hantavirus infection to humans results in two disease forms: HFRS and HCPS. In both syndromes, vascular endothelial cells show increased permeability and both are believed to result from host immune responses to infection, rather than damage caused by the viruses themselves (Khaiboullina and St. Jeor, 2002) .

The incubation period of HFRS is about 3 weeks but can range from 10 days to 6 weeks ( Jonsson et al., 2010; Kramski et al., 2009) . The clinical course is classically subdivided into five overlapping phases: febrile, hypotensive, oliguric, diuretic, and convalescent (Schmaljohn and Hjelle, 1997) . The febrile phase lasts for 3-5 days and is characterized by flu-like symptoms, thirst, nausea, and vomiting. The febrile phase is followed by a hypotensive phase, which lasts for few hours to 2 days, and is characterized by thrombocytopenia and petechial hemorrhage, retroperitoneal edema, and abdominal pain. The subsequent oliguric phase lasts for few days to two weeks. The mortality rate is 15%, with the majority of deaths occurring during the hypotensive and oliguric phases due to the complications of vascular leakage, renal failure, and acute shock. Patients who survive these complications usually progress into the diuretic phase and show improved renal function, with subsequent convalescence and recovery .

The incubation period of HCPS ranges from 9 to 33 days (Young et al., 2000) . The clinical disease course of HCPS is usually divided into prodromal, cardiopulmonary, and diuretic or convalescent phases. The prodromal stage, which usually lasts 3-6 days, is characterized by flu-like symptoms such as fever, headache, chills, and muscle pain. Abdominal pain, nausea, vomiting, and dizziness may occur. This phase usually progresses rapidly to a severe respiratory disease characterized by nonproductive cough and dyspnea. In contrast to HFRS, the increased permeability and fluid leakage occur exclusively in the lungs instead of the kidneys. This bilateral pulmonary edema is usually visible in chest X-rays (Boroja et al., 2002) . Thrombocytopenia, hemoconcentration, and leukocytosis are the most prominent hematologic findings (Simpson et al., 2010) . Rapid deterioration, cardiac insufficiency, and respiratory failure caused by edema and hypotention, shock, and death may occur within 2-10 days after the onset of illness in almost 50% of cases. Patients who survive the acute phase recover within 5-7 days and enter the diuretic phase Zaki et al., 1995) .

Hantavirus infection occurs via inhalation of infectious virus particles into the lungs. The inhaled viruses bind to b3 integrin receptors on the surface of pulmonary endothelial cells, macrophages, and dendritic cells (DCs), where virus replication mainly occurs (Gavrilovskaya et al., 1998) . Capillary leakage is the hallmark of hantavirus infection; however, the sequence of events from inhalation of infectious virus particles until pulmonary capillary leakage remains poorly understood. It was proposed that DCs play the role of a Trojan horse by helping the dissemination of virus throughout the body. Immature DCs located near the respiratory epithelial cells engulf inhaled virus particles, which can replicate in those cells without causing cell death. During their maturation, DCs migrate from the lungs to the lymph nodes where they present the engulfed antigen to immune cells (Raftery et al., 2002) .

The mechanisms by which pathogenic hantaviruses cause capillary leakage is an area of active research. Accumulating pieces of experimental evidences suggest that multiple immunopathologic mechanisms rather than direct viral-induced cytopathic effects are responsible for the disruption of vascular endothelium and the subsequent capillary leakage associated with hantavirus diseases (Borges et al., 2006; Sundstrom et al., 2001) . In PUUV-infected patients, elevated levels of serum lactate dehydrogenase (LDH), indicative of cellular damage, correlated with high levels of serum perforin, granzyme B, and epithelial cell apoptosis markers. These findings suggested that tissue damage is due to an immune reaction and that epithelial apoptosis contributed significantly to the damage (Klingstrom et al., 2006b) .

Some changes in the endothelial cell barrier functions might be related to the use of b3 integrins as receptors. It was shown that the initial interaction of the infecting virus with b3 integrin receptors disturbs the integrin-directed migration of endothelial cells, which is essential for maintaining vascular integrity (Gavrilovskaya et al., 2002) . The cellular immune response to infection involves strong stimulation of hantavirusspecific cytotoxic CD8 T lymphocytes (CTLs), which are released in large numbers into the blood. The severity of the disease was shown to correlate with the number of CTLs in the blood (Kilpatrick et al., 2004) . This vast excess of activated CTLs is partly due to the lack of downregulation of T-cell function as evidenced by low serum levels of cytokines released by regulatory T cells, such as TGF-b, in HCPS patients (Chen and Yang, 1990; Mills, 2004) . The intense antiviral immune response can contribute to the increased permeability of endothelial cells in two ways. First, the elevated levels of inflammatory mediators and cytokines, such as TNF-a, IL-6, and IL-10, released during the acute phase by activated T cells and infected endothelial cells increase the vascular permeability resulting in pulmonary edema (Linderholm et al., 1996) . Second, it was shown in transwell permeability assays that hantavirus-specific cytotoxic T lymphocytes could directly lyse human endothelial cells infected with SNV (Hayasaka et al., 2007) .

The exact mechanism of kidney failure in HFRS is unclear. Renal disease has been attributed to deposition of immune complexes and the presence of inflammatory cell infiltrations with subsequent tubular damage (McCaughey and Hart, 2000; Sironen et al., 2008) .

Diagnosis of hantavirus infection in the clinic relies on the establishment of a history of rodent exposure, symptoms suggestive of respiratory or renal involvement, blood exams showing severe thrombocytopenia, and positive serological tests. Virus isolation from clinical samples is difficult due to the presence of high levels of neutralizing antibodies (McCaughey and Hart, 2000) . High titers of IgM and IgG antibodies against hantavirus N and Gn proteins are detectable in the sera of patients during the acute phase, enabling reliable serological confirmation of infection (Bostik et al., 2000; Elgh et al., 1997; Groen et al., 1994) . SNV Gn antibodies are highly specific and do not cross-react with Gn antigens of other hantaviruses Jenison et al., 1994) . The most widely used serological tests for diagnosis of HCPS are IgM capture and IgG indirect ELISAs (Li et al., 2002) . A rapid test for SNV and SEOV in the form of a strip immunoblot assay has been developed, which relies on antigens derived from recombinant SNV N and Gn proteins and SEOV N protein. The assay was demonstrated to be highly sensitive and specific because it could identify all patients with acute SNV infection during the early course of the disease with no false-positive results . Immunohistochemical detection of hantavirus antigens has been particularly useful in establishing retrospective diagnosis of HCPS from fixed tissue samples . RT-PCR assays have also been used, but results should be always interpreted with caution due to the potential of cross-contamination and have to be always substantiated by positive results from immunodiagnostic assays (Moreli et al., 2004) .

No specific antiviral therapy is currently available for treating hantavirus infections. Management of infected patients relies solely on supportive therapy in an intensive care unit until the virus is cleared by the immune system . Ribavirin, a broad-spectrum nucleoside analogue antiviral drug, has been shown to possess, both in vitro and in vivo, inhibitory activity against hantaviruses (Huggins et al., 1986; Medina et al., 2007; Severson et al., 2003) . A clinical trial showed decreased virus titers, morbidity, and mortality rates in HFRS Chinese patients (Huggins et al., 1991) . Another trial on HFRS patients in Korea showed significant reduction in fatality and improved prognosis when given early in the course of the disease, as well as a reduction in the risk of renal insufficiency (Rusnak et al., 2009) . Intravenous injection of ribavirin was generally well tolerated by HCPS patients (Chapman et al., 1999) . However, a Placebo-controlled, double-blind trial of intravenous ribavirin in HCPS patients was ended prematurely due to low rate of enrollment (Mertz et al., 2004) . Therefore, ribavirin does not seem to have any clinical application in HCPS patients due to the lack of conclusive clinical data. Passive administration of neutralizing antibodies for PUUV has been shown to protect macaques against virus challenge; however, no human clinical trials were conducted (Klingstrom et al., 2005) .

Despite many efforts that have been invested in developing a safe effective vaccine to protect against hantavirus infection, currently, no FDAapproved vaccine is available in the United States . A formalin-inactivated HTNV vaccine (Hantavax) produced from mouse brain-derived virus is licensed for use in Korea since the 1990s. Efficacy studies showed that 97% of human volunteers receiving this vaccine developed high titers of specific neutralizing antibodies after a booster dose; however, the induced humoral immune response was short lived (Cho and Howard, 1999) . Another formaline-inactivated vaccine produced from suspensions of Vero cells elicited a neutralizing antibody response in mice, which was fivefold higher than Hantavax (Choi et al., 2003) . However, it is unlikely that these vaccines can provide protection against all hantaviruses. The N, Gn, and Gc proteins of hantaviruses are highly immunogenic (Bharadwaj et al., 2002) . Therefore, different approaches were taken to develop recombinant vaccines using proteins expressed in baculovirus, vaccinia virus, and the yeast Saccharomyces cerevisiae Schmaljohn et al., 1990; Yoshimatsu et al., 1993) . Recombinant N proteins of PUUV and DOBV expressed in yeast were shown to induce a protective immune response in rodent models (Dargeviciute et al., 2002; Geldmacher et al., 2004) . Moreover, deer mice immunized with recombinant deer mouse cytomegalovirus (PCMV) expressing SNV Gn developed an antibody response to SNV (Rizvanov et al., 2003) . Although highly protective in mouse models, a recombinant vaccinia virus containing the S and M segments of HTNV proved to be inefficient in a phase II clinical trial. It elicited neutralizing antibodies in only 72% of the 142 participating volunteers (McClain et al., 2000) . Several other studies using naked DNA vaccines containing the M or S genome segments of SNV and SEOV have shown similar success in rodent models, but no clinical administration has been reported (Bharadwaj et al., 1999; Hooper et al., 1999) .

Frequent emergence of zoonotic viruses is a serious concern to human health. For example, the recent emergence of H1N1 (swine flu) has created havoc in human lives, especially in pregnant women and young children who were predicted to be most susceptible for this new virus species. History witnesses the emergence and reemergence of numerous pathogens in the past century, with an estimated frequency of one new pathogen every 18 months. Many of these new pathogens were zoonotic RNA viruses, such as hantavirus, H1NI swine flu, Nipah virus, Hendra virus, Ebola virus, West Nile virus, and SARS (severe acute respiratory syndrome cornovirus; Jones et al., 2008; Morens et al., 2004) . Despite their significant devastation to human lives, the ecology and natural history of these zoonotic viruses are still a mystery. The principles that govern their stable maintenance in their natural reservoirs, compulsions for switching their hosts, mechanisms of recombination and reassortment, molecular mechanisms of their transfer, survival and adaptation in the new host, are the interesting areas that remain poorly understood. The natural hosts of these zoonotic viruses continue to be the reservoirs for the generation of new pathogenic strains and probable causes of future pandemics that pose serious threat to human lives.

Hantaviruses cause HFRS and HCPS when transmitted to humans but maintain a persistent infection in their rodent hosts. Coevolutionary existence of hantaviruses and their rodent hosts provides insights into mechanisms by which these zonotic viruses manage to exist in the environment for millions of years with a potential to infect humans upon transmission. However, understanding the mechanisms by which hantaviruses maintain a persistent infection in their rodent hosts without a disease may provide insights into the possible approaches that could be used for the treatment of hantavirus-associated disease in humans. Excessive proinflammatory cytokine (TNF-a) and CD8þ responses in humans are hypothesized to mediate the pathogenesis of human HFRS and HCPS, suggesting that anti-TNFa therapy might be helpful during the treatment.

Since HCPS is comparatively a rare disease, funding for the development of an HCPS vaccine is not a high priority for many countries. However, with the increasing number of HCPS cases, especially in South America, vaccine efforts for HCPS may receive better appreciation. Due to the requirements of high-containment laboratories for HCPScausing viruses, plasmid DNA gene gun-based approaches will be preferred for the development of an HCPS vaccine.

Dissecting the molecular mechanisms of hantavirus replication in host cells will provide insights about the new potential targets for the design of antiviral therapeutic agents for the treatment of HFRS and HCPS. For example, the newly discovered translation initiation mechanism, operated by hantavirus nucleocapsid protein, demonstrates how a single viral protein lures the host cell translation machinery for the preferential translation of viral mRNAs in virus-infected cells where cellular transcripts are competing for the same translation apparatus . Shutting down this viral translation initiation strategy might inhibit virus replication in infected cells. In addition, viral RdRp uses capped RNA primers to initiate the transcription/replication of viral RNA genome. Capped RNA primers are generated from host cell mRNAs by a unique ''cap-snatching mechanism.'' This mechanism is well understood in influenza virus. However, recent studies suggest that hantaviral cap-snatching mechanism might be different from influenza virus . In-depth understanding of cap-snatching mechanism will reveal novel targets that could be used for the design of antiviral agents for the treatment of diseases caused by a broad spectrum of viruses that use cap-snatching mechanism to initiate transcription/replication of viral genome.

Currently, the studies of hantavirus biology and pathogenesis are limited due to the lack of an animal model and a complete reverse genetic system for these viruses, although a minigenome system for HTNV has been reported (Flick et al., 2003) . Reverse genetic system will generate hopes for screening chemical libraries to identify molecules with antiviral therapeutic potential, for the development of attenuated vaccine candidates, and for the investigation of molecular mechanisms involved in hantavirus replication, gene expression, as well as virus assembly and budding.

Hantavirus nucleocapsid protein oligomerization

Hantavirus nucleocapsid protein coiled-coil domains

The pathogenic NY-1 hantavirus G1 cytoplasmic tail inhibits RIG-I-and TBK-1-directed interferon responses

The NY-1 hantavirus Gn cytoplasmic tail coprecipitates TRAF3 and inhibits cellular interferon responses by disrupting TBK1-TRAF3 complex formation

A retention signal necessary and sufficient for Golgi localization maps to the cytoplasmic tail of a Bunyaviridae (Uukuniemi virus) membrane glycoprotein

Comparison of the deduced gene products of the L, M and S genome segments of hantaviruses

Maturation of Hantaan virus glycoproteins G1 and G2

Analysis of oropouche virus L protein amino acid sequence showed the presence of an additional conserved region that could harbour an important role for the polymerase activity

Hantavirus in northern short-tailed shrew

Characterization of Hantaan virus envelope glycoprotein antigenic determinants defined by monoclonal antibodies

Poliovirus RNA-dependent RNA polymerase (3Dpol): Pre-steady-state kinetic analysis of ribonucleotide incorporation in the presence of Mg2þ

Poliovirus RNA-dependent RNA polymerase (3D(pol)). Divalent cation modulation of primer, template, and nucleotide selection

Genetic and antigenic properties of Dobrava virus: A unique member of the Hantavirus genus, family Bunyaviridae

Genetic analysis of wild-type Dobrava hantavirus in Slovenia: Coexistence of two distinct genetic lineages within the same natural focus

Hantavirus in Argentina

How RNA viruses maintain their genome integrity

Outbreak of hantavirus pulmonary syndrome

P bodies, stress granules, and viral life cycles

Intramuscular inoculation of Sin Nombre hantavirus cDNAs induces cellular and humoral immune responses in BALB/c mice

Genetic vaccines protect against Sin Nombre hantavirus challenge in the deer mouse (Peromyscus maniculatus)

Nonviral heterogeneous sequences are present at the 5' ends of one species of snowshoe hare bunyavirus S complementary RNA

Functional L polymerase of La Crosse virus allows in vivo reconstitution of recombinant nucleocapsids

Analysis of hantavirus genetic diversity in Argentina: S segment-derived phylogeny

Hantavirus cardiopulmonary syndrome: Immune response and pathogenesis

Radiographic findings in 20 patients with Hantavirus pulmonary syndrome correlated with clinical outcome

Sin nombre virus (SNV) Ig isotype antibody response during acute and convalescent phases of hantavirus pulmonary syndrome

Shedding and intracage transmission of Sin Nombre hantavirus in the deer mouse (Peromyscus maniculatus) model

Globin mRNAs are primers for the transcription of influenza viral RNA in vitro

Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides

Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs

A structural and primary sequence comparison of the viral RNAdependent RNA polymerases

A mechanism for initiating RNA-dependent RNA polymerization

Hantaviral infections of rodents: Possible scenarios

The L-L oligomerization domain resides at the very N-terminus of the sendai virus L RNA polymerase protein

The phosphoprotein (P) and L binding sites reside in the N-terminus of the L subunit of the measles virus RNA polymerase

Haemorrhagic fever with renal syndrome: Clinical, virological and epidemiological perspectives

Intravenous ribavirin for hantavirus pulmonary syndrome: Safety and tolerance during 1 year of open-label experience

Abnormalities of T cell immunoregulation in hemorrhagic fever with renal syndrome

Effects of hantaviral infection on survival, growth and fertility in wild rat (Rattus norvegicus) populations of

Complete genetic characterization and analysis of isolation of Sin Nombre virus

Antibody responses in humans to an inactivated hantavirus vaccine (Hantavax)

Review of an inactivated vaccine against hantaviruses

Inactivated Hantaan virus vaccine derived from suspension culture of Vero cells

A vaccinia virus-vectored Hantaan virus vaccine protects hamsters from challenge with Hantaan and Seoul viruses but not Puumala virus

Phylogenetic and geographical relationships of hantavirus strains in eastern and western Paraguay

Hantavirus infections in Europe

On the nature of poliovirus genetic recombinants

Implications of high RNA virus mutation rates: Lethal mutagenesis and the antiviral drug ribavirin

Electron microscopy of the segmented RNA genome of La Crosse virus: Absence of circular molecules

Yeast-expressed Puumala hantavirus nucleocapsid protein induces protection in a bank vole model

T-helper and humoral responses to Puumala hantavirus nucleocapsid protein: Identification of Thelper epitopes in a mouse model

Tomato spotted wilt virus L RNA encodes a putative RNA polymerase

Interactions and trafficking of Andes and Sin Nombre Hantavirus glycoproteins G1 and G2

The mechanism of action of T7 DNA polymerase

The distribution of rates of spontaneous mutation over viruses, prokaryotes, and eukaryotes

Transcription of a recombinant bunyavirus RNA template by transiently expressed bunyavirus proteins

Pathogenicity of Hantaan virus in newborn mice: Genetic reassortant study demonstrating that a single amino acid change in glycoprotein G1 is related to virulence

A major antigenic domain for the human humoral response to Puumala virus nucleocapsid protein is located at the amino-terminus

Serological diagnosis of hantavirus infections by an enzyme-linked immunosorbent assay based on detection of immunoglobulin G and M responses to recombinant nucleocapsid proteins of five viral serotypes

Hantaan virus: Identification of virion proteins

Analyses of the mRNA transcription processes of snowshoe hare bunyavirus S and M RNA species

The hantavirus glycoprotein G1 tail contains dual CCHC-type classical zinc fingers

Hantavirus pulmonary syndrome in the United States

Computer analysis suggests a role for signal sequences in processing polyproteins of enveloped RNA viruses and as a mechanism of viral fusion

Reverse genetics system for Uukuniemi virus (Bunyaviridae): RNA polymerase I-catalyzed expression of chimeric viral RNAs

Rescue of Hantaan virus minigenomes

Isolation, characterization and geographic distribution of Cano Delgadito virus, a newly discovered South American hantavirus (family Bunyaviridae)

The 5' ends of Hantaan virus (Bunyaviridae) RNAs suggest a prime-and-realign mechanism for the initiation of RNA synthesis

beta3 Integrins mediate the cellular entry of hantaviruses that cause respiratory failure

Cellular entry of hantaviruses which cause hemorrhagic fever with renal syndrome is mediated by beta3 integrins

Pathogenic hantaviruses selectively inhibit beta3 integrin directed endothelial cell migration

Tyrosine residues direct the ubiquitination and degradation of the NY-1 hantavirus G1 cytoplasmic tail

Hantavirus pulmonary syndrome-associated hantaviruses contain conserved and functional ITAM signaling elements

Yeast-expressed hantavirus Dobrava nucleocapsid protein induces a strong, long-lasting, and highly crossreactive immune response in mice

Characterization of the Golgi retention motif of Rift Valley fever virus G(N) glycoprotein

Association of intraspecific wounding with hantaviral infection in wild rats (Rattus norvegicus)

Poliovirus RNA-dependent RNA polymerase (3Dpol): Structural, biochemical, and biological analysis of conserved structural motifs A and B

Poliovirus RNA-dependent RNA polymerase (3Dpol): Kinetic, thermodynamic, and structural analysis of ribonucleotide selection

Ultrastructural characteristics of Sin Nombre virus, causative agent of hantavirus pulmonary syndrome

Puumala and Dobrava hantaviruses causing hemorrhagic fever with renal syndrome in Estonia

RNA binding of recombinant nucleocapsid proteins of hantaviruses

A major antigenic domain of hantaviruses is located on the aminoproximal site of the viral nucleocapsid protein

Class and subclass distribution of Hantavirus-specific serum antibodies at different times after the onset of nephropathia epidemica

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

Structure of the RNA-dependent RNA polymerase of poliovirus

Hantavirus infections: Epidemiology and pathogenesis

Mutational evidence for a structural model of the Lassa virus RNA polymerase domain and identification of two residues, Gly1394 and Asp1395, that are critical for transcription but not replication of the genome

Increased permeability of human endothelial cell line EA.hy926 induced by hantavirus-specific cytotoxic T lymphocytes

Naturally occurring Sin Nombre virus genetic reassortants

Interactions and oligomerization of hantavirus glycoproteins

The cytoplasmic tails of hantavirus glycoproteins interact with the nucleocapsid protein

Circular forms of Uukuniemi virion RNA: An electron microscopic study

Wounding: The primary mode of Seoul virus transmission among male Norway rats

Dominant glycoprotein epitope of four corners hantavirus is conserved across a wide geographical area

Hantavirus pulmonary syndromerelated virus from Bolivia

Rapid and specific detection of Sin Nombre virus antibodies in patients with hantavirus pulmonary syndrome by a strip immunoblot assay suitable for field diagnosis

The Springer Index of Viruses

DNA vaccination with hantavirus M segment elicits neutralizing antibodies and protects against seoul virus infection

Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: Implications for drug resistance

Ribavirin therapy for Hantaan virus infection in suckling mice

Prospective, double-blind, concurrent, placebo-controlled clinical trial of intravenous ribavirin therapy of hemorrhagic fever with renal syndrome

Electron cryo-microscopy and single-particle averaging of Rift Valley fever virus: Evidence for GN-GC glycoprotein heterodimers

Electron cryotomography of Tula hantavirus suggests a unique assembly paradigm for enveloped viruses

Sin Nombre virus mRNA synthesis

Transmission of Black Creek Canal virus between cotton rats

Rift valley fever virus nonstructural protein NSs promotes viral RNA replication and transcription in a minigenome system

Association of serine in position 1124 of Hantaan virus glycoprotein with virulence in mice

Mutation of the aspartic acid residues of the GDD sequence motif of poliovirus RNA-dependent RNA polymerase results in enzymes with altered metal ion requirements for activity

A cophylogenetic perspective of RNA-virus evolution

Detection of Dobrava hantaviruses in Apodemus agrarius mice in the Transdanubian region of Hungary

Characterization of human antibody responses to four corners hantavirus infections among patients with hantavirus pulmonary syndrome

CspA, the major cold-shock protein of Escherichia coli, is an RNA chaperone

Characterization of Bunyamwera virus S RNA that is transcribed and replicated by the L protein expressed from recombinant vaccinia virus

Hantaan virus enters cells by clathrin-dependent receptor-mediated endocytosis

Laguna Negra virus associated with HPS in western Paraguay and Bolivia

Global trends in emerging infectious diseases

Purification and characterization of the Sin Nombre virus nucleocapsid protein expressed in Escherichia coli

A global perspective on hantavirus ecology, epidemiology, and disease

Polymerase structures and function: Variations on a theme?

Maternal antibodies postpone hantavirus infection and enhance individual breeding success

Primary structural comparison of RNA-dependent polymerases from plant, animal and bacterial viruses

Pathogenesis of puumala and other hantavirus infections

Host switch during evolution of a genetically distinct hantavirus in the American shrew mole (Neurotrichus gibbsii)

Evolutionary insights from a genetically divergent hantavirus harbored by the European common mole (Talpa europaea)

Urine-associated horizontal transmission of Seoul virus among rats

Interaction between molecules of hantavirus nucleocapsid protein

Mapping of the regions involved in homotypic interactions of Tula hantavirus N protein

Oligomerization of Hantavirus N protein: C-terminal alpha-helices interact to form a shared hydrophobic space

Hantavirus immunology

Role of specific CD8þ T cells in the severity of a fulminant zoonotic viral hemorrhagic fever, hantavirus pulmonary syndrome

Hemorrhagic fever with renal syndrome caused by the Seoul virus

Central European Dobrava Hantavirus isolate from a striped field mouse

Hemorrhagic fever with renal syndrome caused by 2 lineages of Dobrava hantavirus

Delayed viremia and antibody responses in Puumala hantavirus challenged passively immunized cynomolgus macaques

Dobrava, but not Saaremaa, hantavirus is lethal and induces nitric oxide production in suckling mice

Loss of cell membrane integrity in puumala hantavirus-infected patients correlates with levels of epithelial cell apoptosis and perforin

A bunyamwera virus minireplicon system in mosquito cells

Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor

Nephropathia epidemica with a 6-week incubation period after occupational exposure to Puumala hantavirus

Priming of influenza viral RNA transcription by capped heterologous RNAs

Are the 5' ends of influenza viral mRNAs synthesized in vivo donated by host mRNAs?

Uukuniemi virus maturation: Accumulation of virus particles and viral antigens in the Golgi complex

Completion of the Tula hantavirus genome sequence: Properties of the L segment and heterogeneity found in the 3' termini of S and L genome RNAs

Tula hantavirus L protein is a 250 kDa perinuclear membrane-associated protein

Localization of Bunyamwera bunyavirus G1 glycoprotein to the Golgi requires association with G2 but not with NSm

Peptide antagonists that inhibit Sin Nombre virus and hantaan virus entry through the beta3-integrin receptor

Observations on natural and laboratory infection of rodents with the etiologic agent of Korean hemorrhagic fever

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

Association of the nucleocapsid protein of the Seoul and Hantaan hantaviruses with small ubiquitin-like modifier-1-related molecules

Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site

Complete nucleotide sequences of the M and S segments of two hantavirus isolates from California: Evidence for reassortment in nature among viruses related to hantavirus pulmonary syndrome

Serologic diagnosis of Hantaan virus infection based on a peptide antigen

Elevated plasma levels of tumor necrosis factor (TNF)-alpha, soluble TNF receptors, interleukin (IL)-6, and IL-10 in patients with hemorrhagic fever with renal syndrome

The Hantaan virus glycoprotein precursor is cleaved at the conserved pentapeptide WAASA

The L protein of Rift Valley fever virus can rescue viral ribonucleoproteins and transcribe synthetic genome-like RNA molecules

Histopathology of Peromyscus leucopus naturally infected with pathogenic NY-1 hantaviruses: Pathologic markers of HPS viral infection in mice

Cellular receptors and hantavirus pathogenesis

Pathogenic hantaviruses elicit different immunoreactions in THP-1 cells and primary monocytes and induce differentiation of human monocytes to dendritic-like cells

Distinction between Bunyaviridae genera by surface structure and comparison with Hantaan virus using negative stain electron microscopy

A signal for Golgi retention in the bunyavirus G1 glycoprotein

Clinical evaluation of a vaccinia-vectored Hantaan virus vaccine

Ribavirin, human convalescent plasma and anti-beta3 integrin antibody inhibit infection by Sin Nombre virus in the deer mouse model

Placebocontrolled, double-blind trial of intravenous ribavirin for the treatment of hantavirus cardiopulmonary syndrome in North America

Accumulation of terminally deleted RNAs may play a role in Seoul virus persistence

Regulatory T cells: Friend or foe in immunity to infection?

Trimeric hantavirus nucleocapsid protein binds specifically to the viral RNA panhandle

The hantavirus nucleocapsid protein recognizes specific features of the viral RNA panhandle and is altered in conformation upon RNA binding

The triplet repeats of the Sin Nombre hantavirus 5' UTR are sufficient in cis for nucleocapsid-mediated translation initiation

Hantavirus N protein exhibits genus-specific recognition of the viral RNA panhandle

A protein that replaces the entire cellular eIF4F complex

Storage of cellular 5' mRNA caps in P bodies for viral cap-snatching

Hantavirus nucleocapsid protein has distinct m7G cap-and RNA-binding sites

Detection of Brazilian hantavirus by reverse transcription polymerase chain reaction amplification of N gene in patients with hantavirus cardiopulmonary syndrome

The challenge of emerging and re-emerging infectious diseases

Cellular entry of Hantaan virus A9 strain: Specific interactions with beta3 integrins and a novel 70 kDa protein

Rift Valley fever virus L segment: Correction of the sequence and possible functional role of newly identified regions conserved in RNA-dependent polymerases

Hantavirus infection

Genetic characterization of new Dobrava hantavirus isolate from Greece

Sin Nombre virus pathogenesis in Peromyscus maniculatus

Structure-function relationships among RNA-dependent RNA polymerases

Genetic identification of a hantavirus associated with an outbreak of acute respiratory illness

Hantavirus pulmonary syndrome in the United States: A pathological description of a disease caused by a new agent

Analysis of RNA-dependent RNA polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure

Bunyaviridae: Recent biochemical developments

Segmented genome and nucleocapsid of La Crosse virus

Nucleotide sequences at the terminal of La Crosse virus RNAs

RNA binding properties of bunyamwera virus nucleocapsid protein and selective binding to an element in the 5' terminus of the negative-sense S segment

The glycoprotein cytoplasmic tail of Uukuniemi virus (Bunyaviridae) interacts with ribonucleoproteins and is critical for genome packaging

Insights into bunyavirus architecture from electron cryotomography of Uukuniemi virus

Hantavirus pulmonary syndrome outbreak in Argentina: Molecular evidence for person-to-person transmission of Andes virus

Complete nucleotide sequence of the M RNA segment of Andes virus and analysis of the variability of the termini of the virus S, M and L RNA segments

Bunyavirus N: eIF4F surrogate and cap-guardian

La Crosse virions contain a primerstimulated RNA polymerase and a methylated cap-dependent endonuclease

The Hantaan virus M-segment glycoproteins G1 and G2 can be expressed independently

Spectrum of hantavirus infection: Hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome

Ribonucleoproteins of Uukuniemi virus are circular

Role of the virus nucleoprotein in the regulation of lymphocytic choriomeningitis virus transcription and RNA replication

A unique cap(m7G pppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription

Genetics of hantaviruses: Implications to taxonomy

Virus evolution and genetic diversity of hantaviruses and their rodent hosts

Puumala hantavirus genome in patients with nephropathia epidemica: Correlation of PCR positivity with HLA haplotype and link to viral sequences in local rodents

Transfection-mediated generation of functionally competent Tula hantavirus with recombinant S RNA segment

Identification of four conserved motifs among the RNA-dependent polymerase encoding elements

Hantavirus infection of dendritic cells

Unusual transcripts in La Crosse virus-infected cells and the site for nucleocapsid assembly

The ends of La Crosse virus genome and antigenome RNAs within nucleocapsids are base paired

New and Old World hantaviruses differentially utilize host cytoskeletal components during their life cycles

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

High rates of molecular evolution in hantaviruses

Hantavirus nucleocapsid protein is expressed as a membrane-associated protein in the perinuclear region

Polarized entry and release in epithelial cells of Black Creek Canal virus, a New World hantavirus

Role of actin microfilaments in Black Creek Canal virus morphogenesis

Generation of a recombinant cytomegalovirus for expression of a hantavirus glycoprotein

Genetic reassortment among viruses causing hantavirus pulmonary syndrome

Genetic relatedness of deer mice (Peromyscus maniculatus) infected with Sin Nombre virus. Vector Borne Zoonotic Dis

La Crosse virus small genome mRNA is made in the cytoplasm

Structure and mechanism of DNA polymerases

Monoclonal antibodies to three strains of hantaviruses: Hantaan, R22, and Puumala

Experience with intravenous ribavirin in the treatment of hemorrhagic fever with renal syndrome in Korea

Coexpression of the membrane glycoproteins G1 and G2 of Hantaan virus is required for targeting to the Golgi complex

Viral RNA replication in association with cellular membranes

Genetic and biochemical evidence for an oligomeric structure of the functional L polymerase of the prototypic arenavirus lymphocytic choriomeningitis virus

Hantaviruses: A global disease problem

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

Coding strategy of the S genome segment of Hantaan virus

Hantaan virus M RNA: Coding strategy, nucleotide sequence, and gene order

Antigenic subunits of Hantaan virus expressed by baculovirus and vaccinia virus recombinants

Hantavirus-induced immunity in rodent reservoirs and humans

Degrons at the C terminus of the pathogenic but not the nonpathogenic hantavirus G1 tail direct proteasomal degradation

Characterization of the Hantaan nucleocapsid protein-ribonucleic acid interaction

Ribavirin causes error catastrophe during Hantaan virus replication

Essential amino acids of the hantaan virus N protein in its interaction with RNA

Golgi localization of Hantaan virus glycoproteins requires coexpression of G1 and G2

Analysis of N-linked glycosylation of hantaan virus glycoproteins and the role of oligosaccharide side chains in protein folding and intracellular trafficking

Mapping the Golgi targeting and retention signal of Bunyamwera virus glycoproteins

Role of the cytoplasmic tail domains of Bunyamwera orthobunyavirus glycoproteins Gn and Gc in virus assembly and morphogenesis

Host-derived 5' ends and overlapping complementary 3' ends of the two mRNAs transcribed from the ambisense S segment of Uukuniemi virus

Hantavirus pulmonary syndrome

Pathology of Puumala hantavirus infection in macaques

The L polymerase protein of parainfluenza virus 3 forms an oligomer and can interact with the heterologous Sendai virus L, P and C proteins

Intragenic complementation and oligomerization of the L subunit of the sendai virus RNA polymerase

Thottapalayam virus, a prototype shrewborne hantavirus

Newfound hantavirus in Chinese mole shrew

Muju virus, a novel hantavirus harboured by the arvicolid rodent Myodes regulus in Korea

Hantavirus maturation

Genome structure and variability of a virus causing hantavirus pulmonary syndrome

Sin Nombre virus glycoprotein trafficking

A unified polymerase mechanism for nonhomologous DNA and RNA polymerases

Primary structure of the large (L) RNA segment of nephropathia epidemica virus strain Hallnas B1 coding for the viral RNA polymerase

Hantavirus infection induces the expression of RANTES and IP-10 without causing increased permeability in human lung microvascular endothelial cells

Lack of vertical transmission of Hantaan virus from persistently infected dam to progeny in laboratory mice

Close structural resemblance between putative polymerase of a Drosophila transposable genetic element 17.6 and pol gene product of Moloney murine leukaemia virus

Completion of the rabies virus genome sequence determination: Highly conserved domains among the L (polymerase) proteins of unsegmented negative-strand RNA viruses

In vitro translation of Uukuniemi virusspecific RNAs: Identification of a nonstructural protein and a precursor to the membrane glycoproteins

Emerging viruses: The case 'hantavirus

Human memory cytotoxic T-lymphocyte (CTL) responses to Hantaan virus infection: Identification of virus-specific and cross-reactive CD8(þ) CTL epitopes on nucleocapsid protein

Mutation analysis of the GDD sequence motif of a calicivirus RNA-dependent RNA polymerase

Hantavirus pulmonary syndrome in Panama: Identification of novel hantaviruses and their likely reservoirs

Bunyamwera virus can repair both insertions and deletions during RNA replication

Degradation and aggresome formation of the Gn tail of the apathogenic Tula hantavirus

MultiCoil: A program for predicting two-and three-stranded coiled coils

Evolutionary aspects of recombination in RNA viruses

Antibody responses to Four Corners hantavirus infections in the deer mouse (Peromyscus maniculatus): Identification of an immunodominant region of the viral nucleocapsid protein

Daxx, a novel Fas-binding protein that activates JNK and apoptosis

The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses

Protective immunity of Hantaan virus nucleocapsid and envelope protein studied using baculovirus-expressed proteins

Characterization of the nucleocapsid protein of Hantaan virus strain 76-118 using monoclonal antibodies

The incubation period of hantavirus pulmonary syndrome

Hantavirus pulmonary syndrome. Pathogenesis of an emerging infectious disease

Rift valley fever virus L protein forms a biologically active oligomer

New ecological aspects of hantavirus infection: A change of a paradigm and a challenge of prevention-A review