Study of Viral Coinfection of the Ixodes persulcatus Ticks Feeding on Humans in a Natural Focus of the South of the Far East

Abstract

The phenomenon of pathogen co-infection detected in a half-fed Ixodes persulcatus tick taken from a human in the south of the Far East was studied. Research was carried out on PEK, Vero, and Vero-E6 cell lines, outbred mice, and chicken embryos using ELISA, PCR, IMFA, plaque formation, and electron microscopy. The tick contained an antigen and a genetic marker of the tick-borne encephalitis virus (TBEV). The patient had post-vaccination antibodies in a titer of 1:200, as a result of which, obviously, an antibody-dependent elimination of TBEV occurred. The tick-borne co-isolate also contained an unknown pathogen (Kiparis-144 virus), which, in our opinion, was a trigger for the activation of chronic infection in suckling white mice. In the laboratory co-isolate, ectromelia virus was present, as evidenced by paw edema during the intradermal infection of mice, characteristic rashes on the chorioallantoic envelope of chicken embryos, and typical plaques on Vero-E6. The Kiparis-144 virus was not pathogenic for white mice and chicken embryos, but it successfully multiplied in the PEK, Vero, and Vero-E6 lines. Viral co-infection was confirmed by electron microscopy. Passaging on mice contributed to an increase in the virulence of the co-isolate, whose titer increased by 10,000 times by the fifth passage, which poses an epidemiological danger.

Keywords: biological properties of co-isolate; coinfection; ectromelia virus; electron microscopy; south of the Russian Far East; tick Ixodes persulcatus; tick-borne encephalitis virus (TBEV); virus isolate.

Figure 1

Verification of antibodies to a new isolate in the blood serum of a patient who removed a tick from himself on the 5th day of bloodsucking. Indirect MFA method on the PEK cell line model. Note: (A) control of uninfected cells; (B) original serum; (C) serum diluted 1:2; (D) serum diluted 1:4; (E) serum diluted 1:8; (F) serum diluted 1:16. ×650.

Figure 2

Clinical manifestations in mice of 2 days of age on the 5th day after intracerebral infection with the isolate (2nd passage).

Figure 3

The death of white mice during intracerebral (i/cer), subcutaneous (s/cut), and intraperitoneal (i/per) infection with the viral isolate of the 5th passage (dilution 10⁻¹, which amounted to 3 lg PFU).

Figure 4

Plaque-forming ability of the bioisolate on cell lines PEK, Vero, and Vero-E6 (on the 5th day after infection).

Figure 5

Specific antigen fluorescence in PEK, Vero, and Vero-E6 cells infected with a viral isolate 3 and 24 h pi. MFA method: PEK cells; Vero cells; Vero-E6 cells. ×650.

Figure 6

Accumulation of the viral isolate in the supernatant in the period from 3 h to 9 days pi of the PEK cell line. The visualization of the antigen on cells using the method of fluorescent antibodies shows the average level of antigen-positive cells (%).

Figure 7

Verification of the viral isolate in the Vero-E6 cell culture 5 days post-infection (pi). Titer of virus isolate before filtration (1st row) and after filtration (2nd row).

Figure 8

Chorion-allantoic envelope of chicken embryos 9–10 days old, infected with a suspension of the original viral isolate (A) and its filtrate (B) on days 1, 3 and 7–9 postinfetion (pi).

Figure 9

The virus co-isolate from the tick I. persulcatus that half-fed on a human. Two viruses of different sizes are visible: one large and smallpox-like, up to 500 nm (double arrow); the other is much smaller, from 60 to 100 nm (arrow). Electron microscopy with negative contrast.

Figure 10

Virus co-isolate from the tick I. persulcatus that half-fed on a human. (A) A significant predominance of large viral particles up to 500 nm in size; (B–D) electron-dense conglomerates, which consist of fused pox-like viral particles with a rim around the periphery, representing adherent particles of a small virus, up to 100 nm in size.