The family Orthomyxoviridae
had previously been comprised of five genera, Influenzavirus A
, Influenzavirus B
, Influenzavirus C
, and Thogotovirus
. Influenza viruses are important human pathogens, causing yearly outbreaks of respiratory infection and periodic severe epidemics resulting in thousands to millions of deaths annually (21
). The genus Isavirus
has only one member, which causes severe anemia in multiple fish strains and is a significant commercial problem. The thogotoviruses consist of three viruses: Thogoto virus, Dhori virus, and Araguari virus. Dhori virus has been reported to cause encephalitis and viral illness after the accidental infection of five laboratory workers (8
). Because of the genetic ability of the orthomyxoviruses within a given genus to undergo reassortment of their genome segments, with the resultant emergence of new virus strains, these viruses pose a continual threat for epidemic outbreaks of disease in humans, poultry, and livestock.
Phylogenetic and sequence analysis of the complete predicted ORFs from both QRFV and JAV demonstrated a clear relationship with members of the family Orthomyxoviridae. Based on the observed phylogenetic distances, we propose that QRFV and JAV should be classified into a novel genus in this family, tentatively named Quarjavirus. This genus designation is further supported by the unique end-terminal sequence seen in the sequencing of both QRFV and JAV. Limited sequencing and serologic analysis support the addition of a third virus, LKCV, as a third member of this genus. The ultrastructural, morphological, and serologic studies presented in this paper further support the classification of these viruses as novel orthomyxoviruses.
The genomes of orthomyxoviruses consist of six to eight segments of negative single-strand RNA. In this study, we have generated sequence data demonstrating that QRFV and JAV are multisegmented RNA viruses. We have identified and completely sequenced five segments from QRFV, including four segments which we predict to encode proteins which are essential to the orthomyxovirus replication machinery: PA, PB1, PB2, and a glycoprotein sequence which is likely to function as the viral entry/fusion protein/HA. We have not definitively identified NP or M, nor have we identified any sequence which shares significant homology with the influenza virus neuraminidase or nonstructural segments. It is possible that the fifth segment encodes one of these functions, although no homology was detected. It is likely that additional segments exist but were not amplified under the conditions we used. One potential reason may be that the end-terminal sequence is not completely conserved among all segments. This has precedent, as the 3′-terminal sequence of Thogoto virus segment 6 is not conserved with the other segments, differing at three nucleotides (17
The ends of orthomyxovirus segments are characterized by conserved sequences which show partial reverse complementarity (14
). The conservation of these terminal sequences is thought to be important to the binding of the viral polymerase subunit, PB1, and activation of a unique process called cap snatching, which is enabled by the polymerase subunit, PB2 (6
). Priming of viral mRNA synthesis in influenza viruses occurs by stealing capped fragments of 10 to 13 nt from the host (these are m7
-containing RNA fragments derived by cleavage of host cell RNA polymerase II transcripts) (21
). In contrast, while Thogoto virus mRNA is capped, 5′ RACE analysis has determined that Thogoto virus mRNAs do not contain any significant heterogeneous sequence (35
). 5′ RACE of mRNA from QRFV identified 9 to 11 nucleotides which are heterogeneous among the different RACE products (Table ). We hypothesize that this observed heterogeneity derives from host sequences captured in a cap-snatching process more similar to that described for influenza A than to that described for Thogoto virus.
While QRFV likely shares features of its mRNA synthesis process with the influenza viruses, it also resembles the thogotoviruses in other aspects, including its growth and tropism features. QRFV and Thogoto virus grow more slowly than the influenza viruses, with significant virus output at 24 h postinfection (7
). QRFV and JAV, like Thogoto and Dhori viruses, are thought to be tick borne. Perhaps because of this difference in tropism, the HA sequence determined for QRFV shares greater sequence similarity with baculovirus glycoproteins and the HA of thogotoviruses, which are also more closely related to the baculovirus glycoproteins, than to the influenza virus HA proteins (Fig. ) (26
). This might also explain why no neuraminidase sequence has been identified. Thus, QRFV shares features with both the influenza viruses and the thogotoviruses, further supporting its classification as a unique orthomyxovirus genus.
QRFV was initially isolated from two children with self-limited viral illness from the area of Quaranfil, Egypt (32
). Serologic studies in the 1960s indicated that up to 8% of the local population had been exposed to QRFV (32
). Aside from these studies, the prevalence of QRFV in the human population has not been assessed, nor has there been extensive effort made to determine the possible contributions of QRFV to human disease. The isolation of QRFV, JAV, and LKCV from ticks and birds in Africa, Central Asia, and the Pacific demonstrates that these viruses can be found over a wide geographic area, so a large fraction of the human population has potential exposure to this group of viruses. Classification and sequencing of these viruses are important first steps to developing assays to determine the extent of human infection caused by them and to understanding the basic virology and pathogenesis of these novel orthomyxoviruses.