Viral Shedding Into Tears of HSV-1 Infected Transgenic C57BL/6 Mice With Human ApoE e4/e4 Knock-in Gene is Independent of High Phenotypic Reactivation Status

Abstract

Purpose: : We evaluated corneal lesions by slit-lamp examination (SLE) and shedding of infectious virus and viral DNA copy numbers by CV-1 plaque assay and quantitative real-time PCR, respectively, in tears of HSV-1 infected transgenic C57BL/6 mice in the comparison of two strains: 17Syn⁺ (high phenotypic reactivator) and its LAT promoter deletion recombinant 17ΔPst (LAT^-) (low phenotypic reactivator) against the human homozygous ApoE e4/e4 genetic background.

Methods: : Female C57BL/6 mice were given 0.5 ml human serum by intraperitoneal (IP) injection to eliminate mortality. One hour post IP, corneas were heavily scarified and inoculation done with 10⁴ plaque forming units (PFU) per eye: 20 eyes [10 ApoE(4/4)] with 17Syn⁺, 20 eyes [10 ApoE(4/4)] with 17ΔPst(LAT^-), 10 eyes (5 parent non-transgenic) with 17Syn⁺ and 10 eyes (5 parent non-transgenic) with 17ΔPst(LAT^-). Prior to SLE, tears were swabbed 1 day post infection (PI) for 7 consecutive days to assess acute infection and up to 30 days PI for latent infection. Virus was eluted from the swab specimens and PFUs determined by standard procedures. DNA copy numbers of the HSV-1 polymerase gene were quantified. Eyes were scored daily for corneal lesions before swabbing.

Results: : Corneal lesions for all mice irrespective of viral strain peaked at 2 days and tapered off after 7 days PI. Some ApoE(4/4) mice shed infectious virus [17Syn+ or 17ΔPst(LAT^-)] up to 14 days PI. No infectious virus was detected in the parent C57BL/6 mice. Daily HSV-1 DNA recovery frequencies per mouse within the 14 days PI were: ApoE(4/4) at 2.25x10² [17Syn⁺ or 17ΔPst(LAT^-)], parent non-transgenic at 2.0x10 (17Syn⁺) and 0.2x10 [17ΔPst(LAT^-)].

Conclusions: : Our current data suggest HSV-1 shedding by transgenic mice is mainly dependent on the human ApoE e4/e4 genetic background and that the phenotypic nature of the virus plays no major role. Alternatively, the presence of the ApoE e4/e4 gene renders the latency silencing to be less effective, allowing higher frequency of viral reactivation.

Keywords: herpes simplex virus • transgenics/knock-outs • protective mechanisms