“Nature” published HKU study: Golden hamster experiment suggests new crown can be aerosol propagation

Since the outbreak, whether the new coronavirus can be transmitted through aerosols has been one of the focus of academic circles. A new study by a team at the University of Hong Kong, a leading international journal, nature, shows that in the Golden Hamster animal model experiment, the new coronavirus can be transmitted by aerosol.

The paper was published online as the Accelerated Article Preview, entitled Pathogenes and transmission of SARS-CoV-2 in golden hamsters. The paper is written by Hui-Ling Yen of the University of Hong Kong’s Li Ka-shing School of Medicine and also by Dr. Pan Liwen, a professor at the School of Public Health at the University of Hong Kong.

In the wake of the new coronavirus, researchers urgently need appropriate small animal models to support the development of vaccines and treatments. The researchers reported the pathogenesis and infectiousness of the new coronavirus (SARS-CoV-2) in golden hamsters (golden Syrian hamsters). Immunohistochemistry showed that 2 and 5 days after the golden hamster was vaccinated against the virus, there were viral antigens in the nasal mucosal membrane, bronchial epithelial cells and pulmonary realist regions. 7 days after being infected with the virus, the virus is quickly cleared and lung cells are hyperplasia. Viral antigens have also been found in the upper skin cells of the golden hamster duo, and viral RNA has been detected in feces. It is worth noting that animal model tests have shown that the new coronavirus can be effectively transmitted from infected golden hamsters to infant hamsters through direct contact and aerosols. The efficiency of vector transmission is less in hamster cages. Although viral RNA was detected in the nasal lotion of the inoculated hamster for 14 consecutive days, the new coronavirus can spread for a short time. Inoculated infected and naturally infected hamsters showed significant weight loss, and all golden hamsters were able to detect neutralizing antibodies when they recovered. The results show that the new coronavirus infection characteristics of golden hamsters are similar to those of mild infection in humans.

The paper points out that suitable animal models are essential for understanding the pathogenesis of neo-coronary pneumonia and evaluating vaccine and treatment candidates. Previous animal studies on SARS-CoV have shown that the interaction between the protoscotensin (S protein) of the virus and the host’s angiotensin-converting enzyme 2 (ACE2) receptors, as well as the age and innate immune state of the infected subjects, play an important role in the pathogenesis. Like SARS-CoV, the S protein of SARS-CoV-2 uses ACE2 receptors ( mainly distributed in epithelial cells in the lungs and small intestine) to enter the cells for viral replication. The combination of SARS-CoV-2 and human ACE2 is good, but the combination with ACE2 in mice is limited, which limits the application of inbred mice in viral research. Genetically modified ICR mice with macaques and expressions of human ACE2 receptors have been shown to be susceptible to SARS-CoV-2; Crab-eating macaques and rhesus monkeys, which were attacked by SARS-CoV-2, showed limited and moderate clinical symptoms, respectively. Infected genetically modified mice showed moderate levels of pneumonia and no significant histological changes in non-breathing tissue. It was reported that genetically modified mice that had previously expressed human ACE2 receptors supported the replication of SARS-CoV in respiratory epithelial cells, but because ACE2 in the brains of genetically modified mice was also high lysed, the mortality rate of mice was also added to the associated variables of neurological lesions.

Golden hamsters are a widely used experimental animal model that has previously shown that SARS-CoV can replicate in its body, but MERS-CoV is not, because MERS-CoV uses the DPP4 protein as the main receptor for the virus into the cell, not ACE2. Previously, a study of SARS-CoV (Urbani strain) in a 5-week-old golden hamster showed that the virus was able to replicate strongly in its body, detecting a spike in viral drops in the lungs two days after the virus was inoculated, and seven days after the virus was inoculated, the golden hamster was able to quickly remove the virus. However, the virus-inoculated golden hamsters did not lose weight or have significant disease conditions. A follow-up study said different SARS-CoV strains were tested in golden hamsters and found differences in the toxicity between SARS-CoV strains. It is reported that THE USE OF SARS-CoV (Frk-1 strain) is fatal to hamsters. The difference between the Frk-1 strain and the non-lethal Urbani strain is the L1148F mutation in the S2 domain. Hamsters can also be infected with other respiratory viruses, including human lung virus, human para-flu virus and influenza A virus, and may support the transmission of influenza through contact or air. The comparison of ACE2 proteins in humans, macaques, mice and hamsters showed that hamster ACE2 may interact more effectively with SARS-CoV-2 S proteins than rat ACE2. Here, the team assessed the pathogenesis and exposure propagation of SARS-CoV-2 in male golden hamsters at 4-5 weeks.

Suggested aerosol-borne animal model experiment

To study the ability of SARS-CoV-2 to spread through aerosols in hamsters, the researchers placed the donor (inoculated with the new coronavirus) hamster and the infant hamster (healthy hamster) in two adjacent iron cages and placed the two cages together for 8 hours, one day after the donor hamster was inoculated with the virus, in the following experiment:

Infectious viruses in the nasal fluids of donor hamsters can detoxify for 6 days, while the RNA of the virus can be detected continuously for 14 days, that is, at a later stage, the infected hamster strains are not infected. As shown in the following image:

Viral RNA can be detected in donor stool samples 2, 4, 6 days after inoculation, but not infectious viruses, as follows:

The researchers found that the new coronavirus was effective in aerosol transmission in hamsters because infectious viruses were detected in all exposed hamster nasal lotions 1 day after exposure, and the viral load peaked 3 days after exposure, as shown in the following image:

Although the infectious virus was not isolated, viral RNA was detected for 14 consecutive days from stool samples of infected aerosol contacts, as shown in the following figure:

Hamsters exposed to aerosols showed maximum weight loss 7 days after exposure (average s.72.5,42%, N-3). Compared to donor hamsters, aerosol-contact hamsters excrete a considerable number of viruses in nasal lotion.

Pathogenesis of SARS-CoV-2 in Golden Hamsters

In the experiment, the researchers gave the golden hamster a negative 4-square TCID50 (50% tissue cell infection) of negative 4 times (Beta-CoV / Hong / VM20001061 / 2020 virus, GISAID EPI_ISL_412028). The virus was isolated from vero E6 cells with nasopharyngeal siphons and pharynx swabs in a patient with new coronary pneumonia in Hong Kong. The researchers collected nasal armor, brain, lung, heart, duodenum, liver, spleen and kidney tissue 2, 5 and 7 days after the golden hamster was vaccinated to monitor viral replication and histopathological changes. On the second day after the virus is inoculated, a peak lung viral load of golden hamsters can be detected, and the viral load begins to decrease 5 days after the virus is inoculated. Although high copies of viral RNA can continue to be detected, no infectious virus has been detected for 7 days after the virus has been inoculated.

Infectious virus load varies significantly between 2 and 7 days after vaccination (P s 0.019, Dunn’s multiple comparison test), but there is no difference in the number of copies of RNA viruses (P s 0.076). Although low-copy viral RNA was detected 2 and 5 days after vaccination, no infectious virus was detected in the kidneys.

Histopathological examination found that 5-10% of inflammatory cells in the lungs increased and changed 2 days after the virus was inoculated.

Five days after the virus is inoculated, 15-35% of the inflammatory cells in the lungs increase.

Monocytol immersion can be observed in areas where viral antigens are detected 2 and 5 days after the virus is inoculated. The immunohistachemical of the N protein of SARS-CoV-2 indicates the presence of viral antigens in the bronchial epithelial cells of golden hamsters (where the arrow in the figure below refers) 2 days after the virus is inoculated.

5 days after the virus is inoculated, it develops into lung cells (the position indicated by the arrow in the figure below).

7 days after the virus, 30-60% of the lungs change, as shown in the following image:

However, no virus antigens have been detected as shown in the following image:

The growth of type 2 lung cells is obvious, as shown in the following image:

CD3-positive T lymphocytes were detected in the area around the bronchial tube s5 days after inoculation, which may help to quickly remove infected cells, as shown in the following image:

Inflammatory cells are soaked in the nasal armor, as shown in the following image:

Viral antigens are detected in olfactory sensory neurons in nasal epithelial cells and nasal mucosa, as shown in the following image:

Infections in olfactory neurons are further confirmed in cells that express SARS-CoV N proteins and neuron-specific beta-III microtubule proteins, as shown in the following image:

Compared to the control group, the number of olfactory neurons in the nasal mucous membrane of golden hamsters 2 days after the virus infection was reduced, as shown in the figure below

7 days after the virus is inoculated, the nasal epithelial cells decay significantly, as shown in the following image:

Tissue repair can then be observed 14 days after vaccination, as shown in the following image:

Although there is no inflammation in the duodenal epithelial cells, as follows:

However, virus antigens were detected from the duodenal epithelial cells 2 days after the virus was inoculated, as shown in the following image:

No significant changes in histopathological disease were observed from the brain, heart, liver or kidneys 5 days after the virus was inoculated, as shown in the following image:

To assess the ability of the new coronavirus to spread in hamsters, the researchers inoculated three donor hamsters with the TCID50 virus of 8 x 10 minus 4 times through their noses. Within 24 hours of inoculation, transfer each donor to a new cage and raise it with a young hamster. The researchers monitored weight changes and clinical signs daily, collecting nasal lotions from donor hamsters and contact hamsters every other day for 14 days. In donor hamsters, although viral RNA can be detected for 14 consecutive days, the peak of the infectious virus occurs early after vaccination and then rapidly decreases, as shown in the following figure:

Six days after sars-CoV-2 was inoculated, hamsters showed the largest average weight loss (average s.SD, -11.97,4.51%, N-6), as shown in the following figure:

The transmission from the donor to the contact is very efficient, sars-CoV-2 can be detected from co-resident hamsters within 1 day of contact, and a peak of viral load is detected in the co-resident hamster nasal lotion 3 days after exposure, as shown in the following figure:

The largest average weight loss in co-resident hamsters occurred 6 days after exposure (average -SD, -10.68,3.42%, N -3), and all animals regained their original weight 11 days after exposure, as shown in the following figure:

Using PRNT analysis, the antibody was neutralized from the donor 14 days after the inoculation (1:640 for all titers) and 13 days after exposure to the co-resident hamster (1:160, 1:320, and 1:160 titage). For 14 consecutive days, the researchers detected viral RNA from the nasal lotion of donor hamsters, but the infectious virus titer dropped rapidly. The researchers repeated the experiment and shared the virus with the infant hamsters for six days after the virus was inoculated. A small amount of viral RNA was detected in the nasal lotion of a common infant hamster 3 days after exposure, and no viral RNA was detected in the nasal lotion 7 days after exposure. Also, no infectious virus has been detected in the nasal lotion, as shown in the following image:

And co-residents of hamsters did not show weight loss, as shown in the following figure:

PRNT analyzed neutralizing antibodies in co-resident hamsters without detection for 12 days after exposure. The results showed that the virus inoculated with SARS-CoV-2 donors could travel for less than 6 days. Subsequent transmission from donor to co-resident contact swashes is associated with the detection of an infectious disease in the donor nasal lotion, not to the detection of viral RNA.

The spread of viruses from donors to co-residents can be mediated through a variety of transmission pathways.