CDC Weekly

Preplanned Studies

Hemorrhagic Fever with Renal Syndrome — Province, China, 1999−2018

Cui Shang1,&; Yingwei Sun2,&; Qiangling Yin1; Xiaoxia Huang1; Xuesheng Liu2; Quanfu Zhang1; Lingling Mao2; Chuan Li1; Aqian Li1; Qin Wang1; Lina Sun1; Mifang Liang1; Shiwen Wang1,#; Dexin Li1,#; Jiandong Li1,#

Seoul virus and sub-genotype VI and VII of Hantaan Summary virus were also detected in Liaoning. The dynamic What is already known on this topic? changing of hotspots, the scale of the outbreaks, and Hemorrhagic fever with renal syndrome (HFRS) is sporadic cases required precise risk-based strategies that endemic in Liaoning Province. Both Seoul and are integrated and regional precise to target the affected Hantaan virus are circulating in rodents, and epidemic people for further improvement of HFRS prevention outbreaks and sporadic cases have been recorded every and control. year since the disease was recognized. Data for HFRS patients were obtained from the What is added by this report? Chinese National Notifiable Disease Surveillance The epidemic trend of HFRS over the past 20 years System (NNDSS), and the data were analyzed with (1999–2018) in Liaoning was analyzed, which showed Excel (version 2013, Microsoft, USA) and SPSS both regional complexity and consistence with the software (version 16.0, SPSS Inc., USA). Geographic epidemic in China. Genetic and antigenic stability of maps were created using ArcGIS Software (ESRI, the circulating hantavirus were demonstrated, which Redlands, CA, USA). suggested the effectiveness of the approved inactivated Rodents were trapped with snap-traps in residential vaccine currently used in China. areas and the fields of national HFRS surveillance sites. What are the implications for public health The rodents serum antibodies against hantavirus were practice? detected using double antigen sandwich ELISA (1). Precise risk-based strategic practices that are integrated The rodents’ lungs were tested for hantavirus antigen and regional are required for further improvement of with fluorescence labelled monoclonal antibody by the prevention and control of HFRS. direct immunofluorescence assay (2) and for viral genomic RNA by real-time RT-PCR (3). Positive test Hemorrhagic fever with renal syndrome (HFRS) is samples were loaded to Vero E6 cells for virus an important public health threat in China, and serious isolation. The antigenicity of new viral isolates was epidemic outbreaks and sporadic cases were recorded evaluated with 30 reference sera collected from in the 1980s and 1990s. The incidence and mortality recovered patients of HFRS using the plaque reduction have declined in the past 20 years in China, but the neutralization test (PRNT) assay. The complete L, M, epidemic characteristics of HFRS are highly variable at and S segments of the viral genome were amplified by both the provincial and county levels. Liaoning overlapping PCR and sequenced with the amplification Province is typically a natural focus of HFRS, and primers using BIG DYE terminator and a capillary endemic HFRS in Liaoning might reflect the sequencer. The hantavirus genomic sequence deposits challenges facing China. A total of 39,352 HFRS cases in GenBank were collected for comparative analysis. with 285 deaths were reported to the Chinese National All the sequences were aligned with the MAFFT Notifiable Disease Surveillance System (NNDSS) from program, version 7 (EMBL-EBI, UK). Comparative 1999 to 2018, and the incidence and case fatality rate analyses of nucleotide sequences and amino acid demonstrated region-specific characteristics and sequences were operated by DNASTAR (Madison, spatiotemporal variation of the HFRS epidemic. Wisconsin, USA). Phylogenetic trees were generated Hantaviruses circulating in rodents were detected that using the neighbor-joining (NJ) method implemented showed the Seoul virus was the major pathogen causing in MEGA6.0 (4). Topologies were evaluated by HFRS in the province. In addition, sub-genotype III is bootstrap analysis of 1000 iterations. the major genotype, and sub-genotype I and IV of A total of 39,352 HFRS cases with 285 deaths were

350 CCDC Weekly / Vol. 2 / No. 20 Chinese Center for Disease Control and Prevention China CDC Weekly reported from 1999 to 2018 in Liaoning. The HFRS varied in different years and places, and the incidence rate varied from 1.71 to 13.05 per 100,000 fall/winter peak was greater in City and persons annually, reaching a peak in 2004, decreasing City. significantly from 2006, reaching a plateau in 2007, The reported cases involved all the 14 cities in the then fluctuating between 1.71–3.0 per 100,000 province, but 7 cities contributed 84% of reported persons (Figure 1). The national incidence rate, cases. These cities are (7,114 cases, 18.08%), meanwhile, varied from 0.65 to 3.93 per 100,000 (5,367 cases, 13.64%), Dandong (5,038 cases, persons between 1999 and 2018, started to decrease 12.80%), (4,631 cases, 11.77%), dramatically since 1999 reaching a plateau in 2007, (4,194 cases, 10.77%), (3,524 cases, 8.96%), then fluctuated at a level of 0.65–0.99 per 100,000 and Fushun (3,191 cases, 8.11%). The fluctuations in (Figure 1). incidence for each city were not synchronous, and the In Liaoning, reported HFRS cases peak twice difference was statistically significant (Figure 2). annually. The spring peak, occurring between March The ages of the 23,554 HFRS cases reported in and May, contributed 35% of cases, and the fall-winter 2004–2018, ranging from infancy to 85 years (median: peak, occurring between October and December, 43 years). The majority of the cases (85.27%) occurred contributed 26.5% of cases overall. In most prefecture- between 15–60 years, while 1.78% were in children level cities, the spring peak was greater than the fall- <14 years and 12.94% were in persons >60 years. In winter peak, however, the monthly distribution of the past 20 years, the proportion of reported cases in

A 60,000 600

50,000 National reported cases 500 National reported deaths 40,000 400

30,000 300 Number of cases

20,000 200 Number of deaths

10,000 100

0 0 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 Year

B 60,000 50

50,000 40 Reported cases in Liaoning 40,000 Reported deaths in Liaoning 30 30,000 20 Number of cases

20,000 Number of deaths

10 10,000

0 0 2018 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 Year

FIGURE 1. The reported cases and deaths of hemorrhagic fever with renal syndrome (HFRS) from 1999 to 2018 in China (A) and Liaoning Province (B).

Chinese Center for Disease Control and Prevention CCDC Weekly / Vol. 2 / No. 20 351 China CDC Weekly

1999 2000 2001 2002 2003

2004 2005 2006 2007 2008

2013 2009 2010 2011 2012 HFRS incidence (1/100,000 persons) 0 0.001–3.000 3.001–5.000 5.001–10.000 2014 2015 2016 2017 2018 10.001–15.000 15.001–25.000 >25.000 N 01530 60 Miles

FIGURE 2. Maps showing prefecture incidence rate of hemorrhagic fever with renal syndrome (HFRS) from 1999 to 2018 in Liaoning Province. the 55–65 years group has increased, while that of the mouse (Mus musculus) was next highest. The captured 30–40 years group has decreased significantly densities of rodents in residential areas was higher than (Supplementary Figure S1, available in http://weekly. in fields. In residential areas, the antigen detection rate chinacdc.cn/), which may be related to the fact that the was about 5.8% (29/500), ranging from 0% (0/50) in old have replaced the young as the main agricultural Benxi County to 14% (7/50) in Yuhong and production work force in most rural areas of China. Haicheng City; and the serum antibody detection rate The ratio of male to female patients was 2.88:1. was about 17.2% (86/500), ranging from 2% (1/50) in Among female patients, the proportion of cases in the Benxi county to 50% (25/50) in Fengcheng County highest age group was higher than that in the male and Dandong City. In the fields, the antigen detection (Supplementary Figure S1). Most patients (62.74%) rate was about 4.6% (23/500), ranging from 0% were farmers, followed by housewives and unemployed (0/50) in to 32% (16/50) in Haicheng (12.94%), and students and non-agriculture workers City; and the serum antibody detection rate was about were the other two occupations at relative high risk of 9.2% (46/500), ranging from 0% (1/50) in Yuhong acquiring HFRS (Supplementary Figure S2, available District to 46% (23/50) in Benxi County. Hantavirus- in http://weekly.chinacdc.cn/). There were 285 deaths antigen positive rodents were further tested by RT- in total from 1999 to 2018 with an average case fatality PCR assay, viral genome RNA were detected from 18 rate of 0.72%, which involved all 14 cities. Areas with rodents using real-time RT-PCR (3), and the complete high mortality were usually areas with low prevalence, genome of L, M and S segments were obtained from such as (4.84%, 3/62), (2.31%, 16 Rattus norvegicus lung samples directly, including 3 7/303), (2.01%, 17/847), and (1.99%, in Fushun, 4 in Shenyang, 2 in Jinzhou, and 7 in 11/553). To some extent, it showed that the experience Tieling. Two viral isolates were obtained from those and awareness of clinical doctors played an important lung samples collected at Tieling, named LNTL001 role in the treatment of HFRS. and LNTL003. No significant antigenicity difference Previous laboratory surveillance data of hantavirus was observed between the two new isolates and the were collected and sorted. In 2018, 867 rodents were reference strain L99 in plaque reduction neutralization trapped with 25,336 snap-traps in residential areas and tests with blood from 30 recovered patients. in fields in the cities and counties of Shenyang, Benxi, Pair-wise alignment and comparison of complete Dandong, Jinzhou, and Anshan. The predominant genome sequences showed that both the nucleotide rodent species was Rattus norvegicus in residential areas and amino acid sequences were closely related to each and Apodemus agrarius in the fields, and the house other, with similarity above 95%. The sequences

352 CCDC Weekly / Vol. 2 / No. 20 Chinese Center for Disease Control and Prevention China CDC Weekly obtained in 2018 shared 95.6%–100% of amino acid The risk of occasional spillover of hantavirus from homology and shared 94%–99.7% of nucleotide rodents to humans showed region-specific homology with sequences obtained previously. No characteristics. Areas with high mortality were observed significant recombination was observed among these in areas with low prevalence, which suggests that lack sequences using recombination detection program of experience and awareness of diagnosis and treatment version 4 ( RDP4 ) software (5). Phylogenetic analyses may cause patients to miss the best period of treatment showed that the hantavirus in Liaoning could be and increase the patient's severity and mortality rate, clustered into the clade of sub-genotype III (S3) and which had been crucial to improving the prevention sub-genotype I (S1) of the Seoul virus, and sub- and treatment of HFRS. In China, the appropriate age for vaccination of current approved HFRS vaccines genotype VI of Hantaan virus (H6) (2) (Figure 3). ranged from 15 to 60 years, while the population out Seoul virus was the predominant pathogen, and S3 was the protection range of vaccination contributed about the major genotype currently circulating in the 15% of total reported cases, which is another challenge province. S4 and H7 of Hantaan virus were also found faced by current prevention and control work, in the province (6–7). especially with an increasing proportion of cases over 60 years old. DISCUSSION Both Seoul and Hantaan virus are circulating in Liaoning. Seoul virus is the main pathogen that cause HFRS is endemic in Liaoning, and the incidence HFRS, and the major sub-genotype is S3. The highly peaked twice a year and varied in a cyclic fashion (8). close relatedness of complete genome sequences

ShenyangRn28 ShenyangW43 HuludaoRn68 LNTL004 A ShenyangW309 B C BjHD01 ShenyangW297 huluisland2014-877-1 51 ShenyangW280 HLD65 HBQ17 HLD78 LNJZ009 ShenyangRn32 changxingisland2012-232-8 47 LNJZ010 ShenyangW281 changxingisland2012-234-3 ShenyangW3 LNFS001 ShenyangRn183 changxingisland2012-321-8 22 ShenyangW37 LNFS003 ShenyangRn19 songmuisland2012-324-1 56 LNFS004 ShenyangW2 songmuisland2012-324-5 ShenyangW289 Jinzhou/14/2005/sewer-rat ShenyangW307 dalian2013-56-3 35 Dandong/4/2005/sewer-rat LNJZ010 ShenyangRn139 changxingisland2014-307-1 Dalian/18/2005/sewer-rat 14 ShenyangW183 LNTL004 Huludao/L31/2005/sewer-rat ShenyangW287 ShenyangRn4 LNFS003 Huludao/X11/2005/sewer-rat ShenyangW301 LNFS004 37 /93/2005/sewer-rat LNJZ009 27 ShenyangRn41 BjHD01 Xingcheng/71/2005/sewer-rat ShenyangW286 xingcheng/33/2005/sewer-rat ShenyangRn137 changxingisland2014-0126-3 ShenyangW300 songmuisland2012-324-7 20 Xingcheng/1/2005/sewer-rat HLD65 /87/2005/sewer-rat ShenyangW27 songmuisland2012-324-3 ShenyangW294 19 Linghai/93/2005/sewer-rat 17 changxingisland2012-322-3 ShenyangW308 Dalian/28/2005/sewer-rat S3 ShenyangW88 changxingisland2012-321-7 ShenyangHu8 10 99 Dalian/25/2005/sewer-rat 49 ShenyangHu7 S3 changxingisland2012-234-1 Shenyang/535/2005/sewer-rat 48 ShenyangHu5 changxingisland2012-232-6 ShenyangHu6 77 Shenyang/560/2005/sewer-rat ShenyangHu4 changxingisland2014-416-3 Shenyang-Yuhong/4/2005/sewer-rat ShenyangHu3 3575 LNSY006 88 huluisland2014-879-1 Shenyang-Yuhong/7/2005/sewer-rat 82 LNSY007 SEOV LNFS001 Fushun/24/2005/sewer-rat 1881 LNSY008 LNSY005 LNJZ010 46 LNSY007 LNTL004 13 LNTL012 BjHD01 changxingisland2014-701-3 37 88 SEOV 26 LNFS004 29 LNTL013 56 changxingisland2014-0126-2 LNFS003 37 LNTL011 86 LNFS001 songmuisland2012-324-6 96 93HBX12 songmuisland2012-324-2 62 LNSY005 HLD12 S3 LNSY008 82 ZT10 changxingisland2012-322-2 Z37 48 74 LNTL002 Pf26 changxingisland2012-321-5 4118 LNTL003 JUN5_14 changxingisland2012-232-10 69 62 ShenyangRn111 LNSY006 ShenyangRn144 dagushanpeninsula2012-323-2 LNTL001 69 ShenyangRn167 30 3027 LNTL011 HBQ17 ZT10 58 LNTL012 changxingisland2014-0126-4 Z37 LNTL013 SEOV 3534 ShenyangRn20 HuludaoRn52 77 ZT71 ShenyangRn131 changxingisland2012-234-2 B-1 ShenyangRn74 13 ShenyangRn75 HuludaoRn100 64 80-39 S4 64 14 73 56 LNTL001 39 Tchoupitoulas LNTL002 HLD78 95 93 LNTL003 82 HLD12 Dandong/1/2005/sewer-rat IR461 S6 63 Shenyang-Yuhong/5/2005/sewer-rat 84 Humber LNJZ009 82 SR-11 Dandong/6/2005/sewer-rat 80_39 S4 50 HuludaoRn50 80 Dandong/7/2005/sewer-rat 94 Tchoupitoulas 28 LNTL001 HuludaoRn101 99 97 L99 LNTL012 S1 99 Hb8610 88 R22 L99 94 S1 LNTL013 R22 63 HuludaoRn101 34 66 ShenyangRn127 LNTL011 28 HB55 ShenyangRn180 57 Gou3 S5 ShenyangRn139 71 K24_e7 99 59 AH09 H1 ShenyangRn19 K24_v2 NC167 87 99 B78 66 LNSY005 H2 61 Humber S6 H5 66 86 99 LNSY008 IR461 76-118 H7 88 LR1 LNSY006 ZJ5 S5 54 Hu 76 99 62 Gou3 CGRn53 H9 LNSY007 81 A9 100 84FLi 99 43 LNTL002 H5 58 A16 H4 SN7 76 Q32 LNTL003 KY 99 B78 22 H5 93HBQ4 H2 99 84FLi 49 H5 4014 Z10 H8 ZT10 88 Z5 45 100 CGAa4MP9 98 Z37 H3 CGAa4MP9 H3 98 CGAa4P15 99 CGAa4P15 ZT71 39 62 CJAp93 61 100 Q32 46 91 H4 18 85 Bao14 80-39 CGRni1 ShenyangAa13 65 S4 28 DandongHu-92 Tchoupitoulas 100 A9 H9 DandongHu-33 HTNV B-1 87 61 DandongHu-88 24 CGRn53 70 DandongHu-84 59 K24_e7 99 10 99 Z10 H8 70 DandongHu-85 30 HTNV DandongHu-78 K24_v2 22 Z5 DandongHu-86 100 76-118 68 99 HuludaoRn101 H7 DandongHu-44 S1 22 DandongHu-83 L99 32 LR1 DandongHu-22 68 90 13 ShenyangRn127 Bao14 48 DandongHu-93 H6 8 98 43 CJAp93 9 DandongHu-29 HB55 77 3 51 5 DandongHu-34 65 DandongHu-42 DandongHu-91 46 R22 7 92 DandongHu-44 62 DandongHu-27 Humber DandongHu-77 S6 DandongHu-83 H6 DandongHu-82 IR461 96 81 DandongHu-77 6 DandongHu-42 ZJ5 80 DandongHu-90 S5 36 DandongHu-82 5 DandongHu-32 83 Gou3 23 DandongHu-47 DandongHu-88 3 HTNV 21 DandongHu-36 24 DandongHu-91 9 DandongHu-89 Puumala DandongHu-28 Puumala Puumala

0.1 0.05 0.05

FIGURE 3. Phylogenetic analysis of hantavirus genomic sequences obtained in Liaoning province. (A) phylogenetic tree based on the Gn segment (1383–1630 nt). (B) phylogenetic tree based on the Gc segment (2000–2317 nt). (C) phylogenetic tree based on the S segment (633–991 nt). Sequences in different cities are marked by different colors, and isolates obtained in this study were marked by .

Chinese Center for Disease Control and Prevention CCDC Weekly / Vol. 2 / No. 20 353 China CDC Weekly obtained from different places in the province for Disease Control and Prevention, , China; 2 Institute for prevention and control of infection and infectious diseases, Center for suggested that Liaoning belong to the same natural Disease Control and Prevention of Liaoning Province, Shenyang, focus of infection. However, several sub-genotypes Liaoning, China. & detected in rodents revealed the complexity of the Joint first authors. natural focus of HFRS in Liaoning. Genetic and Submitted: December 18, 2019; Accepted: March 27, 2020 antigenic stabilities of the detected hantavirus over a long period of time may suggest the effectiveness of the REFERENCES approved inactivated vaccine currently used in China. Hantaviruses are enzootic viruses that maintain 1. Zhang QF, Li JD, Li WH, Li C, Liu QZ, Liang MF, et al. persistent infections in their rodent hosts, and the Development and evaluation of a double antigen sandwich ELISA for spillover of these viruses to humans could occur at any the detection of total antibodies against hemorrhagic fever with renal syndrome virus. Chin J Exp Clin Virol 2007;21(4):386 − 8. time when contact happens between a susceptible http://dx.doi.org/10.3760/cma.j.issn.1003-9279.2007.04.030. (In person and an infective virion (9–10). After decades of Chinese). implementation of comprehensive strategies for HFRS 2. Wang H, Yoshimatsu K, Ebihara H, Ogino M, Araki K, Kariwa H, et al. Genetic diversity of hantaviruses isolated in china and prevention and control, China has brought HFRS characterization of novel hantaviruses isolated from Niviventer from highly common epidemic outbreaks to largely confucianus and Rattus rattus. Virology 2000;278(2):332 − 45. sporadic cases. Further improvement of HFRS http://dx.doi.org/10.1006/viro.2000.0630. 3. Pang Z, Li DX. Laboratory diagnosis of viral hemorrhagic fevers. Chin J prevention and control requires precise risk-based Virol 2013;29(3):349 − 56. http://dx.doi.org/10.13242/j.cnki.bing strategic practices that are integrated and regional to duxuebao.002398. (In Chinese). target highly-affected people in high-risk areas. These 4. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol measures could improve public awareness of HFRS 2013;30(12):2725 − 9. http://dx.doi.org/10.1093/molbev/mst197. transmission, reduce human exposure to infected 5. Martin DP, Murrell B, Golden M, Khoosal A, Muhire B. RDP4: Detection and analysis of recombination patterns in virus genomes. rodents and their excreta, and increase vaccination Virus Evol 2015;1(1):vev003. http://dx.doi.org/10.1093/ve/vev003. among at-risk populations. 6. Che L, Zhang GB, Gao D, Zhao L, Wang XL. Analysis on gene Conflict of interest: No conflicts of interest were sequence characteristic of hantavirus G2 segment in patients with hemorrhagic fever with renal syndrome in Liaoning, 2017. Dis Surveill reported. 2018;33(12):995 − 9. http://dx.doi.org/10.3784/j.issn.1003-9961.2018. Acknowledgements: We thank all persons involved 12.007. (In Chinese). in the work of HFRS surveillance in China but not 7. Geng YZ, Tian J, Liu Y, Wang B, Sun YW, Li X, et al. Genetic subtypes and distribution of Seoul hanta virus in Liaoning province. listed as an author in this paper. Chin J Public Health 2012;28(12):1594 − 6. http://dx.doi.org/10. Funing: This work was supported by National 11847/zgggws-2012-28-12-15. (In Chinese). Mega-Projects for Infectious Disease (2016ZX 8. Zhang S, Wang SW, Yin WW, Liang MF, Li JD, Zhang QF, et al. Epidemic characteristics of hemorrhagic fever with renal syndrome in 10004222-002, 2018ZX10711001), and National Key China, 2006-2012. BMC Infect Dis 2014;14:384. http://dx.doi.org/10. Research and Development Program of China 1186/1471-2334-14-384. (2016YFD0500303). 9. Vaheri A, Strandin T, Hepojoki J, Sironen T, Henttonen H, Mä kelä S, et al. Uncovering the mysteries of hantavirus infections. Nat Rev doi: 10.46234/ccdcw2020.091 Microbiol 2013;11(8):539 − 50. http://dx.doi.org/10.1038/nrmicro # Corresponding authors: Jiandong Li, [email protected]; Dexin Li, 3066. [email protected]; Shiwen Wang, [email protected]. 10. Jiang H, Zheng XY, Wang LM, Du H, Wang PZ, Bai XF. Hantavirus infection: a global zoonotic challenge. Virol Sin 2017;32(1):32 − 43. 1 Institute for Viral Disease Control and Prevention, Chinese Center http://dx.doi.org/10.1007/s12250-016-3899-x. (In Chinese).

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100% 90% 80% 70% 60% Total 50% 40% 30% 20% 10% 0% 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 100% 90% 80% 70% 60% Male 50% 40% 30% 20% 10% 0% 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 100% 90% 80% 70% Female 60% 50% 40% 30% 20% 10% 0% 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 Year 1– 10– 15– 20– 25– 30– 35– 40– 45– 50– 55– 60– 65– 70– 75– 80– ≥85

SUPPLEMENTARY FIGURE S1. Age distribution of reported hemorrhagic fever with renal syndrome (HFRS) cases in Liaoning Province.

Chinese Center for Disease Control and Prevention CCDC Weekly / Vol. 2 / No. 20 S1 China CDC Weekly

80.0 2004 2005 2006 2007 2008 2009 2010 2011 70.0 2012 2013 2014 2015 2016 2017 2018 Average

60.0

50.0

40.0

Proportion (%) 30.0

20.0

10.0

0.0

Other Student Farmer Not clear HerdsmanFishermen Medical staff School teacher Migrant labourer Nursery governessPublic functionary Kids in kindergarden Commercial personnel Kids not in kindergarden Non-agriculture worker Public servantsThe emeritus and staff and retired Food and beverage personnel Housewife and unemployment Seafarers and long-distance pilots

SUPPLEMENTARY FIGURE S2. Occupational distribution of the reported cases of hemorrhagic fever with renal syndrome (HFRS) in Liaoning Province.

S2 CCDC Weekly / Vol. 2 / No. 20 Chinese Center for Disease Control and Prevention