Detection of Chikungunya virus in Saliva and Urine Samples of Patients from Rio de Janeiro, Brazil. A Minimally Invasive Tool for Surveillance

In this study, we collected saliva and urine samples from individuals in the metropolitan region of Rio de Janeiro, Brazil, during the years of 2017 through 2019 and we were able to detect the presence of Chikungunya virus genome in these samples. Our findings reinforce the possibility to monitor Chikungunya virus circulation by analyzing saliva and urine from individuals during inter-epidemic periods.


Introduction
Chikungunya virus (CHIKV) is a mosquito-borne virus, transmitted in the urban area mainly by the Aedes aegypti and Aedes albopictus mosquitoes [1]. CHIKV belongs to family Togaviridae and genus Alphavirus and consists of a single-stranded RNA genome virus, about 70 nm diameter and a phospholipid envelope [2]. This virus was first described in Africa (Tanzania) in 1954 and later identified in Asia, and it was responsible for outbreaks in these two continents from the 1960s to the 1980s [3]. Currently, CHIKV is considered a real threat to countries localized in temperate and tropical zones that are infested by Aedes spp, such as Europe and the Americas [3,4].
Human CHIKV infection results in a spectrum of manifestations and it begins with a silent incubation period lasting 2-4 days on average (range 1-12 days) [5], which can evolve to either an asymptomatic and subclinical outcome or to clinical manifestations. Clinical onset is abrupt, with high fever, headache, back pain, myalgia, and arthralgia; the latter can be intense, affecting mainly the extremities (ankles, wrists, phalanges) but also large joints, referred to as the effect of the incapacitating arthralgia [6].
Our group collected saliva and urine from volunteers presenting symptoms compatible with the disease, except for one healthy individual (male) from which saliva was collected. The individuals that had saliva collected were one male and four females. The individuals that had urine collected were two males and four females. Three females donated both, saliva and urine. The samples were collected from individuals at the Federal University of Rio de Janeiro, using approved Protocol Ethics: 80709 HUCFF/FM/ UFRJ from 2017 through 2019. Volunteers were students, student relatives and staff from the University Campus, and the ages were between 25 and 45 years old.
The saliva samples (n=5) were obtained from five individuals with arbovirus-like symptoms and one asymptomatic individual.
In the urine group (n=6) all individuals were exhibiting arboviruslike symptoms. The total number of samples, therefore, was eleven (n=11) and the total number of individuals was eight (n=8). Individual samples were submitted to RNA extraction by the TRIzol™ method Reagent (Invitrogen, Carlsbad, California, USA), followed by a Reverse Transcriptase (RT) assay by superscript IV (Invitrogen, Carlsbad, California, USA), following the manufacturer recommendation. CHIKV RNA detection was performed by using RT 2-step-qPCR and conventional PCR, using the forward 5'-ctttggagccaacgctatcgctt-3' (SGCK-F) and reverse 5'-tttgtccttgcactctgctgta-3' (SGCK-R) primers in the standardization SYBR Green qPCR assays. For the conventional PCR, Nested methodology was used in the first reaction with the forward 5'-taccgtataagactctagtc-3' (Nestalpha-F) and reverse 5'-tgaatgtccccaaatcttccagg-3' (Nestalpha-R) primers, followed by the second reaction with primers (SGCK-F/SGCK-R). The amplified viral material was visualized on a 1.5% (v/v) agarose gel ( Figure   1D).  Table 1). The qPCR results showed that all urine samples were positive for CHIKV RNA, including the sample from the asymptomatic individual. Four saliva samples were positive, and one was negative. The negative saliva sample was from the donor that had arbovirus-like symptoms but donated saliva only. All two individuals that had symptoms and donated both saliva and urine had both samples positive. Overall, we had ten positive samples for CHIKV and one negative ( Figure   1E) ( Table 1). The generic RT-PCR for alphaviruses showed positive results, and direct sequencing of the viral amplicons showed a CHIKV-specific sequence. The result of the Sanger sequencing was used as query for BLAST searches of NCBI.

Virus Dilution
Average CT in triplicate  In order to identify query sequences were used to search sequences on the CHIKV genome on the NCBI genome database, using BLASTn [7]. Sequences resulting from these searches, showing more than 90% identity were deemed as homologous. All the sequences from the homologous were then used as Datasets to the aligned using the MUSCLE software [8] and then pruned for removal of regions with a high frequency of indels using TrimAL using the "-gappyout" comman [9].
The maximum likelihood (ML) tree topology was inferred with the Iq-Tree 1.6 program [10]. Branch support was assessed by the ultrafast bootstrap implementation of IqTree using 1,000 replicates [11]. IqTree was executed via the command "iqtree -s infile -bb 1000". Because no outgroup was included in our analysis, rooting of the genome CHIKV genealogy was performed using the minimal ancestor deviation method of Tria et al. [ 12]. Figure 2 shows the phylogenetic tree indicating in red the RIO_2019_CHIKV genome sequenced in the work. Our results are similar to those of Musso et. al. [13] where they reported high number of CHIKV-positive samples during a period of high CHIKV prevalence, agreeing with the epidemiological data of the metropolitan region of Rio de Janeiro at the time of collection [4,14]. In the context of endemic co-circulation of other arbovirus diseases transmitted by Ae.
aegypti such as dengue and zika fevers [14,15], a fast, reliable and noninvasive diagnostic method is important for surveillance and early detection of an increase in the number of infections.
In this study, we show that it is possible to detect CHIKV

Ethics Approval and Consent to Participate
The Ethical Committee of RJ/BR Rio de Janeiro (80709 HUCFF/ FM/UFRJ) has approved this study.

Availability of data and material
All the supporting data generated for this manuscript and its conclusions is available in the text. No other supporting material is required.

Competing Interests
The authors declare that they have no competing interests.

Consent for publication
Written consent was obtained from the patients / family for publication.

Funding
This work was supported by the Fundação Carlos Chagas

Author Contributions
All authors collaborated in the study design; TSS, TESG, VGR,