Human-to-Human Transmission of Andes Virus Modeled in Syrian Hamsters

Several occurrences of human-to-human transmission of Andes virus, an etiological agent of hantavirus cardiopulmonary syndrome, are documented. Syrian hamsters consistently model human hantavirus cardiopulmonary syndrome, yet neither transmission nor shedding has been investigated. We demonstrate horizontal virus transmission and show that Andes virus is shed efficiently from both inoculated and contact-infected hamsters.


Human-to-Human Transmission of Andes
Virus Modeled in Syrian Hamster

Study design
To account for unknown ANDV shedding and transmission rates between hamsters, twelve pairs of inoculated-naïve animals were used.To maximize exposure, two hamsters inoculated with ANDV were enclosed in a cage in direct contact with two naïve animals, in a total of 6 cages, as shown in Appendix Figure 1.

Animals and husbandry
Twenty-four Syrian hamsters were purchase from Janvier labs, France, and transported to the BSL-4 facility.Four hamsters of ≈3-weeks old (<100 g) were allocated into each of six IVC GR900 cages (900 cm 2 , Tecniplast Sealsafe).Low-dust bedding, a shelter and nesting material were provided.Animals were acclimated for 12 days and kept in a reversed 12-hour daylight cycle, at 21°C (±1°C) and 50% (±10%) of relative humidity.Water and food were offered ad libitum throughout the experiment.

In vivo infection
ANDV Chile-9717869 was quantified by infecting monolayers of Vero E6 cells in 96well plates with four replicates of six ten-fold dilutions (1x10 −0 -1x10 −6 ).Supernantants were harvested either at 1 hour, or days 1, 2 and 4 post-inoculation.The lowest dilution that demonstrated an increase in ANDV-RNA copies over time was estimated as an equivalent of 1 plaque forming unit (PFU-eq).
Under isoflurane anesthesia, hamsters were weighted, ear-tagged and implanted with a temperature-logging transponder (IPTT-300, Plexx).Two animals from each cage, randomly assigned to the naïve cohort (n = 12), were placed into six fresh cages.Animals from the "inoculated cohort" were inoculated with 200 PFU-eq of ANDV in 100 uL of sterile PBS via intranasal route (i.n.) and returned to their respective cages (Appendix Figure 1).One day postinoculation (dpi), each pair of inoculated animals was placed into a fresh cage with their corresponding pair of naïve animals.All animals were observed daily to detect development of signs of disease and endpoint scoring.Animals that reached the endpoint scoring criteria were euthanized with an overdose of isoflurane and exsanguination by cardiac puncture.Surviving animals were euthanized on dpi 40.Oral and rectal mucosa was sampled every other day under light isoflurane sedation and urine was collected opportunistically.Animals were weighed every third day and the animals were moved to clean cages once per week or when the last surviving inoculated animal was euthanized.

Real-Time Quantitative PCR (RT-qPCR)
Swabs used to collect mucosal or urine samples were placed directly into 560 uL of AVL and inactivated in 700 μL of ethanol 100%.Viral RNA was extracted using a QIAmp Viral RNA Mini Kit (Qiagen) following the manufacturer's instructions.Tissue samples were individually weighed, then homogenized in RLT buffer using a stainless-steel bead and a Tissuelyser II

(
Qiagen) homogenizer, for 10 min at 30 Hz.Samples were centrifuged for 10 minutes at 6000 xg and the supernatant was inactivated in 600 μl of ethanol 70% per 30 mg of tissue.ANDV RNA was quantitated by qRT-PCR.Briefly, AgPath-ID One-Step RT-PCR mix (4387391, Thermo Fisher) was added to 5 μL of extracted RNA sample (elute) and analyzed in an Applied Biosystems 7500 platform.Primers (ANDVf: aaggcagtggaggtggac, ANDVr: ccctgttggatcaactggtt) and probe (ANDVp: FAM-acgggcagctgtgtctacattgga-BBQ) targeting a 162 bp fragment within ANDV-S segment (130 -291 bp from NCBI ref. seq.: NC_003466.1)wereadded to 20 μL of master mix for total volume of 25 μL.Samples were incubated for 15 min at 45°C, 10 min at 95°C followed by 45 cycles of 15 sec at 95°C and 60 sec at 60°C.Samples that had cycle threshold (Ct) values ≤40 were regarded as positive.A standard curve of ANDV in vitro transcripts (10 to 10 7 copies) was included to quantify Ct values, which was calculated using the supplied software (ABI-7500 v2.3).To estimate ANDV copy numbers/g of tissue, the resulting ANDV-S copies per μL of elute used in the PCR mix, obtained from a total extraction