Detection of herpesviruses in neotropical primates from São Paulo

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Brazilian Journal of Microbiology 
https://doi.org/10.1007/s42770-023-01105-z
VETERINARY MICROBIOLOGY - RESEARCH PAPER
Detection of herpesviruses in neotropical primates from São Paulo, 
Brazil
Isabella Naomi Furusato1 · Ketlyn Bolsachini Figueiredo1  · Ana Carolina Souza Ramos de Carvalho1  · 
Camila Santos da Silva Ferreira1  · Juliana Possatto Fernandes Takahashi1,2  · Lidia Midori Kimura1  · 
Camila Siqueira Aleixo1 · Odília Pereira de Brito1 · Adriana Luchs3  · Mariana Sequetin Cunha3  · 
Natália Coelho Couto de Azevedo Fernandes1  · Leonardo José Tadeu de Araújo1  · José Luiz Catão‑Dias4  · 
Juliana Mariotti Guerra1,4 
Received: 2 May 2023 / Accepted: 14 August 2023 
© The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia 2023
Abstract
Transmission of herpesvirus between humans and non-human primates represents a serious potential threat to human health 
and endangered species conservation. This study aimed to identify herpesvirus genomes in samples of neotropical primates 
(NTPs) in the state of São Paulo, Brazil. A total of 242 NTPs, including Callithrix sp., Alouatta sp., Sapajus sp., and Cal-
licebus sp., were evaluated by pan-herpesvirus polymerase chain reaction (PCR) and sequencing. Sixty-two (25.6%) samples 
containing genome segments representative of members of the family Herpesviridae, including 16.1% for Callitrichine 
gammaherpesvirus 3, 6.1% for Human alphaherpesvirus 1, 2.1% for Alouatta macconnelli cytomegalovirus, and 0.83% for 
Cebus albifrons lymphocryptovirus 1. No co-infections were detected. The detection of herpesvirus genomes was signifi-
cantly higher among adult animals (p = 0.033) and those kept under human care (p = 0.008671). These findings confirm the 
importance of monitoring the occurrence of herpesviruses in NTP populations in epizootic events.
Keywords Herpesviridae · Non-human primate · Marmosets · Howler monkey · Titi monkey · Capuchin monkey · HHV-1 · 
CalHV3 · CalbLCV1 · Cytomegalovirus · Gammaherpesvirus · Alphaherpesvirus · Betaherpesvirus
Introduction
Brazil has the most significant number of native primate 
species in the world, with over 150 species found within its 
borders [1]. According to Hirsh et al. (2006), the Brazilian 
Atlantic forest harbors a total of twenty-three primate spe-
cies, of which twenty species are endemic to this particu-
lar ecosystem [2]. Approximately 38% of these species are 
classified as threatened with extinction, and 48% are expe-
riencing population declines attributable to various threats 
including habitat loss, hunting, and infectious diseases [1]. 
In this scenario, the increased interaction between humans 
and wildlife animals has led to a higher chance of the emer-
gence and resurgence of a wide variety of infectious and 
zoonotic pathogens [3, 4].
Herpesviruses are a family of double-stranded DNA 
viruses that infect a wide range of animal species, including 
humans and non-human primates. These viruses are often 
asymptomatic or cause mild lesions in their natural host. 
However, interspecies transmission, through contaminated 
Responsible Editor: Fernando R. Spilki
 * Juliana Mariotti Guerra 
 jumariotti.vet@gmail.com
1 Centro de Patologia, Instituto Adolfo Lutz, Avenida Dr. 
Arnaldo, 351, Pacaembú, São Paulo, SP 01246000, Brazil
2 Programa de Pós-Graduação Em Doenças Infecciosas E 
Parasitárias - Faculdade de Medicina, Universidade Federal 
de Mato Grosso Do Sul, Bairro Universitário, Av. Costa E 
Silva, S/nº, Campo Grande, MS 79070900, Brazil
3 Centro de Virologia, Instituto Adolfo Lutz, Avenida 
Dr. Arnaldo, 351, Pacaembú, São Paulo, SP 01246000, 
Brazil
4 Laboratório de Patologia Comparada (LAPCOM), 
Departamento de Patologia, Faculdade de Veterinária E 
Zootecnia, Universidade de São Paulo, Avenida Professor 
Orlando Marques de Paiva, 70, São Paulo, SP 05508270, 
Brazil
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http://orcid.org/0000-0002-7275-574X
http://orcid.org/0000-0003-3126-7923
http://orcid.org/0000-0002-0386-5486
http://orcid.org/0000-0002-1285-1254
http://orcid.org/0000-0002-0958-3340
http://orcid.org/0000-0003-4131-990X
http://orcid.org/0000-0001-8903-604X
http://orcid.org/0000-0003-4754-4200
http://orcid.org/0000-0002-5427-4053
http://orcid.org/0000-0003-2999-3395
http://orcid.org/0000-0002-0326-7187
 Brazilian Journal of Microbiology
1 3
saliva and respiratory droplets by bites, scratches, or inges-
tion, is associated with severe disease and high mortality 
[4, 5]. Some herpesviruses detected in non-human primates 
(NHPs) can infect humans and vice versa [6], highlighting 
the importance of permanent virus surveillance for wildlife 
conservation and public health.
Focusing on the One Health perspective, this study aimed 
to characterize the circulation of herpesviruses in the neo-
tropical primates (NTPs) population from São Paulo state 
within the Brazilian NTPs Epizootic Events Surveillance 
Program of the Ministry of Health.
Material and methods
Animals
Fresh-frozen liver samples from 242 NTPs were submit-
ted to the Adolfo Lutz Institute as part of the National Sur-
veillance Program of Epizootics of the Brazilian Ministry 
of Health [7] between January 2017 and December 2022. 
The epidemiological data comprised essential information 
regarding the date and the location of occurrence of the epi-
zootic events. The biological data of the NTPs consisted of 
the identification of genus/species, age group, and sex. Addi-
tionally, the living conditions of the animals were consid-
ered, distinguishing between free-ranging animals and those 
under human care, which encompassed animals living in 
domestic environments or housed in rescue or rehabilitation 
facilities for more than 15 days. All datasets were retrieved 
from the submission forms (Information System on Diseases 
of Compulsory Declaration/Sistema Nacional de Agravos 
de Notificação—SINAN) provided by municipal health or 
wildlife and environmental service officials.
All of these procedures were approved by the Animal 
Use Committee and Technical-Research Committee of the 
Adolfo Lutz Institute (CEUA-IAL no. 03/2019 and CTC-
IAL no. 20–2019, respectively), Chico Mendes Institute for 
Biodiversity Conservation (protocol no. 68697), and the 
National System for the Management of Genetic Heritage 
and Associated Traditional Knowledge (no. A1A2A72).
Nucleic acid extraction
Nucleic acids were extracted using the BioGene® DNA/
RNA Viral kit (Quibasa – Química Básica Ltda, Belo Hor-
izonte, MG, Brazil). Approximately 25 mg of fresh-frozen 
liver tissue was disrupted with lysis buffer and MagNA 
Lyser® Green Beads (Roche, Basel, Switzerland) in a tis-
sue homogenizer (Loccus, Cotia, SP, Brazil) for 30 s and 
incubated at 56 °C overnight. After the addition of pro-
teinase K and carrier RNA, the spin-column protocol was 
followed under the manufacturer’s instructions. A nega-
tive extraction control (NEC) was added to each batch of 
reaction (Sigma-Aldrich, Saint Louis, MO, USA) instead 
of the clinical sample. Purified nucleic acids were stored 
at − 20 °C.
Polymerase chain reaction (PCR)
Herpesvirus detection was performed using a nested 
consensus pan-herpesvirus polymerase chain reaction 
(PCR) targeting a fragment of the DNA polymerase gene 
[8–10]. In the first PCR round, three degenerate and dI-
substituted primers were applied: 285 s DFA (5′-GAY TTY 
GCIAGYYTITAYCC-3′), 285 s ILK (5′- TCC TGG ACA 
AGC AGCARIYSGCIMTIAA-3′), and 285as KG1 (5′-
GTC TTG CTC ACC AGITCIACICCYTT-3′). For the sec-
ond round, the primers 286sTGV (5′-TGT AAC TCG GTG 
TAYGGITTYACIGGIGT-3′) and 286-as IYG (5′-CAC 
AGA GTC CGT RTCICCRTAIAT-3′) were used. Each PCR 
mix contained 12.5 µL of GoTaq® Green Master Mix (Pro-
mega, Madison, WI, EUA), 2.0 µL of each primer (20 µM), 
8 µL of extracted nucleic acid in the first round or 5 µL 
of the product from the first round, and ultrapure water 
(Sigma-Aldrich, Saint Louis, MO, USA) to complete a totalvolume of 25 µL of reaction. For both rounds, the cycling 
conditions were 95 °C for 10 min, then 45 cycles of 20 s at 
95 °C, 30 s annealing at 46 °C, and 30 s extension at 72 °C, 
and followed by a final extension step at 72 °C for 10 min. 
Positive controls included DNA extracted from a Human 
alphaherpesvirus type 1 infected sample from the external 
quality proficiency testing (Controllab, Rio de Janeiro, RJ, 
Brazil) and ultrapure water (Sigma-Aldrich, Saint Louis, 
MO, USA), NECs were used as negative controls.
For the detection of the Human alphaherpesvirus 
(HHV) genomes, the primers HSV-U (5′-GAG CCA CTT 
CCA GAA GCG CAG-3′) and HSV1 (5′-GTT CGT CCT CGT 
CCT CCC C-3′) [11] were applied. The 25-µL reaction mix-
ture comprised 2.0 µL of each primer (20 µM), 12.5 µL 
of GoTaq® Green Master Mix (Promega, Madison, WI, 
EUA), 8 µL of extracted nucleic acid, and ultrapure water 
(Sigma-Aldrich, Saint Louis, MO, USA). The amplifica-
tion conditions were 95 °C for 1 min, 45 cycles of 94 °C 
(1 min), 65 °C (1 min), and 72 °C (1 min), followed by a 
final run at 72 °C (10 min).
PCR amplicons were analyzed by electrophoresis in a 
2% (w/v) agarose gel with 5 µL of each reaction product, 
a molecular size marker of 50 bp (Ludwig Biotecnologia, 
Alvorada, RS, Brazil), stained with GelRed® Loading Buffer 
(Biotium Inc., Hayward, CA, USA), and examined under UV 
light using a Gel Doc EZ® Gel Documentation System and 
Image Lab Software (Biorad, Hercules, CA, USA).
Brazilian Journal of Microbiology 
1 3
Sanger sequencing
The amplified PCR products were purified by incubation 
with ExoSAP-IT/Cleansweep (Thermo Fisher Scientific™, 
Waltham, Massachusetts, USA) and then subjected to Sanger 
sequencing. Direct nucleotide sequencing was performed 
in both directions with the products of the second-round 
PCRs using an automatic sequencer, the ABI-Prism 3500® 
Genetic Analyzer, armed with 50-cm capillaries and POP7 
polymer (Applied Biosystems™, Foster City, CA, USA), 
with 2.5 pmol of the primer (286sTGV or 286-as IYG for 
57 cases and HSV-U or HSV1 for 5 cases), and 0.5 mL of 
BigDye™ Terminator v3.1 (Thermo Fisher Scientific™, 
Waltham, Massachusetts, USA) in a final volume of 10 mL.
Sequence alignment and phylogenetic analysis
ABI files containing the sequences were converted into 
FASTA format using FinchTV 1.4.0 and compared with oth-
ers deposited in GenBank using BLAST (Basic Local Align-
ment Search Tool) to identify herpesvirus species that were 
detected in the pan-PCR or specific HHV-1 PCR. Forward 
and reverse sequences of the DNA polymerase region were 
trimmed and edited using MEGA11 [12] to obtain the con-
sensus sequence as a contig. Then, sequences were aligned 
with their respective reference genomes (KU963227.1 
Alouatta macconnelli cytomegalovirus isolate Ase046 DNA 
polymerase gene, partial; AY139027.1 Cebus albifrons 
lymphocryptovirus 1 DNA polymerase gene, partial cds or 
ON513441.1 Human alphaherpesvirus 1 isolate NIVD4 
complete genome) with the ClustalW algorithm in MEGA11 
[12]. A phylogenetic tree was constructed using Geneious 
Prime 2022.2 (Biomatters) with the neighbor-joining algo-
rithm (consensus method: Majority greedy clustering; 1000 
replications of bootstraps).
Data analysis
All data obtained in this study were compiled and tabulated 
in a Microsoft (Redmond, WA) Excel spreadsheet. Subse-
quent analysis was performed using R statistical software 
version 3.5.1. Categorical variables (genus, age group, sex, 
and location) and their association with positive or negative 
PCR for herpesviruses were analyzed with Fisher’s exact. 
p-value < 0.05 was considered statistically significant. The 
map with the distribution of herpesvirus cases was con-
structed using the QGIS Geographic Information System 
software (QGIS version 3.26.1-Buenos Aires, QGIS Devel-
opment Team). The shape files of the map of Brazil and 
São Paulo, including the municipalities used in the QGIS 
software (SIRGAS 2000), were developed by the Brazilian 
Institute of Geography and Statistics (IBGE).
Results
Among the 242 animals evaluated, 195 (80.5%) belonged 
to the genus Callithrix sp., 33 (13.6%) were Alouatta sp., 7 
(2.9%) were Sapajus sp., 3 (1.2%) were Callicebus sp., and 
4 (1.7%) were not identified in the necropsy record. Regard-
ing their age group, 22 (9.1%) were classified as infants, 23 
(9.5%) as juveniles, 92 (38.0%) as adults, and 105 (43.4%) 
were of unspecified age. Regarding the gender, 68 (28.1%) 
were females and 86 (35.5%) males, and in 88 (36.4%) sam-
ples, gender was not specified (Table 1).
In sixty-two (25.6%) samples, genomic fragment repre-
sentatives of the Herpesviridae family were detected through 
PCR assay. The samples in which the herpesvirus genome 
was identified included 52 (83.9%) animals from the genus 
Callithrix sp., 7 (11.3%) from Alouatta sp., and 2 (3.2%) 
and 1 (1.6%) from Sapajus sp. Among these, 23 (37.1%) 
were female and 23 (37.1%) were male, and 16 (25.8%) were 
animals whose sex was not identified. Based on age group, 
2 (3.2%) were infants, 8 (12.9%) were juveniles, 31 (50.0%) 
were adults, and 21 (33.9%) were not specified (Table 1).
No statistically significant differences were observed with 
respect to the genus and sex of the animals, between the group 
in which herpesvirus was detected and the group in which 
the virus was not detected. The detection of the herpesvirus 
genome was significantly higher in adults than in younger ani-
mals (p = 0.033). Regarding the living conditions, a significantly 
higher frequency of detection of genomic fragments of herpesvi-
ruses was observed among the animals kept under human care, 
compared to those in free-ranging environment (p = 0.008671).
The locations with the highest frequency of positive cases 
were in São Paulo, Barueri, Bragança Paulista, Ribeirão 
Preto, Valinhos, and Campinas, according to Fig. 1 and Sup-
plementary Table S1.
Following sequencing, 39 (16.1%) cases were identified 
as Callitrichine gammaherpesvirus 3 (CalHV), all observed 
in animals of the genus Callithrix. Among these cases, 14 
(35.9%) were identified as females, 12 (30.8%) as males, and 
13 (33.3%) had unidentified gender. Sixteen (41.0%) animals 
were classified as adults, 5 (12.8%) as juveniles, 1 (2.6%) as 
infants, and 17 (43.6%) were not-categorized based on age 
group. Furthermore, 5 (12.8%) of these animals were kept 
under human care, 20 (51.3%) were free-ranging, and 14 
(35.9%) had no specification of living conditions.
Sixteen (6.1%) cases were associated to Human alphaher-
pesvirus 1 (HHV-1), twelve identified through pan-herpes-
virus assay, and five by HHV-1-specific PCR. Among these, 
13 (81.3%) were Callithrix sp., 2 (12.5%) were Alouatta sp., 
and one (6.3%) was Callicebus sp. Seven (43.8%) of these 
animals were identified as females, and 9 (56.3%) as males. 
In terms of age groups, 12 (75.0%) were categorized as 
adults, 2 (12.5%) as juveniles, 1 (6.25%) as an infant, and 
 Brazilian Journal of Microbiology
1 3
Table 1 Distribution of the 
cases based on genus, sex, age 
group, living conditions (free-
ranging or under human care), 
and statistical analysis using 
Fisher’s exact test
P-value < 0.05 marked in bold
Positive Negative Total p-value
N % N %
Genus
  Callithrix sp. 52 83.87 143 79.44 195 0.5769
  Alouatta sp. 7 11.29 26 14.44 33 0.6692
  Sapajus sp. 2 3.23 5 2.78 7 1.000
  Callicebus sp. 1 1.61 2 1.11 3 1.000
  Not identified 0 0 4 2.22 4 0.5748
  Total 62 100 180 100 242
Sex
  Female 23 37.1 45 25 68 0.2181
  Male 23 37.1 63 35 86 0.8863
  Not identified 16 25.81 72 40 88 0.06587
  Total 62 100 180 100 242
Age group
  Infant 2 3.23 20 11.11 22 0.0739
  Juvenile 8 12.9 15 8.33 23 0.7946
  Adult 31 50 61 33.89 92 0.03322
  Not identified 21 33.87 84 46.67 105 0.1019
  Total 62 100 180 100 242
Living conditions
  Free-ranging 28 45.16 106 58.89 135 0.3162
  Under human care 8 12.90 5 2.78 12 0.008671
  Not identified 26 41.94 69 38.33 95 0.7833
  Total 62 100 180 100 242Fig. 1 Map of São Paulo State showing the municipalities where 
there were epizootic occurrences and samples collected for herpesvi-
rus PCR testing, from 2017 and 2022. The municipalities with tested 
samples are highlighted in gray. The red dots indicate the number of 
confirmed cases in each city, according to the legend (QGis)
Brazilian Journal of Microbiology 
1 3
1 (6.25%) individual remained unassigned. Furthermore, 3 
(18.75%) animals were kept under human care, 5 (31.25%) 
were observed in their natural habitat, and 8 (50.0%) were 
not identified with respect to their living conditions.
Five (2.1%) cases were recognized as Alouatta macco-
nnelli cytomegalovirus. Among them, two animals were 
males, one was female, and two remained unidentified. Two 
animals were adults, one was juvenile, and two were not 
categorized by age group. Two animals were free-ranging, 
while three had unspecified living conditions.
Two (0.83%) cases of Sapajus sp. were identified as 
Cebus albifrons lymphocryptovirus 1 (CalbLCV1). One 
case involved a free-living, adult male while the other case 
lacked information regarding the gender, age group, or living 
conditions of the animal.
Figure 2 presents the phylogenetic analysis of the genetic 
sequences. Co-infections were not evidenced. Supplemen-
tary Table S2 provides additional information and GenBank 
accession numbers.
Discussion
Neotropical primates are susceptible to infection by a 
wide variety of herpesvirus [6]. Here, genome fragments 
of viruses of three subfamilies of the Herpesviridae were 
Fig. 2 Phylogenetic tree obtained by the neighbor-joining method 
using fragments (about 300 pb) of the DNA polymerase gene from the 
herpesvirus. Bootstrap values  greater than 50% obtained from 1,000 
pseudoreplicates are shown at the appropriate  branch points. Ranid 
herpesvirus 1 (NC_008211) was used as an outgroup (GeneiousPrime 
2022.2, Biomatters). All cases sequenced were are highlighted in 
bold  and identified by the number, the genus of the NTPs (Alou = 
Alouatta, Cali = Callithrix,  Titi = Callicebus, Sap = Sapajus) and 
the municipality of occurrence (SP = São Paulo,  BAR = Barueri, 
BRP= Bragança Paulista, CAM = Campinas; CAJ = Campos do 
Jordão,  COT: Cotia, DIA = Diadema; INB = Inúbia Paulista; ITS 
= Itapecerica da Serra; ITA = Itatiba; ITP = Itupeva; JAG= Jaguar-
iúna; JAR= Jarinú; JUN = Jundiaí, MOC = Mococa, MOG = Mogi 
Guaçu, OSA = Osasco, PED = Pedreira, RP = Ribeirão Preto, SER 
= Serrana, SJP = São José do Rio Preto, SAB = Santa Branca, SOC= 
Socorro, VGS= Vargem Grande do Sul, VOT = Votuporanga)
 Brazilian Journal of Microbiology
1 3
detected in samples of four different genera of NTPs sam-
pled in different regions of the State of São Paulo, Brazil. 
The frequency of the detection of herpesvirus genomes, 
around 25%, was lower than that reported in a study with 
NHPs in the state of Rio de Janeiro, where approximately 
34% of specimens were found to harbor herpevirus genomes, 
out of 283 sampled animals [13]. Significantly, adult animals 
showed a higher frequency of herpesvirus detection than 
other age groups, as observed by Bonfim et al. [13]. This is 
likely due to the fact that adults naturally tend to have more 
opportunities for contracting the infections from infected 
hosts [5, 6] or, even, present a higher viral load as they age. 
Another hypothesis includes that adult carcasses are more 
easily identifiable and subject to necropsy, whereas infants 
and juveniles are more susceptible to predation or autolysis.
A higher frequency of herpesvirus genome detection was 
observed among animals that were kept under human care. 
This population of animals commonly experiences height-
ened exposure to humans, veterinarians, caretakers, keep-
ers, and other staff at rehabilitation centers or zoos, con-
sequently augmenting the risk of interspecies transmission 
[5]. Moreover, their confinement in cages and the continual 
introduction of new individuals of the same species lead to 
increased interactions, potentially including asymptomatic 
carriers with the capacity for viral transmission between 
humans and animals, as well as among animals [6].
The majority of the cases were associated with urban 
centers, such as São Paulo, Campinas, and Ribeirão Preto, 
as observed in the cities of the state of Rio de Janeiro 
[13]. This may be related to a collection bias since these 
municipalities presented a more robust surveillance sys-
tem, or even related to areas with higher population den-
sity and greater contact between humans and animals in 
anthropogenic landscapes.
Among the positive samples, 62.9% were identified as 
Callitrichine Herpesvirus 3 (CalHV3) by sequencing, all in 
marmosets. CalHV3 is a lymphocryptovirus, first isolated 
and sequenced in 2000, from a cell line derived from a com-
mon marmoset B-cell lymphoma [14, 15]. The Lymphocryp-
tovirus genus contains over 50 different species-specific 
gammaherpesviruses that infect non-human primates, all of 
them closely related to the Epstein-Barr virus (EBV), also 
known as Human gammaherpesvirus 4, an indigenous virus 
in the human population [16]. EBV infects human B lym-
phocytes and can maintain latent infections, with the major-
ity of cases asymptomatic. However, the virus can cause 
infectious mononucleosis, and it is also associated with 
neoplastic development, such as Burkitt’s lymphoma, T-cell 
lymphoma, nasopharyngeal carcinoma, oral hairy leukopla-
kia, and non-Hodgkin’s lymphoma in immunocompromised 
individuals [17]. CalHV3 is more closely related to a primi-
tive lymphocryptovirus and has already been documented 
in zoo and laboratory facilities or free-ranging marmosets. 
Almost 35–65% of wild-caught and marmosets kept in cap-
tivity under human care were seropositive for CalHV3 [14, 
15, 18]. Although most infected animals are asymptomatic, 
with rare exception cases describing weight loss, inappe-
tence, diarrhea, lymphoproliferative disease, and B-cell lym-
phoma in the intestinal tract and mesenteric lymph nodes 
[14, 15], the zoonotic potential of the virus is still unknown; 
however, interspecies transfer of some Gammaherpesvirinae 
has already been demonstrated [19–22].
Another lymphocryptovirus identified in this study 
was similar to Cebus albifrons lymphocryptovirus 1 (Cal-
bLCV1), described in 2003 from the lung tissue of Cebus 
albifrons [23]. This virus is not recognized by the Interna-
tional Committee on Taxonomy of Viruses (ICTV, https:// 
talk. ictvo nline. org/, March 2022 release, MSL #37), since 
the complete genome has not yet been characterized, like 
other Cebidae lymphocryptovirus (Cebus albifrons lym-
phocryptovirus 2, Cebus apella lymphocryptovirus 1, C. 
capucinus lymphocryptovirus 1 and 2) [24]. None of these 
viruses has been associated with clinical findings or tissue 
damage. In this context, three novel lymphocryptoviruses 
from free-ranging golden-handed tamarin (Saguinus midas), 
squirrel monkey (Saimiri sciureus), and white-faced saki 
(Pithecia pithecia) were described from French Guiana [25], 
with genetic similarities to CalHV3 and CalbLCV1.
Human alphaherpesvirus 1 was the second most com-
mon herpesvirus detected in this NTP population of São 
Paulo State during the study period. Humans are the natural 
host of the virus and transmission to non-human primates 
can occur by direct contact through saliva and aerosols. As 
an important zooanthroponosis, cases of HSV-1 infection 
have already been described in Old World primates, such 
as eastern lowland gorillas (Gorilla beringei graueri) [26], 
western lowland gorillas (Gorilla gorilla gorilla) [27], chim-
panzees (Pan troglodytes) [28], juvenile orangutan (Pongo 
pygmaeus pygmaeus) [29], bonobos (Pan paniscus) [30], 
and lar gibbons (Hylobates lar) [31]. Platyrrhines are par-
ticularly susceptible to Human alphaherpesvirus infection, 
with systemic and fatal diseases in callitrichids (Callithrix 
jaculus, C. penicillata, and C. geoffroyi) [32–34],white-
faced saki monkeys (Pithecia pithecia pithecia) [35], owl 
monkeys (Aotus trivirgatus) [36, 37], capuchin (Cebus sp.) 
[38, 39], and tamarin species (Saguinus sp.) [40]. The patho-
genesis in NTPs is similar to humans, with primary infection 
typically occurring via mucosal surfaces or skin abrasions, 
leading to clinical diseases, which include vesicular skin 
lesions or mucous membranes, fever, lymphadenopathy, and 
neurological signs [32, 33]. The establishment of latency in 
sensory ganglia with reactivation of the virus can occur in 
response to stress or immunosuppression, especially in Old 
World primates [41].
https://talk.ictvonline.org/
https://talk.ictvonline.org/
Brazilian Journal of Microbiology 
1 3
In this study, the infection by HHV-1 was more preva-
lent among animals of the genus Callithrix. Marmosets 
are commonly found in southeast Brazil, where family 
groups frequently interact with humans, including food 
sharing, thereby facilitating the transmission of infectious 
agents [42]. Callithrix penicillata and C. jacchus were 
both introduced in the state of São Paulo [43], threaten-
ing the original populations of other marmoset species 
such as Callithrix aurita (Geoffroy in Humboldt, 1812), 
Callithrix flaviceps (Thomas, 1903), and Callithrix kuhlii 
(Coimbra-Filho, 1985) [44]. Hybrids of C. penicillata and 
C. jacchus are frequently observed, and species distinction 
based solely on visual inspection is difficult [45].
HHV-1 was detected in two howler monkeys (Alouatta 
sp.). Encephalitis related to infection by this virus has 
already been reported in this genus [46]. Also, the case of 
HHV-1 infection in a black-fronted titi (Callicebus nigri-
frons) kept under human care, whose clinical and patho-
logical findings were recently described [47], was also 
included and characterized in this study.
Alouatta macconnelli cytomegalovirus is a recently 
described Cytomegalovirus (CMVs) in a Guyana red 
howler from French Guiana [48]. Cytomegaloviruses of 
NTP are represented by only three viral entities recog-
nized by the ICTV, from Aotus trivirgatus, Saimiri sciu-
reus, and Cebus sp., despite the wide diversity of platyr-
rhines [49–51]. Generally, CMVs exhibit a limited ability 
to infect hosts, and their presence in healthy, mature indi-
viduals is typically symptomless [52]. These viruses com-
monly act as opportunistic agents, and infection and illness 
typically only manifest in individuals with compromised 
immune systems. Anti-cytomegalovirus antibodies were 
identified in the serum of free-ranging black howler mon-
keys (Alouatta caraya) from Misiones, Argentina [53]. 
Also, two types of CMVs, specifically Callitrichine her-
pesvirus 1 and 2, have been identified in marmosets, but 
they are not known to cause any disease [54].
In conclusion, molecular investigation through pan-
herpesvirus PCR is a useful tool for the detection of a 
wide variety of herpesviruses in neotropical primates. The 
study of herpesviruses in NTPs is extremely important in 
the context of One Health, focusing on the fatal transmis-
sion of viruses between humans and non-human primates. 
Potential zoonotic infections should also be investigated 
and may be included as a complement to the Epizootic 
Surveillance Program of the Ministry of Health, which is 
currently only focused on yellow fever detection.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s42770- 023- 01105-z.
Acknowledgements The authors thank all the professionals directly 
or indirectly involved in the Non-Human Primates Epizootic Events 
Surveillance Program of the Brazilian Ministry of Health in São Paulo 
State; field, surveillance, and laboratory staff, especially those from 
the Centro de Vigilância Epidemiológica Prof. Alexandre Vranjac, the 
Pathology Center, and the Strategic Laboratory of Instituto Adolfo Lutz.
Author contribution INF, CSA, OPB, LMD, JPFT: collected and 
analyzed the data. MSC: carried out the genetic material extraction. 
ACSRC, KBF, CSSF, OPB: performed the PCR tests. AL: conduct the 
sequencing. LJTA: conduct the phylogenetic and mapping analyses. 
JMG, INF: analyzed, interpreted the data, and drafted the manuscript. 
JMG, NCCA, JLCD: elaborated the study design. JLCD: supervised 
JMG in the post-doctoral. All the authors critically revised the manu-
script for intellectual content and approved the final version.
Funding This study was supported by Conselho Nacional de Desen-
volvimento Científico e Tecnológico (CNPq) (process number 
404510/2021) to J.M.G. and the Scientific Initiation Scholarship – 
PIBIC (CNPq) (#116603/2022) for INF and CSA, Grupo de Apoio às 
Políticas de Prevenção e Proteção à Saúde/Fundo Especial de Saúde 
para Imunização em Massa e Controle de Doenças (GAPS/FESIMA 
numbers 040/2019, 28/2020 and 54/2022), the multi-user sequencing 
equipment acquired through PDIP—São Paulo Research Foundation 
(FAPESP) (grant# 2017/50333–7), and the FAPESP research grant 
2020/14786–0 to A.L. JLCD is a research fellow of CNPq (grant # 
304106/2022).
Declarations 
Conflict of interest The authors declare no competing interests.
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	Detection of herpesviruses in neotropical primates from São Paulo, Brazil
	Abstract
	Introduction
	Material and methods
	Animals
	Nucleic acid extraction
	Polymerase chain reaction (PCR)
	Sanger sequencing
	Sequence alignment and phylogenetic analysis
	Data analysis
	Results
	Discussion
	Anchor 13
	Acknowledgements 
	References