Teaching a new mouse old tricks: Humanized mice as an infection model for Variola virus

Christina L Hutson; Ashley V Kondas; Jana M Ritter; Zachary Reed; Sharon Dietz Ostergaard; Clint N Morgan; Nadia Gallardo-Romero; Cassandra Tansey; Matthew R Mauldin; Johanna S Salzer; Christine M Hughes; Cynthia S Goldsmith; Darin Carroll; Victoria A Olson

doi:10.1371/journal.ppat.1009633

. 2021 Sep 21;17(9):e1009633. doi: 10.1371/journal.ppat.1009633

Teaching a new mouse old tricks: Humanized mice as an infection model for Variola virus

Christina L Hutson ^1,^*,^#, Ashley V Kondas ^1,^#, Jana M Ritter ², Zachary Reed ¹, Sharon Dietz Ostergaard ³, Clint N Morgan ¹, Nadia Gallardo-Romero ¹, Cassandra Tansey ³, Matthew R Mauldin ¹, Johanna S Salzer ¹, Christine M Hughes ¹, Cynthia S Goldsmith ², Darin Carroll ¹, Victoria A Olson ¹

Editor: Bernard Moss⁴

¹Poxvirus and Rabies Branch, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America

²Infectious Diseases Pathology Branch, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America

³Comparative Medicine Branch, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America

⁴National Institute of Allergy and Infectious Diseases, UNITED STATES

The authors have declared that no competing interests exist.

^✉

* E-mail: zuu6@cdc.gov

Contributed equally.

Roles

Christina L Hutson: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Ashley V Kondas: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

Jana M Ritter: Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

Zachary Reed: Investigation, Methodology, Writing – review & editing

Sharon Dietz Ostergaard: Investigation, Writing – review & editing

Clint N Morgan: Data curation, Formal analysis, Investigation, Validation, Writing – original draft, Writing – review & editing

Nadia Gallardo-Romero: Conceptualization, Investigation, Methodology, Writing – review & editing

Cassandra Tansey: Investigation, Methodology, Writing – review & editing

Matthew R Mauldin: Investigation, Writing – review & editing

Johanna S Salzer: Investigation, Writing – review & editing

Christine M Hughes: Data curation, Formal analysis, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

Cynthia S Goldsmith: Investigation, Methodology, Validation, Visualization, Writing – review & editing

Darin Carroll: Conceptualization, Funding acquisition, Supervision, Writing – review & editing

Victoria A Olson: Conceptualization, Funding acquisition, Investigation, Methodology, Supervision, Writing – review & editing

Bernard Moss: Editor

PMCID: PMC8454956 PMID: 34547055

Abstract

Smallpox, caused by the solely human pathogen Variola virus (VARV), was declared eradicated in 1980. While known VARV stocks are secure, smallpox remains a bioterrorist threat agent. Recent U.S. Food and Drug Administration approval of the first smallpox anti-viral (tecovirimat) therapeutic was a successful step forward in smallpox preparedness; however, orthopoxviruses can become resistant to treatment, suggesting a multi-therapeutic approach is necessary. Animal models are required for testing medical countermeasures (MCMs) and ideally MCMs are tested directly against the pathogen of interest. Since VARV only infects humans, a representative animal model for testing therapeutics directly against VARV remains a challenge. Here we show that three different humanized mice strains are highly susceptible to VARV infection, establishing the first small animal model using VARV. In comparison, the non-humanized, immunosuppressed background mouse was not susceptible to systemic VARV infection. Following an intranasal VARV challenge that mimics the natural route for human smallpox transmission, the virus spread systemically within the humanized mouse before mortality (~ 13 days post infection), similar to the time from exposure to symptom onset for ordinary human smallpox. Our identification of a permissive/representative VARV animal model can facilitate testing of MCMs in a manner consistent with their intended use.

Author summary

Preparedness activities against highly transmissible respiratory viruses with high mortality have been highlighted during the ongoing COVID-19 pandemic. Smallpox, caused by Variola virus (VARV) infection, is highly transmissible and estimated to have killed 300 million people in the 20th century alone with ~30% mortality. Through an intensive vaccination campaign smallpox was declared eradicated in 1980 and routine smallpox vaccination of individuals ceased. Today’s population has little/no immunity against VARV; if smallpox were to re-emerge the worldwide results would be devastating. Development of antiviral strategies is critical for outbreak response efforts and in order to truly gauge the potential effectiveness of a medical countermeasure (MCM), it must be tested directly against the pathogen. VARV is solely a human pathogen and infections of surrogate animal models have been largely unsuccessful. Here we use the humanized mouse to establish the first small animal model using VARV. We found that the virus spread systemically within humanized mice before mortality (~ 13 days post infection), similar to the time from exposure to symptom onset for ordinary human smallpox. These findings improve upon current MCM efficacy testing methods by providing a means to conduct in vivo evaluations of smallpox MCMs directly against VARV.

Introduction

Variola virus, the causative agent of smallpox, is a solely human pathogen. The World Health Organization (WHO) certified the global eradication of smallpox in 1980 with the last reported naturally occurring case in Somalia in 1977. While the known viral stocks are secure in two laboratories (U.S. Centers for Disease Control and Prevention [CDC] and VECTOR Institute), the threat of unknown sources outside the repositories remains and could be used with malicious intent. Variola virus (VARV) is regulated by the U.S. Federal Select Agent Program, which oversees the possession, use and transfer of biological select agents and toxins, which have the potential to pose a severe threat to public. Forgotten lyophilized VARV vials from the 1950’s were discovered in 2014 at a non-WHO approved variola virus laboratory [1]. Additionally, the procedure for recreating an OPXV has been documented (Horsepox virus) thereby providing means to recreate a human pathogenic OPXV such as VARV [2]. A recent tabletop exercise evaluating what would happen if smallpox was used as a bioweapon indicated potentially devastating consequences [3]. While two vaccines (Vaccinia virus; ACAM 2000 and JYNNEOS) and one anti-viral (TPOXX [ST-246]) have received licensure by the U.S. Food and Drug Administration (FDA) as medical countermeasures (MCMs) for use against smallpox infection, these MCMs were developed after the eradication of human smallpox and have never been tested against the authentic agent in humans. Because VARV causes smallpox only in humans, and thus far no satisfactory VARV animal model has been developed, the U.S. FDA has approved The Animal Model Rule as a pathway for regulatory approval. Under this rule, potential therapeutics must be tested in at least two surrogate Orthopoxvirus animal models, such as Monkeypox virus in non-human primates (NHP), Rabbitpox virus in rabbits and/or Ectromelia virus (ECTV) in mice (21 CFR 601.90). While this route to U.S. FDA licensure is critical for preparedness, the development of a small animal model susceptible to VARV infection would be ideal for testing MCMs directly against the authentic agent.

Humanized mice have become a valuable tool for studying infectious diseases [4–6]. An animal model with a human-like immune system, would be advantageous to the study of VARV, potentially identifying why VARV is a solely human pathogen and providing valuable samples for understanding aspects of the human immune response to VARV infection. Given this, we sought to determine if humanized mice can support a productive VARV infection. Three different strains of humanized mice (hu-PBMC, hu-CD34+, and hu-BLT) were evaluated and compared to the susceptibility of the immunodeficient NSG background mouse. This sequence of studies allowed us to better understand if susceptibility to VARV infection is attributable to a lack of murine immune response, the humanization process or some combination of both. To produce these mice, the NOD scid gamma (NSG) background mouse undergoes different methods of humanization. For humanized-PBMC mice (hu-PBMC), the NSG mouse is engrafted with human peripheral blood mononuclear cells (PBMC) which produces a strong human T cell engraftment (effector and memory T cells). Humanized-CD34+ (hu-CD34+) mice are created by engrafting the NSG mouse with human hematopoietic stem cells (CD34+ cells) which produces circulating human CD4⁺ and CD8⁺ T cells. Humanized-BLT (hu-BLT) mice, which are considered to be the most humanized because they develop human T and B cells, are created by both engrafting CD34+ cells and human fetal liver and thymus under the murine renal capsule. We found that all three strains of humanized mice were susceptible to systemic VARV infection following intranasal (IN) inoculation, while NSG background mice were not. Herein, we present novel humanized mouse models of smallpox that utilize the exact etiologic agent of human disease and provide further evidence that VARV dissemination requires some component of the human immune system.

Results

VARV disease in humanized mice

For part one of the study, humanized mice were challenged intranasally with one of the following: a high dose of VARV, a low dose of VARV, diluent for a non-infectious control, ɣ-irradiated VARV or received no challenge material (Table 1 and Fig 1A). The first clinical signs observed were suspect pox lesions on the hocks of a subset of mice beginning at 7 days post infection (dpi). Swabs of the lesions were collected. The hock lesions were sporadic, with only five infected animals affected, but did include animals of all three strains and no uninfected control animals (Table 1). For hu-CD34+, and hu-BLT infected groups, onset of other clinical signs was variable but generally began between 7–17 dpi (hu-CD34+ high dose), 17–19 dpi (hu-CD34+ low dose), and 12–14 dpi (hu-BLT both doses). The majority of hu-PBMC infected mice did not show clinical signs attributed to viral infection, with only three animals displaying clinical signs close to study end (17–21 dpi for both high (1/4) and low-dose groups (2/4)). The main clinical sign was >20% weight loss. Clinical disease progressed rapidly for hu-CD34+ and hu-BLT and animals were not always able to be euthanized before succumbing to disease. Hu-CD34+ 8 (low dose group) was believed to have an unsuccessful inoculation. While necropsy samples from this animal had very low levels of viral DNA in the spleen, kidney and ovaries (46.0 fg/μl, 50.5 fg/μl and 43.8 fg/μl respectively), this animal displayed no clinical signs and survived to study end. Unsuccessful inoculation was further supported by lack of pathologic lesions, viral immunostaining, and absence of detectable viable virus. Upon review of the clinical record, this mouse experienced a “bubble” during the inoculation supporting the hypothesis that the animal was not successfully inoculated (i.e., entire inoculum not delivered into nares). This animal was excluded from the study.

Table 1. Clinical signs and gross findings.

Humanized mice (hu-BLT, hu-CD34+ and hu-PBMC) were inoculated via the intranasal route with 7x10³ or 7x10⁵ plaque forming units (pfu) of VARV (VARV_JAP51_hrpr (primary clade I)) (n = 4 per group). Clinical signs were recorded daily, and weights were taken at least three times weekly. Euthanasia/pain scores were determined by weight loss, behavior and appearance. We only considered a score of ≥5 as clinical signs attributed to viral infection because a score of 4 was often seen in non-infected controls due to weight loss alone; weight loss was not utilized as a pain score for control mice unless observed for two consecutive weight recording days. At 21 days post infection (dpi) or a pain score of 10, euthanasia was performed and gross findings during necropsy recorded. Clinical signs, mortality, and abnormal gross findings are displayed per group. Hu-CD34+-8 was excluded from this analysis since it was unsuccessfully infected.

		Clinical Signs					Mortality		Necropsy Findings
	Group	>20% Wt Loss	Minimally subdued Activity	Unresponsive when stimulated	Ruffled/ piloerection	Skin lesions	Found Deceased	Did not survive Day 21	Hepatic necrosis	Gallbladder hemorrhage	Splenomegaly	Lymphadenomegaly
hu-PBMC	Diluent Non-infectious Control	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	1/2
hu-PBMC	ɣ-irradiated VARV Non-infectious Control	0/2	0/2	0/2	0/2	0/2	1/2	1/2	0/2	0/2	0/2	0/2
hu-PBMC	VARV 7x10⁵	1/4	1/4	0/4	0/4	0/4	0/4	1/4	0/4	0/4	0/4	0/4
hu-PBMC	VARV 7x10³	2/4	0/4	0/4	2/4	1/4	0/4	0/4	0/4	0/4	0/4	0/4
hu-PBMC	Negative Non-infectious Control	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2
hu-CD34	ɣ-irradiated VARV Non-infectious Control	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	1/2	0/2
hu-CD34	VARV 7x10⁵	4/4	0/4	2/4	4/4	0/4	2/4	4/4	3/4	0/4	0/4	0/4
hu-CD34	VARV 7x10³	3/3	1/3	0/3	1/3	1/3	1/3	3/3	1/3	2/3	2/3	1/3
hu-CD34	Negative Non-infectious Control	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2
hu-BLT	ɣ-irradiated VARV Non-infectious Control	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2
hu-BLT	VARV 7x10⁵	3/4	1/4	2/4	3/4	2/4	1/4	4/4	3/4	0/4	1/4	0/4
hu-BLT	VARV 7x10³	3/4	1/4	0/4	3/4	1/4	4/4	4/4	2/4	0/4	0/4	0/4
hu-BLT	Negative Non-infectious Control	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2

Open in a new tab

Dose-dependent mortality was seen in the hu-CD34+ and hu-BLT mice beginning at day 13 (Fig 1B and 1C). High dose hu-CD34+ mice were significantly more likely to succumb earlier than those in the low dose group (p = 0.02). While differences in survivorship between dose groups for hu-BLT mice were not significant (p = 0.2), there was a trend suggesting the high dose group succumbed earlier than those challenged with the low dose. All hu-BLT and hu-CD34+ mice in the high dose group and low dose group succumbed to disease. The hu-PBMC mice did not have high morbidity, and only one from the high dose group was euthanized before study end (19 dpi). Hu-PBMC mice were significantly more likely to survive VARV infection compared to hu-BLT and hu-CD34+, controlling for dose (high dose p = 0.004, low dose p = 0.008). There was no significant difference in survivorship between hu-BLT and hu-CD34+ mice when controlling for dose (high dose p = 0.20; low dose = 0.63). One hu-PBMC control mouse (challenged with ɣ-irradiated VARV) was found deceased on day 13, which may have been related to anesthesia complications; this was the only control animal to die during the study. All necropsy samples from this animal were negative for VARV DNA.

Pathology and immunohistochemistry

Cutaneous lesions (vesicles or ulcers) were observed on the hocks of 5 mice. At necropsy, the most common gross pathologic observation was multifocal and coalescing regions of hepatic necrosis in hu-CD34+ (n = 4/7 mice) and hu-BLT (n = 5/8) mice. Sporadic splenomegaly and gallbladder hemorrhage were also seen in these strains. Hu-PBMC mice had no remarkable gross lesions (Table 1).

The following tissues were examined by histopathology and immunohistochemistry, when available: brain, lung, liver, kidney, spleen, adrenal gland, lymph nodes, bone marrow, female reproductive tract, gastrointestinal tract, oronasal tissues, and skin from hock lesions when present (the observed lesions were collected for tissue culture propagation, however the surrounding area was examined by histopathology and immunohistochemistry). Findings are summarized in Table 2 and shown in Figs 2 and 3. Hock skin lesions were identified microscopically for 1 hu-PBMC and 1 hu-CD34+ mouse, with histopathological findings including epidermal hyperplasia and hyperkeratosis with epithelial staining by VARV immunohistochemistry at the chronic ulcer margins. Periarticular tissues (synovium, tendon, periosteum) also had mild inflammation and VARV immunostaining (Fig 2A and 2B).

Table 2. Microscopic lesions and VARV immunolocalization in tissues from humanized (PBMC, CD34+, BLT) mice and non-humanized background strain (NSG) mice inoculated intranasally with VARV.

Fractions indicate number of animals with finding divided by number of animals with tissue type collected.

	Mouse strain, VARV dose
Histopathologic findings	PBMC		CD34+		BLT		NSG		VARV immunolocalization^*
Histopathologic findings	Low n = 4	High n = 4	Low n = 3	High n = 4	Low n = 4	High n = 4	Low n = 5	High n = 5	VARV immunolocalization^*
Skin, hock: chronic ulceration	+ (1/1)	0	+ (1/1)	0	- (0/1)	- (0/2)	N/A	N/A	Hyperplastic epidermis at ulcer margin, periarticular mesenchymal cells
Liver: hepatocellular necrosis; rare intracytoplasmic inclusions	+ (1/4)	+ (1/4)	+++ (3/3)	+++ (4/4)	+++ (4/4)	+++ (4/4)	- (0/5)	- (0/5)	Hepatocytes, Kupffer cells, endothelial cells
Adrenal gland: multifocal cortical > medullary necrosis	+ (1/1)	+ (2/3)	+++ (3/3)	+ (2/2)	+++ (3/3)	++ (2/2)	0	0	Necrotic and intact cortical epithelium and medullary chromaffin cells
Spleen: necrosis, hemorrhage, lymphoid depletion	++ (1/1)	- (0/1)	+++ (2/2)	+++ (3/3)	+++ (3/3)	+++ (2/2)	- (0/4)	- (0/4)	Mesenchymal cells, reticuloendothelial cells
Lymph nodes: necrosis, lymphoid depletion	+++ (1/4)	+++ (1/1)	++ (3/3)	+ (1/1)	+++ (1/2)	+++ (2/3)	- (0/5)	- (0/5)	Reticuloendothelial cells
Bone marrow: necrosis, hemorrhage	+ (2/4)	- (0/4)	+++ (3/3)	+++ (4/4)	+++ (4/4)	+++ (4/4)	- (0/5)	- (0/5)	Necrotic hematopoietic cells, endosteum, periosteum
Ovary: multifocal necrosis	++ (2/2)	0	- (0/1)	- (0/2)	- (0/4)	- (0/1)	- (0/5)	- (0/4)	Stromal and follicular cells
Uterus: multifocal to mural necrosis	++ (4/4)	+++ (1/4)	++ (2/3)	++ (1/4)	+ (2/4)	+ (1/3)	- (0/5)	- (0/5)	Stromal and smooth muscle cells
Lung: multifocal bronchial epithelial necrosis; peribronchiolar and perivascular edema; mild interstitial pneumonitis	++ (4/4)	++ (3/4)	+ (1/3)	+ (2/4)	+ (3/4)	+ (1/4)	- (0/5)	- (0/4)	Bronchial epithelium, interstitial cells, inflammatory cells
Nasal cavity: multifocal submucosal necrosis and edema, serous gland atrophy, minimal inflammation	+ (3/4)	++ (2/4)	- (0/3)	- (0/4)	- (0/4)	- (0/4)	+ (1/5)	+ (4/5)	Submucosal stroma, respiratory and serous glandular epithelial cells
Tooth: pulp necrosis	+ (1/3)	+ (1/4)	++ (2/3)	++ (1/4)	++ (1/4)	+ (2/4)	- (0/5)	- (0/5)	Pulp, periodontal ligament
Disseminated bacteremia	- (0/4)	- (0/4)	+++ (3/3)	+++ (4/4)	+++ (4/4)	+++ (4/4)	- (0/5)	- (0/5)	N/A

Open in a new tab

Histopathologic scoring:—negative/not present; + mild/focal; ++ moderate/multifocal; +++ severe/extensive; 0 tissue not collected; N/A not applicable

*Immunostaining localized to foci of necrosis when present; however, immunostaining was also seen in tissues without morphologic alterations. Reported immunolocalization includes both necrotic and morphologically intact foci.

Fig 2 — **A, B**: Chronic skin ulceration over the hock, with epidermal hyperplasia (arrow, A) and VARV immunostaining in skin, tendon, and periosteum (hu-PBMC-11; low dose, day of death 21 dpi). **C, D:** Multifocal adrenal gland necrosis (*, C) with VARV immunostaining (hu-BLT-5; high dose, day of death 13 dpi). **E, F:** Diffuse splenic necrosis with lymphoid depletion, fibrin, and hemorrhage; extensive VARV immunostaining in reticuloendothelial and mesenchymal cells (hu-CD34+-4; high dose, day of death 16 dpi). **G, H**: Nasal mucosa with submucosal edema and mild inflammation (open arrows, G); extensive VARV immunostaining (hu-PBMC-6; high dose, day of death 21 dpi). **I, J:** Tooth with dental pulp necrosis (*, I); VARV immunostaining of pulp and periodontal ligament (hu-CD34+-9; low dose, day of death 20 dpi). **K, L**: Ovary with multifocal stromal and follicular necrosis (*, K) and VARV immunostaining (hu-PBMC-10; low dose, day of death 21 dpi). **M, N:** Uterus with transmural necrosis and VARV immunostaining (PBMC-5; high dose, day of death 13 dpi). **O, P**: Focal necrosis (*, O) in renal subcapsular human fetal thymic graft, with extensive VARV immunostaining (hu-BLT-5; high dose,). A, C, E, G, I, K, M, O (hematoxylin-eosin); B, D, F, H, J, L, N, P (VARV immunohistochemistry, viral antigen labeling in red). Original magnifications: A, B, O, P (x50); C-J, M, N (x100); K, L (x200).

Fig 3 — Hu-CD34+ and hu-BLT mice had similar findings in liver, bone marrow, and lung, which contrasted those seen in hu-PBMC mice. Hu-CD34+ and hu-BLT livers (A, G) had confluent and lobular hepatocyte necrosis with very rare eosinophilic globular inclusions (A, inset), and VARV immunostaining of hepatocytes, Kupffer cells, and occasional endothelial cells (B, H). Hu-CD34+ and hu-BLT bone marrow specimens similarly showed extensive necrosis and hemorrhage associated with VARV immunostaining (C, D, I, J). Hu-PBMC liver (M) and bone marrow (O) showed minimal inflammation (open arrows, M) and no necrosis, and only very rare VARV immunostaining in these tissues (N, P). Conversely, hu-CD34+ and hu-BLT lung tissues showed minimal inflammation (E, K) and VARV immunostaining (F, L), while hu-PBMC lungs (Q) had more prominent peribronchiolar and perivascular inflammation (arrowheads, Q) with VARV immunostaining (R). Hu-CD34+ and hu-BLT mice had disseminated intravascular bacteria (arrows in A, E, G, K), which were not present in hu-PBMC mice. A, B, E, F (hu-CD34⁺-4, high dose, day of death 16 dpi); C, D (hu-CD34⁺-6, high dose, day of death 17 dpi); G, H (hu-BLT-5, high dose, day of death 13 dpi); I, J (hu-BLT-9, low dose, day of death 17 dpi); K,L (hu-BLT-4, high dose, day of death 13 dpi); M, N, Q, R (hu-PBMC-11, low dose, day of death 21 dpi); O, P (hu-PBMC-5, high dose, day of death 19 dpi). A, C, E, G, I, K, M, O, Q (hematoxylin-eosin); B, D, F, H, J, L, N, P, R (VARV immunohistochemistry, viral antigen labeling in red). Original magnifications: A, B, C, D, G, H, I, J, O, P (x100); E, F, K, L, Q, R (x200); M, N (X400).

Histopathologic findings in extracutaneous tissues were generally similar between low and high dose groups for each mouse strain (Table 2 and Figs 2 and 3). Hu-CD34+ and hu-BLT mice had tissue necrosis with minimal inflammation in the liver, adrenal gland, lymphoid tissues, and reproductive tract. Abundant VARV antigen was detected within necrotic foci, but also sometimes within morphologically unaffected tissue, by immunohistochemistry. Livers showed confluent and lobular hepatocellular necrosis, with immunostaining localized prominently within necrotic and intact hepatocytes, as well as scattered Kupffer and endothelial cells. Rarely, eosinophilic globular cytoplasmic material reminiscent of viral inclusions was present within hepatocytes (Fig 3A inset). Adrenal glands had discrete foci of necrosis most commonly in the cortex, and occasionally in the medulla. Poxviral antigen localized to foci of necrosis, as well as other foci of intact cells. Examined spleens uniformly showed diffuse necrosis with severe lymphoid depletion and red pulp expansion by fibrin and hemorrhage. Poxviral antigen localized to macrophages and mesenchymal cells around central arteries in the regions of periarteriolar lymphoid sheath depletion. Lymph nodes from various sites similarly showed lymphoid necrosis and depletion, with poxviral immunostaining in reticuloendothelial cells. Bone marrow showed extensive necrosis and hemorrhage with immunostaining in the hematopoietic compartment, and patchy staining of endosteum and periosteum. Lungs had a mild increase in interstitial cellularity, with scattered interstitial immunostaining. One hu-BLT mouse (hu-BLT-6) also had multifocal bronchiolar epithelial necrosis and immunostaining. Nasal tissues had scattered small foci of submucosal, and rarely respiratory epithelial or serous glandular, immunostaining, without apparent inflammation or necrosis. Teeth had multifocal to extensive immunostaining that was concentrated in the dental pulp, sometimes in association with necrosis, and the periodontal ligament. For available female reproductive tract tissues, the ovarian stroma consistently had scattered viral immunostaining, without overt morphological alterations; follicles also stained to a lesser extent. 2/7 hu-CD34+ uteri, and 3/7 hu-BLT uteri, had moderate to extensive immunostaining of smooth muscle and stromal (including perivascular) cells, variably accompanied by necrosis. Oviduct and vagina had scattered or patchy immunostaining in a pattern similar to that observed in the uterus, without overt necrosis. Gastrointestinal tissues also showed inconsistent and rare, scattered staining in the smooth muscle, serosa, and rarely submucosa. Kidneys showed very rare, scattered immunostaining within glomeruli and interstitial cells. For hu-BLT mice with remnant subcapsular grafts, grafted human tissues showed extensive immunostaining. Brain had no histopathologic findings and no immunostaining, and heart was not available, from any animal of these strains. Disseminated bacteremia was seen histologically in all virally challenged animals of these two strains, with bacterial emboli consistently found in brain, lung, liver, spleen, and kidney. Immunohistochemical testing of a subset of tissues from four animals (2 hu-CD34+ and 2 hu-BLT) detected involvement of multiple, mixed bacteria, including Staphylococcus spp., Enterococcus spp., and Streptococcus spp. Gram-negative bacteria were not identified. Control animals inoculated with phosphate-buffered saline (PBS) or gamma-irradiated virus had no significant histopathologic findings and no VARV immunostaining (S1 Fig).

Hu-PBMC mice had overall similar findings, but with less liver, bone marrow, and tooth involvement, increased nasal and lung involvement, slightly more prominent inflammation overall, and absence of bacteremia (Table 2 and Figs 2 and 3). Lungs from hu-PBMC mice had more consistent and numerous foci of bronchiolar epithelial necrosis accompanied by perivascular and peribronchiolar edema and mild inflammation, and mild interstitial pneumonitis. Immunostaining localized to bronchiolar epithelial cells, peribronchiolar and perivascular stromal and inflammatory cells, and scattered interstitial cells. Nasal tissues had patchy edema, necrosis, and mild inflammation, with necrosis and atrophy of serous glands, with corresponding multifocal to widespread immunostaining of epithelium and submucosal stroma and glands. These changes were more prominent in the high dose hu-PBMC group. One animal in the low dose group (hu-PBMC9) had a fibrinocellular and hemorrhagic exudate, which showed granular intra- and extracellular VARV immunostaining, in the middle nasal meatus. All uninfected, and n = 2/8 infected hu-PBMC animals had systemic, atypical lymphoid proliferation suggestive of the development of graft vs host (GVH) disease. Control animals had no VARV immunostaining (S1 Fig).

A subset of tissues were examined by transmission electron microscopy (Fig 4). Ultrastructural evaluation revealed almost exclusively immature VARV particles in the hepatocytes of the native mouse liver tissue of infected hu-CD34+ (n = 2), hu-BLT (n = 2) and hu-PBMC (n = 1) mice (Fig 4A.); however, both mature and immature particles were located in the sinusoidal endothelial cells and free in the sinusoids of the livers (Fig 4B). In the examined hu-PBMC murine ovary, uterus, and adventitial cells surrounding the bile duct, and in human fetal thymic allograft from a hu-BLT mouse (Fig 4C), both mature and immature particles were present.

Molecular findings

Of the 222 oral swabs that were collected throughout the study, only 4 were positive for viral DNA. Two oral swabs contained viable virus and were from the lower dose groups: hu-BLT-8 on 12 dpi (46.2 pfu/ml) and hu-PBMC-11 on 19 dpi (594 pfu/ml). All hock lesion swabs were negative for viral DNA, but all five hock lesion tissues contained viral DNA and viable virus (Fig 5). Remarkably high loads of viable virus were present in multiple tissues, including heart, kidney, liver, lung, ovaries and spleen, and in some instances reached as high as 1.66 x 10¹¹ pfu/gram of tissue (Fig 5). The hu-BLT mice had the highest viral loads (both dose groups) followed by slightly lower levels in hu-CD34+ mice. Tissue viral load comparisons between hu-BLT and hu-CD34+ were not significantly different when controlling for dose. Despite little mortality in the hu-PBMC mice, high viral loads were detected in most of the tissues tested. Hu-PBMC-5, a mouse in the hu-PBMC high dose group, had the highest viral loads compared to other mice in that group and was the only hu-PBMC euthanized (19 dpi) due to clinical signs. Minimal whole blood euthanasia samples were available for evaluation of viremia. Viral DNA was detected in 9/10 infected animals (3/4 high dose hu-PBMC, 4/4 low dose hu-PBMC, 2/2 high dose hu-BLT) ranging from 1.67x10² to 2.05x10⁵ fg/μl with hu-BLT mice having the highest quantities. Post DNA evaluation, three blood samples had sufficient volume remaining for viral titration, all from the hu-PBMC low dose group, and 2/3 contained viable virus (2.0x10² and 1.39x10³ pfu/ml). To look for evidence of antibodies post infection, serum (when available) was tested for the presence of human IgM and IgG by ELISAs. Serum available for testing included all hu-PBMC mice (excluding one ɣ-irradiated control animal), all hu-CD34+ control mice, two from the high and two from the low dose group, and for hu-BLT mice only serum from the ɣ-irradiated VARV control group, one uninfected animal and four from the high dose group. None of the tested serum samples had detectable levels of human IgM or IgG in ELISAs designed for human serum.

Fig 5 — Humanized-mice (hu-BLT (A), hu-CD34+ (B) and hu-PBMC (C)) were inoculated via the intranasal route with 7x10³ or 7x10⁵ plaque forming units (pfu) of VARV (VARV_JAP51_hrpr (primary clade I)) (n = 4 per group). Animals that succumbed to VARV infection, or were euthanized at 21 dpi, had tissues collected and processed for viral titration (plaque assay). The mean with SEM is shown. An * indicates one or more of that sample had cell culture monolayer destroyed or plaques were present but below the limit of detection for this assay.

Non-humanized NSG mice are not susceptible to systemic VARV disease

Part two of the study determined whether the immunosuppressed NSG background mouse was susceptible to systemic VARV infection. Clinical signs and weight loss were minimal/absent in the NSG mice (both doses) and all NSG animals survived until study end (21 dpi). No abnormal findings were seen during necropsy. On pathologic evaluation, 1/5 in the low dose group, and 4/5 in the high dose group had very mild inflammatory changes associated with VARV immunostaining in the nasal submucosa (S2 Fig). No significant histopathologic changes, and no VARV immunostaining, were seen in extranasal tissues (lung, liver, spleen, kidney, lymphoid tissues, and female reproductive tract) (S2 Fig). The oral and skin swabs were negative for viral DNA. Several necropsy tissues, mainly the lung, liver and nasal cavity, from several NSG mice were positive for viral DNA (S3 Fig). However, the only tissues that contained viable virus were two nasal cavities, one from the low and one from the high dose group, which was the site of inoculation (S3C Fig). All NSG negative control mice were negative for viral DNA and had no significant pathologic findings and no VARV immunostaining (S1 Fig).

We utilized hu-PBMC mice as positive controls during this part of the study; similar to part one of the study, hu-PBMC mice began displaying clinical signs late in the study (~day 19) and one mouse had to be euthanized on day 19. Molecular and pathology results were overall similar to part one of the study, with the virus spreading systemically and high loads of viable virus found throughout most tissues tested (S3D Fig).

Discussion

Due to the discontinuation of smallpox vaccination after disease eradication, the human population is increasingly susceptible to smallpox. Its malicious release could have devastating consequences, making imperative the continued development of preventive and therapeutic countermeasures. Animal models are an invaluable tools for the development of MCMs. For smallpox MCM testing, an ideal animal model would mimic natural human smallpox disease using the authentic agent (VARV), use a realistic infectious dose and aerosol droplet route of infection, have an 8–14 day incubation period, identifiable prodrome, detectable immune response, high mortality and systemic rash 1–4 days post prodrome [7,8]. Various models have successfully fulfilled parts, but not all, of this ideal. Historically, most adult animals have been insusceptible to VARV challenge, even when susceptible to other orthopoxviruses. While CAST/EiJ mice and prairie dogs (Cynomys ludovicianus) are susceptible to an IN Monkeypox virus challenge [9–12], neither developed a systemic infection following IN VARV challenge [13,14]. Similar findings were observed in ICR and SCID mice following IN VARV challenge [15]. NHPs are susceptible to VARV infection, with development of systemic rash illness and mortality; however, this model has several disadvantages including: use of infectious doses much higher (1x10⁸ to 1x10⁹ pfu) than the suspected dose required for human infection, and requires intravenous inoculation, an unnatural route of infection which bypasses the initial local replication and viremia, eliminating the incubation and prodromal periods [16]. In this model, only infectious doses at 1x10⁹ pfu resulted in high mortality which manifested as hemorrhagic smallpox with animals succumbing to the disease as early as 4 dpi [16,17] limiting the window for testing post-exposure MCMs. Due to these difficulties, animal models using surrogate orthopoxviruses have been utilized to test MCMs, but none against the authentic agent VARV.

Here we present the first small animal models of human smallpox, utilizing humanized mice that are highly susceptible to VARV infection. We have shown that these novel humanized mouse models will be useful for studying VARV infection and testing efficacy of MCMs against the virus. While all three humanized mouse strains were susceptible to VARV and supported a productive infection, the more advanced hu-CD34+ and hu-BLT mice are the best candidates for further model characterization. The engraftment of human CD34+ cells in the hu-CD34+ mouse results in the development of a self-renewing naïve human immune system located in the mouse bone marrow as well as circulating in the tissues; it is comprised of multiple human cells such as T, B, natural killer and antigen presenting cells [18] and the lymphocytes are H2 class restricted [18,19]. Further advanced, the hu-BLT is described as a complete immune system with the engraftment of human CD34+ cells and additionally, engraftment of human fetal and liver tissue allowing for the development of not only a variety of human cells but a naïve human immune system that is HLA restricted [18,20] in the mouse. For both strains, high and low doses of VARV administered IN produced systemic disease and high mortality, with pathologic features resembling aspects of the severe, highly lethal hemorrhagic form of human smallpox [8] and also corroborated what has been shown in high dose intravenous inoculation of VARV in macaques [16,17]. Pertinent features include hepatosplenic, lymphoid, and hematopoietic necrosis, with widespread VARV antigen and nucleic acid detection in these and other tissues, and the uniform presence of bacteremia with a variety of gram-positive cocci at the time of death. Systemic bacterial infections due to gram-positive cocci are a common feature of fatal human smallpox [8,21,22], and were also uniformly seen in intravenously inoculated macaques that developed hemorrhagic disease [17]. The role of bacterial infections as potentiators of VARV infection and/or secondary infections being the immediate cause of death in human smallpox has been debated [8,21,23–26], and these mice models may be valuable in investigating this important aspect of severe human smallpox. Although the model developed pathologic features resembling aspects of the hemorrhagic form of human smallpox, the approximate 13-day incubation period in these mice better approximates that of ordinary human smallpox, and allows a broader window for testing potential new MCMs than some other animal models which have a more rapid mortality onset [7,16].

Cutaneous lesions (vesicles and ulcers) were occasionally, but not consistently, identified in these mice, suggesting that their disease progression may most closely resemble that of early hemorrhagic smallpox, in which severe disease develops before cutaneous vesicular lesions. It could be that the animals did not live long enough for a systemic rash to form, or subtle skin lesions were obstructed by the animal’s fur. The presence of infectious virions in the skin was not evaluated in this study (with exception of the collected hock lesions) and should be examined in future studies. Moreover, hemorrhage per se was not a feature of VARV infection in these mice and therefore, disease in this model may not fit neatly into one of the well-defined clinical types of human smallpox [8,16,17]. As with other animal models, the time from onset of clinical signs to death was short in these mice. A defined prodrome was not apparent, and antemortem noninvasive samples (oral swabs, skin swabs) were not useful for confirmation of infection. The absence of mucocutaneous exudates and lack of deroofing prior to swab collection may account for the futility of these samples. Further studies with lower doses of VARV are warranted to attempt to both recreate ordinary smallpox and to increase the prodromal period and potentially identify distinct biomarkers of infection that may be useful when evaluating new antivirals in a post exposure setting. Additional antemortem blood sampling should be considered in future studies as a method for confirmation of infection prior to development of clinical signs and to evaluate hematologic alterations in disease as well as determine if any biomarkers predict outcome. While human IgG and IgM were not detected in the limited serum samples available in this study, future studies should include other detection methods for evaluation of human immune response which would strengthen the model. Subsequent studies to evaluate the detection of human immune response (in particular the hu-BLT model since IgG has been reported [27–29]), as well as studies to understand the viral trafficking of the virus in the host will provide greater understanding of the utility of the model to evaluate MCMs as well as potentially provide valuable samples to understand the human immune response in these animals.

Of the assessed strains of humanized mice, the hu-PBMC mice were deemed the least promising for studying systemic VARV infection. This was based on their apparent delayed disease course evidenced by little mortality despite high viral loads in tissue, and the development of systemic lymphoproliferative lesions, attributed to GVH [19]. GVH develops in all three of these humanized mice strains and has been well described elsewhere [18–20], but onset is earlier (within 4 weeks) in hu-PBMC mice, compared to 20 weeks and 12 months in hu-BLT and hu-CD34+, respectively. However, the hu-PBMC model may be useful for investigating specific aspects of VARV disease, such as respiratory or reproductive tract effects based on histopathology results. Additionally, comparing the hu-PBMC mice to the non-humanized NSG background mice is useful in hypothesizing why the humanized mice are susceptible to VARV infection as it is the least humanized mouse strain used in this study. The non-humanized, immunosuppressed NSG background mice lack an adaptive immune response and have less functional innate responses compared to other immunodeficient mice [19]. We found that after VARV challenge, the NSG mouse supported virus replication only at the site of inoculation (viable virus found in nasal tissue in 2/10 mice at 21 dpi) but was unable to disseminate to additional tissues. The NSG mice are engrafted with human PBMC cells (to create the hu-PBMC mouse strain) resulting in the development of primarily mature human donor HLA-restricted T cells (poor engraftment has been reported of monocytes and B cells [18]). Human effector and memory T cells can be found in secondary lymph organs and circulation [18,20] suggesting VARV infects human T cells and that infection is sufficient to allow for a productive VARV infection in these mice. We can further hypothesize that VARV uses the T cells to spread systematically in the host and replicates in human T cells and host mouse tissue as well. This is supported by detectable viremia and comparable viable virus loads in lymphoid and non-lymphoid organs. Future time course studies should be conducted to determine how the virus traffics in the mouse and what human cell populations are present focusing on early time points. Taken together, it appears that unless VARV is given at extremely high infectious doses intravenously as seen in the VARV NHP model, some component of the human immune system is needed in order to spread systemically.

Of note, compared to traditional laboratory mice, the humanized mice are more variable due to the humanization process, which is a potential limitation in regard to the reproducibility as an animal model. Although not an outbred model the humanization levels differ for each mouse [30]. However it’s important to note that quality control (QC) is performed (by the company) on the human marker levels using flow cytometry before animals are approved for use in studies. Due to the more advanced humanization methodology used to create the hu-BLT mice, which includes engraftment of human tissue within the mouse, it is reasonable to assume that the variation within this mouse strain is most pronounced. This was observed when comparing the viral titers between the hu-BLT mice, which did vary. Hu-CD34+ mice were more similar in regard to viral load, which might be explained by the more simplified humanization process which does not entail human tissue engraftment. However, tissue viral load comparisons between hu-BLT and hu-CD34+ were not significantly different when controlling for dose. The relatively consistent titers seen in the hu-CD34+ may be a benefit of this mouse strain for future studies. Regardless of trends in titer variation, for both hu-BLT and hu-CD34+ mice, the end result of mortality was the same for all mice. The high VARV challenge dose for both mouse strains resulted in similar days until death while more variation was seen in the time until death for the hu-BLT group challenged with a low dose. The hu-CD34+ mice challenged with a low dose had a consistent time until death, like that seen with the high challenge group. The observed 100% mortality for both mouse strains make them suitable for the purpose of therapeutic testing regardless of any variation in viral titers. Hu-PBMC mice viral titers are harder to compare due to delayed viral disease progression and viral dissemination; however, we are not considering these for future studies due to GVH as mentioned above.

We hypothesize the development, prevalence and location of human immune cells within these three mouse strains [18,20] are contributing to the differences we observed in the time course of disease, with the least humanized (hu-PBMC) mice having a delay in clinical signs and mortality. In vitro studies with ECTV using mouse primary cell lines such as macrophages and fibroblasts have pointed towards an important role of the mitochondria during viral replication and morphogenesis [31–33]. Performing similar VARV in vitro studies with different human immune primary cell lines, or human CD34+ material that is used to engraft the mice, to compare both viral progression and gene expression within the cells would be informative. Using the findings from our in vivo studies combined with these proposed additional in vitro studies could help in determining the differences observed between the three humanized mice strains and importantly which human cell(s) are needed for VARV infection when the inoculum is not given as a high dose via a systemic (e.g. intravenous) route.

Numerous studies have been performed looking at the VARV genome in order to help in understanding why this virus is solely a human pathogen [34–37]. When analyzing the VARV genome and other closely related poxviruses, authors detected a “hotspot” of genome variation within the VARV ortholog of the vaccinia virus O1L gene, at the level of single nucleotide polymorphisms [38]. The O1L gene has been shown to be necessary for efficient replication of Vaccinia virus in human cells [39]. This gene is non-functional in the two most closely related viruses to VARV (Camelpox virus and Taterapox virus), which rarely infect humans, and typically cause self-limiting infections when they do [38,40,41]. Authors note that the VARV O1L ortholog has been retained in all VARV isolates, therefore it is possible that the gene has undergone subsequent selection for optimal function in human cells and may be critical for dissemination and establishment of VARV within human hosts. When investigating the evolution of the VARV genome, investigators recently hypothesized genetic losses that have occurred during evolution of the virus [42]. Comparison of the genetic differences between modern VARV isolates compared to ancient isolates could identify potential genetic elements involved in the evolution of smallpox to becoming a solely human pathogen. In addition to these types of genetic studies, this newly characterized humanized mouse model could allow identification of human components necessary for VARV systemic spread. Further studies, as suggested above, should be done to explore which human characteristic(s) contribute to a productive infection in these humanized mice.

In conclusion, our results indicate that hu-CD34+ and hu-BLT mice will be valuable as models of human smallpox for continued development of pre- and post-exposure treatments. There is currently only one antiviral compound licensed by the U.S. FDA (TPOXX, SIGA) for treatment of smallpox infection. Previous in vivo studies have found that multi-drug treatment (ST-246 and CMX-001) has higher levels of protection from mortality than single drug therapy [43,44] and resistance has been reported with single-drug treatment [45]. The WHO Advisory Committee on Variola Virus Research (ACVVR) has recommended the licensure of at least two therapeutics for treatment of smallpox prior to destruction of all viral stocks. While surrogate models of smallpox infection have utility, they lack in vivo testing against the authentic agent of smallpox, VARV. Additionally, surrogate animal models do not allow investigation of the role of the human immunologic response in VARV infection. Our results showed that non-humanized NSG mice were not susceptible to systemic disease, indicating that a human component is required for the virus to spread from the inoculation site and cause severe smallpox disease development. Future studies that further characterize and develop these models may both strengthen the correlation of animal model and human disease (i.e. aid in identifying why VARV is solely a human pathogen), and also provide the novel opportunity to investigate the role of human-specific immunologic responses in VARV infection and smallpox disease.

Methods

Ethics statement

Animal care and use procedures were approved and followed according to the U.S. CDC Institutional Animal Care and Use Committee (IACUC) under protocol number 2671GALMOUC.

Mice

Female Hu-PBMC, hu-CD34+ and hu-BLT (NSG background) and NSG mice, were purchased from The Jackson Laboratory (JAX) (additional information can be found: hu-BLT [46] and on the JAX website for hu-PBMC and hu-CD34+: https://www.jax.org/). Hu-CD34+ and hu-BLT were received at 15 weeks old (12 weeks post engraftment) while hu-PBMC mice were 8 weeks old (3 days post engraftment) during part one of the study. For part two, NSG mice were received at 7 weeks old and hu-PBMC mice were 7 weeks old (~1 week post engraftment). Precautions were taken including the use of sterile drinking water and sterilization by autoclaving of bedding, enrichment and food used during the study. Mice were grouped housed (n = 3–5) upon being received and acclimated for at least 3 days prior to beginning the studies. After acclimation but prior to inoculation, serum was collected via the submandibular vein (U.S. CDC IACUC policy 026).

Challenge virus

Work with live VARV is conducted in the Biosafety Level 4 laboratory (BSL-4), approved by the WHO ACVVR, is done in accordance with all applicable U.S. Federal Select Agent regulations (42 CFR part 73) and all inactivation procedures used had been reviewed and approved by the U.S. CDC Laboratory Safety Review Board. These studies are reviewed biannually as part of the United States Government Policy for Oversight of Life Sciences Dual Use Research of Concern.

A semi-purified preparation of VARV_JAP51_hrpr (primary clade I) was selected as the challenge virus, as it has been used in historic NHP studies [16,17,47] as well as other mouse studies [13]. This virus was isolated from a U.S. solider in Japan in 1951 and was part of the U.S. Army repository before being transferred to the U.S. CDC VARV collection [16]. The isolate underwent 5 passages on chorioallantoic membrane before being passaged twice in African green monkey kidney cells (BSC-40) and undergoing purification as previously described [48–53]. Inoculum was diluted in PBS. For ɣ-irradiation inactivation, the virus was treated with 5 x 10⁶ rads. All challenge doses were confirmed via standard cell culture techniques (back-titration) immediately following inoculation.

Animal inoculation

All mice were inoculated via the IN route to mimic the natural route of human smallpox infection. Viral inoculation was done while animals were maintained under 3–5% inhalation isoflurane anesthesia. Inoculum was diluted in PBS and 0.05% bovine serum albumin. Animal group sizes are defined as “n”. For part one of the study, Hu-PBMC, hu-CD34+ and hu-BLT mice were inoculated with 7x10³ or 7x10⁵ plaque forming unit (pfu) (n = 4 per group). To serve as controls, two hu-PBMC mice were inoculated with diluent and two of each mouse strain were inoculated with ɣ-irradiated VARV_JAP51_hrpr equivalent to 7x10⁵ pfu. For part two of the study, NSG mice were inoculated with 4x10⁶ or 5x10⁴ pfu (n = 5 per group). For negative controls, NSG mice were uninfected or mock-infected with diluent (n = 3 per group). Three hu-PBMC mice were also included in the part two of the study and challenged with 4x10⁶ pfu to serve as positive controls.

Animal sampling, observations and euthanasia

Clinical signs were recorded daily, and oral swabs, physicals and weights were taken three times weekly under 3–5% inhalation isoflurane anesthesia. Euthanasia/pain scores were determined by weight loss, behavior and appearance, with clinical scoring performed twice daily once animals reached a score of ≥5. We only considered a score of ≥5 as clinical signs attributed to viral infection because a score of 4 was often seen in non-infected controls due to weight loss alone; weight loss was not utilized as a pain score for control mice unless observed for two consecutive weight recording days. At 21 dpi or a pain score of 10, euthanasia was performed via exsanguination and cervical dislocation under 5% inhalation isoflurane anesthesia. Oral swab (polyester frozen dry), tissues, whole blood and serum (Starstedt) were collected upon euthanasia. The following tissues were collected: nose, lung, liver, spleen, ovaries, heart, kidney and any other tissue with abnormal appearance. Between samples, necropsy tools were decontaminated in 5% micro-chem, scrubbed with a brush and rinsed with water. All samples were frozen at -80°C for future analysis.

Sample processing and DNA extraction

Swabs were processed as previously described [10]. Tissues and whole blood were thawed on ice. Aliquots of 1 mm zirconia/silica beads (Biospec) and PBS for tissue homogenizing were made by mixing 0.5 ml and 0.7 ml, respectively. Each beads/PBS tube was weighed prior to pouring contents into the tissue tube; the tissue tube was weighed and change in readings was used as an approximate tissue weight. Samples were homogenized using the Mini-BeadBeater-16 (Biospec) by grinding twice for one minute, and icing samples for one minute between runs. Sample inactivation (per internally validated/approved inactivation method) and DNA extraction were performed as previously described except samples were heated at ≥56°C [10]. The remaining sample was re-frozen at -80°C.

Pathology and immunohistochemistry

During necropsy, a portion of each collected tissue and the remaining mouse carcass were placed into 10% neutral-buffered formalin for 7 days for virus inactivation (per internally validated/approved inactivation method) and tissue fixation and were transferred to a BSL-2 laboratory. Tissues were processed for routine paraffin histology, and sections were cut at 4 microns and stained with hematoxylin-eosin (H&E). Immunohistochemical (IHC) detection of VARV was performed as previously described [54], using a rabbit polyclonal anti-VARV antibody (U.S. CDC, Atlanta, GA). Formalin-fixed, ɣ-irradiated, paraffin-embedded monkey kidney cells infected with VARV were used as a positive control. Negative control utilized normal rabbit serum in place of the primary antibody. Tissues for electron microscopic examination were either formalin-fixed and placed in phosphate buffer and then buffered with 2.5% glutaraldehyde or were processed by the on-slide embedding technique [55]. Samples were then embedded in Epon/Araldite by standard methods [56].

Molecular assays

Real-time PCR was performed as previously described [57]. A standard dilution series of semi-purified VARV DNA was assayed on each PCR plate in order to quantify DNA levels within samples. Two fg viral DNA/rxn was the positive cut-off value. Samples that crossed the threshold (C_t value) before cycle 38 in duplicate underwent a modified version of titration with a 96 hour incubation [10]. Samples were thawed on ice prior to sonication at 40% output twice at one-minute intervals, with 10 seconds of icing between sonication. Samples were titrated on BSC-40 cells with a 96hour incubation prior to staining with crystal violet stain. All samples were serially diluted and titrated in duplicate. The following quality control measures were used to determine if sample needed to be repeated: ≥25% of the CV stained monolayer was missing in the countable wells, if samples did not dilute out as anticipated when conducting serial dilutions, and if counts could not be determined in any dilution due to diluting out too far. The limit of detection for this assay was pre-determined as an average of at least ≥3 plaques in the lowest dilution (10⁻¹). Detection of anti-Orthopoxvirus IgG and IgM was performed as previously described [58].

Statistics

Comparisons of the tissue viral load (pfu/ml) between mouse strains and dose were made using the Wilcoxon rank-sum test, as the data are not normally distributed. Differences in mortality rates for three humanized mouse strains were compared using Fisher’s exact test, due to small sample size. Kaplan-Meier survivorship curves were calculated and compared using the log-rank test. A p-value < 0.05 was considered statistically significant. Data analysis was performed using SAS version 9.4 (SAS Institute).

Supporting information

S1 Fig. VARV immunohistochemistry in representative tissues from control mice of each strain inoculated with PBS only or with gamma-irradiated VARV.

No VARV immunostaining (viral antigen labeling in red) is present in liver, kidney, lung, or bone marrow from hu-PBMC mouse inoculated with PBS only (top row, hu-PBMC-1, day of death 21 dpi); hu-PBMC mouse inoculated with gamma-irradiated VARV (second row, hu-PBMC-3 day of death 13 dpi); hu-CD34+ mouse inoculated with gamma-irradiated VARV (third row, hu-CD34+-1); hu-BLT mouse inoculated with gamma-irradiated VARV (fourth row, hu-BLT-1, day of death 21 dpi); NSG mouse inoculated with gamma-irradiated VARV (fifth row, NSG-11, day of death 21 dpi); NSG mouse inoculated with PBS only (bottom row, NSG-16, day of death 21 dpi). Original magnifications: liver, kidney, lung (x40); bone marrow (x100).

(TIF)

Click here for additional data file.^{(4.1MB, tif)}

S2 Fig. Representative histopathology and VARV immunohistochemistry in non-humanized NSG mice.

A, B: Nasal tissue with mild inflammatory changes and immunostaining of VARV (NSG-9, high dose, day of death 21 dpi). C, D: Liver without histopathologic changes or immunostaining of VARV (NSG-9, high dose). E, F: Lung without histopathologic changes or immunostaining of VARV (NSG-5, low dose). A, C, E (hematoxylin-eosin); B, D, F (VARV immunohistochemistry, viral antigen labeling in red). Original magnification: all images (x100).

(TIF)

Click here for additional data file.^{(4.1MB, tif)}

S3 Fig. NSG background mouse was not susceptible to systemic variola virus infection.

NSG mice were inoculated with 4x10⁶ or 5x10⁴ pfu for a high and low dose group (n = 5 per group). Three hu-PBMC mice were also included and challenged with 4x10⁶ pfu to serve as positive controls. On 21 dpi, animals were euthanized. (A,B): Several necropsied tissues from NSG mice contained low levels of viral DNA mainly the lung, liver and nasal cavity in both challenge groups. (C) Only two nasal cavities, one animal from the low (NSG-1) and one from the high dose group (NSG-6) contain viable virus. An * indicates one or more of that sample had cell culture monolayer destroyed or plaques were present but below the LOD for this assay. (D) In contrast, virus spread systemically through the n = 3 hu-PBMC mice leading to clinical signs and one animal had to be euthanized on day 19 due to a clinical score of 10; high loads of viable virus were found throughout most tissues tested from the hu-PBMC mice.

(TIF)

Click here for additional data file.^{(2.7MB, tif)}

Acknowledgments

Michael Townsend, Jillybeth Burgado, Brock Martin, Zachary Weiner, Scott Smith, Theodora Khan, Todd Smith and Felix Jackson for various supportive roles throughout the study; and Heather Hayes and Brigid Bollweg for histology and immunohistochemistry support.

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This study was funded by Biomedical Advanced Research and Development Authority (VO). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

References

1.Reardon S. NIH finds forgotten smallpox store: Nature; 2014. Available from: https://www.nature.com/news/nih-finds-forgotten-smallpox-store-1.15526. [DOI] [PubMed] [Google Scholar]
2.Noyce RS, Lederman S, Evans DH. Construction of an infectious horsepox virus vaccine from chemically synthesized DNA fragments. PLoS One. 2018;13(1):e0188453. Epub 2018/01/20. doi: 10.1371/journal.pone.0188453; PubMed Central PMCID: PMC5774680. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.MacIntyre CR, Heslop D.J., Nand D., Schramm C., Butel M., Rawlinson W., Baker M., Kiedrzynski T., Nelson C., Rosewell A., Fotualii L., Yeo K., Elsgaard J., Fonua L. and Lane J.M. White Paper on response to a smallpox bioterrorism release in the Pacific. Global Biosecurity. 2019;1(1):91–105. 10.31646/gbio.10 [DOI] [Google Scholar]
4.Kim KC, Choi BS, Kim KC, Park KH, Lee HJ, Cho YK, et al. A Simple Mouse Model for the Study of Human Immunodeficiency Virus. AIDS Res Hum Retroviruses. 2016;32(2):194–202. Epub 2015/11/14. doi: 10.1089/AID.2015.0211 ; PubMed Central PMCID: PMC4761813. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Bird BH, Spengler JR, Chakrabarti AK, Khristova ML, Sealy TK, Coleman-McCray JD, et al. Humanized Mouse Model of Ebola Virus Disease Mimics the Immune Responses in Human Disease. J Infect Dis. 2016;213(5):703–11. Epub 2015/11/20. doi: 10.1093/infdis/jiv538 ; PubMed Central PMCID: PMC4747627. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Jaiswal S, Smith K, Ramirez A, Woda M, Pazoles P, Shultz LD, et al. Dengue virus infection induces broadly cross-reactive human IgM antibodies that recognize intact virions in humanized BLT-NSG mice. Exp Biol Med (Maywood). 2015;240(1):67–78. Epub 2014/08/16. doi: 10.1177/1535370214546273 ; PubMed Central PMCID: PMC4446989. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Chapman JL, Nichols DK, Martinez MJ, Raymond JW. Animal models of orthopoxvirus infection. Vet Pathol. 2010;47(5):852–70. Epub 2010/08/05. doi: 10.1177/0300985810378649 . [DOI] [PubMed] [Google Scholar]
8.Fenner F, Henderson D.A., Arita I., Jezek Z., Ladnyi I.D. Smallpox and its eradication: World Health Organization; 1988. [Google Scholar]
9.Americo JL, Moss B, Earl PL. Identification of wild-derived inbred mouse strains highly susceptible to monkeypox virus infection for use as small animal models. J Virol. 2010;84(16):8172–80. Epub 2010/06/04. doi: 10.1128/JVI.00621-10 ; PubMed Central PMCID: PMC2916512. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Hutson CL, Carroll DS, Gallardo-Romero N, Drew C, Zaki SR, Nagy T, et al. Comparison of Monkeypox Virus Clade Kinetics and Pathology within the Prairie Dog Animal Model Using a Serial Sacrifice Study Design. Biomed Res Int. 2015;2015:965710. Epub 2015/09/18. doi: 10.1155/2015/965710; PubMed Central PMCID: PMC4561332. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Hutson CL, Carroll DS, Gallardo-Romero N, Weiss S, Clemmons C, Hughes CM, et al. Monkeypox disease transmission in an experimental setting: prairie dog animal model. PLoS One. 2011;6(12):e28295. Epub 2011/12/14. doi: 10.1371/journal.pone.0028295; PubMed Central PMCID: PMC3229555. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Earl PL, Americo JL, Cotter CA, Moss B. Comparative live bioluminescence imaging of monkeypox virus dissemination in a wild-derived inbred mouse (Mus musculus castaneus) and outbred African dormouse (Graphiurus kelleni). Virology. 2015;475:150–8. Epub 2014/12/03. doi: 10.1016/j.virol.2014.11.015 ; PubMed Central PMCID: PMC4280325. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Gallardo-Romero NF, Hutson CL, Carroll D, Kondas AV, Salzer JS, Dietz-Ostergaard S, et al. Use of live Variola virus to determine whether CAST/EiJ mice are a suitable surrogate animal model for human smallpox. Virus Res. 2020;275:197772. Epub 2019/10/09. doi: 10.1016/j.virusres.2019.197772. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Carroll DS, Olson VA, Smith SK, Braden ZH, Patel N, Abel J, et al. Orthopoxvirus variola infection of Cynomys ludovicianus (North American black tailed prairie dog). Virology. 2013;443(2):358–62. Epub 2013/07/03. doi: 10.1016/j.virol.2013.05.029 . [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Titova KA, Sergeev AA, Zamedyanskaya AS, Galahova DO, Kabanov AS, Morozova AA, et al. Using ICR and SCID mice as animal models for smallpox to assess antiviral drug efficacy. J Gen Virol. 2015;96(9):2832–43. Epub 2015/06/13. doi: 10.1099/vir.0.000216 . [DOI] [PubMed] [Google Scholar]
16.Jahrling PB, Hensley LE, Martinez MJ, Leduc JW, Rubins KH, Relman DA, et al. Exploring the potential of variola virus infection of cynomolgus macaques as a model for human smallpox. Proc Natl Acad Sci U S A. 2004;101(42):15196–200. Epub 2004/10/13. doi: 10.1073/pnas.0405954101 ; PubMed Central PMCID: PMC523454. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Wahl-Jensen V, Cann JA, Rubins KH, Huggins JW, Fisher RW, Johnson AJ, et al. Progression of pathogenic events in cynomolgus macaques infected with variola virus. PLoS One. 2011;6(10):e24832. Epub 2011/10/15. doi: 10.1371/journal.pone.0024832; PubMed Central PMCID: PMC3188545. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Shultz LD, Brehm MA, Garcia-Martinez JV, Greiner DL. Humanized mice for immune system investigation: progress, promise and challenges. Nat Rev Immunol. 2012;12(11):786–98. Epub 2012/10/13. doi: 10.1038/nri3311 ; PubMed Central PMCID: PMC3749872. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Shultz LD, Ishikawa F, Greiner DL. Humanized mice in translational biomedical research. Nat Rev Immunol. 2007;7(2):118–30. Epub 2007/01/30. doi: 10.1038/nri2017 . [DOI] [PubMed] [Google Scholar]
20.Shultz LD, Keck J, Burzenski L, Jangalwe S, Vaidya S, Greiner DL, et al. Humanized mouse models of immunological diseases and precision medicine. Mamm Genome. 2019;30(5–6):123–42. Epub 2019/03/09. doi: 10.1007/s00335-019-09796-2 ; PubMed Central PMCID: PMC6610695. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Councilman WT, Magrath GB, Brinckerhoff WR. The Pathological Anatomy and Histology of Variola. J Med Res. 1904;11(1):12–135. Epub 1904/02/01. ; PubMed Central PMCID: PMC2104636. [PMC free article] [PubMed] [Google Scholar]
22.Lillie RD. Smallpox and Vaccinia The Pathologic Histology. Archives of Pathology. 1930;10. [Google Scholar]
23.Downie AW, Macdonald A. Smallpox and related virus infections in man. Br Med Bull. 1953;9(3):191–5. Epub 1953/01/01. doi: 10.1093/oxfordjournals.bmb.a074354 . [DOI] [PubMed] [Google Scholar]
24.Martin DB. The cause of death in smallpox: an examination of the pathology record. Mil Med. 2002;167(7):546–51. Epub 2002/07/20. . [PubMed] [Google Scholar]
25.Bras G. The morbid anatomy of smallpox. Doc Med Geogr Trop. 1952;4(4):303–51. Epub 1952/12/01. . [PubMed] [Google Scholar]
26.Moore ZS, Seward JF, Lane JM. Smallpox. Lancet. 2006;367(9508):425–35. Epub 2006/02/07. doi: 10.1016/S0140-6736(06)68143-9 . [DOI] [PubMed] [Google Scholar]
27.Biswas S, Chang H, Sarkis PT, Fikrig E, Zhu Q, Marasco WA. Humoral immune responses in humanized BLT mice immunized with West Nile virus and HIV-1 envelope proteins are largely mediated via human CD5+ B cells. Immunology. 2011;134(4):419–33. Epub 2011/11/03. doi: 10.1111/j.1365-2567.2011.03501.x ; PubMed Central PMCID: PMC3230796. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Lan P, Tonomura N, Shimizu A, Wang S, Yang YG. Reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and CD34+ cell transplantation. Blood. 2006;108(2):487–92. Epub 2006/01/18. doi: 10.1182/blood-2005-11-4388 . [DOI] [PubMed] [Google Scholar]
29.Melkus MW, Estes JD, Padgett-Thomas A, Gatlin J, Denton PW, Othieno FA, et al. Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1. Nat Med. 2006;12(11):1316–22. Epub 2006/10/24. doi: 10.1038/nm1431 . [DOI] [PubMed] [Google Scholar]
30.Pearson T, Greiner DL, Shultz LD. Creation of "humanized" mice to study human immunity. Curr Protoc Immunol. 2008;Chapter 15:Unit 15 21. Epub 2008/05/21. doi: 10.1002/0471142735.im1521s81; PubMed Central PMCID: PMC3023233. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Gregorczyk KP, Wyzewski Z, Szczepanowska J, Toka FN, Mielcarska MB, Bossowska-Nowicka M, et al. Ectromelia Virus Affects Mitochondrial Network Morphology, Distribution, and Physiology in Murine Fibroblasts and Macrophage Cell Line. Viruses. 2018;10(5). Epub 2018/05/19. doi: 10.3390/v10050266; PubMed Central PMCID: PMC5977259. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Szulc-Dabrowska L, Wyzewski Z, Gregorczyk-Zboroch KP, Toka FN, Szczepanowska J, Struzik J, et al. Mitochondria-related gene expression profiles in murine fibroblasts and macrophages during later stages of ectromelia virus infection in vitro. Acta Virol. 2020;64(3):307–24. Epub 2020/09/29. doi: 10.4149/av_2020_305 . [DOI] [PubMed] [Google Scholar]
33.Wong PS, Sutejo R, Chen H, Ng SH, Sugrue RJ, Tan BH. A System Based-Approach to Examine Cytokine Response in Poxvirus-Infected Macrophages. Viruses. 2018;10(12). Epub 2018/12/20. doi: 10.3390/v10120692; PubMed Central PMCID: PMC6316232. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Yadav VN, Pyaram K, Mullick J, Sahu A. Identification of hot spots in the variola virus complement inhibitor (SPICE) for human complement regulation. J Virol. 2008;82(7):3283–94. Epub 2008/01/25. doi: 10.1128/JVI.01935-07 ; PubMed Central PMCID: PMC2268448. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Lee SH, Jung JU, Means RE. ’Complementing’ viral infection: mechanisms for evading innate immunity. Trends Microbiol. 2003;11(10):449–52. Epub 2003/10/15. doi: 10.1016/j.tim.2003.08.004 . [DOI] [PubMed] [Google Scholar]
36.Rosengard AM, Liu Y, Nie Z, Jimenez R. Variola virus immune evasion design: expression of a highly efficient inhibitor of human complement. Proc Natl Acad Sci U S A. 2002;99(13):8808–13. Epub 2002/05/30. doi: 10.1073/pnas.112220499 ; PubMed Central PMCID: PMC124380. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Van Vliet K, Mohamed MR, Zhang L, Villa NY, Werden SJ, Liu J, et al. Poxvirus proteomics and virus-host protein interactions. Microbiol Mol Biol Rev. 2009;73(4):730–49. Epub 2009/12/01. doi: 10.1128/MMBR.00026-09 ; PubMed Central PMCID: PMC2786582. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Smithson C, Purdy A, Verster AJ, Upton C. Prediction of steps in the evolution of variola virus host range. PLoS One. 2014;9(3):e91520. Epub 2014/03/15. doi: 10.1371/journal.pone.0091520; PubMed Central PMCID: PMC3953476. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Schweneker M, Lukassen S, Spath M, Wolferstatter M, Babel E, Brinkmann K, et al. The vaccinia virus O1 protein is required for sustained activation of extracellular signal-regulated kinase 1/2 and promotes viral virulence. J Virol. 2012;86(4):2323–36. Epub 2011/12/16. doi: 10.1128/JVI.06166-11 ; PubMed Central PMCID: PMC3302380. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Bera BC, Shanmugasundaram K, Barua S, Venkatesan G, Virmani N, Riyesh T, et al. Zoonotic cases of camelpox infection in India. Vet Microbiol. 2011;152(1–2):29–38. Epub 2011/05/17. doi: 10.1016/j.vetmic.2011.04.010 . [DOI] [PubMed] [Google Scholar]
41.Khalafalla AI, Abdelazim F. Human and Dromedary Camel Infection with Camelpox Virus in Eastern Sudan. Vector Borne Zoonotic Dis. 2017;17(4):281–4. Epub 2017/01/06. doi: 10.1089/vbz.2016.2070 . [DOI] [PubMed] [Google Scholar]
42.Muhlemann B, Vinner L, Margaryan A, Wilhelmson H, de la Fuente Castro C, Allentoft ME, et al. Diverse variola virus (smallpox) strains were widespread in northern Europe in the Viking Age. Science. 2020;369(6502). Epub 2020/07/25. doi: 10.1126/science.aaw8977. [DOI] [PubMed] [Google Scholar]
43.Quenelle DC, Prichard MN, Keith KA, Hruby DE, Jordan R, Painter GR, et al. Synergistic efficacy of the combination of ST-246 with CMX001 against orthopoxviruses. Antimicrob Agents Chemother. 2007;51(11):4118–24. Epub 2007/08/29. doi: 10.1128/AAC.00762-07 ; PubMed Central PMCID: PMC2151443. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Chen N, Bellone CJ, Schriewer J, Owens G, Fredrickson T, Parker S, et al. Poxvirus interleukin-4 expression overcomes inherent resistance and vaccine-induced immunity: pathogenesis, prophylaxis, and antiviral therapy. Virology. 2011;409(2):328–37. Epub 2010/11/13. doi: 10.1016/j.virol.2010.10.021 ; PubMed Central PMCID: PMC3008208. [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Lederman ER, Davidson W, Groff HL, Smith SK, Warkentien T, Li Y, et al. Progressive vaccinia: case description and laboratory-guided therapy with vaccinia immune globulin, ST-246, and CMX001. J Infect Dis. 2012;206(9):1372–85. Epub 2012/08/21. doi: 10.1093/infdis/jis510 ; PubMed Central PMCID: PMC3529603. [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Aryee KE, Shultz LD, Brehm MA. Immunodeficient mouse model for human hematopoietic stem cell engraftment and immune system development. Methods Mol Biol. 2014;1185:267–78. Epub 2014/07/27. doi: 10.1007/978-1-4939-1133-2_18 ; PubMed Central PMCID: PMC4476265. [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Huggins J, Goff A, Hensley L, Mucker E, Shamblin J, Wlazlowski C, et al. Nonhuman primates are protected from smallpox virus or monkeypox virus challenges by the antiviral drug ST-246. Antimicrob Agents Chemother. 2009;53(6):2620–5. Epub 2009/04/08. doi: 10.1128/AAC.00021-09 ; PubMed Central PMCID: PMC2687232. [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Esposito J, Condit R, Obijeski J. The preparation of orthopoxvirus DNA. J Virol Methods. 1981;2(3):175–9. Epub 1981/02/01. doi: 10.1016/0166-0934(81)90036-7 . [DOI] [PubMed] [Google Scholar]
49.Esposito JJ, Knight JC. Orthopoxvirus DNA: a comparison of restriction profiles and maps. Virology. 1985;143(1):230–51. Epub 1985/05/01. doi: 10.1016/0042-6822(85)90111-4 . [DOI] [PubMed] [Google Scholar]
50.Esposito JJ, Obijeski JF, Nakano JH. Orthopoxvirus DNA: strain differentiation by electrophoresis of restriction endonuclease fragmented virion DNA. Virology. 1978;89(1):53–66. Epub 1978/08/01. doi: 10.1016/0042-6822(78)90039-9 . [DOI] [PubMed] [Google Scholar]
51.Olson VA, Laue T, Laker MT, Babkin IV, Drosten C, Shchelkunov SN, et al. Real-time PCR system for detection of orthopoxviruses and simultaneous identification of smallpox virus. J Clin Microbiol. 2004;42(5):1940–6. Epub 2004/05/08. doi: 10.1128/JCM.42.5.1940-1946.2004 ; PubMed Central PMCID: PMC404623. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Ropp SL, Jin Q, Knight JC, Massung RF, Esposito JJ. PCR strategy for identification and differentiation of small pox and other orthopoxviruses. J Clin Microbiol. 1995;33(8):2069–76. Epub 1995/08/01. doi: 10.1128/jcm.33.8.2069-2076.1995 ; PubMed Central PMCID: PMC228337. [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Sarmiento M, Haffey M, Spear PG. Membrane proteins specified by herpes simplex viruses. III. Role of glycoprotein VP7(B2) in virion infectivity. J Virol. 1979;29(3):1149–58. Epub 1979/03/01. doi: 10.1128/JVI.29.3.1149-1158.1979 ; PubMed Central PMCID: PMC353275. [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Guarner J, Johnson BJ, Paddock CD, Shieh WJ, Goldsmith CS, Reynolds MG, et al. Monkeypox transmission and pathogenesis in prairie dogs. Emerg Infect Dis. 2004;10(3):426–31. Epub 2004/04/28. doi: 10.3201/eid1003.030878 ; PubMed Central PMCID: PMC3322777. [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Martines RB, Ritter JM, Matkovic E, Gary J, Bollweg BC, Bullock H, et al. Pathology and Pathogenesis of SARS-CoV-2 Associated with Fatal Coronavirus Disease, United States. Emerg Infect Dis. 2020;26(9):2005–15. Epub 2020/05/22. doi: 10.3201/eid2609.202095 ; PubMed Central PMCID: PMC7454055. [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Goldsmith CS, Whistler T, Rollin PE, Ksiazek TG, Rota PA, Bellini WJ, et al. Elucidation of Nipah virus morphogenesis and replication using ultrastructural and molecular approaches. Virus Res. 2003;92(1):89–98. Epub 2003/02/28. doi: 10.1016/s0168-1702(02)00323-4 . [DOI] [PubMed] [Google Scholar]
57.Kondas AV, Olson VA, Li Y, Abel J, Laker M, Rose L, et al. Variola virus-specific diagnostic assays: characterization, sensitivity, and specificity. J Clin Microbiol. 2015;53(4):1406–10. Epub 2015/02/13. doi: 10.1128/JCM.03613-14 ; PubMed Central PMCID: PMC4365196. [DOI] [PMC free article] [PubMed] [Google Scholar]
58.Karem KL, Reynolds M, Braden Z, Lou G, Bernard N, Patton J, et al. characterization of acute-phase humoral immunity to monkeypox: use of immunoglobulin M enzyme-linked immunosorbent assay for detection of monkeypox infection during the 2003 North American outbreak. Clin Diagn Lab Immunol. 2005;12(7):867–72. Epub 2005/07/09. doi: 10.1128/CDLI.12.7.867-872.2005 ; PubMed Central PMCID: PMC1182207. [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS Pathog. doi: 10.1371/journal.ppat.1009633.r001

Decision Letter 0

Erle S Robertson, Bernard Moss

30 Oct 2020

Dear Dr. Hutson,

Thank you for submitting your manuscript "Can you teach a new mouse old tricks? Humanized mice as a model for Variola virus". Your manuscript was fully evaluated at the editorial level and by three independent peer reviewers. As you will see, the reviewers appreciated the importance of the topic but identified numerous aspects of the manuscript that must be improved before final consideration for publication. Most of the comments of Reviewers 1 and 3 can be dealt with by modifications of the text except for the queries regarding replication of the virus infectivity assays and negative control staining of anti-VARV antibodies. Reviewer 2 provided a much more critical response, which I largely agree with. Although the reviewer is correct in the assessment that replication of the results would greatly improve the study, I have decided that this will not be a condition of acceptability. Nevertheless, changes in the presentation of the data as outlined are needed to evaluate the significance of the work. I confirmed that Hu-BLT mice could not be found in a search of the JAX website and this mouse strain needs to be fully described

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Bernard Moss, M.D., Ph.D.

Guest Editor

PLOS Pathogens

Erle Robertson

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Background

Smallpox was a human disease caused by variola virus (VARV) and small animal models of smallpox are not available. Due to fear of bioterrorism or re-emergence of smallpox, the WHA authorised research with VARV to develop drugs, diagnostics and a safer vaccine. Testing of such medical countermeasures (MCMs) has been hindered by the lack of a suitable small animal model. Nonetheless, in 2018 the US FDA approved use of an anti-smallpox drug (tecovirimat) using data derived from animal models with surrogate orthopoxviruses. Given that orthopoxviruses can become resistant to tecovirimat, another drug with a different mechanism of action is needed. Here, the authors have explored 3 different humanized mice as possible models in which to test promising new antivirals.

Summary

The study describes the use of humanized mice for infection with VARV and shows that BLT and CD34+ humanized mice support VARV replication and systemic spread to develop disease with some similarity to human smallpox. The authors provide evidence of VARV infection in different tissues by histological and immunohistological analyses and show the presence of orthopoxvirus particles in infected cells by electron microscopy and the presence of VARV DNA by PCR.

Reviewer #2: The manuscript by Hutson presents a new mouse model for variola virus infection using mice that have a humanized hematopoietic system. The results are striking and interesting in that a systemic disease with some features similar to smallpox is shown in some individual mice, especially for the HuCd34+ and HuBLT mice. The study presents data that one would expect to see in the characterization of a new model and there is little doubt that the infection and pathogenesis are unique in the mice engrafted with human hematopoietic cells. However the majority of the data are derived from a single experiment that has a small number of mice per group (4 or 3 only). There is also a focus on presentation of histological findings that are qualitative by nature, which is helpful, but also tends to lend the feel of a case study to the paper, rather than a more rigorously quantitative approach. Even the virus loads are presented on a per-animal basis instead of being compiled in a way that makes basic statistical parameters easy to assess mean/median and a representation of error or distribution. If this model is to be used to test new drugs for smallpox, it is necessary that there is a better understanding of the range of all parameters that might be measured across a group of mice so that the study can be adequately powered. With the current data and especially the way it is presented, this is difficult to glean. On the one hand it is important to acknowledge that the work that is presented here, which requires BSL4 containment, is extremely difficult to do. However on the other, the intent is for other studies to follow the path that is set by this publication, so it is imperative that this not simply leave an impression of what is happening, but provide some firm quantitative information. In balancing these considerations, more data and at least one independent replicate for each strain/dose that the authors would advocate for future use would help greatly to cement the value of the study.

Reviewer #3: The authors describe new mouse models for smallpox. Three humanized mice (hu-PBMC, hu-CD34+ and hu-BLT) were infected with variola virus (VARV) and shown to be susceptible to infection. The infection was lethal for hu-CD34+ and hu-BT mice, whereas the virus replicated in hu-PBMC mice but caused a mild disease with no mortalities. A description of the pathology and virus replication is provided.

The finding of mouse models of smallpox that allow the study of virus replication and pathological consequences of the infection is of high interest in the field. These models will be useful to evaluate candidate anti-viral drugs, although the study of vaccines and immune response will be limited (human IgG and IgM were not detected in the initial studies). Also, they bring fundamental and interesting questions on the human susceptibility to VARV and the factors that confer resistance to infection in mice. The manuscript is mainly descriptive (necessary in the first report of these models) and the discussion could be expanded, even if some hypotheses may need additional experimentation.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: This reviewer is not requesting further experimentation, unless the virus infectivity titrations in figure 5 have only been conducted once without replicate assays.

Reviewer #2: As noted at the end of the general comments. There needs to be independent replication of the experiments and a total of more mice such that a better assessment of the frequency of all features of pathology in a group of each type of mouse and each dose can be presented.

Reviewer #3: One of the most interesting questions is to identify which components of the human immune system are required for VARV to spread in these mice. Further discussion on the component of the human immune system recreated in hu-PBMC mice (showing mild disease) as compared to hu-BLT and hu-CD34+ mice (showing severe disease) would be interesting and maybe informative.

VARV infects not only human immune cells but also mouse tissues, with very high virus titers in different organs. This is an interesting result since human cells may help virus spread to tissues that can support virus replication. More discussion on the infection of mouse tissues by VARV in this model and the existing evidence of the susceptibility of mouse cells to VARV would be helpful. This would emphasize the role of human immune cells in the establishment of a systemic infection in these mice.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Could the authors please consider the following points?

1. Title. It is established that mice can be engineered to become susceptible to infection by different viruses to which normally they are resistant. This was shown first with polio virus and more recently there are other examples as mentioned in the introduction. Given this, the question posed in the first half of the title is already answered. So the title should be shortened by removal of the first part.

2. In several places the paper is drafted for a USA audience who may understand what a “select agent” is and what CDC and FDA are. For the benefit of the international readership of PLOS Pathogens could these terms be defined / explained more fully? e.g. US FDA, US CDC (there is also a CDC in Europe) and the nation from which terms such as “select agent” and “select agent regulations” derive be defined.

3. The abstract concludes that the model described may be useful for 3 things: testing MCMs for smallpox, identifying why VARV is a pathogen only of humans, and provides samples for understanding the human immune response to VARV infection. This reviewer agrees with the first point, but not the latter two and recommends these should be deleted.

4. Lines 54-55. It is not clear what “current recommendations” are referred to. Suggest deletion of “improves upon current recommendations by”.

5. Line 65-66. Reconstruction of horsepox virus. It may be misleading to suggest that horsepox virus (which is considered a strain of VACV) is closely related to VARV. Although all OPXVs are closely related, within the OPXV genus VACV and VARV are quite distinct and taterapox virus and camelpox virus are closest to VARV.

6. Line 96. define IACUC

7. Line 114. Specify if anesthesia was used. Similarly, line 129, define “5% inhalation anesthesia” – inhalation of what at 5%?

8. Line 155. “As” is missing from this sentence.

9. In general the figure legends i) do not provide enough detail of what was done or what is shown, and ii) contain too much interpretation of material presented, which should instead (mostly) be contained in the results narrative.

10. Fig. 1. The labeling and presentation of this figure needs improvement. Data presented in panels A and B are also included in C and D. So delete A and B. Please relabel figure with increased font size. In the legend define what high and low mean - this could easily be included on the figure itself. Change “survivorship” to “survival” (in text also) Day = day post infection.

11. Lines 178 – 184. If this animal was not infected correctly it should be removed from all data presented and the group size redefined.

12. Line 194. Please explain what “gamma-irradiated control hu-PBMC mouse” is. There is no mention of gamma-irradiation in the Fig. legend.

13. Fig. 2. Please re-write legend to describe clearly which animals these samples are from, what dose of virus was given, the day post infection the samples were harvested, and what VARV positive staining looks like. In the histological images please use arrows to point out the features to which the text refers. Ditto Fig. 3. In Fig. 3 arrows are referred to, but were not visible to this reviewer. Specify which panel(s) they are in.

14. Line 254 Define “select tissues”.

15. Fig. 5. Define “processed for viral culture”. Plaque assay, or something else? The number of replicate samples, the number of times the plaque assay (or other assay) was repeated need defining, and error bars should be added. A statistical analysis should be included to justify what is “significant”.

16. The legend to Fig. 5 defines a *. But no * is visible in the figure. Define “LOD”.

17. Line 282. Explain what “Gross findings were absent during necropsy” means.

18. Line 297. Consider changing “extremely” to “increasingly”.

19. Lines 318-321. Sentence repetition. This reviewer feels this sentence overstates what has been shown. The report shows that these mice can be infected with VARV and develop a disease with some similarity to human smallpox. The report has not shown that this model will be useful for studying VARV infection, or potentially features of the human immune response to infection. On the other hand, it is reasonable to propose that this model may be useful for evaluating the effectiveness of drugs against smallpox. Similar comment for last part of discussion.

20. In the discussion, the claim that the model has features similar to hemorrhagic smallpox, or ordinary smallpox, is not well founded base on data presented, are unnecessary and should be deleted. This will help reduce the discussion, which is rather too long.

21. Line 372 typos “pathogenby compareing”

22. Lines 371-83. Discussion on why VARV is a pathogen only of humans should include consideration of gene loss during VARV evolution, see Science, 2020 DOI: 10.1126/science.aaw8977. The focus on the O1L gene is more about why other related viruses do not infect man, and so might also be deleted to shorten the discussion.

Reviewer #2: 1) The paper is not written in a conventional way in which the results section has a narrative so that readers can easily follow what has been done. Rather this information has to be obtained by reading the Materials and Methods before the Results. This is unwieldy and will be more so if the manuscript was formatted with the Methods after the Discussion as per the journal style. Further, the data on the numbers of mice with different findings is put in supplementary tables, when they are key to understanding what is happening across each group of mice. These need to be in the main paper. In the second table the groups should be separated by dose as they are in the first table.

2) The issue of just how many mice were in each group is made more difficult to follow because the authors use “n” to indicate the number of mice with a particular finding and not the total group size as defined conventionally in statistics. Further statements about the frequency of signs of illness that aggregates this across the three different types of humanized mice is not helpful in understanding whether one model would be preferred over another. As noted above, key data like the virus titers need to be presented in a more conventional group-wise manner. Individual data can and should still be shown (as points), but with the titers from the same organ (and experimental group) plotted together. This would allow comparisons across organs or mouse types.

3) A key feature piece of information that is required to truly understand this model is the extent to which virus replication is supported in mouse cells versus human cells. This is accessible to some extent from the histopathology, immunohistochemistry and descriptions, that find in at least some cases that VARV is indeed replicating in mouse tissues (e.g. to cause hock lesions), but is not addressed systematically. For example it would be interesting to know how much of the virus titer in organs may have been contributed by the human cells in transit. Some assessment of this could be made by qPCR for human markers to see is virus levels correlated with human cell levels. Perhaps mice were perfused to limit this complication, but this is not mentioned. In any case, adding more data that explores this issue would add more broad scientific interest to the paper.

4) The different humanized mice are not adequately introduced to the reader, nor are the results discussed in the context of the different biology of these models in a lot of depth. Indeed, the acronymns are not spelt out even in the materials and methods and a search of the Jax catalogue for “Hu-BLT” yeilds no results (the other strains could be found, but are given their full name, which is what is required in the manuscript). The discussion touches on differences in GVH (but again this is not explained) in these mouse strains, but there are also difference in the types of cells that develop in these mice. Given the role of macrophages/monocytes as an important cell for dissemination of ectromelia virus, the fact that these cells tend not to develop well in Hu-PBMC mice also be significant.

Reviewer #3: Line 89. It is mentioned that hu-BLT mice inoculated with low dose group succumbed to disease, but hu-CD34 mice also succumbed at low dose (Table S1 and Fig.1).

Negative control staining of anti-VARV antibodies against uninfected mouse tissues should be shown.

I could not see the legends to Supplementary Figures.

Table S1 should be included in the manuscript since it has relevant information.

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure Files:

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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/plospathogens/s/submission-guidelines#loc-materials-and-methods

PLoS Pathog. 2021 Sep 21;17(9):e1009633. doi: 10.1371/journal.ppat.1009633.r002

Author response to Decision Letter 0

10 Feb 2021

Attachment

Submitted filename: Answers to Reviewers.docx

Click here for additional data file.^{(19.8KB, docx)}

PLoS Pathog. doi: 10.1371/journal.ppat.1009633.r003

Decision Letter 1

Michael H Malim, Bernard Moss

22 Mar 2021

Dear Dr. Hutson,

Thank you very much for submitting a revised manuscript "Can you teach a new mouse old tricks? Humanized mice as a model for Variola virus" for consideration at PLOS Pathogens. Although the reviewers felt that the manuscript was improved, some comments still need to be addressed. It would help and speed up the review process, if you responded in a point-by-point manner and more specifically indicated the changes made. We would also like to suggest a change in the title as humanized mice are an infection model for variola virus rather than a model for variola virus as stated. Also. rather than questioning whether you can teach a new mouse old tricks, you might change the title to the more affirmative "Teaching a new mouse old tricks: Humanized mice as an infection model for variola virus" as an example.

Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed (point-by-point) responses to all review comments, and a description of the changes you have made in the manuscript.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Sincerely,

Bernard Moss, M.D., Ph.D.

Guest Editor

PLOS Pathogens

Michael Malim

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: (No Response)

Reviewer #2: In this revised manuscript, the authors have addressed most of the issues raised about the presentation of the data (noting some exceptions below), but not the substantive point made in my original review that the study remains limited by the very small group sizes and lack of replication, neither was this rebutted in any way. The new presentation of the virus titers further reinforce that variation is a substantial issue here that may make these models difficult to use. Interestingly the HuCD34+ mice seemed more promising in this regard with relatively consistent titers from most tissues rather than viral loads spanning many orders of magnitude as seen with the other types of mice.

Reviewer #3: The authors have addressed the concerns raised by the reviewers.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Reviewer #1: (No Response)

Reviewer #2: There remains no replication of the experiments and no argument was made as to why this is not necessary.

Reviewer #3: None

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: 1) The intranasal route of infection should be noted in the first sentence describing the experiment in the Results section and in at least the legend for the first figure. An outline of the experiment as a panel in the first figure showing a timeline with infection, starting numbers, when mice succumbed and sampling time would also make things clearest.

2) Statistical comparisons made in the text remain difficult to assess from the data shown in figures because often the groups being compared are separated on different graphs. E.g. a statistical comparison of mortality is made between doses for each models in the text, but the groups are plotted on two sets of axes in the figure, with the two doses on separate graphs.

3) The authors still use “n” to refer both to the total group size and the number of mice with a particular finding or are ambiguous in numerical descriptions. In one case this is “n=2/8”. In another sentence “involving up to 50% of liver in hu-CD34+ (n=3) and hu-BLT (n-=5)” – does this mean 50% of the livers in affected mice, or livers in 50% of the mice (but if the group sizes are 3 and 5, how does this make sense). The use of “n” should be exclusively for the group size. When reporting findings “1/5” as used in some places (or 1 of 5) should be used consistently. The reader should not be having to go to the tables to make sense of the text.

4) Do the virus loads correlate in any way to clinical findings? This should be explored and discussed.

5) Variation in the data should be discussed as this has an impact on the usability of these models to test drugs etc.

Reviewer #3: None

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure Files:

Data Requirements:

Reproducibility:

References:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript.

PLoS Pathog. 2021 Sep 21;17(9):e1009633. doi: 10.1371/journal.ppat.1009633.r004

Author response to Decision Letter 1

10 May 2021

Attachment

Submitted filename: Round 2 reviewer comments.docx

Click here for additional data file.^{(20KB, docx)}

PLoS Pathog. doi: 10.1371/journal.ppat.1009633.r005

Decision Letter 2

Michael H Malim, Bernard Moss

11 May 2021

Dear Dr. Hutson,

We are pleased to inform you that your manuscript 'Teaching a new mouse old tricks: Humanized mice as an infection model for Variola virus' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Bernard Moss, M.D., Ph.D.

Guest Editor

PLOS Pathogens

Michael Malim

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):

PLoS Pathog. doi: 10.1371/journal.ppat.1009633.r006

Acceptance letter

Michael H Malim, Bernard Moss

29 Jun 2021

Dear Dr. Hutson,

We are delighted to inform you that your manuscript, "Teaching a new mouse old tricks: Humanized mice as an infection model for Variola virus," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. VARV immunohistochemistry in representative tissues from control mice of each strain inoculated with PBS only or with gamma-irradiated VARV.

(TIF)

Click here for additional data file.^{(4.1MB, tif)}

S2 Fig. Representative histopathology and VARV immunohistochemistry in non-humanized NSG mice.

(TIF)

Click here for additional data file.^{(4.1MB, tif)}

S3 Fig. NSG background mouse was not susceptible to systemic variola virus infection.

(TIF)

Click here for additional data file.^{(2.7MB, tif)}

Attachment

Submitted filename: Answers to Reviewers.docx

Click here for additional data file.^{(19.8KB, docx)}

Attachment

Submitted filename: Round 2 reviewer comments.docx

Click here for additional data file.^{(20KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

[ppat.1009633.ref001] 1.Reardon S. NIH finds forgotten smallpox store: Nature; 2014. Available from: https://www.nature.com/news/nih-finds-forgotten-smallpox-store-1.15526. [DOI] [PubMed] [Google Scholar]

[ppat.1009633.ref002] 2.Noyce RS, Lederman S, Evans DH. Construction of an infectious horsepox virus vaccine from chemically synthesized DNA fragments. PLoS One. 2018;13(1):e0188453. Epub 2018/01/20. doi: 10.1371/journal.pone.0188453; PubMed Central PMCID: PMC5774680. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref003] 3.MacIntyre CR, Heslop D.J., Nand D., Schramm C., Butel M., Rawlinson W., Baker M., Kiedrzynski T., Nelson C., Rosewell A., Fotualii L., Yeo K., Elsgaard J., Fonua L. and Lane J.M. White Paper on response to a smallpox bioterrorism release in the Pacific. Global Biosecurity. 2019;1(1):91–105. 10.31646/gbio.10 [DOI] [Google Scholar]

[ppat.1009633.ref004] 4.Kim KC, Choi BS, Kim KC, Park KH, Lee HJ, Cho YK, et al. A Simple Mouse Model for the Study of Human Immunodeficiency Virus. AIDS Res Hum Retroviruses. 2016;32(2):194–202. Epub 2015/11/14. doi: 10.1089/AID.2015.0211 ; PubMed Central PMCID: PMC4761813. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref005] 5.Bird BH, Spengler JR, Chakrabarti AK, Khristova ML, Sealy TK, Coleman-McCray JD, et al. Humanized Mouse Model of Ebola Virus Disease Mimics the Immune Responses in Human Disease. J Infect Dis. 2016;213(5):703–11. Epub 2015/11/20. doi: 10.1093/infdis/jiv538 ; PubMed Central PMCID: PMC4747627. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref006] 6.Jaiswal S, Smith K, Ramirez A, Woda M, Pazoles P, Shultz LD, et al. Dengue virus infection induces broadly cross-reactive human IgM antibodies that recognize intact virions in humanized BLT-NSG mice. Exp Biol Med (Maywood). 2015;240(1):67–78. Epub 2014/08/16. doi: 10.1177/1535370214546273 ; PubMed Central PMCID: PMC4446989. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref007] 7.Chapman JL, Nichols DK, Martinez MJ, Raymond JW. Animal models of orthopoxvirus infection. Vet Pathol. 2010;47(5):852–70. Epub 2010/08/05. doi: 10.1177/0300985810378649 . [DOI] [PubMed] [Google Scholar]

[ppat.1009633.ref008] 8.Fenner F, Henderson D.A., Arita I., Jezek Z., Ladnyi I.D. Smallpox and its eradication: World Health Organization; 1988. [Google Scholar]

[ppat.1009633.ref009] 9.Americo JL, Moss B, Earl PL. Identification of wild-derived inbred mouse strains highly susceptible to monkeypox virus infection for use as small animal models. J Virol. 2010;84(16):8172–80. Epub 2010/06/04. doi: 10.1128/JVI.00621-10 ; PubMed Central PMCID: PMC2916512. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref010] 10.Hutson CL, Carroll DS, Gallardo-Romero N, Drew C, Zaki SR, Nagy T, et al. Comparison of Monkeypox Virus Clade Kinetics and Pathology within the Prairie Dog Animal Model Using a Serial Sacrifice Study Design. Biomed Res Int. 2015;2015:965710. Epub 2015/09/18. doi: 10.1155/2015/965710; PubMed Central PMCID: PMC4561332. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref011] 11.Hutson CL, Carroll DS, Gallardo-Romero N, Weiss S, Clemmons C, Hughes CM, et al. Monkeypox disease transmission in an experimental setting: prairie dog animal model. PLoS One. 2011;6(12):e28295. Epub 2011/12/14. doi: 10.1371/journal.pone.0028295; PubMed Central PMCID: PMC3229555. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref012] 12.Earl PL, Americo JL, Cotter CA, Moss B. Comparative live bioluminescence imaging of monkeypox virus dissemination in a wild-derived inbred mouse (Mus musculus castaneus) and outbred African dormouse (Graphiurus kelleni). Virology. 2015;475:150–8. Epub 2014/12/03. doi: 10.1016/j.virol.2014.11.015 ; PubMed Central PMCID: PMC4280325. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref013] 13.Gallardo-Romero NF, Hutson CL, Carroll D, Kondas AV, Salzer JS, Dietz-Ostergaard S, et al. Use of live Variola virus to determine whether CAST/EiJ mice are a suitable surrogate animal model for human smallpox. Virus Res. 2020;275:197772. Epub 2019/10/09. doi: 10.1016/j.virusres.2019.197772. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref014] 14.Carroll DS, Olson VA, Smith SK, Braden ZH, Patel N, Abel J, et al. Orthopoxvirus variola infection of Cynomys ludovicianus (North American black tailed prairie dog). Virology. 2013;443(2):358–62. Epub 2013/07/03. doi: 10.1016/j.virol.2013.05.029 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref015] 15.Titova KA, Sergeev AA, Zamedyanskaya AS, Galahova DO, Kabanov AS, Morozova AA, et al. Using ICR and SCID mice as animal models for smallpox to assess antiviral drug efficacy. J Gen Virol. 2015;96(9):2832–43. Epub 2015/06/13. doi: 10.1099/vir.0.000216 . [DOI] [PubMed] [Google Scholar]

[ppat.1009633.ref016] 16.Jahrling PB, Hensley LE, Martinez MJ, Leduc JW, Rubins KH, Relman DA, et al. Exploring the potential of variola virus infection of cynomolgus macaques as a model for human smallpox. Proc Natl Acad Sci U S A. 2004;101(42):15196–200. Epub 2004/10/13. doi: 10.1073/pnas.0405954101 ; PubMed Central PMCID: PMC523454. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref017] 17.Wahl-Jensen V, Cann JA, Rubins KH, Huggins JW, Fisher RW, Johnson AJ, et al. Progression of pathogenic events in cynomolgus macaques infected with variola virus. PLoS One. 2011;6(10):e24832. Epub 2011/10/15. doi: 10.1371/journal.pone.0024832; PubMed Central PMCID: PMC3188545. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref018] 18.Shultz LD, Brehm MA, Garcia-Martinez JV, Greiner DL. Humanized mice for immune system investigation: progress, promise and challenges. Nat Rev Immunol. 2012;12(11):786–98. Epub 2012/10/13. doi: 10.1038/nri3311 ; PubMed Central PMCID: PMC3749872. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref019] 19.Shultz LD, Ishikawa F, Greiner DL. Humanized mice in translational biomedical research. Nat Rev Immunol. 2007;7(2):118–30. Epub 2007/01/30. doi: 10.1038/nri2017 . [DOI] [PubMed] [Google Scholar]

[ppat.1009633.ref020] 20.Shultz LD, Keck J, Burzenski L, Jangalwe S, Vaidya S, Greiner DL, et al. Humanized mouse models of immunological diseases and precision medicine. Mamm Genome. 2019;30(5–6):123–42. Epub 2019/03/09. doi: 10.1007/s00335-019-09796-2 ; PubMed Central PMCID: PMC6610695. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref021] 21.Councilman WT, Magrath GB, Brinckerhoff WR. The Pathological Anatomy and Histology of Variola. J Med Res. 1904;11(1):12–135. Epub 1904/02/01. ; PubMed Central PMCID: PMC2104636. [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref022] 22.Lillie RD. Smallpox and Vaccinia The Pathologic Histology. Archives of Pathology. 1930;10. [Google Scholar]

[ppat.1009633.ref023] 23.Downie AW, Macdonald A. Smallpox and related virus infections in man. Br Med Bull. 1953;9(3):191–5. Epub 1953/01/01. doi: 10.1093/oxfordjournals.bmb.a074354 . [DOI] [PubMed] [Google Scholar]

[ppat.1009633.ref024] 24.Martin DB. The cause of death in smallpox: an examination of the pathology record. Mil Med. 2002;167(7):546–51. Epub 2002/07/20. . [PubMed] [Google Scholar]

[ppat.1009633.ref025] 25.Bras G. The morbid anatomy of smallpox. Doc Med Geogr Trop. 1952;4(4):303–51. Epub 1952/12/01. . [PubMed] [Google Scholar]

[ppat.1009633.ref026] 26.Moore ZS, Seward JF, Lane JM. Smallpox. Lancet. 2006;367(9508):425–35. Epub 2006/02/07. doi: 10.1016/S0140-6736(06)68143-9 . [DOI] [PubMed] [Google Scholar]

[ppat.1009633.ref027] 27.Biswas S, Chang H, Sarkis PT, Fikrig E, Zhu Q, Marasco WA. Humoral immune responses in humanized BLT mice immunized with West Nile virus and HIV-1 envelope proteins are largely mediated via human CD5+ B cells. Immunology. 2011;134(4):419–33. Epub 2011/11/03. doi: 10.1111/j.1365-2567.2011.03501.x ; PubMed Central PMCID: PMC3230796. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref028] 28.Lan P, Tonomura N, Shimizu A, Wang S, Yang YG. Reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and CD34+ cell transplantation. Blood. 2006;108(2):487–92. Epub 2006/01/18. doi: 10.1182/blood-2005-11-4388 . [DOI] [PubMed] [Google Scholar]

[ppat.1009633.ref029] 29.Melkus MW, Estes JD, Padgett-Thomas A, Gatlin J, Denton PW, Othieno FA, et al. Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1. Nat Med. 2006;12(11):1316–22. Epub 2006/10/24. doi: 10.1038/nm1431 . [DOI] [PubMed] [Google Scholar]

[ppat.1009633.ref030] 30.Pearson T, Greiner DL, Shultz LD. Creation of "humanized" mice to study human immunity. Curr Protoc Immunol. 2008;Chapter 15:Unit 15 21. Epub 2008/05/21. doi: 10.1002/0471142735.im1521s81; PubMed Central PMCID: PMC3023233. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref031] 31.Gregorczyk KP, Wyzewski Z, Szczepanowska J, Toka FN, Mielcarska MB, Bossowska-Nowicka M, et al. Ectromelia Virus Affects Mitochondrial Network Morphology, Distribution, and Physiology in Murine Fibroblasts and Macrophage Cell Line. Viruses. 2018;10(5). Epub 2018/05/19. doi: 10.3390/v10050266; PubMed Central PMCID: PMC5977259. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref032] 32.Szulc-Dabrowska L, Wyzewski Z, Gregorczyk-Zboroch KP, Toka FN, Szczepanowska J, Struzik J, et al. Mitochondria-related gene expression profiles in murine fibroblasts and macrophages during later stages of ectromelia virus infection in vitro. Acta Virol. 2020;64(3):307–24. Epub 2020/09/29. doi: 10.4149/av_2020_305 . [DOI] [PubMed] [Google Scholar]

[ppat.1009633.ref033] 33.Wong PS, Sutejo R, Chen H, Ng SH, Sugrue RJ, Tan BH. A System Based-Approach to Examine Cytokine Response in Poxvirus-Infected Macrophages. Viruses. 2018;10(12). Epub 2018/12/20. doi: 10.3390/v10120692; PubMed Central PMCID: PMC6316232. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref034] 34.Yadav VN, Pyaram K, Mullick J, Sahu A. Identification of hot spots in the variola virus complement inhibitor (SPICE) for human complement regulation. J Virol. 2008;82(7):3283–94. Epub 2008/01/25. doi: 10.1128/JVI.01935-07 ; PubMed Central PMCID: PMC2268448. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref035] 35.Lee SH, Jung JU, Means RE. ’Complementing’ viral infection: mechanisms for evading innate immunity. Trends Microbiol. 2003;11(10):449–52. Epub 2003/10/15. doi: 10.1016/j.tim.2003.08.004 . [DOI] [PubMed] [Google Scholar]

[ppat.1009633.ref036] 36.Rosengard AM, Liu Y, Nie Z, Jimenez R. Variola virus immune evasion design: expression of a highly efficient inhibitor of human complement. Proc Natl Acad Sci U S A. 2002;99(13):8808–13. Epub 2002/05/30. doi: 10.1073/pnas.112220499 ; PubMed Central PMCID: PMC124380. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref037] 37.Van Vliet K, Mohamed MR, Zhang L, Villa NY, Werden SJ, Liu J, et al. Poxvirus proteomics and virus-host protein interactions. Microbiol Mol Biol Rev. 2009;73(4):730–49. Epub 2009/12/01. doi: 10.1128/MMBR.00026-09 ; PubMed Central PMCID: PMC2786582. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref038] 38.Smithson C, Purdy A, Verster AJ, Upton C. Prediction of steps in the evolution of variola virus host range. PLoS One. 2014;9(3):e91520. Epub 2014/03/15. doi: 10.1371/journal.pone.0091520; PubMed Central PMCID: PMC3953476. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref039] 39.Schweneker M, Lukassen S, Spath M, Wolferstatter M, Babel E, Brinkmann K, et al. The vaccinia virus O1 protein is required for sustained activation of extracellular signal-regulated kinase 1/2 and promotes viral virulence. J Virol. 2012;86(4):2323–36. Epub 2011/12/16. doi: 10.1128/JVI.06166-11 ; PubMed Central PMCID: PMC3302380. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref040] 40.Bera BC, Shanmugasundaram K, Barua S, Venkatesan G, Virmani N, Riyesh T, et al. Zoonotic cases of camelpox infection in India. Vet Microbiol. 2011;152(1–2):29–38. Epub 2011/05/17. doi: 10.1016/j.vetmic.2011.04.010 . [DOI] [PubMed] [Google Scholar]

[ppat.1009633.ref041] 41.Khalafalla AI, Abdelazim F. Human and Dromedary Camel Infection with Camelpox Virus in Eastern Sudan. Vector Borne Zoonotic Dis. 2017;17(4):281–4. Epub 2017/01/06. doi: 10.1089/vbz.2016.2070 . [DOI] [PubMed] [Google Scholar]

[ppat.1009633.ref042] 42.Muhlemann B, Vinner L, Margaryan A, Wilhelmson H, de la Fuente Castro C, Allentoft ME, et al. Diverse variola virus (smallpox) strains were widespread in northern Europe in the Viking Age. Science. 2020;369(6502). Epub 2020/07/25. doi: 10.1126/science.aaw8977. [DOI] [PubMed] [Google Scholar]

[ppat.1009633.ref043] 43.Quenelle DC, Prichard MN, Keith KA, Hruby DE, Jordan R, Painter GR, et al. Synergistic efficacy of the combination of ST-246 with CMX001 against orthopoxviruses. Antimicrob Agents Chemother. 2007;51(11):4118–24. Epub 2007/08/29. doi: 10.1128/AAC.00762-07 ; PubMed Central PMCID: PMC2151443. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref044] 44.Chen N, Bellone CJ, Schriewer J, Owens G, Fredrickson T, Parker S, et al. Poxvirus interleukin-4 expression overcomes inherent resistance and vaccine-induced immunity: pathogenesis, prophylaxis, and antiviral therapy. Virology. 2011;409(2):328–37. Epub 2010/11/13. doi: 10.1016/j.virol.2010.10.021 ; PubMed Central PMCID: PMC3008208. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref045] 45.Lederman ER, Davidson W, Groff HL, Smith SK, Warkentien T, Li Y, et al. Progressive vaccinia: case description and laboratory-guided therapy with vaccinia immune globulin, ST-246, and CMX001. J Infect Dis. 2012;206(9):1372–85. Epub 2012/08/21. doi: 10.1093/infdis/jis510 ; PubMed Central PMCID: PMC3529603. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref046] 46.Aryee KE, Shultz LD, Brehm MA. Immunodeficient mouse model for human hematopoietic stem cell engraftment and immune system development. Methods Mol Biol. 2014;1185:267–78. Epub 2014/07/27. doi: 10.1007/978-1-4939-1133-2_18 ; PubMed Central PMCID: PMC4476265. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref047] 47.Huggins J, Goff A, Hensley L, Mucker E, Shamblin J, Wlazlowski C, et al. Nonhuman primates are protected from smallpox virus or monkeypox virus challenges by the antiviral drug ST-246. Antimicrob Agents Chemother. 2009;53(6):2620–5. Epub 2009/04/08. doi: 10.1128/AAC.00021-09 ; PubMed Central PMCID: PMC2687232. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref048] 48.Esposito J, Condit R, Obijeski J. The preparation of orthopoxvirus DNA. J Virol Methods. 1981;2(3):175–9. Epub 1981/02/01. doi: 10.1016/0166-0934(81)90036-7 . [DOI] [PubMed] [Google Scholar]

[ppat.1009633.ref049] 49.Esposito JJ, Knight JC. Orthopoxvirus DNA: a comparison of restriction profiles and maps. Virology. 1985;143(1):230–51. Epub 1985/05/01. doi: 10.1016/0042-6822(85)90111-4 . [DOI] [PubMed] [Google Scholar]

[ppat.1009633.ref050] 50.Esposito JJ, Obijeski JF, Nakano JH. Orthopoxvirus DNA: strain differentiation by electrophoresis of restriction endonuclease fragmented virion DNA. Virology. 1978;89(1):53–66. Epub 1978/08/01. doi: 10.1016/0042-6822(78)90039-9 . [DOI] [PubMed] [Google Scholar]

[ppat.1009633.ref051] 51.Olson VA, Laue T, Laker MT, Babkin IV, Drosten C, Shchelkunov SN, et al. Real-time PCR system for detection of orthopoxviruses and simultaneous identification of smallpox virus. J Clin Microbiol. 2004;42(5):1940–6. Epub 2004/05/08. doi: 10.1128/JCM.42.5.1940-1946.2004 ; PubMed Central PMCID: PMC404623. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref052] 52.Ropp SL, Jin Q, Knight JC, Massung RF, Esposito JJ. PCR strategy for identification and differentiation of small pox and other orthopoxviruses. J Clin Microbiol. 1995;33(8):2069–76. Epub 1995/08/01. doi: 10.1128/jcm.33.8.2069-2076.1995 ; PubMed Central PMCID: PMC228337. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref053] 53.Sarmiento M, Haffey M, Spear PG. Membrane proteins specified by herpes simplex viruses. III. Role of glycoprotein VP7(B2) in virion infectivity. J Virol. 1979;29(3):1149–58. Epub 1979/03/01. doi: 10.1128/JVI.29.3.1149-1158.1979 ; PubMed Central PMCID: PMC353275. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref054] 54.Guarner J, Johnson BJ, Paddock CD, Shieh WJ, Goldsmith CS, Reynolds MG, et al. Monkeypox transmission and pathogenesis in prairie dogs. Emerg Infect Dis. 2004;10(3):426–31. Epub 2004/04/28. doi: 10.3201/eid1003.030878 ; PubMed Central PMCID: PMC3322777. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref055] 55.Martines RB, Ritter JM, Matkovic E, Gary J, Bollweg BC, Bullock H, et al. Pathology and Pathogenesis of SARS-CoV-2 Associated with Fatal Coronavirus Disease, United States. Emerg Infect Dis. 2020;26(9):2005–15. Epub 2020/05/22. doi: 10.3201/eid2609.202095 ; PubMed Central PMCID: PMC7454055. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref056] 56.Goldsmith CS, Whistler T, Rollin PE, Ksiazek TG, Rota PA, Bellini WJ, et al. Elucidation of Nipah virus morphogenesis and replication using ultrastructural and molecular approaches. Virus Res. 2003;92(1):89–98. Epub 2003/02/28. doi: 10.1016/s0168-1702(02)00323-4 . [DOI] [PubMed] [Google Scholar]

[ppat.1009633.ref057] 57.Kondas AV, Olson VA, Li Y, Abel J, Laker M, Rose L, et al. Variola virus-specific diagnostic assays: characterization, sensitivity, and specificity. J Clin Microbiol. 2015;53(4):1406–10. Epub 2015/02/13. doi: 10.1128/JCM.03613-14 ; PubMed Central PMCID: PMC4365196. [DOI] [PMC free article] [PubMed] [Google Scholar]

[ppat.1009633.ref058] 58.Karem KL, Reynolds M, Braden Z, Lou G, Bernard N, Patton J, et al. characterization of acute-phase humoral immunity to monkeypox: use of immunoglobulin M enzyme-linked immunosorbent assay for detection of monkeypox infection during the 2003 North American outbreak. Clin Diagn Lab Immunol. 2005;12(7):867–72. Epub 2005/07/09. doi: 10.1128/CDLI.12.7.867-872.2005 ; PubMed Central PMCID: PMC1182207. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Teaching a new mouse old tricks: Humanized mice as an infection model for Variola virus

Christina L Hutson

Ashley V Kondas

Jana M Ritter

Zachary Reed

Sharon Dietz Ostergaard

Clint N Morgan

Nadia Gallardo-Romero

Cassandra Tansey

Matthew R Mauldin

Johanna S Salzer

Christine M Hughes

Cynthia S Goldsmith

Darin Carroll

Victoria A Olson

Roles

Abstract

Author summary

Introduction

Results

VARV disease in humanized mice

Table 1. Clinical signs and gross findings.

Fig 1. Study design, survival table and survivorship curves for mice and dosage groups.

Pathology and immunohistochemistry

Table 2. Microscopic lesions and VARV immunolocalization in tissues from humanized (PBMC, CD34+, BLT) mice and non-humanized background strain (NSG) mice inoculated intranasally with VARV.

Fig 2. Representative histopathology and immunohistochemistry of VARV infection in humanized mice.

Fig 3. Representative differences in pathologic findings among three strains of humanized mice with VARV infection.

Fig 4. Electron microscopic images of VARV in a humanized mouse.

Molecular findings

Fig 5. High viable virus loads were detected in all three types of humanized mice.

Non-humanized NSG mice are not susceptible to systemic VARV disease

Discussion

Methods

Ethics statement

Mice

Challenge virus

Animal inoculation

Animal sampling, observations and euthanasia

Sample processing and DNA extraction

Pathology and immunohistochemistry

Molecular assays

Statistics

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Erle S Robertson

Bernard Moss

Roles

Author response to Decision Letter 0

Decision Letter 1

Michael H Malim

Bernard Moss

Roles

Author response to Decision Letter 1

Decision Letter 2

Michael H Malim

Bernard Moss

Roles

Acceptance letter

Michael H Malim

Bernard Moss

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases