In December 2019, a new disease caused by a virus belonging to the Coronaviridae family was first detected in the city of Wuhan, China, and was named coronavirus disease (COVID-19) (Wu et al. 2020). The etiologic agent—Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)—is highly transmissible among humans through droplets of saliva and quickly spread across the planet. In March 2020, the virus reached every continent which led the World Health Organization to declare COVID-19 as a pandemic (World Health Organization).

Initial studies of epidemiology and genetic comparison showed that the new virus has a high similarity to other coronaviruses found in bats and pangolin (96 and 91% similarity, respectively) (Zhang et al. 2020) and that the “spillover,” that is, the “jump” between hosts, might have occurred in the vicinity of the popular market in Wuhan, where these animals are traditionally traded for human consumption (Wu et al. 2020). Thenceforth, it has been questioned whether SARS-CoV-2 would be able to perform a “spillback”—transmission from humans to wild animals—which could generate wild cycles of the virus, making it even more difficult to face.

In this regard, concern about the infection of non-human primates (NHPs) has grown since the biological, genetic and biochemical similarities between humans and these species could facilitate the transmission of SARS-CoV-2 to NHPs. Comparative studies have indicated that the cellular receptor angiotensin-converting enzyme 2 (ACE2), used by this virus for adsorption to cells, is similar between humans and NHPs, especially in Old World species; as such, these have been used as experimental models for the study of SARS-CoV-2 (Melin et al. 2020). In the Americas, there is a great concern about the risks of SARS-CoV-2 spillback from humans to wildlife, including neotropical NHPs. This could have at least two impacts: a) establishment of a sylvatic cycle with serious implications on control or eradication efforts, and: b) unforeseeable impact on biodiversity, especially if endangered NHP species become involved. The historical comparison with the yellow fever virus (YFV), whose sylvatic cycle was first recognized almost a century ago after a spillback from humans to NHPs, exemplifies the potential damage that may be associated with such impacts (Possas et al. 2018).

To our knowledge, the only neotropical primate species experimentally infected with SARS-CoV-2 was the marmoset Callithrix jacchus. Infected individuals had pyrexia and viral RNA loads detectable for several days post-infection in oral, nasal and anal swabs, as well as in blood and stool samples (Lu et al. 2020). However, little is known about the possibility of viral infection in natural conditions and in other neotropical primates’ families or species. Worryingly, in many Brazilian cities where cases of COVID-19 are currently reaching unprecedented levels, Callithrix spp. are adapted to synanthropic environments, often kept as pets and feeding on human foods. Such coexistence might represent a significant potential source of infection (Longa et al. 2011). In addition, the rapid dissemination of SARS-CoV-2 currently verified throughout Brazil can lead to the exposure of other NHPs, including wild species.

The present study was carried out in search of virological evidence of SARS-CoV-2 infections in NHPs from urban, sylvatic and rural areas of two Brazilian states, Minas Gerais (MG) and Rio Grande do Sul (RS). The current study is part of a wider NHP sampling effort whose main goal is to contribute to the national program for epidemiological surveillance of yellow fever.

Neotropical Primates’ samplings were performed before (November 2019) and during (July 2020–February 2021) the COVID-19 pandemic in two Brazilian states. Initially in MG (Brazil’s Southeast region), where the savannah-like biome predominates, captures occurred in four municipalities (Fig. 1a). Later on in RS (Brazil’s South region), which is covered by Atlantic Forest and Pampa biomes, captures were conducted in seven municipalities (Fig. 1b) (Andrade et al. 2021). Human COVID-19 cases per 100.000 inhabitants up to the sampling efforts are highlighted by the colors of the municipalities on the map (SES-MG 2020; SES-RS 2021).

Figure 1
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Municipalities where sampling of NHPs was performed. a Minas Gerais state; b Rio Grande do Sul state. The number of COVID-19 human cases in each municipality was counted until the sample collection date. Data were obtained from state health departments (SES-MG 2020; SES-RS 2021). The figure was done using QGIS software, version 3.10.

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Live marmosets were captured with Tomahawk traps, according to the protocol previously described (Abreu et al. 2019a). Dead or sick marmosets and howler monkeys were also examined. In MG, they were found based on an information network made up of health professionals, farmers and other people in contact with natural environments, as described elsewhere (Abreu et al. 2019b) while in RS, staff from the State Health Department found and reported the epizootics. Serum and/or oral swab (of live animals), as well as whole blood and/or viscera (lung, liver or kidney of dead animals), were collected, preserved in RNAlater (Thermo Fisher™) and stored in liquid nitrogen at − 196°C. Our methods and protocols followed the collection routine of yellow fever surveillance program and were previously approved by the Institutional Ethics Committee for Animal Experimentation (Protocol CEUA/IFNMG nº14/2019) and by the Brazilian Ministry of the Environment (SISBIO no. 71,714-2).

RNA extractions were performed using QIAamp Viral RNA Mini Kit (QIAGEN® (for serum) and TRIzol™ Reagent (Thermo Fisher™) (for viscera) following manufacturer’s instructions. Detection of the SARS-CoV-2 viral RNA in samples from MG was performed using a real-time RT-PCR kit (CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel, which targets the N1 (2019-nCoV_N1 Combined Primer/Probe Mix) and N2 (2019-nCoV_N2 Combined Primer/Probe Mix) genes of the SARS-CoV-2 (CDC Division of Viral Diseases 2020). In RS, viral RNA detection was performed using the Allplex™ 2019-nCoV (Seegene®), which targets genomic regions within the E, RdRP and N genes. Commercial positive control (2019-nCoV_N_Positive Control, catalog number: 10006622) containing the complete nucleocapsid gene sequence (IDT Technologies) as well as samples from known SARS-CoV-2-positive human patients were used as positive controls during the molecular assays.

In total, 51 NHPs belonging to two different families (Callitrichidae and Atelidae) and four species (Callithrix penicillata, C. jacchus, Alouatta caraya and A. guariba clamitans) were examined (Table 1). Seven NHPs were captured before the reported virus introduction (August–November/2019) and 44 after the introduction (July 2020–February 2021). Different tissues were analyzed, totalizing 89 samples: 30 serum, 20 oral swabs, 19 liver, 14 kidney, 3 lung and 3 whole blood (Table 1). No SARS-CoV-2 positive samples were detected, regardless of NHP species, tissue, habitat, municipalities or state/biome tested (Table 1).

Table 1 Description of Samples Tested by Species, Tissue, Habitat, Date, Municipality (City) and State.
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This was the first study evaluating the possibility of natural infection of free-living neotropical NHPs with SARS-CoV-2. We found no evidence of SARS-CoV-2 infection in NHPs from urban, rural or sylvatic habitats regardless of the tissues or species surveyed. Despite that, in view of the widespread dissemination of SARS-CoV-2, it is important to maintain and expand the surveillance of wildlife, aiming at the early detection of spillovers or spillbacks among human beings and other animals.

Since its first detection, reports of natural SARS-CoV-2 infection of several animals have accumulated, including domestic animals like cats (Sailleau et al. 2020; Carlos et al. 2021), dogs (Sit et al. 2020) and wild captive animals like tigers (McAloose et al. 2020) and minks (Hammer et al. 2021). Minks have also been described as a source of infection for humans (Hammer et al. 2021). In addition, experimental infection studies have reported susceptibility of several animals such as ferrets, bats, hamsters (Shi et al. 2020; Imai et al. 2020) and mainly Old World NHPs (Zheng et al. 2020; Lu et al. 2020). As a result, concern about the possibility of SARS-CoV-2 spillback for Neotropical Primates—as happened with YFV in the last century and probably with ZIKA in the last decade—has grown (Terzian et al. 2018; Yunes Guimarães et al. 2020). However, the only report of experimental infection in a neotropical NHP published to date examined the dynamics of SARS-CoV-2 infection in Callithrix jacchus, the common marmoset. Viral genomes were detected in oral, nasal and anal swabs as well as in serum and feces, despite the lower susceptibility of that species when compared to Old World Primates. (Lu et al. 2020). In the present study, all tissues sampled from the 44 free-living primates collected after the introduction of the virus in Brazil presented no evidence of SAR-CoV-2 infection, including samples from NHP living in rural and urban environments, in close contact with humans.

The cellular receptor ACE2 used by the virus for adsorption to cells is a major determinant in the susceptibility of animals to SARS-CoV-2 infection. The ACE2 receptor of cells from Old World NHPs have up to 99% identity with human ACE2 protein, which might contribute significantly to the high susceptibility that such species display to infection with this virus (Shi et al. 2020; Melin et al. 2020). Neotropical NHPs, such as those sampled in the present study, have ACE2 receptors that share about 92% identity to human ACE2, with four differences across the twelve binding sites regarded as essential for the ACE2 protein to allow binding of SARS-CoV-2 (Melin et al. 2020; Joyraj Bhattacharjee et al. 2021). This may be related to species’ differences in susceptibility and, possibly, with our negative results, even in NHPs living in urban areas with high SARS-CoV-2 human incidence. Even so, it is important to investigate whether the new variants of SARS-CoV-2 could increase their infectivity in wild animals.

Brazil is the country with the greatest biodiversity on the planet and concentrates the largest number of NHP species, many of these are endangered due to anthropogenic effects such as deforestation which, in addition to drastically reducing habitats, increases the risks of zoonotic transmissions (Paglia et al. 2012; Guthid et al. 2020). The explosive spread of SARS-CoV-2 in the country, particularly in areas where contact between humans and native fauna is frequent, provides conditions for spillovers and spillback which increases the need for surveillance for early identification and contingency of the potentially harmful effects (Gryseels et al. 2020). In this sense and despite the small sampling, our study contributes to shedding light on the subject by providing the first results about the absence of circulation of SARS-CoV-2 in Neotropical Primates. It is urgent to expand the sampling of NHP living in different environments, to include other primate species and even other mammals to carry out active surveillance of wild animals and understand the epidemiology of SARS-CoV-2 in natural environments.