Newborn mouse-based model established that enables transmission of clinical SARS-CoV-2 isolates

Newborn mouse-based model established that enables transmission of clinical SARS-CoV-2 isolates

In a recent study published in bioRxiv * Preprint server: Researchers have developed a newborn mouse model that enables transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Studie: Ein neonatales Mausmodell charakterisiert die Übertragbarkeit von SARS-CoV-2-Varianten und zeigt eine Rolle von ORF8. Bildquelle: Dotted Yeti/Shutterstock

*Important NOTE:bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, intended to guide clinical practice/health-related behavior, or treated as established information.

background

Hamsters and ferrets are routinely used to model the pathogenesis of SARS-CoV-2; However, both animal models lack the potential for genetic manipulation to assess host-determining factors of virus transmission. Therefore, it is unclear which SARS-CoV-2 VOC-specific amino acid substitutions and SARS-CoV-2-induced host mechanisms contribute to transmission.

Mice are low-cost and widely available options with versatile genetic and reagent toolkits and fewer challenges in husbandry and regulation; However, transmission of SARS-CoV-2 in adult mice has not been documented. The authors of the present study previously established four- to seven-day-old newborn mice as effective models for IAV (influenza A virus) transmission.

About the study

In the present study, researchers extended their previous analysis and presented a K18 human angiotensin-converting enzyme 2 (hACE2)-expressing neonatal mouse model that transmitted SARS-CoV-2 and examined past and currently circulating SARS-CoV-2 VOC transmission.

The team characterized the SARS-CoV-2 tropism, replication and transmission of the SARS-CoV-2 strain Wuhan-Hu-1 (WA-1) compared to the VOCs Alpha, Beta, Gamma, Delta and Omicron. They also characterized the transmission of two recombinant SARS-CoV-2 viruses that lack the additional open reading frame 6 (ORF6) or ORF8 proteins. C57BL/6 (hACE2-/-) female and C57BL/6 K18-hACE2+/+ male mice and mice were combined to generate K18-hACE2+/- offspring permissive to WA-1. Four- to seven-day-old index case pups were infected intranasally with WA-1.

Pup survival and weight were monitored daily and excretory samples (nasal secretions) were collected longitudinally from the index pups and the pups in their close contact. Upper respiratory tract excretion kinetics (URT) were used to assess viral infections, and weight loss was termed SARS-CoV-2-induced morbidity. Nasal secretion samples were subjected to quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assays and plaque assays were performed on VeroE6 cells overexpressing the transmembrane protease serine 2 (TMPRSS2) and ACE2.

Immunohistochemical (IHC) analysis was performed on the nasopharyngeal tissues of index pups stained for SARS-CoV-2 nucleocapsid (N) protein. Infectious SARS-CoV-2 particles in retrotracheal lavages and lung homogenates were measured to assess viral tropism for the URT and lower respiratory tract (LRT), respectively. The cytokines present in the URT excretion samples from SARS-CoV-2-infected newborn mice were also analyzed.

To assess the transmission kinetics of SARS-CoV-2, daily molting of contact mice was used as an indicator of successful virus replication. The excretory samples were analyzed using multiplex enzyme-linked immunosorbent assays (ELISA). In addition, mice were infected with SARS-CoV-2 without ORF6 (WA-1 ΔORF6) or without ORF8 (WA-1 ΔORF8), and their excretion samples, retrotracheal lavage fluids and lung samples were collected to assess shed SARS-CoV-2, URT replication and LRT replication, respectively. The amount of infectious virus was quantified using plaque assays.

Results

K18-hACE2-expressing neonatal mice efficiently supported the transmission of SARS-CoV-2 WA-1, and SARS-CoV-2 VOCs showed pronounced tropism and replication dynamics in the index neonates. ORF8 proved to be crucial for the successful transmission of SARS-CoV-2. Morbidity and mortality were balanced by two to three days in contact mice compared to index mice, and SARS-CoV-2 ribonucleic acid (RNA) was detected in contact mice, indicating SARS-CoV-2 transmission.

At four dpi, SARS-CoV-2 particles were shed by all contact pups, representing 100% efficiency of WA-1 transmission, and SARS-CoV-2 detection in the newborn mice shedding samples suggested a robust SARS-CoV-2 infection in the upper respiratory tract. Furthermore, SARS-CoV-2-infected cells were identified in the upper olfactory epithelial cells, indicating WA-1 replication in the URT.

Mice infected with Omicron excreted small amounts of SARS-CoV-2. For the WA-1 strain, the excreted SARS-CoV-2 titers were 105 plaque-forming units (PFU)/ml in the upper respiratory tract, but significantly higher titers (5 x 106 PFU/ml) in the lung tissue. At peak replication titers, the tropism of the WA-1 strain progressed toward LRT. On the contrary, alpha replication favored URT. Viral loads in all excretory samples and the corresponding retrotracheal lavage samples were similar. With the exception of Omicron, the model allowed various VOCs of SARS-CoV-2 to replicate and excrete at levels similar to WA-1.

Seven days post-infection (dpi), contact mice with alpha and delta infections showed 100% mortality, while the mortality rates for the WA-1 strain, beta-VOC and gamma-VOC infections were 75%, 86% and 56%. respectively. It is likely that a threshold value for the index shedding titer (≥ 1.2 x 104 PFU/ml) existed in the model to estimate the success of transmission to contacts. WA-1 strain, beta-VOC, and gamma-VOC infections, whose transmission differed in onset, time of completion, and degree of completion, resulted in overall similar cytokine responses.

WA-1 ΔORF8 shed viral loads and URT viral loads were 5 x 102 and 1 x 103 PFU/ml, respectively, significantly lower than those for WA-1 or WA-1 ΔORF6, and infectious viruses were still detectable in index mice at five dpi, indicating a slower slowing of viral clearance in the absence of ORF8. The transmission of WA-1 ΔORF8 was incomplete until the end of the experiment. Furthermore, the maximum virus titer in WA-1-ΔORF8 contact mice was 7.5 × 103 PFU/ml, which was lower than that for WA-1-ΔORF6 and WA-1.

Diploma

Overall, the study results showed that the non-invasive neonatal mouse model can reveal the transmission dynamics of SARS-CoV-2 VOCs except Omicron and that ORF8 is crucial for SARS-CoV-2 transmission.

*Important NOTE:bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, intended to guide clinical practice/health-related behavior, or treated as established information.

Reference:

• Vorläufiger wissenschaftlicher Bericht.
  Rodriguez-Rodriguez, B. et al. (2022) „Ein neonatales Mausmodell charakterisiert die Übertragbarkeit von SARS-CoV-2-Varianten und zeigt eine Rolle für ORF8.“ bioRxiv. doi: 10.1101/2022.10.04.510658. https://www.biorxiv.org/content/10.1101/2022.10.04.510658v1