Sand Fly-Associated Phlebovirus with Evidence of Neutralizing Antibodies in Humans, Kenya

Abstract

We describe a novel virus, designated Ntepes virus (NPV), isolated from sand flies in Kenya. NPV has the characteristic phlebovirus trisegmented genome architecture and is related to, but distinct from, Gabek Forest phlebovirus. Diverse cell cultures derived from wildlife, livestock, and humans were susceptible to NPV, with pronounced permissiveness in swine and rodent cells. NPV infection of newborn mice caused rapid and fatal illness. Permissiveness for NPV replication in sand fly cells, but not mosquito cells, suggests a vector-specific adaptation. Specific neutralizing antibodies were found in 13.9% (26/187) of human serum samples taken at the site of isolation of NPV as well as a disparate site in northeastern Kenya, suggesting a wide distribution. We identify a novel human-infecting arbovirus and highlight the importance of rural areas in tropical Africa for arbovirus surveillance as well as extending arbovirus surveillance to include hematophagous arthropods other than mosquitoes.

Keywords: Gabek Forest phlebovirus; Kenya; Ntepes virus; arbovirus; neutralizing antibodies; phlebovirus; sand flies; virus discovery; viruses.

Figure 1

Geographic location of sand fly collection site (Ntepes) and district hospitals of Marigat and Sangailu, where human serum samples were collected, Kenya.

Figure 2

Genome organization of novel sand fly–associated phlebovirus Ntepes virus identified in Kenya. Sequence length of the L, M, and S segments (in bp) and encoded predicted proteins RdRp, Gn, Gc, N, and nonstructural proteins NSm and NSs (in kDa) are indicated; ORF positions (length in bp) are also indicated. GFV, Gabek Forest virus; L, large segment (encoding the RdRp protein); M, medium segment (encoding the nonstructural protein NSm and the 2 glycoproteins Gn and Gc); N, nucleocapsid protein; ORF, open reading frame; RdRp, RNA-dependent RNA polymerase; S, small segment (encoding the N protein and nonstructural protein NSs in an ambisense manner); vRNA, virus RNA.

Figure 3

Phylogenetic relationship of novel sand fly–associated phlebovirus Ntepes virus from Kenya (red bold text) in relation to other selected members of the Phlebovirus genus. A) RNA-dependent RNA polymerase; B) nucleocapsid protein; C) glycoprotein Gn; D) glycoprotein Gc. The phylogenetic trees were inferred based on complete large, medium, and small protein sequences, applying maximum likelihood analysis in PhyML version 3.0 (http://www.atgc-montpellier.fr/phyml/versions.php) using the LG substitution model. Statistical support of the tree topology was evaluated by bootstrap resampling of the sequences 1,000 times. Sequences are identified by virus name and branch colors. Bootstrap values >70 are indicated at the nodes. Scale bar represents numbers of substitutions per site.

Figure 4

Neutralizing activity of novel sand fly–associated phlebovirus Ntepes virus from Kenya in relation to other selected members of the Phlebovirus genus. Anti-GFV and anti-KARV samples were tested along with 26 human serum samples. GFV, Gabek Forest virus; H, human; KARV, Karimabad virus; NPV, Ntepes virus; NT, neutralizing test; RVFV, Rift Valley fever virus.

Figure 5

In vitro growth kinetics of novel sand fly–associated phlebovirus Ntepes virus from Kenya in different cell lines. A) Insects: LL-5, sand fly; C6/36, mosquito. B) Human: HEK293-T. C) Peridomestic wildlife: hamster, BHK-21; primate, VeroE6/7; mouse, MEF; bat, EidNi. D) Livestock: swine, PK-15; goat, ZN-R; chicken, DF-1; cattle, KN-R. Cells were infected with a multiplicity of infection of 0.1; supernatants were collected every 24 h for 7 d postinfection. Viral genome copies were measured at indicated timepoints by real-time reverse transcription PCR. E) Pathogenicity of Ntepes virus infection in mice. Litters of 2-day-old Swiss Albino suckling mice (8 mice/litter) were intracerebrally inoculated using the indicated virus titers or cell culture media as a control. Animals were monitored daily for signs of disease. Titers are shown in PFU/mL.