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. 2018 Mar 29:9:557.
doi: 10.3389/fmicb.2018.00557. eCollection 2018.

Identification of Unequally Represented Founder Viruses Among Tissues in Very Early SIV Rectal Transmission

Jian Chen  1 , Yanqin Ren  1   2 , Lance Daharsh  3 , Lu Liu  1 , Guobin Kang  3 , Qingsheng Li  3 , Qiang Wei  4 , Yanmin Wan  1   3 , Jianqing Xu  1
Affiliations

Identification of Unequally Represented Founder Viruses Among Tissues in Very Early SIV Rectal Transmission

Jian Chen et al. Front Microbiol. .

Abstract

Characterizing the transmitted/founder (T/F) viruses of multi-variant SIV infection may shed new light on the understanding of mucosal transmission. We intrarectally inoculated six Chinese rhesus macaques with a single high dose of SIVmac251 (3.1 × 104 TCID50) and obtained 985 full-length env sequences from multiple tissues at 6 and 10 days post-infection by single genome amplification (SGA). All 6 monkeys were infected with a range of 2 to 8 T/F viruses and the dominant variants from the inoculum were still dominant in different tissues from each monkey. Interestingly, our data showed that a cluster of rare T/F viruses was unequally represented in different tissues. This cluster of rare T/F viruses phylogenetically related to the non-dominant SIV variants in the inoculum and was not detected in any rectum tissues, but could be identified in the descending colon, jejunum, spleen, or plasma. In 2 out of 6 macaques, identical SIVmac251 variants belonging to this cluster were detected simultaneously in descending colon/jejunum and the inoculum. We also demonstrated that the average CG dinucleotide frequency of these rare T/F viruses found in tissues, as well as non-dominant variants in the inoculum, was significantly higher than the dominant T/F viruses in tissues and the inoculum. Collectively, these findings suggest that descending colon/jejunum might be more susceptible than rectum to SIV in the very early phase of infection. And host CG suppression, which was previously shown to inhibit HIV replication in vitro, may also contribute to the bottleneck selection during in vivo transmission.

Keywords: CG dinucleotide; Chinese rhesus macaque; SIV; rectal transmission; transmitted/founder virus; very early virological events; zinc finger antiviral protein.

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Figures

Figure 1
Figure 1
Experimental design and the genetic make-up of SIVmac251 inoculum. (A) Three groups of Chinese rhesus macaques (3 monkeys/group) were enrolled in this study. Two groups were intrarectally infected with a single high dose of SIVmac251 (3.1 × 104 TCID50/monkey) and euthanized at 6 or 10 dpi. (B,C) Tissue viral RNA loads were measured by quantitative PCR (B) and virus replication in rectal mucosa was detected by in situ hybridization (C). (D) The inoculum stocks of SIVmac251 were evaluated for env diversity by NJ phylogeny and Highlighter plots. Reading frame intact env sequences were shown in parallel in NJ phylogeny and Highlighter plots. Nucleotide polymorphisms in the highlighter plot are indicated by a colored mark. Thymine is represented in red, guanine in orange, adenine in green, cytosine in blue, pink filled circles denote APOBEC signatures, open diamonds represent G-to-A conversions, and gaps are shown in gray in the highlighter plots. Bar length indicates 0.001 nucleotide substitutions per site. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 2
Figure 2
Divergence and diversity analyses of transmitted/founder viruses isolated from different tissue compartments (A). The numbers of base substitutions per site from averaged over all sequence pairs between the inoculum and each tissue compartment (divergence from inoculum, calculated using MEGA7). The number of transmitted variants varied among different tissue compartments at both 6 dpi (B) and 10 dpi (C). Although no significant difference was observed, the number of T/F lineages in rectum tended to be lower than other tissue compartments. Moreover, the numbers of virus lineages in tissues were all less than the inoculum, which contained at least 17 lineages.
Figure 3
Figure 3
N-J tree and Highlighter plots of SGA-derived env nucleotide sequences from 6 dpi macaque Rh070419. Sequences derived from different tissue compartments are shown in N-J phylogeny and Highlighter plots. In the phylogeny plot, the inoculum viruses are depicted in closed gray circles, rectum viruses in closed yellow circles, jejunum viruses in green circles, descending colon viruses in blue squares, spleen viruses in purple downward triangles, and peripheral blood viruses in red upward triangles. The red branches highlight a cluster of variants that was absent from rectum. The right bracket indicates the dominant variants. Identical sequences in the dominant cluster are represented by only one sequence for each tissue compartment. Actual number of sequences is shown in the bracket following the seq ID. Bar length represents 0.001 nucleotide substitutions per site.
Figure 4
Figure 4
N-J tree and Highlighter plots of SGA-derived env nucleotide sequences from 10 dpi macaque Rh060319. Sequences derived from different tissue compartments are shown in N-J phylogeny and Highlighter plots. In the phylogeny plot, the inoculum viruses are depicted in closed gray circles, rectum viruses in closed yellow circles, jejunum viruses in green circles, descending colon viruses in blue squares, spleen viruses in purple downward triangles, and peripheral blood viruses in red upward triangles. The red branches highlight a cluster of variants that was absent from rectum. The right bracket indicates the dominant variants. Identical sequences in the dominant cluster are represented by only one sequence for each tissue compartment. Actual number of sequences is shown in the bracket following the seq ID. Bar length represents 0.001 nucleotide substitutions per site.
Figure 5
Figure 5
The unequally represented T/F variants were animal-specific and their average CG dinucleotide frequency was high. (A) The unequally represented T/F variants identified in Figures 3, 4, and Figures S7, S8 were pooled together with inoculum virus sequences for N-J phylogeny and Highlighter analyses. Identical variants identified at both the descending colon/jejunum and the inoculum are highlighted with red branches. (B) Mean CG dinucleotide frequencies of the T/F and inoculum viruses were also calculated and compared. The data showed this cluster of unequally represented T/F variants had significantly higher average CG frequency.
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