Diversity of Primate Lentiviruses Rebooted

Highlight: The present report reveals for the first time natural lentiviral infection of wild Indian NHPs, rhesus monkeys (Macaca mulatta) and langurs (Semnopithecus entellus) by SIVs that are phylogenetically diverse from all known SIVs, including “SIVmac”, which infects captive rhesus monkeys. The novel SIVs are intriguingly homologous to HIV-1, based on serology and partial lentiviral genomic sequence analyses. Diverse lentiviruses infect human and nonhuman primates (NHPs). There are more than 45 different “species-specific” simian immunodeficiency viruses (SIVs) that infect their cognate NHP hosts in natural habitats in Africa. Indian NHPs are not known to be infected by SIVs in the wild. Conventionally SIVs are named after their natural hosts, except for SIVmac, which infects captive rather than wild rhesus macaques. SIVmac is therefore a misnomer. It is a genetic variant of the African SIVsmm, which infects wild African sooty mangabey monkeys. SIVsmm is the progenitor of human immunodeficiency virus (HIV-2), while SIVcpz that infects wild chimpanzees is the progenitor of HIV-1. Although natural infections cannot be easily studied in wild NHP populations, we have previously reported co-infection of wild Indian NHPs by other retroviruses: simian retroviruses (SRVs) and Simian Foamy viruses (SFV). Apart from zoonosis, transmission of pathogens from humans to animals: anthroponosis, has also been reported in literature.


Introduction
Diverse lentiviruses infect human and nonhuman primates (NHPs). Zoonotic pathogens represent 60% of all known human pathogens [1]. Crossspecies transmission has led to many devastating epidemics which remains a poorly understood phenomenon. Presence of zoonotic pathogens in humans relies on human-animal interface and their environments. The interhuman barrier determines the ability of zoonotic pathogens to efficiently transmit among humans, causing outbreaks, epidemics, or pandemics [2]. Once cross-species transmission of pathogens has occurred, interhuman transmission maintains the infection in the population. For successful cross species transmission, only "spillover" into a single individual of a new host species is not sufficient. For a zoonotic virus to become endemic, sustained chain of transmission in the new host population ("host switching") is required [3].
While the history of HIV-1 as a result of zoonosis of SIVcpz, and HIV-2 due to zoonotic transmission of SIVsmm is documented, there are over 45 different simian lentiviruses that infect different species of African NHPs, but human equivalent of these SIVs are not reported. Bushmeat hunting and consumption constitutes the primary risk factor for zoonotic disease transmission of simian retroviruses including SIVs in Africa.
According to the current understanding, lentiviruses do not infect Indian NHPs in the wild [4]. The present report however reveals natural lentiviral infection of wild Indian NHPs, rhesus monkeys (Macaca mulatta) and langurs (Semnopithecus entellus) based on serology and partial lentiviral genomic sequences. The SIVs are phylogenetically diverse from all known African SIVs, including SIVmac that infects captive rhesus monkeys and are intriguingly homologous to HIV-1. Exogenous lentiviruses appear to have far more extensive history of host switching than previously appreciated. Rhesus monkeys belonging to subfamily cercopithecae and langurs, to colobinae subfamily of Old World monkeys (OWM) are widely distributed in India [8]. Occasionally, the simians aggressively attack, bite and scratch unsuspecting humans to grab food items. Such interactions can lead to zoonosis as well as anthroponosis, when infected blood or saliva from the human and NHP hosts are inadvertently mixed. This can lead to the emergence of unreported infectious diseases [9]. As the human-animal connection escalates, so does the threat for pathogen spread. transcription has been shown to require all domains of TRIM5α, each providing a unique tool for host immunity [10].TRIM5α of wild rhesus monkeys was found to be identical to equivalent sequence of captive rhesus monkeys ( Figure   1(a) and Figure 1

Materials and Methods
Even though viral infections cannot be easily studied in wild NHP population, field studies of SIV-infected hosts are necessary to unravel the phenomenon of natural infection.  Serology: Serological screening of plasma samples with commercial HIV-1/2 Rapid test and WB assay was performed to detect cross reacting antibody to HIVs as a test for lentiviral infection. Commercial SIV serology tests were not available locally so HIV-1/2 serology tests were used to analyze the simian plasma samples. Such diagnostic approach has been used for screening of plasma samples of African NHPs [13]. Immunoblot test was also performed with specific monoclonal antibody against HIV-1 p24 antigen and SIVmac p27 antigen for detection of HIV/SIV specific antigens.
Extraction of DNA and RNA: DNA was extracted from peripheral blood mononuclear cells (PBMC) using Tri reagent (MRC Inc. Cincinnati). Plasma samples were used for extraction of viral RNA using QIA amp viral RNA kit (Qiagen, California) for subsequent DNA PCR and RT-PCR assays. PBMC was also used for virus isolation by co-culture with primary human PBMC from healthy individuals as well as human T-cell lines. Purified PBMC and plasma samples were labeled and stored in screw capped tubes in deep freezes (−80˚) until shipment to the USA on dry ice by designated shipping Company with necessary permit from the Center for Disease Control and Prevention (CDC), Atlanta (USA) and US Department of Fisheries and Wildlife (USFWS). Export permit was obtained by the Veterinary authorities of Rajasthan.
Laboratory experiments: After the collection of samples at the field sites, downstream laboratory experiments and sequencing of partial lentiviral genes were performed at the HIV Program of the National Cancer Institute, Frederick, MD. TRIM5α related investigation was performed at the Dept of Microbiology & Immunology of the Albert Einstein College of Medicine, Bronx, NY.
Amplification of viral genomic by Polymerase Chain Reaction (PCR): To molecularly characterize the lentiviruses infecting wild Indian langurs and rhesus monkeys, partial lentiviral genes were amplified by PCR, DNA PCR to detect integrated proviruses and RT-PCR for detection of replicating lentiviruses.
All primate lentiviruses share three structural genes: group specific antigen, gag, viral polymerase, pol and envelope gene, env, which encode the basic struc- While the accessory proteins are not required for basic replication of the virus in most cell lines, their importance in vivo has been proven by both experimental and naturally occurring accessory gene deletion mutants [14].
In the first instance, degenerate primers from the conserved RT region of the lentiviral pol gene were employed that could amplify both SIV and HIV genomes. Other lentiviral genomic regions that were also amplified using relevant primer pairs designed from sequence information from the Los Alamos National Laboratory (LANL) HIV database (US), included the structural genes gag and env as well as the lentiviral accessory genes vif, vpr, nef and LTR region. The  (Table 1).
Partial lentiviral gene sequences were next used for phylogenetic analyses using standard genetic software programs. Prior to the phylogenetic analyses, nucleotide substitution saturation was tested in Dambe by plotting the observed transitions and transversions versus the evolutionary distance. Due to saturation

Result
The SPRY domain of TRIM5α of wild and captive rhesus monkeys were found to be identical and different from the host factor from other species of NHPs ( Figure 1(a) and Figure 1(b)).
Screening of simian plasma samples by serology revealed the presence of antibody that cross reacted with HIV-1 antigen. A total of 15 rhesus macaques out of 35 samples analyzed (45%), and 6 of 9 (66.6%) langur samples were found to be seropositive by HIV-1WB assay. In the collection of 2016, the lone sample from rhesus monkey was negative by serological and PCR assay (Figure 1(c)).
Two of 3 langur samples were sero-reactive, while 1 langur sample gave a distinct product amplified with primers from vif and 3'LTR region that was smaller than the control HIV-1 product, probably representing a quasispecies ( Figure   1(c)). Mitochondrial DNA analyses confirmed the simian host species of origin, rhesus monkey and langur (Figure 1(d)). Extreme precaution was taken at every step of laboratory experimental process to avoid laboratory contamination during PCR experiments. The reference strain HXB2of subtype B HIV-1was not used in any of the experiments.
When tested by specific immunoblot assay, 15/35 rhesus monkeys and 6 of 9 langur samples revealed the presence of antigens that cross reacted with HIV-1 Gag monoclonal antibody, p24, but similar assay with SIVmac p27 monoclonal antibody were seronegative (data not shown).
Molecular characterization of the novel SIVs: We first performed phylogenetic analysis of the conserved gag genes (Figure 2(a)). In the predicted amino acid Gag sequences, the novel SIVs revealed unexpected homology to HIV-1subtype B sequences from north India with >80% bootstrap support (subtype B HIV-1, Genbank Accession no: EF694037).
Partial Gag protein of the SIVs infecting wild rhesus macaques clustered together with >74% bootstrap support. One sequences from Langur clustered with subtype B HIV-1sequences from China (Figure 2(a)). In the phylogenetic tree  African SIV sequences including SIVmac, SIVagm, SIVsmm, clustered as separate branch, highlighted in green.
Since langurs belong to the colobinae subfamily of primates, we aligned the conserved Gag region of SIVwrc, that infects wild red colobine from Africa with equivalent sequences of SIV infecting langur L4 (Figure 2(b)). Only 52% identity in the translated amino acid sequences of Gag protein was observed compared to nearly 84% identity with Gag p24 sequences of subtype B HIV-1 (Figure 2 Alu-LTR PCR: Integration of lentivirus in the monkey chromosome was confirmed by amplification of the integration site by Alu-LTR repeat PCR, with genomic DNA extracted from PBMC of langurs and rhesus monkey (data not shown).

Discussion
The majority of human emerging infectious diseases are zoonotic, with viruses that originate in wild mammals being of particular concern, like HIV and Ebola viruses. Understanding patterns of viral diversity and transmission in wildlife species are key goals for pandemic surveillance programs, and of vital public health importance, which is the basis for the present investigation. The study in- The putative viral coevolution during primate speciation suggests that many of these zoonotic transmissions were ancient that have been acted on by natural selection for millions of years. The nature of the host-virus relationship is highly variable for primate lentiviruses, with some viruses switching hosts often while others maintain strict host specificity.
Infection of feral Indian langurs and rhesus monkeys by novel exogenous SIVs has not been reported before. The SIVs are not related to any African SIV but were unexpectedly homologous to subtype B HIV-1, based on experimental evidence presented here confirm that the data are clearly not speculative or biologically impossible.
The evidence for a novel animal reservoir of HIV-1 in wild Indian simian population has implications for Public Health and conservation of primate species.
In future, whole genome sequence of the novel SIV genomes would be determined by using the Next Generation Sequencing (NGS) approach for a more robust identification of these unique lentiviruses from the forests of Rajasthan.
The on-going viral dynamics, host restriction factors and virus-host interaction in wild simian population infected by multiple retroviruses are important areas of investigation.

Limitations and Strengths
The limitations of the study are the small sample size, due to reasons inherent with field conditions. The hitherto unreported findings open up a new chapter in primate lentivirus epidemiology. Additional sampling and whole genome sequencing data of the novel SIV genomes by Next Generation Sequencing (NGS) will cer-Journal of Biosciences and Medicines tainly establish the phylogenetic identity further and will be perused in future.

Conclusion
In summary, we propose a counter intuitive reverse transmission of HIV-1 from infected humans to simians through aggressive monkey bite, given the close man-monkey interaction existing in India. From the present report, it cannot be stated if the reverse transmission events represent more than one transmission from man-to-monkey or if this was a single transmission event which led to subsequent transmission between langurs and macaques living in close proximity in the natural habitat. Only further detailed epidemiological and virological studies will address these questions.