Simian Retrovirus Transmission in Rhesus Macaques

Abstract

Historically, to generate Simian Retrovirus (SRV) positive control materials, we performed in vivo passage by inoculating uninfected rhesus macaques with whole blood from an SRV-1 infected (antibody and PCR positive) macaque. However, recent attempts using this approach have failed. This study reports observations and explores why it has become more difficult to transmit SRV via in vivo passage.

Introduction

Simian betaretroviruses (SRV) naturally infect macaques. SRV belongs to the family Retroviridae which are single-stranded enveloped RNA viruses [1]. There are at least seven distinct serotypes that are characterized by gag, pro, pol, and env genes [2]. Retroviruses utilize reverse transcriptase during replication to integrate a DNA provirus into the host cell genome [1]. The proviral integration allows simian retroviruses to establish a lifetime persistent infections in their hosts [3]. SRV transmission can occur by direct contact between infected monkeys and uninfected monkeys through natural exchange of body fluids, in particular blood, saliva, urine, by biting or scratching [4]. Infections can also indirectly occur through contact with contaminated instruments and equipment [5]. Inoculation or transfusion of these SRV infected fluids to uninfected monkeys has transmitted the infection and potentially disease [2]. SRV infections are mainly a problem in captive settings. Infected monkeys develop a disease with many of the characteristics of acquired immune deficiency syndrome [3]. Historically, to generate SRV-1 positive control material from rhesus macaques, we performed in vivo passage by inoculating uninfected macaques with whole blood from SRV-1 infected (antibody and PCR positive) macaques. However, recent attempts using this approach have failed. This case study investigates why it has become seemingly more difficult to transmit SRV.

Materials and Methods

Animals used in this study were all Indian origin rhesus macaques (Macaca mulatta), housed at the California National Primate Research Center, and maintained in accordance with the Animal Welfare Act, Regulations, and the Guide for the Care and Use of Laboratory Animals [6]. Animals used and all procedures involved in this study were approved by our institution’s IACUC and Biological Use Authorization (BUA) [6]. In 2016, acid dextrose (ACD) citrate anticoagulated blood from Donor 1 (antibody and PCR positive on day of transfusion) was successfully transfused into Recipient 1 and resulted in SRV-1 infection after 1 week as confirmed by both antibody and PCR tests. In 2021, 30 ml of ACD blood was collected from SRV-1 infected monkey, Recipient 1 (antibody and PCR positive confirmed at transfusion) and transfused into a naïve macaque, Recipient 2. Animal care and veterinary staff monitored the health condition of animals during and immediately following transfusion and then daily. Blood samples were collected for SRV antibody and SRV PCR testing at days 0, 4, 7, 11, 14, 21, and 28. Plasma and DNA were isolated from heparinized blood to assay for the presence of SRV antibodies using multiplex microbead immunoassay coupled with viral lysate and recombinant transmembrane antigens from Charles River Labs, and viral DNA using real-time PCR (qPCR) for the SRV env gene [7, 9], respectively. After 4 weeks without detection of SRV-1 infection, 5 ml of tissue culture derived SRV-1 was intravenously inoculated into Recipient 2. Blood samples were collected on days 0, 4, 14, 18, 21, 28, 53, 83, and 88 for antibody and PCR testing. A 402 bp PCR amplified fragment from the env gene including the neutralization epitope region; and a 383 bp from the gag gene were generated using the Quantstudio 12K Flex (Thermo Fisher Scientific). The PCR fragment was purified using the Qiagen Qiaquick PCR Purification Kit, then sequenced by the ABI 3730 Genetic Analyzer. Sequencing results were evaluated and analyzed by using the Chromas software. Emboss Needle software from EMBL-EBI was used for both DNA and protein sequence alignments.

Results

In these transmission studies, resource limitations did not allow for viral load quantification in the transfused blood; thus we are providing qualitative Ct values of the qPCR; for comparison, known SRV DNA-positive samples have generally Ct values in the 30’s. Lower Ct values indicate thus higher amounts of SRV DNA in the tested sample. As shown in figure 1A, Recipient 1 transfused with SRV antibody/PCR positive blood with a Ct=25 from Donor 1 resulted in SRV infection as confirmed by SRV antibody/PCR positive results for all sample dates. In contrast, following transfusion with blood of Recipient 1 which had tested SRV positive by PCR with a Ct=36 and antibody assay at the time of transfusion, Recipient 2 blood samples were negative at all time points for four weeks. Following the unsuccessful transfusion attempt from Recipient 1, Recipient 2 was next inoculated with tissue culture derived SRV-1 supernatant and SRV infection was detected by PCR and antibody tests on Day 14. The 400 bp PCR fragment of the SRV-1 neutralizing region of the env gene found in Donor 1 and Recipient 1 were identical but different from GenBank (Accession number M11841) sequence. That same different sequence and the GenBank sequence were both identified in Recipient 2 and the tissue culture virus. Figure 1B shows these nonsynonymous mutations resulted with D265N and C269Y mutants. Subsequent sequencing in other regions of the env and gag genes from PCR positive DNA of Recipient 1 ranging from (2017–2021) produced ambiguous results.

Fig 1. Transfusion and inoculation of SRV-1.

Rhesus macaques were infected with citrate anticoagulated blood or SRV-1 derived tissue culture, respectively. (A) ACD blood from Donor 1 positive for SRV-1 transfused into Recipient 1 and was infected with SRV-1 after 1 week of transfusion in 2016. Recipient 1 ACD blood positive for SRV-1 transfused into Recipient 2 and was SRV-1 negative after 4 weeks of transfusion in 2021. Recipient 2 inoculated with SRV-1 positive culture and infected with SRV-1 after 2 weeks of inoculation in 2021. (B) Nonsynonymous mutations in the neutralizing region of the env of SRV-1 resulted with D265N and C269Y mutants.

Discussion

Viral transmission was unsuccessful when ACD blood of a SRV-1 antibody and PCR positive animal was transfused into an uninfected animal. This observation contrasts with the historical description of ready transmission of this retrovirus [8]. Recent published transmission failures suggest the possibilities that antibody positive and PCR negative profiles do not specify an SRV infection but could be a serological assay artifact or host immune reactivity to an endogenous virus [6]. Endogenous virus sequences complicate the design of an assay that detects all SRV serotypes but not endogenous or other viruses [9]. Our case study presented sequencing results from Donor 1 (2016), Recipient 1 (2016), and Recipient 2, and the SRV-1 positive culture all showing nonsynonymous mutations which produced D265N and C269Y mutants in the epitope neutralizing region of the env gene when compared to the Genbank sequence. We found a mixed population of mutants in Recipient 2 and SRV-1 positive culture when compared to Donor 1, Recipient 1, and Genbank. The ambiguous sequencing results from the PCR fragments generated from other regions of the env and gag genes of Recipient 1 (2017–2021) further confounded this investigation. Here we report sequence differences from the prototype Genbank sequence observed during in vivo and in vitro serial passage experiments. Further studies are needed to elucidate the effects of this genetic diversity on viral fitness, including transmissibility.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.