Genus Hypovirus

Type Species	Cryphonectria hypovirus 1	(CHV-1)

Virion Properties

Morphology

No true virions are associated with members of this family. Pleomorphic vesicles 50-80  nm in diameter, devoid of any detectable viral structural proteins but containing dsRNA and polymerase activity are the only virus-associated particles that can be isolated from infected fungal tissue (Fig. 1).

Physicochemical and Physical Properties

Mr of vesicles is unknown. They have a buoyant density in CsCl of approximately 1.27-1.3  g/cm³ and sediment through sucrose as a broad component of approximately 200S. Their pH stability is unknown. The vesicles can be purified in pH 5.0 buffer and resuspended in pH 7.0 buffer. pH optimum for polymerase activity in vitro is 8.0; the optimum Mg⁺⁺ for polymerase activity is 5  mM. Activity decreases dramatically at pH less than 7.0 or more than 9.0. The vesicles are unstable when heated, or in lipid solvents. Optimal temperature for polymerase activity is 30°C; temperatures over 40°C inactivate polymerase activity. Deoxycholate at concentrations of more than 0.5% inactivates polymerase activity.

Nucleic Acid

Vesicles contain linear dsRNA, approximately 9-13  kbp in size. The genome of the type species, Cryphonectria hypovirus 1 (CHV-1), is 12,712  nts. Apparently only one strand is employed in transcription. The coding (positive) strand contains a short 3-poly(A) tail, which is 20-30 residues in length when analyzed as a component of the dsRNA. Synthetic RNA representing the positive strand of the full-length dsRNA segment is sufficient for infection. Presence of shorter-than-full-length dsRNA deletion molecules is common among some members, and satellite-like dsRNAs are present in other members. No function has been ascribed to any ancillary dsRNA. The 5 terminus of the positive strand of dsRNA from CHV-1 is blocked, but the blocking group is unknown. The 5 terminus of the negative strand is unblocked. Both 5 termini of dsRNA from Cryphonectria hypovirus 3-GH2 (CHV-3/GH2), are unblocked.

Proteins

No structural proteins have been described for members of this family. Functions have been assigned to several nonstructural polypeptides encoded by members of the family. CHV-1/EP713 dsRNA encodes P29, a presumptive NS protein identified in vitro and in vivo. P29 has papain-like protease activity and has been shown by DNA-mediated transformation to contribute to suppression of pigmentation, reduced sporulation, and reduced laccase accumulation. RNA-dependant RNA polymerase activity is associated with isolated vesicles of CHV-1/EP713. The calculated size of the ORF B product, which contains putative RNA polymerase and helicase domains, is approximately 250 10³ based on deduced amino acid sequence from cDNA clones, but no protein of that size has yet been isolated from vesicles. Smaller virus-encoded proteins have been identified in the vesicle-associated polymerase complex, suggesting extensive processing of replication proteins and that ORF B processing occurs in vivo. There are no known external viral proteins. The polymerase transcribes ssRNA molecules in vitro that correspond in size to full-length dsRNA. Approximately 90% of the polymerase products in vitro are of positive polarity.

Lipids

Host-derived lipids make up the vesicles that encapsulate the viral dsRNA.

Carbohydrates

Carbohydrates similar to those involved in fungal cell wall synthesis are associated with vesicles.

Genome Organization and Replication

A 5-leader of approximately 300-500  nts, including several AUG triplets, precedes the AUG codon that initiates the first long ORF. The viral coding region may be expressed from a single long ORF, or may be divided into two ORFs. If two ORFs are present, the shorter, 5 proximal ORF is designated ORFA. Its product may or may not be autocatalytically cleaved, depending on the virus. The UAA termination sequence at the end of ORFA is part of the pentanucleotide UAAUG in all members with two ORFs investigated to date. The AUG of the UAAUG pentanucleotide initiates the other long ORF, ORFB. The N-terminal product of ORFB is a papain-like cysteine protease that autocatalytically releases from the growing polypeptide chain (e.g., P48 for CHV-1/EP713 or P52 for CHV-2/NB58). No further processing in vitro has been demonstrated for the remaining 300 10³ polypeptide from this ORF (Fig. 2). Phylogenetic relatedness to members of the positive-sense, ssRNA genus Potyvirus has been demonstrated by comparisons of protease, polymerase, and helicase domains, although these domains are positioned differently in the two famillies.

Antigenic Properties

No antibody has ever been raised from virus particle preparations. Anti-dsRNA antibodies have been used to confirm the genomic constituent. Chimeric -galactosidase/EP713 ORFA fusion proteins have successfully been used to raise antiserum that is immunoreactive with a virus-specific protein in the infected fungal host, but the location of the protein in the cell is unknown. Antibodies directed against the conserved RNA polymerase domain of ORFB, expressed in bacteria, were used to identify an 87 10³ protein in a CHV-1/EP713 infected isolate.

Biological Properties

Confirmed members infect the chestnut blight fungus, Cryphonectria parasitica. Confirmed members result in reduced virulence (hypovirulence) on chestnut trees and altered fungal morphology in culture, but many possible family members have little or no discernible effect on the fungal host. Some possible members infect other filamentous fungi, e.g., Sclerotinia sclerotiorum. Infection of fungal mycelium is known only through fusion, or anastomosis, of infected with uninfected hyphae. The transmission rate through asexual spores (conidia) varies from a few to close to 100%. Transmission through sexual spores (ascospores) is not known to occur. Transmission via cell-free extracts has not been demonstrated but transfection of protoplasts with full-length synthetic transcripts has been successful for CHV-1/EP713. Confirmed members have been identified throughout chestnut growing areas of Europe, North America, and Asia. dsRNA-containing vesicles have been associated with abnormal Golgi apparatus in freeze-substituted thin sections. No nuclear or mitochondrial associations, nor virus-associated inclusions, have been noted.

List of Species Demarcation Criteria in the Genus

Species are differentiated based on major differences in genetic organization or genome expression, which are accompanied by differences in nucleic acid sequence identity. Thus, CHV-1 differs from CHV-2 in the presence or absence, respectively, of a papain-like proteinase in ORFA. CHV-1 and CHV-2 share less than 70% overall sequence identity. Infection by CHV-1 strains results in a white or near-white phenotype in the fungus; CHV-2 infection results in an orange-brown phenotype.

List of Species in the Genus

Official virus species names are in italics. Tentative virus species names, alternative names ( ), strains or serotypes are not italicized. Virus names, genome sequence accession numbers [ ], and assigned abbreviations ( ) are:

Species in the Genus

Cryphonectria hypovirus 1		(CHV-1)
Cryphonectria hypovirus 1/EP713 (Hypovirulence-associated virus)	[M57938]	(CHV-1/EP713)
Cryphonectria hypovirus 1-EP747		(CHV-1/EP747)
Cryphonectria hypovirus 2		(CHV-2)
Cryphonectria hypovirus 2/NB58	[L29010]	(CHV-2/NB58)

Tentative Species in the Genus

Cryphonectria hypovirus 3/GH2	(CHV-3/GH2)
Cryphonectria hypovirus 4/SR2	(CHV-4/SR2)

Unassigned Viruses in the Family

None reported.

Phylogenetic Relationships in the Family

Hierarchy of relationships within this family is unknown. Many strains and isolates of confirmed and tentative members of the family have been identified by RNA hybridization analysis or nucleotide sequence analysis. CHV-1 and CHV-2 are more closely related to each other than they are to tentative members CHV-3 or CHV-4.

Similarity with Other Taxa

Deduced amino acid sequences of polymerase, helicase, and protease motifs of members of the family Hypoviridae are most closely related to the genus Bymovirus of the family Potyviridae.

Derivation of Names

Hypo: from hypovirulence