Tick-borne Nyamanini virus (NYMV) is the prototypic member of a recently discovered genus in the order Mononegavirales , designated Nyavirus . The NYMV genome codes for six distinct genes. Sequence similarity and structural properties suggest that genes 1, 5, and 6 encode the nucleoprotein (N), the glycoprotein (G), and the viral polymerase (L), respectively. The function of the other viral genes has been unknown to date. We found that the third NYMV gene codes for a protein which, when coexpressed with N and L, can reconstitute viral polymerase activity, suggesting that it represents a polymerase cofactor. The second viral gene codes for a small protein that inhibits viral polymerase activity and further strongly enhances the formation of virus-like particles when coexpressed with gene 4 and the viral glycoprotein G. This suggests that two distinct proteins serve a matrix protein function in NYMV as previously described for members of the family Filoviridae . We further found that NYMV replicates in the nucleus of infected cells like members of the family Bornaviridae . NYMV is a poor inducer of beta interferon, presumably because the viral genome is 5′ monophosphorylated and has a protruding 3′ terminus as observed for bornaviruses. Taken together, our results demonstrate that NYMV possesses biological properties previously regarded as typical for filoviruses and bornaviruses, respectively.

Nyamanini virus (NYMV) and Midway virus (MIDWV) are unclassified tick-borne agents that infect land birds and seabirds, respectively. The recent molecular characterization of both viruses confirmed their already known close serological relationship and revealed them to be nonsegmented, single- and negative-stranded RNA viruses that are clearly related to, but quite distinct from, members of the order Mononegavirales (bornaviruses, filoviruses, paramyxoviruses, and rhabdoviruses). A third agent, soybean cyst nematode virus 1 (SbCNV-1, previously named soybean cyst nematode nyavirus), was recently found to be an additional member of this new virus group. Here, we review the current knowledge about all three viruses and propose classifying them as members of a new mononegaviral family, Nyamiviridae.

bWe established a reverse genetics system for Nyamanini virus (NYMV) and recovered green fluorescent protein (GFP)-expressing virus from full-length cDNA. Using this technology, we assessed the functions of two poorly characterized viral genes. NYMV lacking open reading frame 2 (ORF2) could not be rescued, whereas virus lacking ORF4 was replication competent. ORF4-deficient NYMV readily established a persisting noncytolytic infection but failed to produce infectious viral particles, supporting the view that ORF4 represents an essential factor for NYMV particle assembly. N yamanini virus (NYMV) is the prototype member of a novel genus in the order Mononegavirales, designated "Nyavirus" (1). Recently, Kuhn and coworkers (2) proposed classifying NYMV as a member of a new mononegaviral family, designated "Nyamiviridae." NYMV replicates in the nucleus of infected cells (3), and viral budding was proposed to occur at the plasma membrane (1). The NYMV genome contains six major open reading frames (ORFs). The viral nucleoprotein N, the glycoprotein G, and the polymerase L are encoded by ORF1, ORF5, and ORF6, respectively. We recently showed that ORF3 codes for a polymerase cofactor which is required for NYMV polymerase activity (3). The functions of the proteins encoded by ORF2 and ORF4 remain largely unknown. ORF2 negatively regulates NYMV polymerase activity, presumably through interaction with the ORF3 product (3). ORF2 was further shown to promote production of infectious virus-like particles by an unknown mechanism (3). Formation of virus-like particles required the simultaneous presence of the viral G protein, the product of ORF4, and the product of ORF2, indicating that ORF2 and ORF4 both play a role in NYMV particle assembly (3). It remains unclear, however, whether ORF4 represents the NYMV-specific equivalent of a matrix protein typically found in negative-strand RNA viruses. No substantial homology was detected between ORF4 of NYMV and matrix proteins of other members of the order Mononegavirales (1).Matrix (M) proteins of negative-strand RNA viruses orchestrate the assembly of viral particles by binding to both the viral ribonucleoprotein (RNP) complex and cellular membranes (reviewed in references 4 and 5). The importance of M proteins for the assembly and budding of negative-strand RNA viruses was directly demonstrated for rabies virus, measles virus, and Sendai virus by employing reverse genetics approaches (6-8). M-deficient variants of these viruses showed extensive cell-to-cell spreading but failed to produce cell-free infectious particles.To further characterize the ORF2 and ORF4 genes of NYMV, we set out to establish a reverse genetics system for recovering recombinant virus from cloned cDNA. Using cDNA fragments generated by PCR from RNA of NYMV-infected Vero cells, we constructed a rescue plasmid containing the putative full-length NYMV antigenome. As recently described for reverse genetics systems of Borna disease virus and measles virus (9), the full-length NYMV cDNA was inserted into the vector p...