• View in gallery

    Map of clinical study sites. Samples were collected from six sites: four in the tropical Terai region (Bharatpur Hospital, Chitwan, Bhawani Hospital, Birganj, Narayani Hospital, Birganj, and University College of Medical Science, Bhairahawa), and two in the hilly, temperate climate of the Kathmandu Valley (Sukraraj Tropical and Infectious Disease Hospital and Siddhi Poly Clinic).

  • View in gallery

    Multiple sequence alignment of the DENV2 full genome sequences. Multiple sequence alignment of the DENV2 full genome sequences from the 2015 dengue outbreak in Nepal and 1,311 DENV2 full genome sequences present in the National Center for Biotechnology Information (NCBI) was carried out using a fast Fourier transformation method in MAFFT v. 6.940b. The approximately maximum likelihood phylogenetic tree was generated using the generalized time-reversible model of nucleotide evolution in FastTree v. 2.1.7. FastTree uses SH-like local supports with 1,000 resamples to estimate and validate the reliability of each split in the tree. From the tree, the branch containing the sequences from the Nepal 2015 outbreak (NP64|Serum|2015, NP65|Serum|2015 and 39 full genome sequences from the NCBI) was selected, and a more robust maximum likelihood phylogenetic tree was created using RAxML with 1,000 bootstrap replications. Trees were visualized using FigTree v. 1.4.2. Results of the phylogenetic analysis suggest a strong relationship between the Nepal strains and isolates from the 2009–2010 DENV2 outbreak in India. DENV = dengue virus.

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Whole Genome Sequencing of Dengue Virus Serotype 2 from Two Clinical Isolates and Serological Profile of Dengue in the 2015–2016 Nepal Outbreak

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  • 1 Division of Inflammation Biology, La Jolla Institute for Immunology, La Jolla, California;
  • | 2 Central Department of Biotechnology, Tribhuvan University, Kirtipur, Nepal;
  • | 3 Department of Medicine, School of Medicine, University of California San Diego, La Jolla, California;
  • | 4 Bhawani Hospital, Birganj, Nepal;
  • | 5 Department of Experimental Medicine, School of Medicine, University of California San Francisco, California;
  • | 6 Saw Swee Hock School of Public Health, National University of Singapore, Singapore, Singapore;
  • | 7 Department of Pharmacy, National University of Singapore, Singapore;
  • | 8 Program in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore

ABSTRACT

Dengue virus (DENV) is the cause of one of the most prevalent neglected tropical diseases, and up to half of the world’s population is at risk for infection. Recent results from clinical trials have shown that DENV vaccination can induce the development of severe dengue disease and/or prolong hospitalization after natural infection in certain naive populations. Thus, it is crucial that vaccine development takes into account the history of DENV exposure in the targeted population. In Nepal, DENV infection was first documented in 2004, and despite the increasing prevalence of DENV infection, the population remains relatively naive. However, it is not known which of the four DENV serotypes circulate in Nepal or whether there is evidence of repeated exposure to DENV in the Nepali population. To address this, we studied 112 patients who presented with symptomology suspicious for DENV infection at clinics throughout Nepal during late 2015 and early 2016. Of the 112 patients examined, 39 showed serological and/or genetic evidence of primary or secondary DENV infection: 30 were positive for DENV exposure by IgM/IgG ELISA, two by real-time reverse-transcription PCR (RT-PCR), and seven by both methods. Dengue virus 1–3, but not DENV4, serotypes were detected by RT-PCR. Whole genome sequencing of two DENV2 strains isolated from patients with primary and secondary infections suggests that DENV was introduced into Nepal through India, with which it shares a porous border. Further study is needed to better define the DENV epidemic in Nepal, a country with limited scientific resources and infrastructure.

INTRODUCTION

Dengue virus (DENV) is a member of the Flavivirus genus of single-stranded RNA viruses that include Zika, yellow fever, West Nile, and Japanese encephalomyelitis viruses. Dengue virus is primarily spread via the bite of infected Aedes species mosquitoes and is most commonly found circulating in tropical and subtropical climates. Four antigenically distinct serotypes of DENV have been identified (DENV 1–4). Infection with one serotype provides long-term protective immunity against reinfection with the same serotype; however, secondary infection with a different (heterotypic) serotype can induce “severe dengue,” which manifests as hemorrhage, shock, and, in the most severe cases, death. The precise etiology of severe dengue has yet to be fully elucidated, but the most commonly accepted mechanism is that it is mediated and/or exacerbated by antibody-dependent enhancement of infection. Antibody-dependent enhancement occurs when anti-DENV antibodies generated during a primary infection fail to neutralize the secondary infection and instead actively facilitate viral entry into and replication in host cells, leading to increased viremia and severe disease.1,2 Paradoxically, serotype-specific and cross-reactive anti-DENV antibodies and T cells are also known to play a protective role against secondary homotypic and heterotypic disease.3 Thus, the precise contribution of the immune system to the development of severe dengue remains far from clear.

The high mortality associated with severe dengue is of particular concern, given the large proportion of the global population at risk for DENV infection. Of the approximately 390 million people infected by DENV each year, about 96 million (25%) develop symptomatic disease.4 Asian countries, most notably India, China, and Indonesia, account for up to 70% of infections worldwide.4 Dengue virus is the most common cause of febrile illness in travelers returning from Asia or Central/South America who seek medical care.5 The first known case of DENV in Nepal was described in 2004 in a Japanese traveler who became febrile on route to returning to his country after visiting Nepal.6 Since that time, all four serotypes have been detected in Nepal7 and have moved as far north as the temperate Kathmandu Valley (approximately 4,600 ft. above sea level). The first autochthonous case was reported in 2010.8 Of note, Aedes aegypti and Aedes albopictus mosquitoes, the primary vectors for DENV, are found in most regions of Nepal, including the Kathmandu Valley.9

From the little we know about circulation of DENV in Nepal, a tendency for outbreaks occur in 3-year cycles, with particularly high incidences being noted in 2010, 2013, and 2016.10 Concomitant outbreaks were described in DENV-endemic countries nearby, including Bhutan and China, as well as in Sri Lanka.1114 At present, the infrastructure in Nepal is insufficient to support a wide-scale surveillance program and, despite case reports and local surveillance, the clinical, pathological, and epidemiological characteristics of DENV outbreaks in the country remain poorly characterized.

In this study, we report on a cohort of patients who presented with fever during the 2015 post-monsoon season in Nepal and were suspected of having dengue. We document the incidence of DENV 1–4 infection and the proportion of primary versus secondary infections. In addition, we report the first full-length sequence of DENV serotype 2 isolates from Nepal. New information about the phylogenetic characteristics of DENV circulating in Nepal will improve our ability to monitor outbreaks in the future and to predict virologic shifts throughout the region.

MATERIALS AND METHODS

Ethics statement.

The study was approved by the Ethics Review Boards of the Nepal Health Research Council of Nepal; Tribhuvan University, Nepal; and La Jolla Institute for Immunology, USA. The study was performed in accordance with the ethical standards noted in the 1964 Declaration of Helsinki and its amendments. All patients provided informed consent by signature, mark, or fingerprint after reading or hearing the consent form read to them in a local language.

Patients and sample collection.

We enrolled a total of 112 patients presenting with dengue disease–like symptoms at four medical centers in the Terai region (Bharatpur Hospital, Chitwan; Bhawani Hospital, Birganj, Narayani Hospital, Birganj, and University College of Medical Science, Bhairahawa) and two centers in the Kathmandu Valley (Sukraraj Tropical and Infectious Disease Hospital and Siddhi Poly Clinic) between August 2015 and January 2016 (Figure 1). All, but one of the enrolled patients, were from the Terai region. Samples were tested by serology or real-time reverse-transcription PCR (RT-PCR) in the same year they were collected. A total of 356 cases of DENV infection were reported in Nepal during 2014, primarily in residents of Chitwan, Parsa, Rupandehi, and Kathmandu. Therefore, we considered 112 patients to be a representative sample size for the 6-month period studied. The enrollment criteria were 1) fever (> 38°C) either at presentation or by patient history and 2) clinical suspicion of dengue disease at presentation based on at least one of the following signs or symptoms: myalgia, arthralgia, vomiting, diarrhea, fatigue, mucosal bleeding, rash, or 3) thrombocytopenia < 50,000/μL. Clinical data were collected by the attending physician. Participants could not be categorized with the WHO dengue disease severity classification as not all relevant clinical data were recorded at the time of enrollment.

Figure 1.
Figure 1.

Map of clinical study sites. Samples were collected from six sites: four in the tropical Terai region (Bharatpur Hospital, Chitwan, Bhawani Hospital, Birganj, Narayani Hospital, Birganj, and University College of Medical Science, Bhairahawa), and two in the hilly, temperate climate of the Kathmandu Valley (Sukraraj Tropical and Infectious Disease Hospital and Siddhi Poly Clinic).

Citation: The American Journal of Tropical Medicine and Hygiene 104, 1; 10.4269/ajtmh.20-0163

Venous blood samples (5 mL) were collected into ethylenediamine tetraacetic acid-containing tubes at the time of presentation and centrifuged on-site. The sera were removed and stored at −20°C until transported to the Central Department of Biotechnology at Tribhuvan University for analysis.

Dengue virus disease diagnosis.

All serum samples were tested for DENV-specific IgM and IgG and DENV nonstructural protein 1 (NS1) using specific ELISA kits (DENV Detect IgM [Food and Drug Administration], DENV Detect IgG [Research Use Only], and DENV Detect NS1 [Food and Drug Administration]; all from InBios International, Seattle, WA) according to the manufacturer’s instructions. The presence of active infection was assessed by RT-PCR using primers specific for the DENV 1–4 serotypes provided by the CDC (Atlanta, GA).

Because convalescent sera from the same patients were not available for comparison with acute serum samples, primary and secondary diseases were defined as previously described.15 Primary disease was considered IgM+ or NS1+ or PCR+ and IgM:IgG > 1.2; secondary disease was considered IgM+ or NS1+ or PCR+ and IgM:IgG ratio < 1.2. Patients with negative tests for IgM and NS1 and PCR were considered to have no active infection.

Dengue virus2 sequencing.

Sequences were obtained both from sera directly and after passage on baby hamster kidney-21 cells in vitro. Illumina libraries were constructed from total RNA using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) according to the manufacturer’s instructions. Mag-Bind RxnPure Plus beads (Omega Bio-Tek, Norcross, GA) were used to obtain a library size between 400 and 600 nucleotides. Libraries were purified with the MinElute PCR Purification Kit (Qiagen, Germantown, MD) and quantified by Bioanalyzer High-Sensitivity DNA Assay (Agilent, Santa Clara, CA). Targeted DENV genome enrichment was achieved using custom-designed biotinylated 120-mer xGen Lockdown baits (Integrated DNA Technologies, Newark, NJ) with complementarity to DENV 1–4, Zika virus and chikungunya virus following the procedures described in Kamaraj et al.16 In brief, the biotinylated baits were hybridized to viral genome fragments and immobilized on magnetic streptavidin Dynabeads (Life Technologies, Carlsbad, CA). Unbound library fragments were then washed away using SeqCap EZ hybridization (SeqCap, Madison, WI) and wash kit (Roche, Indianapolis, IN) according to the manufacturer’s instructions. A post-capture PCR amplification of 20 cycles with P1 and P2 primers (Illumina, San Diego, CA) was performed, and the enriched library was purified using the MinElute PCR Purification Kit (Qiagen). Genome assembly was performed using the VIPR4 pipeline (https://github.com/nf-core/vipr/).

Statistical analysis.

Descriptive statistics was used to summarize the data. Calculations were performed in GraphPad Prism 7.0a software (GraphPad, San Diego, CA). Age and gender group mean differences were compared using Student’s t-test.

RESULTS

Patient demographics and symptomatology at presentation.

A total of 112 patients with suspected dengue were enrolled in the study between August 2015 and January 2016. Most of the patients were male (86/112, 77%) and most were in the 20- to 59-year age range (95/112, 85%). The median age was 35.5 (range, 11–65) years. Most patients presented with fever and myalgia (95% and 88%, respectively), and more than half (61%) presented with arthralgia (Table 1). Vomiting, fatigue, and diarrhea were less common (16%, 5%, and 1%, respectively). The time between onset of symptoms and presentation was not recorded. The complete absence of rash at presentation is striking and should be noted, given that rash is generally considered to be a more common manifestation of dengue disease compared with other causes of acute febrile illness.17,18 Among the 112 patients, 73 were negative for DENV infection, despite presenting with a constellation of symptoms similar to those associated with dengue. The symptoms may have resulted from several other diseases that circulate in the region but are not routinely tested for, including scrub typhus,19 Japanese encephalitis virus,20 and chikungunya.21 Also of note, Zika virus, which is closely related to DENV, has not been documented in Nepal to date, and no diagnostic test was available for Zika virus in Nepal during the 2015 season.

Table 1

Prevalence of symptoms at presentation with suspected dengue (n = 112)

CharacteristicNumber% of total
Fever (≥ 38°C)10695
Myalgia9988
Arthralgia6861
Vomiting1816
Platelets < 50,000/μL1211
Fatigue65
Mucosal bleeding44
Diarrhea11
Skin rash00

Serologic evaluation and classification of disease stage.

All sera were evaluated by anti-DENV IgM and IgG ELISA, DENV NS1 ELISA, and DENV serotype-specific RT-PCR to identify the incidence of serotypes and infection status (Table 2). A total of 39 patients were confirmed to have primary or secondary disease based on the tests used: 30 had positive results in NS1 ELISA and/or DENV RT-PCR and nine had positive results in IgM or IgG ELISA. A total of 30 patients were positive by ELISA only, two by RT-PCR only, and seven by both methods. Of the 39, six patients had primary disease and 33 had secondary disease (IgM:IgG > 1.2 or < 1.2, respectively) (Table 2). Nine patients were DENV-positive by RT-PCR, with DENV 1, 2, and 3 detected in 1, 5, and 3 patients, respectively. Seventy-three symptomatic patients were considered to have no active infection based on negative results in RT-PCR or IgM and NS1 ELISA (Table 2). Active infection was slightly, but not significantly, more common among females than males (11/26, 42% versus 27/86, 31%, P = 0.33).

Table 2

Patient diagnosis and DENV immunity status at diagnosis according to age and gender

Age-group (years)GenderNumber of patients (n = 112)
Primary diseaseSecondary diseaseNo active infection
≤ 19M0111
F002
20–59M52145
F11013
≥ 60M012
F000
Total63373

DENV = dengue virus; F = female; M = male; NS1= nonstructural protein 1. Primary disease: positive DENV-specific IgM or NS1 or PCR, and IgM:IgG > 1.2; secondary disease: positive DENV-specific IgM or NS1 or PCR, and IgM:IgG < 1.2; no active infection: negative DENV-specific IgM and NS1 and PCR, and positive or negative IgG.

Phylogenetic and nucleotide sequence analysis of a DENV2 isolate.

We sequenced isolates from two patients, one each with primary (NP64) and secondary infection (NP65), who were positive for DENV2 by RT-PCR. Multiple sequence alignment of the full genome sequences with 1,311 full genome DENV2 sequences in the National Center for Biotechnology Information (NCBI) database was carried out using a fast Fourier transformation method in MAFFT v. 6.940b. The estimated maximum likelihood phylogenetic tree was generated using a generalized time-reversible model of nucleotide evolution in FastTree v. 2.1.7. FastTree uses Shimodaira-Hasegawa (SH)-like local supports with 1,000 resamples to estimate and validate the reliability of each split in the tree. From the tree, the branch containing the sequences from the present cohort (NP64|Serum|2015, NP65|Serum|2015) and 39 full genome sequences from NCBI was selected, and a more robust maximum likelihood phylogenetic tree was created using RAXML with 1,000 bootstrap replications. Trees were visualized using FigTree v. 1.4.2.

Phylogenetic analysis of the full DENV-2 genomes indicates that both isolates from the current study belong to the cosmopolitan genotype IV and are most closely related to a DENV2 strain isolated from India in 2009 (JX475906) (Figure 2, Supplemental Table 1). The 2016 Nepalese DENV2 strains share 99.1% and 99.7% identity with the 2009 Indian DENV2 strain at the nucleotide and amino acid levels, respectively. Amino acid changes relative to the 2009 Indian isolate were observed in the prM (I127T), NS1 (P903H and D1065N), NS2a (S1305T), NS2b (A1464V), NS3 (A1643T), NS4a (M2174V), and NS5 (I2518T, E2870K, and S3167G) (Supplemental Table 2). In addition to the 2009 Indian DENV2 strain, several other strains from India (1996–2010), Sri Lanka (1996), China (2000–2001), and Pakistan (2008) also share a high degree of nucleotide identity ranging from 95.9% to 98.9% (Supplemental Table 1).

Figure 2.
Figure 2.

Multiple sequence alignment of the DENV2 full genome sequences. Multiple sequence alignment of the DENV2 full genome sequences from the 2015 dengue outbreak in Nepal and 1,311 DENV2 full genome sequences present in the National Center for Biotechnology Information (NCBI) was carried out using a fast Fourier transformation method in MAFFT v. 6.940b. The approximately maximum likelihood phylogenetic tree was generated using the generalized time-reversible model of nucleotide evolution in FastTree v. 2.1.7. FastTree uses SH-like local supports with 1,000 resamples to estimate and validate the reliability of each split in the tree. From the tree, the branch containing the sequences from the Nepal 2015 outbreak (NP64|Serum|2015, NP65|Serum|2015 and 39 full genome sequences from the NCBI) was selected, and a more robust maximum likelihood phylogenetic tree was created using RAxML with 1,000 bootstrap replications. Trees were visualized using FigTree v. 1.4.2. Results of the phylogenetic analysis suggest a strong relationship between the Nepal strains and isolates from the 2009–2010 DENV2 outbreak in India. DENV = dengue virus.

Citation: The American Journal of Tropical Medicine and Hygiene 104, 1; 10.4269/ajtmh.20-0163

DISCUSSION

Although the first case of DENV infection in Nepal was documented in 2004, lack of testing in the area at that time makes it unclear whether DENV was circulating before then. Compared with neighboring countries such as India, DENV infections in Nepal have not yet been well characterized by symptomatology, disease severity, incidence, or phylogenetics. In this study, we document cases of locally transmitted DENV and provide the first full sequence of locally transmitted DENV2 in Nepal. Most DENV-infected patients in our cohort were 20–59 years of age, which is consistent with a previous report that the median age of dengue patients in Nepal is 29.5 years.8 Disease in the 20- to 59-year age group portends a significant impact on the productivity of the working population. Although the precise loss of productivity due to DENV infection is unknown, the disability-adjusted life year has been calculated in other Southeast Asian, countries such as Thailand, where each family is estimated to have lost approximately US $61 during the 2005 outbreak, which is more than the average monthly income in Thailand at that time.22

Most dengue cases in our study were males (28/39) and were because of secondary infections (33/39). The male-to-female ratio is more difficult to account for but is likely representative of the inequitable access to health care for women in Nepal compared with men. The increased morbidity of secondary infection often leads to presentation at hospitals, whereas primary infections may not be severe enough for patients to ever enter the medical system, especially in resource-limited settings. However, the collection of samples at single time points in the present study has limited our ability to precisely classify primary and secondary diseases without relying on IgM-to-IgG ratios.

In our patient samples, we were not able to identify a predominant serotype of the virus, which is likely related to the relatively low number of patients enrolled in the study. We were able to detect serotypes 1, 2, and 3 in patients and sequenced two isolates of DENV-2. No DENV-4 was identified by PCR in patients. Historically, the index case of DENV in Nepal was serotype 2, but there was a shift to DENV-1 serotype as the predominant circulating strain in 2010.23 In both 2013 and 2014, serotype 2 was again identified as the predominant strain.

The full genome sequencing analysis suggest that the 2015 DENV2 isolates from the current study are most closely related to a DENV2 strain isolated from Andhra Pradesh, India, in 2009. Travelers from India and China (118,249 and 104,005, respectively) represented the largest share of international tourists to Nepal in 2015 followed by the United States, the United Kingdom, and Sri Lanka (53,645, 46,295, and 57,521, respectively).24 However, these totals may underrepresent the number of people crossing the border from India. India is Nepal’s largest trading partner, accounting for 64% of Nepal’s total trade in 2016/2017 (US $6.36 billion).25 In addition, India and Nepal have a bilateral agreement that allows citizens of each country to cross the border by car for daylong visits without the payment of any levy or tax.26 Given this porosity in the southern border and the volume of trade/international travelers, the observed relatedness between the 2015 DENV2 isolates from Nepal and a strain from India is congruent. The high degree of nucleotide identity with DENV2 strains isolated from Sri Lanka, China, and Pakistan suggests that the virus is circulating across borders throughout this region and that dengue control efforts in Nepal should factor in the current burden of disease in these neighboring countries.

The index case of autochthonous DENV was detected in Nepal in 2010 which has enabled the local and international communities to studying DENV in a population with relatively naive hosts, allowing for the capture of the population’s transition into endemicity for the virus. It is possible, and in fact likely, that DENV was circulating in the southern regions of Nepal well before 2004. However, DENV diagnostic tools were not widely available at that time. With the initial identification, the Nepal government has scaled up educational and public health efforts to capture cases occurring throughout the country. Dengue virus rapid diagnostic test kits (IgM, IgG, and NS1) are now available without cost in government hospitals and are concentrated in the southern, more tropical regions where most cases have been reported. The available InBios tests are known to cross-react with other viruses, especially flaviviruses, which may have affected our results for patients who were positive by IgM/IgG ELISA, but not by NS1 ELISA or PCR. Measurement of IgM and IgG over time would have helped clarify the case definitions. The DENV epidemic in Nepal is likely to be even more widespread than we still know, and further work is needed to more accurately capture the incidence of dengue-related disease in Nepal.

Recent studies have shown a shift in the population of mosquitoes in the Kathmandu Valley from the secondary vector DENV Ae. albopictus to the primary vector Ae. aegypti.27 Understanding the role of climate shifts in virus–vector interactions and the subsequent effect on infections would also provide valuable information about DENV in Nepal that cannot be easily obtained in countries that are already DENV endemic. This information could have broader implications for many countries that are experiencing warmer temperatures but are currently unaffected by DENV, particularly large parts of the Southern United States.

Supplemental tables

ACKNOWLEDGMENTS

We are thankful to the Karius Inc. and La Jolla Institute for Immunology SPARK program for contributing to our project entitled “Dengue and dengue-like infections in the patients visiting to the hospitals in Nepal.” We also would like to thank InBios International Inc. for their generous contribution of DENV IgM, IgG, and NS1 ELISA kits to our study.

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    • Export Citation
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    Takasaki T, Kotaki A, Nishimura K, Sato Y, Tokuda A, Lim CK, Ito M, Tajima S, Nerome R, Kurane I, 2008. First isolation of dengue virus from Nepal. J Travel Med 15: 4649.

    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
  • 9.

    Gautam I, Dhimal M, Shrestha SR, Tamrakar AS, 2009. First record of Aedes aegytpi (L.) vector of Dengue virus from Kathmandu, Nepal. J Nat Hist Mus 24: 156164.

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Author Notes

Address correspondence to Krishna Das Manandhar, Head of Department, Tribhuvan University, Kirtipur 44618, Nepal, E-mail: krishna.manandhar@biotechtu.edu.np or October M. Sessions, Tahir Foundation Building, National University Singapore, 12 Science Drive 2, #09-01N, Singapore 117549, E-mail: october.sessions@nus.edu.sg or Sujan Shresta, La Jolla Institute for Immunology, 9420 Athena Circle, La Jolla, CA 92037, E-mail: sujan@lji.org.

Financial support: K. D. M. was supported by the Fulbright Scholar Program, and M. M. was also supported by the UCSD Infectious Diseases Clinical T32 Fellowship Program (AI 007036). S. S. was supported by the NIH (AI116813). O. M. S. was supported by the Singapore Ministry of Education (https://www.moe.gov.sg) with a Startup grant.

Authors’ addresses: Krishna Das Manandhar, Division of Inflammation Biology, La Jolla Institute for Immunology, La Jolla, CA, and Central Department of Biotechnology, Tribhuvan University, Kirtipur, Nepal, E-mail: krishna.manandhar@biotechtu.edu.np. Melanie McCauley and Sujan Shresta, Division of Inflammation Biology, La Jolla Institute for Immunology, La Jolla, CA, and Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, E-mails: mccauleymelaniemccauley@gmail.com and sujan@lji.org. Birendra Prasad Gupta, Anh-Viet Nguyen, and Annie Elong Ngono, Division of Inflammation Biology, La Jolla Institute for Immunology, La Jolla, CA, E-mails: bgupta@lji.org, anhviettrannguyen@gmail.com, and aelong@lji.org. Roshan Kurmi, Bhawani Hospital, Birganj, Nepal, E-mail: roshankurmi@gmail.com. Anurag Adhikari, Central Department of Biotechnology, Tribhuvan University, Kirtipur, Nepal, E-mail: adhikari.anurag.m@gmail.com. Simona Zompi, Department of Experimental Medicine, School of Medicine, University of California, San Francisco, San Francisco, CA, E-mail: simona.zompi@gmail.com. October M. Sessions, Saw Swee Hock School of Public Health, National University of Singapore, Singapore, Singapore, Department of Pharmacy, National University of Singapore, Singapore, Singapore, and Program in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore, E-mail: october.sessions@nus.edu.sg.

These authors contributed equally to this work.

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