After the emergence of new coronavirus, researchers urgently need appropriate small animal models to support the development of vaccines and treatments. The researchers reported the pathogenesis and infectivity of new coronavirus (sars-cov-2) in golden hamster (golden Syrian hamster). Immunohistochemistry showed that there were virus antigens in the nasal mucosa, bronchial epithelial cells and lung consolidation areas of golden hamster 2 and 5 days after inoculation. 7 days after the infection, the virus cleared rapidly and the lung cells proliferated. Virus antigen was also found in duodenal epithelial cells of golden hamster, and virus RNA was detected in feces. It is worth noting that animal model tests show that the new coronavirus can be effectively transmitted from infected golden hamster to immature golden hamster through direct contact and aerosol. The efficiency of vector transmission in hamster cage is low. Although the virus RNA was detected in the nasal lotion inoculated with hamster for 14 days, the new coronavirus can spread for a short time. Inoculated and naturally infected hamsters showed significant weight loss, and neutralizing antibodies were detected in all golden hamsters after recovery. The results showed that the new coronavirus infection characteristics of golden hamster were similar to that of human mild infection.
The animal models are novel coronaviruses for novel coronavirus pneumonia and for evaluating vaccines and therapeutic candidates. Previous animal studies on SARS CoV have shown that the interaction between the spike protein (s protein) of the virus and the hosts ACE2 receptor, as well as the age and innate immune status of the infected subjects play an important role in the pathogenesis. Like SARS CoV, S protein of SARS cov-2 also uses ACE2 receptor (mainly distributed in epithelial cells of lung and small intestine) to enter cells for virus replication. The combination of sars-cov-2 with human ACE2 is good, but with mouse ACE2 is limited, which limits the application of inbred mice in virus research. Macaques and transgenic ICR mice expressing human ACE2 receptor have been shown to be susceptible to sars-cov-2; however, these animal models are not so easy to obtain. Macaque and rhesus monkeys attacked by sars-cov-2 showed limited and moderate clinical symptoms, respectively. The infected transgenic mice showed moderate pneumonia and no significant histological changes in non respiratory tissues. It has been reported that although the previously generated transgenic mice expressing human ACE2 receptor support the replication of SARS CoV in respiratory epithelial cells, but because of the high expression of ACE2 in the brain of the transgenic mice, the death rate of the mice is also added to the related variables of pathological changes in the nervous system.
Jincang mouse is a widely used experimental animal model. Previous studies have shown that SARS CoV can replicate in its body, but mers cov can not. This is because mers cov uses DPP4 protein as the main receptor of the virus into the cell, rather than ACE2. Previously, the inoculation of 5-week-old golden hamster with SARS CoV (Urban virus strain) showed that the virus had a strong ability of replication in vivo, and the peak of virus titer could be detected in the lungs after 2 days of inoculation; after 7 days of inoculation, golden hamster could quickly eliminate the virus. However, the golden hamster inoculated with the virus did not lose weight or have obvious disease status. A follow-up study reported that different strains of SARS CoV were tested in golden hamsters and found differences in virulence among them. It is reported that SARS CoV (frk-1 strain) is lethal to hamsters. The difference between frk-1 strain and non lethal urbani strain is l1148f mutation in S2 domain. Hamsters can also be infected with other respiratory viruses, including human metapneumovirus, human parainfluenza virus and influenza A virus, and may support influenza transmission through exposure or air. The comparison of ACE2 protein of human, macaque, mouse and hamster showed that ACE2 of hamster may be more effective in interacting with S protein of sars-cov-2 than ACE2 of mouse. Here, the team assessed the pathogenesis and contact transmission of sars-cov-2 in 4-5 week old male golden hamsters.
It suggests that the animal model experiment of aerosol propagation
In order to study the transmission ability of sars-cov-2 through aerosols in hamsters, the researchers put the donor (inoculated with new coronavirus) hamsters and the immature hamsters (healthy hamsters) in two adjacent cages respectively. One day after the donor was inoculated with the virus, the two cages were put together for 8 hours. The experiment is as follows:
Although no infectious virus has been isolated, rna14 days can be detected from fecal samples of infected aerosol contacts for 14 consecutive days, as shown in the following figure:
The hamsters exposed to aerosols showed the greatest weight loss (mean u00b1 SD- seven point seven two u00b15,42uff05uff0cN=3uff09u3002 Compared with donor hamsters, aerosol exposed hamsters excreted a considerable amount of virus in the nasal wash.
The pathogenesis of sars-cov-2 in golden hamster
In the experiment, the new coronavirus (beta cov / HONGKONG / vm20001061 / 2020, gisaid EPI ufe63 ISL ufe63 412028) was infected in the nose of Jincang mice with 8 u00d7 10 negative 4-power TCID50 (50% of tissue cell infection). A novel coronavirus pneumonia virus was isolated from VeroE6 cells from nasopharyngeal aspirates and throat swabs in a new crown pneumonia in Hongkong. The researchers collected the tissues of turbinate, brain, lung, heart, duodenum, liver, spleen and kidney at 2, 5 and 7 days after inoculation of golden hamster to monitor the virus replication and histopathological changes. At the second day after inoculation, the peak of lung virus load of golden hamster was detected, and at the fifth day after inoculation, the virus load began to decrease. Although high copy viral RNA could be detected continuously, no infectious virus was detected 7 days after inoculation.
There was a significant difference in infectious virus load between 2 and 7 days after inoculation (P = 0.019, Dunnes multiple comparison test), but there was no difference in RNA virus copy number (P = 0.076). Although low copy viral RNA was detected 2 and 5 days after inoculation, no infectious virus was detected in the kidney.
Histopathological examination showed that 5-10% of the lungs had inflammatory cells and consolidation 2 days after inoculation.
5 days after inoculation, 15-35% of the lungs showed an increase in inflammatory cells.
Five days after the virus was inoculated, it developed into lung cells (the arrow in the figure below points to the location).
7 days after inoculation, 30-60% of the lungs became solid, as shown in the following figure:
The proliferation of type 2 lung cells is obvious, as shown in the following figure:
There is inflammatory cell infiltration in the turbinate, as shown in the following figure:
Virus antigens were detected in olfactory sensory neurons of nasal epithelial cells and nasal mucosa, as shown in the following figure:
Infection in olfactory neurons was further confirmed in cells expressing N protein and neuron specific u03b2 - III tubulin of SARS CoV, as shown in the following figure:
Compared with the control group, the number of olfactory neurons in the nasal mucosa of golden hamster decreased 2 days after inoculation, as shown in the following figure
7 days after inoculation, the nasal epithelial cells attenuated significantly, as shown in the following figure:
Although there is no inflammation in duodenal epithelial cells, as shown below:
However, virus antigens were detected from duodenal epithelial cells 2 days after inoculation, as shown in the following figure:
In order to evaluate the transmission ability of the new coronavirus in hamsters, the researchers inoculated three donor hamsters with TCID50 virus of 8 u00d7 10-4 power through the nose. 24 hours after inoculation, each donor was transferred to a new cage and raised with a young hamster. The researchers monitored body weight changes and clinical signs every day, and collected nasal lotion from donor hamsters and contact hamsters every other day for 14 days. In the donor hamster, although virus RNA can be detected for 14 consecutive days, the peak value of infectious virus appears in the early stage after inoculation, and then drops rapidly, as shown in the following figure:
The transmission from the donor to the contact is very efficient. Sars-cov-2 can be detected from the co habited hamster one day after contact, and the peak viral load can be detected in the co habited hamster nasal wash three days after contact, as shown in the following figure:
The largest mean weight loss (mean u00b1 SD) was observed in CO habited hamsters at 6 days after exposure- ten point six eight + three point four two %, n = 3), 11 days after exposure, all animals returned to their original weight, as shown in the following figure:
And co habited hamsters did not show weight loss, as shown in the following figure:
No neutralizing antibody (< 1:10) was detected by PRNT analysis. The results showed that the transmission time of sars-cov-2 was shorter than 6 days. The follow-up transmission from donor to co contact was related to the detection of infectious diseases in the donors nasal wash, but not to the detection of viral RNA.