Quaranfil, Johnston Atoll, and Lake Chad viruses are novel members of the family Orthomyxoviridae

Abstract

Arboviral infections are an important cause of emerging infections due to the movements of humans, animals, and hematophagous arthropods. Quaranfil virus (QRFV) is an unclassified arbovirus originally isolated from children with mild febrile illness in Quaranfil, Egypt, in 1953. It has subsequently been isolated in multiple geographic areas from ticks and birds. We used high-throughput sequencing to classify QRFV as a novel orthomyxovirus. The genome of this virus is comprised of multiple RNA segments; five were completely sequenced. Proteins with limited amino acid similarity to conserved domains in polymerase (PA, PB1, and PB2) and hemagglutinin (HA) genes from known orthomyxoviruses were predicted to be present in four of the segments. The fifth sequenced segment shared no detectable similarity to any protein and is of uncertain function. The end-terminal sequences of QRFV are conserved between segments and are different from those of the known orthomyxovirus genera. QRFV is known to cross-react serologically with two other unclassified viruses, Johnston Atoll virus (JAV) and Lake Chad virus (LKCV). The complete open reading frames of PB1 and HA were sequenced for JAV, while a fragment of PB1 of LKCV was identified by mass sequencing. QRFV and JAV PB1 and HA shared 80% and 70% amino acid identity to each other, respectively; the LKCV PB1 fragment shared 83% amino acid identity with the corresponding region of QRFV PB1. Based on phylogenetic analyses, virion ultrastructural features, and the unique end-terminal sequences identified, we propose that QRFV, JAV, and LKCV comprise a novel genus of the family Orthomyxoviridae.

FIG. 1.

Growth and CPE of QRFV. (A) Uninfected 3T12 fibroblasts. (B) QRFV-infected fibroblasts at 5 days postinfection with multiplicity of infection of 0.01. (C) Multistep growth of QRFV in several cell types. The data shown are pooled from three independent experiments. Error bars indicate standard deviations. (D) Single-step growth for QRFV in 3T12 cells. The data shown are from one representative of two independent experiments.

FIG. 2.

Electron microscopy of QRFV. (A and B) Resin-embedded QRFV-infected 3T12 fibroblasts at 5 days postinfection. (C) Cryo-immunoelectron microscopy of uninfected 3T12 fibroblasts labeled with anti-QRFV antibody. (D to F) Cryo-immunoelectron microscopy of QRFV-infected 3T12 fibroblasts labeled with anti-QRFV antibody. Cy, cytoplasm; Nu, nucleus. Bars, 200 nm.

FIG. 3.

Phylogenetic analysis of the conserved viral proteins. Phylogenetic trees were constructed with the sequences for homologous proteins. (A) PB1; (B) HA; (C) PA; (D) PB2. Trees were generated using the maximum-parsimony and maximum-likelihood methods with 1,000 bootstrap replicates. The most parsimonious trees are shown. The numbers at the branches show bootstrap values for both methods (maximum parsimony, maximum likelihood). FluC, influenza C virus (C/Ann Arbor/1/50); FluB, influenza B virus (B/Lee/40); Th, Thogoto virus (SiAr 126); Dh, Dhori virus (Indian/1313/61); Isa, infectious salmon anemia virus; Quaranfil, QRFV (EG T 377); Johnston Atoll, JAV (South Pacific strain). For the polymerase sequences, the five reference strains of influenza A virus (A/Puerto Rico/8/34 [H1N1], A/New York/392/2004 [H3N2], A/Hong Kong/1073/99 [H9N2], A/Goose/Guangdong/1/96 [H5N1], and A/Korea/426/68 [H2N2]) grouped closely, and for clarity, only FluA is indicated.