PLoS ONE
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Diagnostic system for the detection of severe fever with thrombocytopenia syndrome virus RNA from suspected infected animals
DOI: 10.1371/journal.pone.0238671, Volume: 16, Issue: 1
Article Type: research-article, Article History
Abstract

Background

Severe fever with thrombocytopenia syndrome virus (SFTSV) causes severe hemorrhagic fever in humans and cats. Clinical symptoms of SFTS-infected cats resemble those of SFTS patients, whereas SFTS-contracted cats have high levels of viral RNA loads in the serum and body fluids. Due to the risk of direct infection from SFTS-infected cats to human, it is important to diagnose SFTS-suspected animals. In this study, a reverse transcription polymerase chain reaction (RT-PCR) was newly developed to diagnose SFTS-suspected animals without non-specific reactions.

Methodology/principle findings

Four primer sets were newly designed from consensus sequences constructed from 108 strains of SFTSV. A RT-PCR with these four primer sets successfully and specifically detected four clades of SFTSV. Their limits of detection are 1–10 copies/reaction. Using this RT-PCR, 5 cat cases among 56 SFTS-suspected animal cases were diagnosed as SFTS. From these cats, IgM or IgG against SFTSV were detected by enzyme-linked immunosorbent assay (ELISA), but not neutralizing antibodies by plaque reduction neutralization titer (PRNT) test. This phenomenon is similar to those of fatal SFTS patients.

Conclusion/significance

This newly developed RT-PCR could detect SFTSV RNA of several clades and from SFTS-suspected animals. In addition to ELISA and PRNT test, the useful laboratory diagnosis systems of SFTS-suspected animals has been made in this study.

Park, Fujita, Kimura, Hotta, Imaoka, Shimojima, Saijo, Maeda, Morikawa, and Ikegami: Diagnostic system for the detection of severe fever with thrombocytopenia syndrome virus RNA from suspected infected animals

Introduction

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral hemorrhagic fever that was first identified in China [1], with cases since reported in Japan, South Korea, Vietnam and Taiwan [25]. Seven to eight clades of SFTSV are reportedly spreading throughout Japan, China and South Korea [6]. SFTS virus (SFTSV) has been identified as a causative virus of SFTS belonging to the genus Banyangvirus, family Phenuiviridae, order Bunyavirales.

SFTS has been considered to be mainly transmitted by tick bites. Ticks infest a variety of animals, while viral RNA and antibodies against SFTSV have been detected in wild animals, domestic animals and companion animals, such as dogs and cats [711]. Since these animals show no clinical symptoms, they have been considered subclinically infected with SFTSV. In recent years, SFTS patients without a history of tick bites have been reported, and they are considered to have received the virus through transmission from animals, such as cats and dogs [10, 12]. Furthermore, it has been shown that cheetahs [13], cats [14] and dogs can contract SFTS (manuscript in preparation). Thus, it is important to diagnose SFTS-suspected animals.

In Japan, conventional one-step RT-PCR, ELISA and the isolation of SFTSV are performed to diagnose human SFTS cases [15]. However, there are some limits with this system due to the non-specific reactions in case of animal SFTS cases. Thus, in this study, a reverse transcription polymerase chain reaction (RT-PCR) was developed to establish a laboratory diagnosis system for detecting several clades of SFTSV in the specimens of SFTS-suspected animals.

Materials and methods

Serum samples

The samples, including serum and oral and rectal swabs, were collected from SFTS-suspected cats (n = 36) and dogs (n = 19) at veterinary hospitals throughout Japan for routine SFTS diagnostic work from August 2017 to March 2019. Cats exhibiting a fever (>39 °C), leukocytopenia (<2000 /μL), thrombocytopenia (<200,000 /μL) and elevated levels of AST, ALT and CK were suspected of having SFTS. Dogs showed anorexia, depression, fever and some gastrointestinal tract symptoms, including diarrhea and vomiting. Clinical information was provided by veterinarians.

A phylogenic analysis for primer design

The nucleotide sequences of 100 strains of SFTSV S segment and M segment were selected randomly from Genbank to cover all the clades and aligned and phylogenetically analyzed (S1 Fig). In brief, phylogenic trees were constructed using with the maximum likelihood method with the Tamura-Nei model using the MEGA 7 software program [16]. The robustness of the resulting branching patterns was tested using the bootstrap method with 1,000 replicates. From this analysis, it was confirmed that the seven to eight clades of SFTSV correlate with their geographical location, as has been reported previously [6]. The nucleotide identity, determined using the Bioedit sequence alignment editor [17], was 94.1%-99.1% in S segment, and 93%-99.7% in M segment among clusters. The consensus sequences of S segment and M segment among the different strains were selected using the Bioedit program, and primers were designed by the NCBI Primer-BLAST [18] from the consensus sequences.

RT-PCR

RNAs from three strains of SFTSV belonging to different clades—SPL010 (J1 clade, accession No. AB817999), cat#1 (C4 clade, accession No. DRA007207) and HB29 (C3 clade, accession No. NC_018137)—were used as positive controls. The copy numbers of RNA samples used as positive controls have been measured by real time RT-PCR according to our previous study [14]. Then, RNAs were diluted by 10-fold with diethylpyrocarbonate (DEPC)-treated distilled water (DW) (Nippon Gene Co., Ltd., Tokyo, Japan). DEPC-treated DW was used as a negative control. RNA was extracted using a High Pure Viral RNA Kit (Roche, Mannheim, Germany) as previously reported [14]. RT-PCR was performed using the Superscript III one-step RT-PCR system with platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA) under the following conditions: RT at 55 °C for 30 min, inactivation at 95 °C for 2 min, and then 40 cycles of PCR at 94 °C for 30 s, 52 °C for 30 s, and 68 °C for 30 s, followed by extension at 68 °C for 5 min.

SFTSV detection from samples using four designated primers

Total RNAs were extracted from the specimens of dogs and cats using ISOGEN (Wako, Osaka, Japan) and a precipitation carrier (Ethachinmate; Wako), according to the manufacturer’s instructions. RT-PCR was performed using a Superscript III one-step RT-PCR system with platinum Taq DNA polymerase (Invitrogen) with four sets of specific primers. Samples with more than two positive bands were considered SFTSV RNA-positive.

Amplification of viral genome of S segment and phylogenetic analysis

Sequencing and phylogenetic analysis was performed on the viral S gene segment for all RT-PCR positive samples. RT-PCR was performed using a Superscript III one-step RT-PCR system with platinum Taq DNA polymerase (Invitrogen) with primers covering the entire S segment region according to a previously reported study [6]. The PCR products were determined by electrophoresis on 1% agarose gels with GR Red Loading Buffer (GRR-1000, Bio-Craft, Tokyo, Japan). The PCR products were then extracted and purified using an illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare, Buckinghamshire, UK). The samples were sequenced using the general Sanger sequencing technique.

The nucleotide sequences determined in this study were deposited in the DDBJ GenBank databases. For the phylogenetic analysis, three nucleotide data points per cluster were selected. The sequence alignment was computed using the Clustal W program of MEGA 7 software program. The phylogenetic tree was constructed using the maximum likelihood method based on the Tamura-Nei model of the MEGA program. The confidence of the tree was tested using 1000 bootstrap replications.

Detection of IgM and IgG in cats by an enzyme-linked immunosorbent assay (ELISA)

Antibodies against SFTSV were detected by an ELISA, essentially performed as in the previously described study [19]. In brief, SFTSV- or mock-infected Huh7 cells were lysed in 1% NP40 in phosphate-buffered saline (PBS), ultraviolet (UV)-irradiated to completely inactivate SFTSV, and then clarified by centrifugation at 12,000 rpm for 10 min. The lysates were coated onto the ELISA plate (Nunc-Immuno plate; Thermo Fisher Scientific, Roskilde, Denmark). The antigen-coated wells were then blocked with 20% Blocking One (Nacalai Tesque, Inc., Kyoto, Japan) in PBS (blocking solution) at room temperature for 1 h. Sera of cats and dogs were inactivated at 56 °C for 30 min and serially 4-fold diluted from 1:100 to 1:6400 in the blocking solution at 37 °C for 1 h. Horseradish (HRD)-conjugated goat anti-feline IgG Fc and HRD-conjugated goat anti-feline IgM (Novus biologicals), and HRD-conjugated goat anti-dog IgM(μ) and HRD-conjugated sheep anti-dog IgG(H) were used to detect IgM and IgG antibodies in cats and dogs, respectively. The reaction was finally visualized by a substrate for HRP (ABST, 2, 2azinobis (3-ethylbenzthiazolinesulfonic acid); Roche, Mannheim, Germany) for 30 min at room temperature. The optical density (OD) at 405 nm was measured with an iMark microplate reader (Bio-Rad, Tokyo, Japan). The OD values in the mock-antigen coated well were subtracted from the OD value in the respective SFTSV-antigen coated wells. The cut-off OD value was set as the average subtracted OD value plus three times the standard deviation (SD), that is, mean + 3SD, of SFTS-negative serum that had been confirmed by an indirect immunofluorescent antibody assay using SFTSV-infected Vero cells. The sera were considered positive when the OD values were above the cut-off value.

The 50% plaque reduction neutralization titer (PRNT50)

The PRNT test was performed to determine the neutralizing antibodies against SFTSV using Vero cells (ATCC), according to previously reported studies. Approximately 100 plaque-forming units of the HB29 strain of SFTSV were mixed with serially diluted heat-inactivated sera and incubated for 1 h at 37 °C and then inoculated into confluent monolayers of Vero cell in 12-well plates for 1 h at 37 °C. The inocula were removed, and the cells were washed once with DMEM containing 2% FBS and kanamycin and then cultured at 37 °C in a 5% CO2 incubator with DMEM containing 2% FBS, Kanamycin and 1% methylcellulose for 1 week. Cultured cells were fixed with 10% buffered formalin and exposed to UV radiation to inactivate the virus. The cells were permeabilized with 0.1% Triton X-100, followed by incubation with rabbit antibodies against SFTSV-N as primary antibodies and HRP-conjugated recombinant protein A/G (Cat. No. 32490, Thermo Scientific, Rockford, IL, USA) as secondary antibodies. Plaques were visualized with 3, 3’-diaminobenzidine tetrahydrochloride (Peroxidase stain DAB kit [Brown stain]; Nacalai Tesque). The PRNT50 value was determined as the reciprocal of the highest dilution at which the number of the plaques was below 50% of the number calculated without cat serum.

Results

Four primer pairs (2 for the S segment and 2 for the M segment) successfully detected the RNAs of the three SFTSV strains belonging to different clusters with a detection limit of 1–10 copies/reaction (Fig 1 and Table 1). For the detection of SFTSV RNA from samples, these four primer sets were used.

RT-PCR electrophoresis for the confirmation of the detection limit.
Fig 1
(A) Primer 2. Viral RNA was extracted from the SPL010 strain. 103−100 copies/reaction and mock sample (from left to right). A PCR product of 125 bp was observed. (B) Primer 4. Viral RNA was extracted from HB29 strain. 103−100 copies/reaction and mock sample (from left to right). A PCR product of 179 bp was observed.RT-PCR electrophoresis for the confirmation of the detection limit.
Table 1
Information of primers.
No. of primerSegmentSiteProduct size (bp)Nucleotide sequenceLimit of detection
1S segment1347–13692015’-TGCTGCAGCACATGTCCAAGTGG-3’1~10
1524–14965’-GACACAAAGTTCATCATTGTCTTTGCCCT-3’
2S segment1028–10481255’-GCCATCTGTCTTCTTTTTGCG-3’1~10
1131–11525’-AGTCACTTGCAAGGCTAAGAGG-3’
3M segment2422–24421855’-AGGCAAGGTTGGAGAGATACA-3’1~10
2586–26065’-CCCCAATAGTGGTGGGTATGG-3’
4M segment373–3931795’-AGTTCCTGGGCCTTCATACAA-3’1~10
530–5515’-CATCACCTATCCAGAGAACCCT-3’

From August 2017 to March 2019, 56 cases were collected, and RT-PCR was performed to detect SFTSV RNA (Fig 2). Among them, SFTSV RNA was detected in the sera of five cats. The PCR products were confirmed with RT-PCR using all four sets of primer pairs (Fig 3). Five SFTS contracted cats showed clinical symptoms including depression, loss of appetite and jaundice. They showed body weight loss, fever, leukocytopenia (2180~7540/μL), thrombocytopenia (15,000~120,000/μL) and high total bilirubin level. All of the five cats were kept both indoor and outdoor and four of the five cats had tick-bite history. Samples were collected within one week after disease onset. Two cats were dead and three cats recovered (Table 3). The positive samples were evaluated to determine the nucleotide sequence of the S segment (Table 2). These nucleotide sequences of the S segment from five cases were phylogenetically analyzed with the corresponding segment of the Heartland virus as an outgroup (Fig 4). As a result, four strains were clustered into genotype J1, and one strain was clustered into genotype J3 of the Japanese clade. Totally, RT-PCR with four primer pairs successfully and specifically detected SFTSV belonging to four clades, including positive controls.

Location of sample collected and diagnosed as SFTS.
Fig 2
The map indicates the provinces/prefectures (yellow) where samples were collected and where cats were diagnosed as SFTS (red).Location of sample collected and diagnosed as SFTS.
RT-PCR electrophoresis of five SFTS cat cases.
Fig 3
The PCR product bands were observed. I and I’: sample RNA, II: positive control, III: negative control. (A) No. 1, (B) No. 2, (C) No. 3, (D) No. 4, and (E) No. 5.RT-PCR electrophoresis of five SFTS cat cases.
Phylogenic trees of detected SFTSV genome in this study (red) and reference SFTSV genome (black) for the S segments.
Fig 4
Phylogenic trees of detected SFTSV genome in this study (red) and reference SFTSV genome (black) for the S segments.
Table 2
The accession number of positive cases.
StrainThe accession No.
JDVS17LC514461
JDVS22LC514462
JDVS26LC514463
JDVS41LC514464
JDVS47LC514465
Table 3
Information of five cats.
No. of case12345
SFTSV RNA+++++
IgM+
(1:400)
+
(1:400)
-+
(1:1600)
+
(1:100)
IgG+
(1:6400)
+
(1:100)
+
(1:100)
+
(1:1600)
+
(1:100)
ProvinceKagoshimaMieMieMieMie
Animalcatcatcatcatcat
Disease onsetJanuary 2018May 2018June 2018August 2018October 2018
Sampling time pointUnknownafter 4 daysafter 7 daysafter 4 daysafter 6 days
Age9 m10 m3 y14 y15 y 5 m
Sex♀x
Conditionindoor & outdoorindoor & outdoorindoor & outdoorindoor & outdoorindoor & outdoor
Body weight (kg)N.D.2.92.82.84.9
Body temperature (°C)39.339.1>4038.339.4
RBC (104/μl)301101.51191831388
WBC (/μl)40002180420075403600
Platelet (/μl)56000150004400012000018000
ALT/GPT (I/U)806959N.D.135
AST/GOT (I/U)N.D.N.D.N.D.N.D.N.D.
CK (I/U)N.D.N.D.N.D.N.D.N.D.
T-bil (mg/dl)5.9N.D.2.9N.D.2
FeLVnegativeN.D.N.D.N.D.negative
FIVnegativeN.D.N.D.N.D.positive
SymptomsDepression, Anorexia, Intussusception (ileum to colon),
GI hemorrhage, vomit, jaundice
Depression, Anorexia, jaundiceDepression, Anorexia, jaundiceDepression, Anorexia, jaundiceDepression, Anorexia
Tick-bite historytick-bite scarnegativepositivepositivepositive
PrognosisDeadRecoveredRecoveredRecoveredDead

In addition to SFTSV RNA, antibodies against SFTSV were detected in the sera of the five cats that were PCR-positive (Table 3). Three samples had IgM and IgG against SFTSV, and two had IgM or IgG, respectively (Table 3). Serum samples were collected at a one-week interval from one case. IgM was detected in these two interval sera, and IgG was detected in the serum collected one week later. This seroconversion pattern was similar to that of our previous study. Antibodies were not detected in the RT-PCR-negative animals.

The neutralizing antibodies against SFTSV were then measured with the PRNT50 according to our previous study [14]. The titer of neutralizing antibodies was below the limit of detection, indicating that the antibodies detected by the ELISA could not neutralize SFTSV, similar to those of fatal human cases.

Discussion

In Japan, for the diagnosis of human and animal SFTS cases, conventional one-step RT-PCR to detect the SFTSV genome, ELISA to detect the antibodies against SFTSV and the isolation of SFTSV are performed [15]. However, non-specific reactions were obtained from some SFTS-suspected animal cases, especially dogs, in the one-step RT-PCR using primers for human SFTS diagnosis. For the simple, rapid and specific detection of SFTSV RNA from SFTS-suspected animal cases, primer sets were newly designed in this study. Four primer sets were able to detect SFTSV RNA belonging to four genotypes with a low detection limit. Some genotypes were not tested since these were not available. However, the primers used in this study might detect other clades since the sequence of these was identical to the primer sequences. Two pairs were specific for the S segment, and two pairs were specific for the M segment. SFTSV RNAs were detected from five cases using these primers. Four positive bands were observed in all five cases. These positive cases were distributed in the same region where human SFTS cases have been reported (Table 4). The sites at which two cases were detected were close to each other. The genotype of these strains was J1 and J3, showing 99.3%-99.7% homology. These findings are believed to establish the hot spot and circulation of SFTSV among ticks and animals. In Japan, several phylogenetic clades of SFTSV are circulating in the SFTS endemic region, thus it is of interest to clarify if the genomic reassortment event occurred in animal derived SFTS viruses in future. The ages of the cats ranged from 9 months to 15 years old. The period of disease onset was from January to October. All of these cats were kept both indoors and outdoors. In addition, four cases had a tick-bite history, indicating the transmission of SFTSV by tick. Their clinical symptoms were similar to those described in previous reports. Thus, these cats were diagnosed with SFTS. After cats were diagnosed as SFTS, veterinarians were informed; 1) they should wear personal protective equipment (PPE), such as gloves, mask, face shield, goggle and gown, to treat SFTS-diagnosed cats since SFTSV could be secreted from blood, saliva, urine and feces for about two or three weeks, 2) they should consult a doctor if they have a direct contact with these blood, saliva, urine and feces and then have clinical symptoms, such as, fever, 3) they should inform owners of the animals not to contact without PPE.

Table 4
Distribution of positive cases.
Area
SFTS patients reportedSFTS patients not reported
Animal casesNegative cases
Cats1912
Dogs712
Positive cases
Cats50
Dogs00

Five cats had IgM and/or IgG against SFTSV, determined by an ELISA. However, the titer of neutralizing antibodies was below the limit of detection. The serum specimens of the cats were collected within one week after the onset. It takes approximately two weeks to see a robust immune response, the samples collected in this study might be too early to assess the neutralizing antibodies in the naturally infected animals.

In conclusion, the RT-PCR approach developed in the present study and the IgM- and IgG-ELISA performed to detect SFTS-specific antibodies were useful for making a laboratory diagnosis of SFTS-suspected cats and dogs.

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28 Oct 2020

PONE-D-20-25871

Development of a reverse transcription polymerase chain reaction for the detection of severe fever with thrombocytopenia syndrome virus from suspected infected animals

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Additional Editor Comments:

[Independent review by academic editor]

The manuscript entitled “Development of a reverse transcription polymerase chain reaction for the detection of severe fever with thrombocytopenia syndrome virus from suspected infected animals” describes the development of conventional RT-PCR for the detection of S-segment (125 or 178bp) or M-segment (185 or 179bp) of SFTSV. The new RT-PCR detected SFTSV RNA from 3 positive controls (SFTSV culture sup: J1, C4, and C3 clades) and 5 of 56 animal samples (dogs and cats), which were IgM and/or IgG positive for SFTSV. Positive clinical samples were all from cats, and the partial S-segment nucleotide sequences of those positive samples were phylogenetically grouped into J1 (Cat47, 41, 26, 17) or J3 (Cat22) clades. This study showed the presence of viremic cats harboring SFTSV in Japan.  

Major points:

    • The title of this study does not reflect additional SFTS case identification in cats in Japan. The study in this manuscript identified five cats with detectable SFTSV RNA, and specific IgG / IgM. The development of conventional RT-PCR not sufficiently novel for publication by itself. Please consider changing the title, while revising the abstract and conclusion accordingly.
    • Figure 1 and 2 do not provide the rationale of given primer designs, which should be removed or moved to Supplementary materials, if required. The alignment of each primer sequence to representative strains in those clades might be alternatively shown.
    • Line 59: Authors should provide the number of animals, sampling locations, and status of cat and dog cases in this study.
    • Table 3: Information of five cat cases should be individually listed in the Table.
    • Table 4 requires further clarification. It is not clear about “type of assay”, “animal species”, and “type of samples” from the Table information. Corresponding text in line 179 is not apparently relevant to the Table information.
    • In Discussion section, authors should describe current diagnostic options for SFTSV in humans and animals with citations. Although this study used conventional RT-PCR, it is important to provide the rationale to use the classical approach over the real-time PCR.

Minor points:

    • Figure 3 and 4 are not labelled for size or samples. Authors may combine two images in a single Figure.
    • Line 86-88: Authors should describe the preparation of RNA samples with serial RNA copy numbers: i.e., in vitro transcribed RNA with known copy numbers? If the reference control is DNA, the measurement indicates the cDNA detection limit.
    • Please describe about the content of negative controls in the Materials and Methods and the Figure 3, 4, and 5.
    • Line 133: “RT-PCR was performed to detect in clinical animal specimen” should be deleted, because this section is described about IgM and IgG detection.
    • Authors should clarify if they also tried to isolate SFTSV from those cat samples.
    • Additional analysis of M-segment or other genetic information (e.g., NGS) should better characterize viral isolates. It might be reassortant strains if several strains are co-circulating within the region.
    • Please provide further information of positive cats, in terms of the follow-up isolation from owners or treatment after the diagnosis, in Discussion section.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

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Reviewer #1: N/A

**********

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Reviewer #1: Yes

**********

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Reviewer #1: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this article, the authors describe the development and characterization of 2 sets of primers that detect SFTSV in cell supernatants and animal samples. This is a straightforward paper with strong methodology and is fairly well written, with only some minor grammatical changes required.

One change I would like to see is a better description of the impact and contribution of this assay. For example, some of the points that could be added include:

- please include a paragraph describing the clinical presentation of the cats in the study, the timelines of presentation and sampling, and whether they survived or not.

- Table 3 - since there are only five cats it would be nice to display data for each individual cat

- please describe why the authors developed this assay. Does the human rtPCR assay not work in animals? Does the animal rtPCR developed by the authors work in human samples?

- why was the L segment not chosen as a PCR target? It is highly conserved and would be a good choice for detecting as many clades as possible

- please provide background and discussion about how many serotypes exist for SFTSV. How do you know which clade to use for the neutralizing assays? Can antibodies from one clades cross-neutralize other clades? Since you only used one clade, could a serotype mismatch be a possible reason for the lack of neutralization?

Line 16 - remove the word "to"

Line 17 - change "shows" to "have"

Line 21 - change "by" to "from"

Line 23 - remove "were"

Line 23 - change "several" to the specific number of clades the PCR detects

Line 24 - change "by" to "using"

Line 29 - change "clades from" to "clades and from"

Line 25 - change to "where a high number of SFTS patients have been reported, indicating circulating SFTSV in the environment. This diagnostic assay could be a helpful tool in detecting SFTS in animals."

Line 51-52 - I would move this sentence to Line 42. Change to "...are reportedly spreading throughout..."

Line 54 - in your abstract you say the assay worked against several clades, here you say all clades. please pick the correct one and then be consistent.

Line 60 - delete "the"

Lines 86-88 - delete. these are results and do not belong in the methods section.

Line 98 - change to "Sequencing and phylogenetic analysis was performed on the viral S gene segment for all RT-PCR positive samples."

Line 159 - change "nucleotide" to "nucleotide sequence"

Line 163 - delete "according to previous studies"

Line 165 - change "positive on RT-PCR" to "PCR-positive"

Line 172 - the antibodies are still functional, they just are not neutralizing. please modify this.

Line 178 - please include a figure of the four positive bands for the five cats

Line 189 - neutralizing antibodies rarely appear before 7 days after infection, and it usually takes 10-14 days to see a robust response. in this study, your samples were taken too early to really assess whether neutralizing antibodies were induced.

**********

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Reviewer #1: No

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20 Nov 2020

Editor comments

Major points:

The title of this study does not reflect additional SFTS case identification in cats in Japan. The study in this manuscript identified five cats with detectable SFTSV RNA, and specific IgG / IgM. The development of conventional RT-PCR not sufficiently novel for publication by itself. Please consider changing the title, while revising the abstract and conclusion accordingly.

1. Figure 1 and 2 do not provide the rationale of given primer designs, which should be removed or moved to Supplementary materials, if required. The alignment of each primer sequence to representative strains in those clades might be alternatively shown.

Answer : Primers in this study were designed using all strains of figure 1 and 2. Since the consensus sequence for the primer design were made with these strains, we showed the phylogenic tree (figure 1) to cover all clades. Figure 2 might be moved to supplementary material.

2. Line 59: Authors should provide the number of animals, sampling locations, and status of cat and dog cases in this study.

Answer : The number of animals were added, cats are 37 cases and dogs are 19 cases (line 62). Samples were sent from all around Japan and we described the sites using maps (Figure 2). Also we added the status of dog cases as “Dogs showed anorexia, depression, fever and some gastrointestinal tract symptoms, including diarrhea and vomit (line 65-66).

3. Table 3: Information of five cat cases should be individually listed in the Table.

Answer : Information of five cat cases were listed individually in the Table 3.

4. Table 4 requires further clarification. It is not clear about “type of assay”, “animal species”, and “type of samples” from the Table information. Corresponding text in line 179 is not apparently relevant to the Table information.

Answer : We changed the Table 4.

5. In Discussion section, authors should describe current diagnostic options for SFTSV in humans and animals with citations. Although this study used conventional RT-PCR, it is important to provide the rationale to use the classical approach over the real-time PCR.

Answer : We added sentences in discussion section. “In Japan, for the diagnosis of human and animal SFTS cases, conventional one-step RT-PCR to detect the SFTSV genome, ELISA to detect the antibodies against SFTSV and the isolation of SFTSV are performed. However, non-specific reactions were obtained from some SFTS-suspected animal cases, especially dogs, using primers for human SFTS diagnosis. For the simple, rapid and specific detection of SFTSV RNA from SFTS-suspected animal cases, primer sets were newly designed in this study.” (Line 186-191)

Minor points:

1. Figure 3 and 4 are not labelled for size or samples. Authors may combine two images in a single Figure.

Answer : Figure 3 and 4 were labelled and combined as a single figure.

2. Line 86-88: Authors should describe the preparation of RNA samples with serial RNA copy numbers: i.e., in vitro transcribed RNA with known copy numbers? If the reference control is DNA, the measurement indicates the cDNA detection limit.

Answer : Because the copy numbers of RNA samples have been measured in our previous study, RNA samples with known copy numbers were diluted by 10-fold. We corrected sentences as “RNAs from three strains of SFTSV belonging to different clades - SPL010 (J1 clade, accession No. AB817999), cat#1 (C4 clade, accession No. DRA007207) and HB29 (C3 clade, accession No. NC_018137) - were used as positive controls. The copy numbers of RNA samples used as positive controls have been measured by real time RT-PCR according to our previous study. Then, RNA were diluted by 10-fold with diethylpyrocarbonate (DEPC)-treated distilled water (DW).” (Line 82-87)

3. Please describe about the content of negative controls in the Materials and Methods and the Figure 3, 4, and 5.

Answer : The content of negative control was added. “DEPC-treated DW was used as a negative control.” (Line 87)

4. Line 133: “RT-PCR was performed to detect in clinical animal specimen” should be deleted, because this section is described about IgM and IgG detection.

Answer : This sentence were deleted.

5. Authors should clarify if they also tried to isolate SFTSV from those cat samples.

Answer : We did not try to isolate SFTSV from those positive samples.

6. Additional analysis of M-segment or other genetic information (e.g., NGS) should better characterize viral isolates. It might be reassortant strains if several strains are co-circulating within the region.

Answer : It is of interest to clarify if the genomic reassortment event occurred in animal derived SFTS viruses, however, this is not an purpose of the manuscript so that the following sentence was added in Discussion section,. i.e.,

“In Japan, several phylogenetic clades are circulating in the SFTS endemic region, thus it is of interest to clarify if the genomic reassortment event occurred in animal derived SFTS viruses in future." (Line 198-201)

7. Please provide further information of positive cats, in terms of the follow-up isolation from owners or treatment after the diagnosis, in Discussion section.

Answer : We added the information in Discussion section, i.e., “After cats were diagnosed as SFTS, veterinarians were informed; 1) they should wear personal protective equipment (PPE), such as gloves, mask, face shield, goggle and gown, to treat SFTS-diagnosed cats since SFTSV could be secreted from blood, saliva, urine and feces for about two or three weeks, 2) they should consult a doctor if they have a direct contact with these blood, saliva, urine and feces and then have clinical symptoms, such as, fever, 3) they should inform owners of the animals not to contact without PPE.” (Line 205-210).

Review Comments to the Author

Reviewer #1: In this article, the authors describe the development and characterization of 2 sets of primers that detect SFTSV in cell supernatants and animal samples. This is a straightforward paper with strong methodology and is fairly well written, with only some minor grammatical changes required.

1. please include a paragraph describing the clinical presentation of the cats in the study, the timelines of presentation and sampling, and whether they survived or not.

Answer : We added sentences as “Five SFTS cat cases showed clinical symptoms including depression, loss of appetite and jaundice. They had body weight loss, fever, leukocytopenia (2180~7540/μL), thrombocytopenia (15,000~120,000/μL) and high total bilirubin level. All of five cats were indoor and outdoor and four of five cats had tick-bite history. Samples were collected within one week after disease onset. Two cats were dead and three cats recovered (Table 3).” (Line 165-169).

2. Table 3 - since there are only five cats it would be nice to display data for each individual cat

Answer : We added the information of five cats in table 3.

3. please describe why the authors developed this assay. Does the human rtPCR assay not work in animals? Does the animal rtPCR developed by the authors work in human samples?

Answer : Some researchers including us tried the human rtPCR assay to detect the animal SFTS cases. However, there were lots of non-specific results, especially in dogs. We performed the sequence analysis those cases, and found that some genomes of dogs could react in the human rtPCR assay. We described that in Discussion section (line 186-191).

4. why was the L segment not chosen as a PCR target? It is highly conserved and would be a good choice for detecting as many clades as possible

Answer : Because we thought that two segments are enough to diagnose animal SFTS cases, we chose two segments, S segment and M segment.

5. please provide background and discussion about how many serotypes exist for SFTSV. How do you know which clade to use for the neutralizing assays? Can antibodies from one clades cross-neutralize other clades? Since you only used one clade, could a serotype mismatch be a possible reason for the lack of neutralization?

Answer : We added about how many serotypes exist for SFTSV (line43-44). We tried the neutralization antibody assay using neutralizing antibody positive sera and SFTSV strain belonging to other clades. Although the rate of neutralization in one dilution point was different a little, the titer of neutralization antibody was not different. So, we performed the final neutralization antibody assay using one clade and described the results. Anyway, the titer of neutralizing antibody of five cats were under the limit of detection.

6. Line 16 - remove the word "to"

Answer : We removed the word “to”.

7. Line 17 - change "shows" to "have"

Answer : We changed “show” to “have”.

8. Line 21 - change "by" to "from"

Answer : We changed “by” to “from”.

9 . Line 23 - remove "were"

Answer : We removed “were”.

10. Line 23 - change "several" to the specific number of clades the PCR detects

Answer : We changed “several” to “six”.

11. Line 24 - change "by" to "using"

Answer : We changed “by” to “using”.

12. Line 29 - change "clades from" to "clades and from"

Answer : We changed “clades from” to “clades and from”.

13. Line 35 - change to "where a high number of SFTS patients have been reported, indicating circulating SFTSV in the environment. This diagnostic assay could be a helpful tool in detecting SFTS in animals."

Answer : We changed the sentence.

14. Line 51-52 - I would move this sentence to Line 42. Change to "...are reportedly spreading throughout..."

Answer : We moved the sentence to Line 43 and changed.

15. Line 54 - in your abstract you say the assay worked against several clades, here you say all clades. please pick the correct one and then be consistent.

Answer : We changed to “several clades” to be consistent.

16. Line 60 - delete "the"

Answer : We deleted “the”.

17. Lines 86-88 - delete. these are results and do not belong in the methods section.

Answer : We deleted these.

18. Line 98 - change to "Sequencing and phylogenetic analysis was performed on the viral S gene segment for all RT-PCR positive samples."

Answer : We changed as your advice (line 104-105).

19. Line 159 - change "nucleotide" to "nucleotide sequence"

Answer : We changed to “nucleotide sequence” (line 170).

20. Line 163 - delete "according to previous studies"

Answer : We deleted the phrase.

21. Line 165 - change "positive on RT-PCR" to "PCR-positive"

Answer : We changed to “PCR-positive” (line 176).

22. Line 172 - the antibodies are still functional, they just are not neutralizing. please modify this.

Answer : We changed to “could not neutralize SFTSV” (line 183).

23. Line 178 - please include a figure of the four positive bands for the five cats

Answer : We included all five positive bands in the Figure 5.

24. Line 189 - neutralizing antibodies rarely appear before 7 days after infection, and it usually takes 10-14 days to see a robust response. in this study, your samples were taken too early to really assess whether neutralizing antibodies were induced.

Answer : We changed the sentence like this.

“It takes approximately two weeks to see a robust immune response, the samples collected in this study might be too early to assess the neutralizing antibodies in the naturally infected animals.” (Line 213-215)

Submitted filename: Response to Editor reviewers comments.docx

2 Dec 2020

PONE-D-20-25871R1

Diagnostic system for the detection of severe fever with thrombocytopenia syndrome virus from suspected infected animals

PLOS ONE

Dear Dr. Morikawa,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The revised manuscript entitled “Diagnostic system for the detection of severe fever with thrombocytopenia syndrome virus from suspected infected animals” significantly improve the presentation of contents. Please consider a few additional minor changes.

Minor points:

    • Line 15: “those of SFTS patients, whereas SFTS-contracted cats….”
    • Line 22: “specifically detected six clades of SFTSV” in the Abstract was not shown in the Result section. Please consider the revision.
    • Line 35: “indicating the cat-to-human transmission of SFTSV”?
    • Line 46: a variety of animals, while viral RNA and antibodies…”
    • Line 64: “vomiting”
    • The rationale of new RT-PCR appeared in Discussion (lines 182 – 187). A similar brief rationale should be given in Introduction and Abstract sections.
    • Title: “Diagnostic system for the detection of severe fever with thrombocytopenia syndrome virus RNA from suspected infected animals” or “Diagnostic system for the RNA detection of severe fever with thrombocytopenia syndrome virus from suspected infected animals”

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.


4 Dec 2020

1. Line 15: “those of SFTS patients, whereas SFTS-contracted cats….”

Response : We corrected the sentence according to the comment (line 15-16).

2. Line 22: “specifically detected six clades of SFTSV” in the Abstract was not shown in the Result section. Please consider the revision.

Response : “six clades” were changed to “four clades” since two clades were not included in the clinical samples in the present study. However, the primers designed in the completely conserved region among eight clades, thus the PCR might detect all the eight clades. In this regard, we added the contents in the Result and Discussion section (line 173-174, 192-194).

3. Line 35: “indicating the cat-to-human transmission of SFTSV”?

Response : The sentence was corrected according to the comment (line 36).

4. Line 46: a variety of animals, while viral RNA and antibodies…”

Response : The sentence was corrected according to the comment (line 46).

5. Line 64: “vomiting”

Response : The sentence was corrected according to the comment (line 67).

6. The rationale of new RT-PCR appeared in Discussion (lines 182 – 187). A similar brief rationale should be given in Introduction and Abstract sections.

Response : The brief rationale were added in the Abstract and Introduction sections (line 18-19, 53-55).

7. Title: “Diagnostic system for the detection of severe fever with thrombocytopenia syndrome virus RNA from suspected infected animals” or “Diagnostic system for the RNA detection of severe fever with thrombocytopenia syndrome virus from suspected infected animals”

Response : Title was changed to “Diagnostic system for the detection of severe fever with thrombocytopenia syndrome virus RNA from suspected infected animals”.

Submitted filename: Response to Reviewers.docx

7 Dec 2020

Diagnostic system for the detection of severe fever with thrombocytopenia syndrome virus RNA from suspected infected animals

PONE-D-20-25871R2

Dear Dr. Morikawa,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Tetsuro Ikegami

Academic Editor

PLOS ONE


15 Dec 2020

PONE-D-20-25871R2

Diagnostic system for the detection of severe fever with thrombocytopenia syndrome virus RNA from suspected infected animals

Dear Dr. Morikawa:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

Dr. Tetsuro Ikegami

Academic Editor

PLOS ONE

Supplementary materials
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This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
https://www.researchpad.co/tools/openurl?pubtype=article&doi=10.1371/journal.pone.0238671&title=Diagnostic system for the detection of severe fever with thrombocytopenia syndrome virus RNA from suspected infected animals&author=&keyword=&subject=Research Article,Biology and Life Sciences,Molecular Biology,Molecular Biology Techniques,Artificial Gene Amplification and Extension,Polymerase Chain Reaction,Reverse Transcriptase-Polymerase Chain Reaction,Research and Analysis Methods,Molecular Biology Techniques,Artificial Gene Amplification and Extension,Polymerase Chain Reaction,Reverse Transcriptase-Polymerase Chain Reaction,Research and Analysis Methods,Immunologic Techniques,Immunoassays,Enzyme-Linked Immunoassays,Biology and Life Sciences,Organisms,Eukaryota,Animals,Vertebrates,Amniotes,Mammals,Cats,Biology and Life Sciences,Zoology,Animals,Vertebrates,Amniotes,Mammals,Cats,Biology and Life Sciences,Veterinary Science,Veterinary Medicine,Veterinary Diagnostics,Biology and Life Sciences,Organisms,Eukaryota,Animals,Vertebrates,Amniotes,Mammals,Dogs,Biology and Life Sciences,Zoology,Animals,Vertebrates,Amniotes,Mammals,Dogs,Biology and Life Sciences,Molecular Biology,Molecular Biology Techniques,Artificial Gene Amplification and Extension,Polymerase Chain Reaction,Research and Analysis Methods,Molecular Biology Techniques,Artificial Gene Amplification and Extension,Polymerase Chain Reaction,Biology and Life Sciences,Evolutionary Biology,Evolutionary Systematics,Phylogenetics,Phylogenetic Analysis,Biology and Life Sciences,Taxonomy,Evolutionary Systematics,Phylogenetics,Phylogenetic Analysis,Computer and Information Sciences,Data Management,Taxonomy,Evolutionary Systematics,Phylogenetics,Phylogenetic Analysis,Biology and Life Sciences,Physiology,Immune Physiology,Antibodies,Biology and Life Sciences,Immunology,Immune System Proteins,Antibodies,Medicine and Health Sciences,Immunology,Immune System Proteins,Antibodies,Biology and Life Sciences,Biochemistry,Proteins,Immune System Proteins,Antibodies,