Virulent infection of outbred Hartley guinea pigs with recombinant Pichinde virus as a surrogate small animal model for human Lassa fever

Figures & data

Figure 1. Mortality and clinical symptoms of recombinant Pichinde virus (rP2 and rP18) infections in outbred Hartley guinea pigs. Guinea pigs were mock infected (PBS) or infected via IP route with 10,000 pfu of rP2 or rP18. (a) Survival curve plotted for rP2 (n = 25) and rP18 (n = 57). (b) Rectal temperature (°C) monitored during the experiment. Temperature > 39.5°C was considered feverish and identified by a dashed line. (c) Body weight monitored during the experiment. Body weight of each animal was normalized to its body weight at day 0 (set as 1.0). Data shown were the average of animals in replicate studies using fresh batches of recombinant viruses that were conducted between 2008 and 2015, with error bars representing standard deviation.

Figure 2. Gross Pathology of rP2 and rP18-infected guinea pigs.

For rP2 infection, images of liver, stomach, intestine, and skin are from two rP2-infected animals, which stayed healthy and were euthanized at day 14 pi. For rP18 infection, the images were from animals euthanized after reaching terminal points. The liver was from a guinea pig euthanized at day 11 pi; the stomach was from a guinea pig euthanized at day 12 pi; the large intestine was from a guinea pig euthanized at day 13 pi; the skin was from a guinea pig euthanized at day 14 pi.

Figure 3. H&E staining of liver, lung, and intestine.

These images were from one rP2-infected guinea pig that stayed healthy and was euthanized at Day 18 pi, and from one rP18-infected guinea pig that was euthanized at Day 15 pi after reaching terminal points.

Table 1. Complete blood count (CBC) of recombinant Pichinde (rP2 or rP18) virus-infected outbred Hartley guinea pigs*.

Virus strains	RBC 10^6/mm^3	PLT 10^3/mm^3	WBC 10^3/mm^3	LYMF 10^3/mm^3	LYMF %	GRAN 10^3/mm^3	GRAN %	MONO 10^3/mm^3	MONO %
rP18	3.9 ± 0.9	76.5 ± 33.7	3.0 ± 1.8	0.7 ± 0.7	24.5 ± 8.1	1.8 ± 0.9	61.7 ± 8.4	0.5 ± 0.3	13.9 ± 0.7
rP2	4.6 ± 0.8	318 ± 94.4	11.2 ± 4.0	5.1 ± 1.9	46.7 ± 12.4	4.6 ± 2.2	40.9 ± 9.0	1.4 ± 0.7	12.4 ± 4.0

* Blood were collected at the terminal points of rP18-virus infected animals and from rP2-virus infected animals on the same day. Data shown were the average from at least 3 independent experiments. RBC, red blood cells; PLT, platelets; WBC, white blood cells; LYMF, lymphocytes; GRAN, granulocytes; MONO, monocytes. Lymphocytes (LYMF), granulocytes (GRAN), and monocytes (MONO) are shown in number (10³/mm³) and in percentage of white blood cells (WBCs).

Table 2. Coagulation assays of blood collected from recombinant (rP2 or rP18) virus-infected outbred Hartley guinea pigs*.

Animal ID	Virus	Morbidity	PTT (sec)	PT (sec)	Fibrinogen (mg/dl)	D-dimer (ng/ml)
855956	rP2	Healthy	11.4	31.2	215	<250 (normal)
855959	rP18	Moribund	>100	>50	113	<250 (normal)
855961	rP18	Moribund	>100	>50	113	<250 (normal)

* Blood were collected at day 15 post-infection from two rP18-virus infected guinea pigs and one rP2-virus infected animal and analyzed by Antech Diagnostics.

Table 3. Viral titers (PFU/g) in tissues of rPICV-infected outbred Hartley guinea pigs*.

	Heart	Lung	Liver	Spleen	Stomach	Pancreas	Adrenal gland	Kidney	Lymph node
rP2	<20	<20	<20	<20	<20	<20	<20	<20	<20
rP18	6.5E+06	6.7E+07	1.9E+08	3.0E+07	5.6E+07	6.5E+06	3.2E+08	3.9E+06	3.1E+06

* The tissues were collected at the terminal points of rP18-virus infected animals and from rP2-virus infected animals at day 15. Virus titers in tissue homogenates were determined by plaque assay and shown as plaque-forming unit (PFU) per gram of tissue. Data shown were the average from at least 3 independent experiments.

Figure 4. Kinetics of virus replication in rP2- and rP18-infected animals. Sera, livers, and spleens from the rP2- and rP18-infected animals at different days during the course of the experiment or at terminal points were collected for viral titer quantification by plaque assay.

Figure 5. IHC staining of liver, lung, spleen, and intestine from rP18-infected guinea pig and LASV-infected human. (a) Mouse anti-PICV serum was used to validate its specificity to detect PICV antigens via IHC of PICV-infected Vero cells (bottom panel, red color) versus mock (uninfected) Vero cells (top panel). The same mouse anti-PICV serum was used in IHC to detect PICV antigens in guinea pig tissue sections prepared from a representative rP18-infected guinea pig that was euthanized at day 15 pi after reaching terminal points. Immunostaining of PICV antigens is seen in hepatocytes and sinusoidal lining cells in liver; mesothelial cells on pleural surface of lung; dendritic cells in spleen; endothelial cells in submucosa of intestine. The examples of immunolocalization in each photomicrograph are highlighted by arrowheads.