Long-term infection and vertical transmission of a gammaretrovirus in a foreign host species

Abstract

Increasing evidence has indicated natural transspecies transmission of gammaretroviruses; however, viral-host interactions after initial xeno-exposure remain poorly understood. Potential association of xenotropic murine leukemia virus-related virus (XMRV) in patients with prostate cancer and chronic fatigue syndrome has attracted broad interests in this topic. Although recent studies have indicated that XMRV is unlikely a human pathogen, further understanding of XMRV xenoinfection would allow in vivo modeling of the initial steps of gammaretroviral interspecies transmission, evolution and dissemination in a new host population. In this study, we monitored the long-term consequences of XMRV infection and its possible vertical transmission in a permissive foreign host, wild-derived Mus pahari mice. One year post-infection, XMRV-infected mice showed no notable pathological changes, while proviral DNA was detected in three out of eight mice. XMRV-infected mice remained seropositive throughout the study although the levels of gp70 Env- and p30 capsid-specific antibodies gradually decreased. When vertical XMRV transmission was assessed, no viremia, humoral immune responses nor endogenization were observed in nine offspring from infected mothers, yet one offspring was found PCR-positive for XMRV-specific sequences. Amplified viral sequences from the offspring showed several mutations, including one amino acid deletion in the receptor binding domain of Env SU. Our results therefore demonstrate long-term asymptomatic infection, low incidence of vertical transmission and limited evolution of XMRV upon transspecies infection of a permissive new host, Mus pahari.

Figure 1. Sustained anti-XMRV humoral immunity in mice chronically infected with XMRV.

(A) Neutralization assay was carried out using GFP-carrying XMRV as described previously , . Flow cytometry analysis of virus neutralization activities of plasma samples from XMRV-infected mice (P1F and P2F) is compared to those from a control mouse (6M). (B) Results from neutralization assay are summarized in percentage which was calculated as the reciprocal of infectivity with a maximum infectivity being determined by incubation of the virus with an uninfected mouse serum. Data from Parent 1 female (P1F), P1 male (P1M), P2F, P3F, and P3M are shown. (C) XMRV proteins were detected by Western blotting with plasma samples of XMRV-infected mice. Results from five (P1F, P1M, P2F, P3F, and P3M) mice and a control (p5F) are shown. Viral proteins, envelope, Env (gp70), capsid, CA (p30), envelope transmembrane, TM (p15E), and matrix, MA (p15) are indicated. XMRV antigen negative and positive are indicated as − and +, respectively. Blood samples were collected at 8 wk, 12 wk, 16 wk, 24 wk and 1 year p.i.

Figure 2. Detection of anti-XMRV antibodies in the offspring from XMRV-infected mice.

(A) Flow cytometry data from 293T cells is indicated as XMRV negative (−) control. XMRV-GFP virus was pre-treated with plasma samples from infected mice (XMRV positive (+) anti-XMRV), non-infected mice (XMRV positive (+) control), and offspring (B1, B7, and B9) at a dilution of 1∶20, and then used to infect 293T cells. Numbers shown in parentheses are the percentages of neutralization, which was calculated as the reciprocal of infectivity with a maximum infectivity being determined by incubation of the virus with an uninfected mouse serum. NA, not applicable. (B) XMRV proteins were detected by Western blotting. Data from mice infected with XMRV after 8 and 24 weeks p.i. as positive controls and three offspring (B1, B7, and B9) are shown. Sizes of expected viral proteins are indicated to the left side of the Western blotting: envelope glycoprotein, Env (gp70), capsid, CA (p30), transmembrane, TM (p15E), and matrix, MA (p15).

Figure 3. Sequence analysis of env gene from XMRV proviral DNA-positive offspring samples.

(A) Nested-PCR, cloning, and sequence analysis were performed as described previously . The number of mutations in the env gene from lymph node and heart of the mouse B1 are summarized. A total of 2751 bp and 4366 bp were sequenced from lymph node and heart, respectively. (B) Schematic representation of XMRV Env with partial amino acid sequence of receptor binding domain (RBD) is shown. One Env isolate from heart (B1: GenBank accession no. JN987227) has one amino acid deletion of serine at position 148 in SU as boxed in blue. Amino acids from 1 to 33, from 34 to 444, and from 445 to 645 indicate signal peptide (SP), surface protein (SU), and transmembrane protein (TM), respectively. Variable regions (VRA, VRB, and VRC) are highlighted in gray. (C) Protein expression of wild-type XMRV Env and XMRV Env with S148 mutation was analyzed by Western blotting. Env protein was visualized with goat anti-MLV antibody (1∶2000, ATCC). Actin (1∶2000, sigma) was also detected as a loading control. Statistical analysis showed that protein expression was not statistically significant (p = 0.31). (D) Retroviral vectors pseudotyped with XMRV Env or XMRV Env with S148 mutation were produced by transient transfection in 293T cells. The transduction titer of retrovirus pseudotyped with XMRV Env or XMRV Env with a serine mutation is statistically significant (p = 0.009). Error bars indicate 95% confidence intervals.