Seoul Virus Infection and Spread in United States Home-Based Ratteries: Rat and Human Testing Results from a Multistate Outbrea

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The Journal of Infectious Diseases MAJOR ARTICLE

Seoul Virus Infection and Spread in United States Home-Based Ratteries: Rat and Human Testing Results From a Multistate Outbreak Investigation Barbara Knust,1, Shelley Brown,1 Annabelle de St. Maurice,1, Shannon Whitmer,1 Sarah E. Koske,2 Elizabeth Ervin,1 Ketan Patel,1 James Graziano,1 Maria E. Morales-Betoulle,1 Jennifer House,3 Deborah Cannon,1 Janna Kerins,1,4 Stacy Holzbauer,5 Connie Austin,6 Suzanne Gibbons-Burgener,2 Leah Colton,3 John Dunn,7 Sara Zufan,1 Mary Joung Choi,1 William R. Davis,1 Cheng-Feng Chiang,1 Craig R. Manning,1 Linda Roesch,1 Trevor Shoemaker,1 Lawrence Purpura,1 Jennifer McQuiston,1 Dallin Peterson,8 Rachel Radcliffe,9 Ann Garvey,9 Ellen Christel,10 Laura Morgan,11 Joni Scheftel,5 James Kazmierczak,2 John D. Klena,1 Stuart T. Nichol,1 Pierre E. Rollin1; on behalf of the Multistate Seoul Virus Outbreak Investigation Team Downloaded from https://academic.oup.com/jid/article/222/8/1311/5850525 by guest on 27 September 2021 1United States Centers for Disease Control and Prevention, Atlanta, Georgia, USA, 2Wisconsin Department of Health Services, Madison, Wisconsin, USA, 3Colorado Department of Public Health and Environment, Denver, Colorado, USA, 4Chicago Department of Public Health, Chicago, Illinois, USA, 5Minnesota Department of Health, St. Paul, Minnesota, USA, 6Illinois Department of Public Health, Springfield, Illinois, USA,7 Tennessee Department of Health, Nashville, Tennessee, USA, 8Utah Department of Health, Salt Lake City, Utah, USA, 9South Carolina Department of Health and Environmental Control, Columbia, South Carolina, USA, 10Iowa Department of Public Health, Des Moines, Iowa, USA, 11Manitowoc County Health Department, Manitowoc, Wisconsin, USA

(See the Editorial Commentary by Fill, on pages 1247–9.) Background. During 2017, a multistate outbreak investigation occurred after the confirmation of Seoul virus (SEOV) infections in people and pet rats. A total of 147 humans and 897 rats were tested. Methods. In addition to immunoglobulin (Ig)G and IgM serology and traditional reverse-transcription polymerase chain reaction (RT-PCR), novel quantitative RT-PCR primers/probe were developed, and whole genome sequencing was performed. Results. Seventeen people had SEOV IgM, indicating recent infection; 7 reported symptoms and 3 were hospitalized. All patients recovered. Thirty-one facilities in 11 US states had SEOV infection, and among those with≥ 10 rats tested, rat IgG prevalence ranged 2%–70% and SEOV RT-PCR positivity ranged 0%–70%. Human laboratory-confirmed cases were significantly associated with rat IgG positivity and RT-PCR positivity (P = .03 and P = .006, respectively). Genomic sequencing identified >99.5% homology between SEOV sequences in this outbreak, and these were >99% identical to SEOV associated with previous pet rat infections in England, the Netherlands, and France. Frequent trade of rats between home-based ratteries contributed to transmission of SEOV between facilities. Conclusions. Pet rat owners, breeders, and the healthcare and public health community should be aware and take steps to prevent SEOV transmission in pet rats and to humans. Biosecurity measures and diagnostic testing can prevent further infections. Keywords. Hantavirus; Seoul virus; Zoonotic.

Seoul virus (SEOV), an Old World Orthohantavirus, is globally Humans can become infected with SEOV through aerosol endemic due to the worldwide distribution of its primary ro- exposure to virus shed in rodent urine, saliva, or feces or via dent hosts: Rattus norvegicus, the Norway rat [1], and Rattus direct inoculation (eg, rodent bites or scratches, or contact with rattus, the black rat [2]. Trapping studies of wild rats in many mucous membranes) [12]. Human infections with SEOV and parts of the world have found SEOV ribonucleic acid (RNA) [3, other Old World hantaviruses are classically characterized as 4], infectious virus [5], and antibodies [6, 7]. Infected rats shed hemorrhagic fever with renal syndrome (HFRS). The HFRS virus in urine, saliva, and feces for periods from 1 month to symptoms include fever, headache, muscle aches, and abdom- >4 months [8], and they can develop antibodies while they are inal pain. Clinical manifestations can include acute kidney in- actively infected and shedding virus [9]. Rats become infected jury that may progress to oliguric renal failure, conjunctivitis, through bites or from exposure to infectious urine and feces of and hemorrhage in severe cases [13]. Mortality for SEOV pa- other rats [10, 11]. There is no known clinical disease exhibited tients with HFRS is approximately 2%. Mild or unapparent ill- by rats infected with SEOV. ness with SEOV infection is also reported [14]. Hantavirus infections from exposure to wild rodents are

most common in Asia, with more than 10 000 cases per year Received 29 January 2020; editorial decision 27 April 2020; accepted 31 May 2020; published reported in China alone [15]. Numerous SEOV outbreaks in online June 2, 2020. Correspondence: B. Knust, DVM, MPH, 1600 Clifton Rd. NE, MS H16-4, Atlanta, GA 30333 several countries have been reported among laboratory workers ([email protected]). who directly handled or were in the vicinity of infected labora- The Journal of Infectious Diseases® 2020;222:1311–9 tory rats, or those who worked with infected tissue culture lines Published by Oxford University Press for the Infectious Diseases Society of America 2020. This work is written by (a) US Government employee(s) and is in the public domain in the US. [16–18]. Effective biosecurity and test-and-cull strategies were DOI: 10.1093/infdis/jiaa307 developed to eradicate infection from research rat colonies, and,

US Pet Rat Seoul Virus Outbreak • jid 2020:222 (15 October) • 1311 currently, diagnostic testing for SEOV is recommended for rou- Pathogens diagnostic laboratory as previously described [27] tine rodent health monitoring [19]. In the United States, SEOV (Supplementary Appendix). A specimen was IgM positive when infections are uncommonly reported [20]. In previous studies, the adjusted optical density (OD) of at least one 400-fold dilu- SEOV cases were reported in patients with occupational expo- tion was greater than 0.1 and the sum of the adjusted OD for sure to wild rats in Texas [21] and Maryland [22] and recently the specimen was greater than 0.45. A specimen was IgG posi- in Washington DC [23]. tive when the adjusted OD of at least one 400-fold dilution was In 2013, SEOV infections were reported in “fancy rat” greater than 0.2 and the sum of the adjusted OD for the spec- breeders and colonies in the United Kingdom [24] and Sweden imen was greater than 0.95. A specimen was negative if it failed [25]. Animal trade between breeding colonies contributed to to meet either criterion. SEOV spread. In 2017, the US public health community inves- Ribonucleic acid was extracted from blood and tissue using an tigated a multistate outbreak of SEOV in pet rats. After initial automated magnetic bead separation system (MagMax). A pan- laboratory confirmation of 2 symptomatic case-patients from hantavirus L segment-nested RT-PCR assay was performed on a Wisconsin home-based rattery [26], trace-back and trace- extracted RNA. Sequences obtained from the second-round de- Downloaded from https://academic.oup.com/jid/article/222/8/1311/5850525 by guest on 27 September 2021 forward testing of rats and humans with rat contact found a oxyribonucleic acid fragments were used to develop a strain- total of 31 rat-owning households or ratteries (“facilities”) in specific, real-time RT-PCR (Supplementary Appendix). 11 states with human and/or rat SEOV infection [26]. In this report, we summarize the results of the epidemiologic inves- Ribonucleic Acid Sequencing and Phylogenetic Analysis tigation and laboratory testing of humans and rats during the Extracted SEOV RNA from multiple facilities and states were outbreak. selected for next-generation sequencing (NGS) using MiSeq and MiniSeq systems (Illumina), and reads were assembled MATERIALS AND METHODS to multiple full-length hantavirus reference genomes using

Epidemiologic Investigation Geneious mapper. Consensus sequences, excluding reference sequences, were generated in Geneious 9.1.4 using the highest Many details of the epidemiologic investigation have been de- quality threshold (Supplementary Appendix). scribed previously [26]. In brief, facilities that had either re- In June 2018, 15 months after the last positive rat was detected ceived rats from (trace-forward) or sent rats to (trace-backward) in the 2017 outbreak investigation, a rat carcass suspected of a facility with laboratory-confirmed SEOV infections in people SEOV infection was submitted to the CDC for testing and was or rats (confirmed facility) were suspected of having rats with RT-PCR positive. The RNA from this rat was included in NGS SEOV infection (suspected facility) (Supplementary Appendix analysis. Viral genomes were assembled using viral-ngs (Broad Figure 1). Testing of rats or exposed persons was offered to es- Institute) with a custom database consisting of pan-hantavirus tablish whether SEOV infection was present (Supplementary genomes. The SEOV genomes were deposited into GenBank Appendix), and exposed persons were interviewed about recent (MK360773-98). Nearly full-length genomes for each segment illness. If serology or reverse-transcription polymerase chain were aligned using MUSCLE in Geneious/v11.1.2, and phyloge- reaction (RT-PCR) testing found evidence of SEOV infection netic trees were constructed (Supplementary Appendix). in either rats or humans, the facility was classified as confirmed, and additional trace-forward and trace-backward investiga- Statistical Analysis tions occurred. If all tests were negative, the facility was no All human and rodent data, specimen type, and related test longer suspected and was considered “cleared.” Infection con- results were compiled in Microsoft Excel. Data were analyzed trol measures included quarantine of rats from confirmed and using SAS version 9.3. Statistical analysis included χ 2 test for cat- suspected facilities and euthanasia of infected rats. Guidance egorical comparisons and Wilcoxon rank-sum and Spearman was provided to rat owners on handling and cleaning methods correlation tests for continuous variable comparisons. P < .05 to prevent virus transmission (Supplementary Appendix). was considered statistically significant. Rat facilities that had ≥10 Recent or current SEOV infection in a human was confirmed rats tested were included for analyses comparing seroprevalence through detection of specific anti-SEOV immunoglobulin (Ig) and RT-PCR-positive prevalence and associations with human M or SEOV RNA. Acute SEOV infection was defined as a person SEOV infections. Rat movement networks were constructed ret- having contact with rats from a confirmed or suspected facility rospectively by entering trace-back information using UCINET who had either fever or other symptoms compatible with SEOV 6.636 and visualized using NetDraw 1.161 (Harvard, MA). infection and confirmed laboratory test results.

Serology and Reverse-Transcription Polymerase Chain Reaction RESULTS Human and rat anti-SEOV IgG and human IgM enzyme-linked Human Testing immunosorbent assay (ELISA) was performed at the Centers A total of 209 blood specimens from 176 individuals were re- for Disease Control and Prevention (CDC)’s Viral Special ceived for SEOV testing from December 2016 to May 2017.

1312 • jid 2020:222 (15 October) • Knust et al Of all 176 people tested, 65% were female, and the median age platelet values 143, 115; reference range 150–400), leukopenia was 27 years (range = 1–66). Forty-five persons were associated (n = 2; nadir value 3.5; reference range 4.5–10.8), hypergly- with confirmed rat facilities, whereas the remaining 131 per- cemia (n = 3; peak values 118, 124, 125; reference range 70–99), sons were associated with facilities that did not have confirmed elevated creatinine (n = 2; peak values 1.13; reference range SEOV infection. Four persons (9%) had only IgG detected and 0.5–1.1), and mild elevations in aspartate aminotransferase no symptoms, indicating previous exposure to SEOV. Of these (n = 3; peak values 64, 50, 41; reference range 10–40), alanine 4, 1 was associated with a facility that had positive rats and the aminotransferase (n = 2; peak values 62; reference range 10–50), remaining 3 persons were associated with 2 facilities that did and prothrombin time (n = 2; peak values 15.4 and 12.4; refer- not have rats available for testing at the time of the investigation. ence ranges 11.6–15.2 and 9.7–11.8, respectively). In addition, Seventeen of the 45 (38%) exposed persons had anti-SEOV partial thromboplastin time was elevated in 1 patient where it IgM, indicating recent infection—all other people tested were was measured (value = 31; reference range 22–30). Detailed IgM negative. Seven of these 17 individuals (41%) reported medical records were not available for 1 additional hospitalized illness that met the case definition for acute SEOV infection, patient who had pulmonary infiltrates noted on chest x-ray and Downloaded from https://academic.oup.com/jid/article/222/8/1311/5850525 by guest on 27 September 2021 4 sought healthcare when ill, and 3 (18%) were hospitalized. also experienced renal failure. All case-patients recovered from Three persons with symptoms did not seek healthcare, and the their illnesses with supportive care. remaining 10 individuals with IgM reported no symptoms. Seoul virus RT-PCR was performed on 3 laboratory- Rat Testing confirmed patients who were acutely ill at the time of specimen Overall, 1947 rat specimens were tested by the CDC, which in- collection, and 2 were both IgM positive and RT-PCR positive, cluded 1377 blood specimens from 91 rat facilities. Tests per- at 2 and 4 days postonset. For 2 case-patients with serial ELISAs formed varied, depending on the specimen types submitted and performed, IgM persisted beyond 83 and 115 days postonset, the blood volume available. Serology was performed on all blood respectively, but was undetectable at 145 days (Supplementary specimens received, whereas RT-PCR was only performed if ad- Appendix Table 3). Sin Nombre virus (SNV) ELISA was per- equate volume was available. Evidence of rats with SEOV infec- formed on 25 specimens from 21 SEOV antibody-positive tion was found in 24 facilities (26%), and the number of rats patients; although cross-reactivity was observed for some indi- tested ranged from 1 to 127. Among 897 total rats tested from viduals that had high IgG or IgM titers, the SNV ELISA did not confirmed facilities, 243 (25%) had evidence of SEOV infec- meet the criteria for positivity in 18 of 23 (78%) IgM-positive tion either by IgG or RT-PCR positivity or both (Table 2). Four specimens and 18 of 23 (78%) IgG-positive specimens, with an facilities confirmed rats’ SEOV infection by testing at a com- estimated sensitivity of 22%. mercial laboratory, and 3 facilities had humans test positive but Among symptomatic case-patients, illness onsets occurred no rats were tested, so the total number of confirmed facilities between early December 2016 and late April 2017 (Figure 1). in the US outbreak was 31. Prevalences of ELISA-positive and Commonly reported symptoms included fever, headache, RT-PCR-positive rats varied considerably by facility: 2%–70% and muscle aches (Table 1). Medical records were available IgG positivity and 0%–70% RT-PCR positivity in facilities with for 3 case-patients who sought healthcare during their illness; at least 10 rats tested (n = 18 facilities) (Table 2). Seoul virus all 3 had proteinuria detected, and 2 had microhematuria. was not isolated in any virus culture attempts (Supplementary Additional physical exam and laboratory abnormalities in- Appendix). cluded tachycardia (n = 2; 133 beats per minute), tachypnea Of 547 rat carcasses tested by both RT-PCR and ELISA, 29% (n = 1; 25 breaths per minute), thrombocytopenia (n = 2; nadir were RT-PCR positive and 30% were IgG positive; 23% had both IgG and RNA, whereas 6% were RT-PCR positive only and 7% were IgG positive only (Table 3). Reverse-transcription pol- 2 ymerase chain reaction was performed on antemortem venous blood specimens and postmortem lung tissue from 41 rats at 2 confirmed facilities. A total of 28 rats (68%) were RT-PCR pos-

1 itive in lung tissue, whereas only 12 (29%) were also positive in venous blood. The sensitivity of venous blood in detecting

Cases Reported SEOV RNA compared with lung tissue was 43% (Table 4). There was no relationship between facility size (approximated 0 04-Dec-16 01-Jan-17 05-Feb-17 05-Mar-17 02-Apr-17 by number of rats tested) and seroprevalence or RT-PCR prev- Week of Illness Onset alence (IgG Spearman coefficient = −0.29, P = .24; RT-PCR Spearman coefficient = 0.21, P = .44). Figure 1. Count of symptomatic laboratory-confirmed human Seoul virus case- A total of 12 confirmed facilities had both humans and≥ 10 rats patients by week of illness onset (n = 7). tested by IgG and 10 by RT-PCR. Among these, the median rat

US Pet Rat Seoul Virus Outbreak • jid 2020:222 (15 October) • 1313 Table 1. Reported Symptoms by 7 Case-Patients With Laboratory-Confirmed Recent SEOV Infection (IgM Detected)

Symptom Cases Reporting (%) Symptom Cases Reporting (%) Symptom Cases Reporting (%)

Muscle aches 6 (86) Joint pain 3 (43) Hematuria 1 (14) Headache 6 (86) Chills 3 (43) Ocular hyperemia 1 (14) Fever 5 (71) Diarrhea 3 (43) Blurred vision 1 (14) Decreased appetite 4 (57) Sore throat 2 (29) Back pain 1 (14) Nausea 4 (57) Dizziness 2 (29) Chest pain 1 (14) Abdominal pain 3 (43) Shortness of breath 2 (29) Sweating 1 (14) Cough 3 (43) Weight loss 2 (29) Drowsiness 1 (14) Abbreviations: IgM, immunoglobulin M; SEOV, Seoul virus.

SEOV IgG seroprevalence in facilities with at least 1 IgM positive in facilities where humans tested negative was 4%, a significant human was 38%, whereas median rat SEOV IgG seroprevalence association (Wilcoxon rank sum, P = .01). Downloaded from https://academic.oup.com/jid/article/222/8/1311/5850525 by guest on 27 September 2021 in facilities where humans tested negative was 13%. This differ- ence was significant by Wilcoxon rank sum (P = .03). The median Ribonucleic Acid Sequencing prevalence of RT-PCR-positive rats in facilities with IgM-positive In agreement with Kim et al [28], most SEOV genome humans was 42%, whereas median RT-PCR-positive prevalence sequences analyzed clustered together by geographic origin.

Table 2. SEOV ELISA and RT-PCR Results for Humans and Rats Tested at 31 Confirmed Facilities

Rats RT-PCR Rats RT-PCR Humans IgM/ Humans IgM Facility ID Rats IgG Tested Totala Rats IgG Positive (%) Tested Total Positive (%) IgG Tested Total Positive (%)

1 127 65 (51%) 129 48 (37%) 2 2 (100%) 2 107 4 (4%) 65 0 0 3 101 2 (2%) 9 0 0 4 98 27 (28%) 98 23 (23%) 5 1 (20%) 5 94 29 (31%) 94 42 (45%) 8 2 (25%) 6 50 2 (4%) 0 0 7 43 2 (5%) 43 4 (9%) 1 0 8 35 5 (14%) 7 2 (29%) 2 1 (50%) 9 34 7 (21%) 34 1 (3%) 1 0 10 29 11 (38%) 29 9 (31%) 2 1 (50%) 11 26 15 (58%) 26 16 (62%) 4 4 (100%) 12 26 17 (65%) 11 4 (36%) 0 13 26 1 (4%) 24 0 1 0 14 25 2 (8%) 16 1 (6%) 0 15 19 6 (32%) 19 1 (5%) 1 0 16 16 1 (6%) 1 0 0 17 15 1 (7%) 0 2 0 18 10 7 (70%) 10 7 (70%) 2 1 (50%) 19 6 2 (33%) 6 3 (50%) 0 20 3 2 (67%) 0 0 21 2 2 (100%) 2 2 (100%) 2 1 (50%) 22 2 1 (50%) 2 1 (50%) 5 0 23 2 1 (50%) 2 1 (50%) 3 0 24 1 1 (100%) 1 1 (100%) 1 0 25b 0 0 3 0 26 0 0 1 1 (100%) 27b 0 0 2 0 28b 0 0 2 0 29b 0 0 3 0 30 0 0 2 2 (100%) 31 0 0 1 1 (100%) Total 897 213 (24%) 628 166 (26%) 56 17 (30%)

Abbreviations: ELISA, enzyme-linked immunosorbent assay; ID, identification; Ig, immunoglobulin; RT-PCR, reverse-transcription polymerase chain reaction; SEOV, Seoul virus. aRepresents total number of individual animals tested per facility. bSEOV infection was confirmed in rats via testing at a commercial laboratory.

1314 • jid 2020:222 (15 October) • Knust et al Table 3. Comparison of SEOV blood IgG ELISA and Carcass RT-PCR 201802480) suggests that the outbreak strain continues to circu- Results Performed in Parallel on 547 Rats late in the United States.

RT-PCR-Positive Lung RT-PCR-Negative Test Result Tissue Lung Tissue Total Trace-Back Findings and Rat Facilities

IgG-positive blood 128 (23%) 36 (7%) 164 (30%) Thirty-one facilities in 11 states were confirmed to have SEOV IgG-negative blood 31 (6%) 352 (64%) 383 (70%) infection through testing of rats and/or humans. Affected facil- Total 159 (29%) 388 (71%) 547 ities were located throughout the United States, with the greatest Abbreviations: ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; RT-PCR, reverse-transcription polymerase chain reaction; SEOV, Seoul virus. number of confirmed facilities in Illinois (n = 12) and Wisconsin (n = 5). Exchange of rats with Canadian facilities was noted, and the Public Health Agency of Canada’s investigation identified Table 4. Comparison of SEOV Blood and Carcass RT-PCR Results additional confirmed ratteries and 1 recent human case [26]. Performed in Parallel on 41 Rats Confirmed US facilities included home-based ratteries, local Downloaded from https://academic.oup.com/jid/article/222/8/1311/5850525 by guest on 27 September 2021 RT-PCR-Positive RT-PCR-Negative pet stores, and homes. Rats were sold and traded as household Test Result Lung Tissue Lung Tissue Total pets, breeding animals, and for feeding to carnivores (snakes RT-PCR-positive blood 12 (29%) 0 12 (29%) or raptors). No commercial ratteries supplying national or re- RT-PCR-negative blood 16 (39%) 13 (31%) 29 (71%) gional chain pet stores were apparently involved. Trace-back in- Total 28 (68%) 13 (31%) 41 vestigations found that rats were commonly exchanged between Abbreviations: RT-PCR, reverse-transcription polymerase chain reaction; SEOV, Seoul virus. facilities, mainly for breeding purposes (Figure 3). Given the complex movement of infected rats between facil- L, M, and S segment sequences from the 2017 outbreak were ities, we were unable to determine a single source of infected nearly identical (99.565% L, 99.793% M, 99.509% S minimum animals. However, the close match of sequence to the Cherwell pairwise identity) (L phylogenetic tree [Figure 2]; M phyloge- strain as previously described suggests possible importation netic tree and S phylogenetic tree [Supplemental Figures 2 and to the United States and Canada from the United Kingdom 3]) and clustered on the same clade as a sequence from a pet rat or Europe. Some confirmed facilities did not have available or in Cherwell, England in 2013 (99.436% L, 99.586% M, 99.167% complete records of rat transactions with other facilities, and S minimum pairwise identity) [29]. Sequences on the Cherwell so ascertainment of all infected rats was incomplete. Details clade are distinct from SEOV sequences collected from wild rats of public health measures, education, and outbreak investiga- in the United Kingdom (2012, Humber strain), wild rats in the tion coordination are described further in the Supplementary United States (2002, Northeast Baltimore), and rats collected Appendix. in the Netherlands in 2013 (Rn84). The inferred phylogenetic DISCUSSION relationships support a rapid and broad geographic expansion of the Cherwell clade that occurred internationally and within Altogether, the 2017 US SEOV outbreak investigation identi- the United States in a short time frame. Furthermore, a se- fied 31 facilities with infected rats or people, and 17 people with quence derived from a pet rat in Illinois in June 2018 (Specimen evidence of recent infection. Although SEOV was described in

AF288297 SEOVL99 CHINA XXX China, North Korea Group A L 78 China, France Group B 93 China, South Korea, USA Group C 88 MK360809 SEOV 201700420 IL USA 23-Jan-2017 MK360808 SEOV 201701022 IL USA 23-Jan-2017 MK360807 SEOV 201700423 IL USA 23-Jan-2017 MK360806 SEOV 201701261 WI USA 02-Feb-2017 MK360800 SEOV 201802480 IL USA 05-Jun-2018 MK360810 SEOV 201700683 IL USA 25-Jan-2017 MK360813 SEOV 201700860 WI USA 24-Jan-2017 MK360811 SEOV 201701321 CO USA 14-Jan-2017 MK360812 SEOV 201701593 CO USA 02-Feb-2017 MK360803 SEOV 201701093 IL USA 29-Jan-2017 83 MK360802 SEOV 201701367 TN USA 03-Feb-2017 MK360801 SEOV 201702096 IA USA 01-Mar-2017 MK360806 SEOV 201701554 UT USA 02-2017 Group E 98 MK360804 SEOV 201701555 UT USA Feb-2017 KM948594 SEOV Cherwell UK 2013 MG764083 SEOV SEOV/NL/Rn2147/2016 Netherlands Sep-2016 88 92 MG764082 SEOV SEOV/NL/Rn2125/2016 Netherlands Sep-2016 JX879770 SEOV Humber UK 2012 85 100 KM948596 SEOV Humber UK 2012 98 MG972931 SEOV Rn84 Netherlands 2013 KT897724 SEOV Northeast Baltimore USA 2002 79 KX079474 SEOV IR162 UK 1984 100 KX079471 SEOV IR33 UK 1984 94 KM948595 SEOV IR461 UK 1984 85 KX079468 SEOV IR473 UK 1984 KP900346 SEOV JiangxiXianjianRn-07-2011 China 2011 Group D KC490924 ANJO Anjozorobe/Rr/MDG/2009/ATD261 Madagascar 2009

0.02 Substitutions/site

Figure 2. Phylogenetic tree of L segment of Seoul virus (SEOV) sequences from 2017 US outbreak and select reference SEOV strains.

US Pet Rat Seoul Virus Outbreak • jid 2020:222 (15 October) • 1315 13 A 3 28 2 17 8 B

27 26 7 9

24 11 Downloaded from https://academic.oup.com/jid/article/222/8/1311/5850525 by guest on 27 September 2021 18 5 12

4 10 1

16 31 25

15 23 Confirmed USA Facility 6 19 20 Confirmed Canadian Facility 14 22 Rat movement between facilities

30 Unconfirmed rat movement

Figure 3. Transmission chain of Seoul virus-infected rats between confirmed facilities. wild Norway rats in the United States [7, 20, 21, 30–33], it was Gathering a patient’s history of animal exposures and travel is not reported previously in pet rats. It is likely that SEOV was re- important in the diagnosis of rare infectious diseases, and this cently introduced into this pet rat population, considering that is especially crucial for identifying hantavirus disease. The non- 81% of antibody-positive people in this outbreak had IgM de- specific clinical symptoms of the first patient and many others tected, indicating recent exposure to SEOV (<6 months) despite in the outbreak could have easily been overlooked by clinicians; long-term rat contact. Many case-patients with evidence of re- fortunately, history of rodent exposures was noted, which led to cent infection were asymptomatic. Fewer than half (7) had acute hantavirus testing [26]. Hantavirus infections are nationally no- illness symptoms, and 3 were hospitalized and recovered after tifiable in the United States. The CDC, several state laboratories, supportive care. Among case-patients with medical records and a commercial diagnostic laboratory provide diagnostic se- available, proteinuria was universally detected, as was previ- rological testing for hantavirus infection, and the assays used ously reported in 51 SEOV patients in China [34]. Proteinuria are primarily directed toward detection of antibodies to New is a highly sensitive and universal clinical indicator for HFRS World orthohantaviruses (eg, SNV and other related viruses). [13]. One patient had acute renal failure, a frequent feature of Although antibody cross-reactivity was observed between Old severe HFRS, and also pulmonary infiltrates, which has been World and New World orthohantavirus assays, the SNV IgM described in both New World and Old World hantavirus infec- and IgG ELISAs most frequently used are poorly sensitive. If tions [13, 34, 35]. The relatively mild clinical course of illness in SEOV infection is suspected, the clinician should specifically these patients stands in contrast to most New World hantavirus request SEOV antibody testing, which is available from the infections, where intubation and treatment for hypotension are CDC. The CDC routinely performs both SEOV and SNV anti- a frequent feature [1]. body ELISA on all specimens submitted for hantavirus testing.

1316 • jid 2020:222 (15 October) • Knust et al There was strong bootstrap support for inferred relatedness frequent practice of trading rats between ratteries for breeding of US pet rat SEOV strains to the Cherwell strain [29], which purposes. Rattery owners can reduce the risk of SEOV infection points to international trade of pet rats as the likely source of in both their animals and in people by observing biosecurity this outbreak in the United States and Canada. The Cherwell measures that will reduce infection transmission between an- strain has also appeared in France and the Netherlands asso- imals. Biosecurity methods include a 4-week quarantine and ciated with pet or feeder rats in 2014 and 2016 [36, 37]. The serological testing of new animals before commingling with molecular evidence supports the spread of the Cherwell strain the animals in the colony, cohorting animal groups, regular to rats in 5 countries to date. cleaning and disinfection of enclosures, and recordkeeping of Currently, the only regulation governing the international animal acquisitions. Resources regarding SEOV testing, preven- trade of rodents into the United States is a ban on rodents tion, and best management practices are available from several originating from Africa, which was developed after the 2003 sources [30, 44, 45]. Previous interviews with pet rat owners in monkeypox outbreak that affected 41 people in 5 states [38, the United Kingdom found that education and communication

39]. Parallels between the monkeypox and SEOV outbreak about SEOV should center on protecting the health and purity Downloaded from https://academic.oup.com/jid/article/222/8/1311/5850525 by guest on 27 September 2021 can be found: both outbreaks could be linked to imported in- of the pet rat colony through biosecurity measures [46]. fected pet rodents, and both outbreaks were focused in the Observational results reported here have limitations because upper Midwestern United States, with spread through trade of different specimens were available for testing from different animals across state lines. Aside from SEOV and Monkeypox, ratteries, and rats were not sampled randomly. Furthermore, we frozen rodents imported to the United Kingdom and the United had limited information of the timeframe during which SEOV States have also been linked to outbreaks of Salmonella [40, 41], infection was introduced into a given facility or the number of and other zoonotic diseases may be spread by rodents across in- infected animals introduced. ternational borders, such as plague, tularemia, and lymphocytic choriomeningitis virus. After the 2003 US ban on importation CONCLUSIONS of African rodents, rodent importations to the United States Despite extensive efforts to identify all facilities with SEOV- from other international regions continue to rise, particularly infected rats, we were not able to trace the outbreak to its ori- rodents originating from Europe [42]. gins, nor completely trace all potentially infected rats from all Seoul virus antibody testing for rat blood specimens is avail- confirmed facilities, which contributed to gaps in identifying able from commercial diagnostic laboratories, as is RT-PCR for and controlling further spread of SEOV. Since this outbreak blood, tissue, and environmental swab specimens. We found investigation concluded in May 2017, no further cases of pet that IgG positivity is a moderately sensitive (80.5%) indicator rat-associated SEOV were identified in humans. However, in of an individual animal’s current SEOV infection status. Blood June 2018, an infection was detected in a pet rat from Illinois RT-PCR had relatively low diagnostic sensitivity (43%). We (Specimen 201802480; Figure 2), confirming the Cherwell did not test any environmental swab specimens during the in- strain’s ongoing presence. As a result, we expect that SEOV is vestigation, and we are not aware of any validation studies to still circulating in US pet rats. Pet rat owners and breeders, vet- measure the suitability of environmental swabs to detect SEOV erinarians, and public health officials should be prepared for fu- shed by infected rats. Until evidence can be provided, we would ture cases. Use of biosecurity practices and testing can prevent not recommend testing environmental swab specimens as a re- further transmission in pet rats, whereas clinical awareness of liable indicator of SEOV infection. Currently, serology remains symptoms and gathering of pertinent animal exposure history the best antemortem diagnostic test to determine whether a rat can help identify human cases. Prompt reporting of suspected has or had SEOV infection. human cases and laboratory confirmation will facilitate identi- Rat seroprevalence and RT-PCR positivity varied widely fication and response. among facilities with ≥10 rats tested; the highest IgG prevalence observed was 70%, and the highest RT-PCR prevalence was 62%. Supplementary Data Comparably high prevalences have been previously observed in Supplementary materials are available at The Journal of Infectious United Kingdom pet rat facilities [29] and in wild rat capture Diseases online. Consisting of data provided by the authors to studies [7, 11]. There were associations between increasing rat benefit the reader, the posted materials are not copyedited and IgG prevalence and RT-PCR tissue prevalence and the occur- are the sole responsibility of the authors, so questions or com- rence of human SEOV infections. This could be due to more ments should be addressed to the corresponding author. rodents with active infections and viral shedding in the environment, leading to increased exposure risk for rat handlers. Notes Outbreak facilities were largely home-based pet rat breeding Disclaimer. The findings and conclusions in this report are operations, and they included households with pet rats and those of the authors and do not necessarily represent the offi- small pet stores. An important factor in the virus spread was the cial position of the Centers for Disease Control and Prevention.

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