Three asymptomatic animal infection models of hemorrhagic fever with renal syndrome caused by hantaviruses

Abstract

Hantaan virus (HTNV) and Puumala virus (PUUV) are rodent-borne hantaviruses that are the primary causes of hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia. The development of well characterized animal models of HTNV and PUUV infection is critical for the evaluation and the potential licensure of HFRS vaccines and therapeutics. In this study we present three animal models of HTNV infection (hamster, ferret and marmoset), and two animal models of PUUV infection (hamster, ferret). Infection of hamsters with a ~3 times the infectious dose 99% (ID99) of HTNV by the intramuscular and ~1 ID99 of HTNV by the intranasal route leads to a persistent asymptomatic infection, characterized by sporadic viremia and high levels of viral genome in the lung, brain and kidney. In contrast, infection of hamsters with ~2 ID99 of PUUV by the intramuscular or ~1 ID99 of PUUV by the intranasal route leads to seroconversion with no detectable viremia, and a transient detection of viral genome. Infection of ferrets with a high dose of either HTNV or PUUV by the intramuscular route leads to seroconversion and gradual weight loss, though kidney function remained unimpaired and serum viremia and viral dissemination to organs was not detected. In marmosets a 1,000 PFU HTNV intramuscular challenge led to robust seroconversion and neutralizing antibody production. Similarly to the ferret model of HTNV infection, no renal impairment, serum viremia or viral dissemination to organs was detected in marmosets. This is the first report of hantavirus infection in ferrets and marmosets.

Fig 1. HTNV and PUUV infect hamsters in a dose dependent manner.

Syrian hamsters were challenged with varying concentrations of either HTNV (A) or PUUV (B) through the i.m. (left) or i.n. (right) route. Between 28 and 35 days post infection hamsters were terminally bled and N-ELISA endpoint titers (log₁₀) were used to determine infection status. The mean titer is displayed for each group, and the limit of detection (2log₁₀) is depicted as a dashed line.

Fig 2. Kinetics of seroconversion in low dose hamster models.

Syrian hamsters were infected either 10 PFU HTNV i.m., 500 PFU HTNV i.n., 1000 PFU PUUV i.m., or 1000 PFU PUUV i.n. Hamsters were terminally bled at various points post infection and N-ELISA endpoint titers (log₁₀) were used to determine infection status. The mean titer (A-D), and specific OD sum ± standard error of the mean (SEM) (E-F) for the N-ELISA is displayed for each group. The limit of detection (2log₁₀) for the N-ELISA is depicted as a dashed line. The specific OD sum represents the area under the titer curve.

Fig 3. Viral genome is detectable in the organs and sera of infected hamsters.

Syrian hamsters were infected either 10 PFU HTNV i.m., 500 PFU HTNV i.n., 1000 PFU PUUV i.m., or 1000 PFU PUUV i.n. Hamsters were euthanized at various points post infection and heart (A), lung (B), liver (C), spleen (D), kidney (E), brain (F), sera (G), and urine (H) were evaluated by RT-PCR for the presence of viral genome. The mean ± SEM is displayed at each time point. The dashed line indicates the limit of detection (1log₁₀).

Fig 4. Infectious virus was isolated from the organs of HTNV i.m. infected animals.

Syrian golden hamsters were infected with 10 PFU HTNV i.m. and sacrificed at various time points post infection. Heart (A), lung (B), liver (C), spleen (D), kidney (E), and brain (F) from the time range where the organ was RT-PCR positive were sampled by plaque assay for the presence of infectious virus. The mean ± SEM is displayed at each time point. The limit of detection (1.7 log₁₀) is depicted as a dashed line.

Fig 5. Ferrets are susceptible to HTNV and PUUV infection.

Ferrets were challenged with either 2,000 PFU HTNV i.n., or 2,000 PFU PUUV i.n. (low dose challenge arrow) and did not seroconvert. The same animals were subsequently challenged with either 200,000 PFU HTNV (A,D,G,J), 94,000 PFU PUUV Beaumont (B,E,H,K), or 164,000 PFU of PUUV Seloignes (C,F,I,L) i.m. on day 0, and immunosuppressed with 30 mg/kg cyclophosphamide on day 41. The daily weight (g) for each ferret is displayed (A-C). Seroconversion of ferrets was monitored by N-ELISA specific OD sum (D-F), and neutralizing antibody production by PsVNA₅₀ (G-I) and PRNT₅₀ (J-L). The dashed line represents the limit of detection for the PRNT (20) and PsVNA (20) assay.

Fig 6. PUUV infected ferrets experience weight loss, but no other clinical signs of infection.

Ferrets were infected with 94,000 PFU PUUV Beaumont i.m. and sera was collected twice weekly. Weight (A), temperature (B), N-ELISA specific OD sum (C), N-ELISA titer (log₁₀), (D) PsVNA₅₀ titers (E), and PRNT₅₀ titers (F). The dotted line represents the limit of dectection for the PRNT assay (20), the PsVNA assay (20) and the N-ELISA (2log₁₀).

Fig 7. PUUV infected ferrets do not exhibit signs of kidney impairment.

Ferrets were infected with 94,000 PFU PUUV Beaumont i.m. Blood and urine were collected twice weekly for analysis of kidney function by assaying proteinuria (A), hematuria (B), blood urea nitrogen (C), and creatinine (D) levels. Shaded gray areas represent the normal range [48].

Fig 8. Marmosets are susceptible to HTNV infection.

Three marmosets were infected with 1,000 PFU HTNV i.m. Sera was collected weekly to monitor seroconversion. N-ELISA titer (log₁₀) (A) N-ELISA specific OD sum (B) PRNT₅₀ (C) and PsVNA₅₀ titers(D) are displayed. Serum viremia was assayed by RT-PCR (E). The limit of detection for N-ELISA titer (2log₁₀), PRNT (20), PsVNA (20) and RT-PCR (1.0log₁₀) is depicted by the dotted line.