动物实验,新冠 z-bo 2020-09-14 318

　　Since the outbreak, whether the new coronavirus can spread through aerosols has been one of the focuses of the academic community. The world's top journal "Nature" recently published a study by the Hong Kong University team showing that in the golden hamster animal model experiment, the new coronavirus can be transmitted through aerosols.

　　The paper was published online on May 14th, local time in the form of "Accelerated Article Preview", entitled Pathogenesis and transmission of SARS-CoV-2 in golden hamsters. The corresponding author of the paper is Hui-Ling Yen (Hui-Ling Yen), University of Hong Kong Li Ka-shing School of Medicine, and the author of the paper also includes Professor Pan Liewen of the School of Public Health of the University of Hong Kong.

　　After the emergence of the new coronavirus, researchers urgently need suitable 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 the golden hamsters had virus antigens in their nasal mucosa, bronchial epithelial cells, and lung consolidation areas 2 and 5 days after virus inoculation. Seven days after virus inoculation, the virus cleared rapidly and lung cells proliferated. Viral antigens were also found in duodenal epithelial cells of golden hamsters, and viral RNA was detected in feces.

　　It is worth noting that animal model experiments have proved that the new coronavirus can effectively spread from infected golden hamsters to naive golden hamsters through direct contact and aerosols. In hamster cages, the transmission efficiency is low. Although viral RNA was detected in the nasal wash of inoculated hamsters for 14 consecutive days, the new coronavirus can spread for a short time. Hamsters vaccinated with infection and naturally infected showed significant weight loss, and neutralizing antibodies were detected in all golden hamsters after recovery. This result shows that the new coronavirus infection characteristics of golden hamsters are similar to those of mild human infections.

　　The paper pointed out that a suitable animal model is essential for understanding the pathogenesis of new coronary pneumonia and evaluating vaccines and therapeutic candidates. Previous animal studies on SARS-CoV have shown the interaction between the virus’s spike protein (S protein) and the host’s angiotensin converting enzyme 2 (ACE2) receptor, as well as the age and innate immune status of the infected subject It plays an important role in pathogenesis. Like SARS-CoV, the S protein of SARS-CoV-2 also uses ACE2 receptors (mainly distributed in the epithelial cells of the lung and small intestine) to enter cells for virus replication. SARS-CoV-2 binds well to human ACE2, but has limited binding to murine ACE2, which limits the application of inbred mice in virus research. Rhesus monkeys and transgenic ICR mice expressing the human ACE2 receptor have been shown to be susceptible to SARS-CoV-2; however, these animal models are not so easily available. The cynomolgus macaques and rhesus monkeys attacked by SARS-CoV-2 showed limited and moderate clinical symptoms, respectively. The infected transgenic mice showed moderate pneumonia and no obvious histological changes in non-respiratory tissues. According to reports, although the previously produced transgenic mice expressing human ACE2 receptor support SARS-CoV replication in respiratory epithelial cells, the transgenic mice also have high expression of ACE2 in the brain, resulting in the death of mice. Variables related to the disease of the system.

　　Golden hamster is a widely used experimental animal model. Previous studies have shown that SARS-CoV can replicate in its body, but MERS-CoV cannot. This is because MERS-CoV uses the DPP4 protein as the main receptor for the virus to enter cells. Body, not ACE2. Previously, the SARS-CoV (Urbani strain) inoculation studies on 5-week-old golden hamsters showed that the virus has a strong ability to replicate in the body, and the peak virus titer can be detected in the lungs 2 days after inoculation; After 7 days from the virus, the golden hamster can quickly clear the virus. However, the golden hamsters vaccinated with the virus did not have weight loss or obvious disease conditions. A follow-up study reported that different SARS-CoV strains were tested in golden hamsters, and differences in virulence between SARS-CoV strains were found. It is reported that SARS-CoV (Frk-1 strain) is lethal 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 by other respiratory viruses, including human metapneumovirus, human parainfluenza virus, and influenza A virus, and may support the spread of influenza through contact or air. The comparison of the ACE2 protein of human, rhesus monkey, mouse and hamster indicated that hamster ACE2 may interact with the S protein of SARS-CoV-2 more effectively than murine ACE2. Here, the research team evaluated the pathogenesis and contact transmission ability of SARS-CoV-2 in male golden hamsters aged 4-5 weeks.

　　prompted aerosol transmission animal model experiment

　　In order to study the aerosol transmission ability of SARS-CoV-2 in hamsters, the researchers placed the donor (receiving the new crown virus inoculation) hamster and the naive hamster (healthy hamster) in two adjacent iron cages. One day after the hamster was inoculated with the virus, the two cages were placed together for 8 hours.

　　The infectious virus in the donor hamster nasal wash can be detoxified for 6 days, while the viral RNA can be detected continuously for 14 days, which means that in the later stage, the infected hamster is not infectious.

　　Viral RNA can be detected in donor stool samples 2 days, 4 days, and 6 days after virus inoculation, but it is not an infectious virus.

　　Researchers have found that the aerosol transmission of the new coronavirus in hamsters is effective, because the infectious virus was detected in all exposed hamster nasal washes one day after contact, and the viral load reached a peak 3 days after contact.

　　Although no infectious virus has been isolated, viral RNA can be detected in stool samples from infected aerosol contacts for 14 consecutive days.

　　Hamsters exposed to aerosol showed the greatest weight loss 7 days after exposure (mean ± SD, -7.72 ± 5,42%, N = 3). Compared with donor hamsters, aerosol contact hamsters excreted a considerable amount of virus in the nasal wash.

　　In order to evaluate the other transmission ability of the new coronavirus in hamsters, the researchers inoculated 3 donor hamsters intranasally with 8×10 minus 4 power of TCID50 virus. Twenty-four hours after inoculation, each donor was transferred to a new cage and raised with a young hamster. The researchers monitored weight changes and clinical signs every day, and collected nasal washes from donor hamsters and contact hamsters every other day for 14 days. In donor hamsters, although viral RNA could be detected for 14 consecutive days, the peak of infectious virus appeared in the early stage after virus inoculation, and then dropped rapidly.

　　6 days after SARS-CoV-2 inoculation, hamsters showed the greatest average weight loss (mean ± SD, -11.97 ± 4.51%, N = 6).

　　The transmission from the donor to the contact is very efficient. SARS-CoV-2 can be detected from cohabiting hamsters one day after contact, and the peak viral load is detected in cohabiting hamster nasal wash 3 days after contact.

　　co-living hamsters showed the greatest average weight loss 6 days after exposure (mean ± SD, -10.68 ± 3.42%, N = 3), 11 days after exposure, all animals recovered their original body weight.

　　Use PRNT analysis to detect neutralizing antibodies from donors 14 days after virus inoculation (all titers are 1:640) and 13 days after co-living hamster contact (at 1:160, 1:320 and 1:160 titers) . The researchers detected viral RNA in the nasal wash of donor hamsters for 14 consecutive days, but the infectious virus titer dropped rapidly. The researchers repeated the experiment and cohabited with the naive hamsters 6 days after the virus inoculation. A small amount of viral RNA was detected in the nasal wash of the co-living naive hamsters 3 days after the contact, and no viral RNA was detected in the nasal wash 7 days after the contact. Moreover, no infectious virus was detected in the nasal wash.

　　and cohabiting hamsters showed no weight loss.

　　PRNT analysis did not detect neutralizing antibodies of cohabiting hamsters 12 days after exposure (<1:10). The results showed that the virus from donors vaccinated with SARS-CoV-2 can spread for less than 6 days. The subsequent transmission from the donor to the co-living contacts is related to the detection of infectious diseases in the donor's nasal wash, and has nothing to do with the detection of viral RNA.

　　The pathogenesis of SARS-CoV-2 in golden hamsters

　　In the experiment, the researchers infected golden hamsters intranasally with a new coronavirus (Beta-CoV / Hong Kong / VM20001061/2020 virus, GISAID#EPI_ISL_412028) with a minus 4 power TCID50 (50% tissue cell infection) of 8 x 10. The virus was isolated from Vero E6 cells from nasopharyngeal aspirates and throat swabs from a Hong Kong patient with new coronary pneumonia. The researchers collected turbinate, brain, lung, heart, duodenum, liver, spleen, and kidney tissues at 2, 5, and 7 days after the golden hamsters were inoculated with the virus to monitor virus replication and histopathological changes. On the second day after virus inoculation, the peak viral load in the lungs of golden hamsters can be detected, and the viral load begins to decrease 5 days after inoculation. Although high copies of viral RNA could continue to be detected, no infectious virus was detected 7 days after virus inoculation.

　　There was a significant difference in infectious viral load between 2 and 7 days after virus inoculation (P = 0.019, Dunn's multiple comparison test), but there was no difference in RNA virus copy number (P = 0.076). Although low-copy viral RNA was detected at 2 and 5 days after virus inoculation, no infectious virus was detected in the kidney.

　　Histopathological examination revealed that 5-10% of lungs had increased and consolidated inflammatory cells 2 days after virus inoculation.

　　5 days after virus inoculation, 15-35% of lungs have increased inflammatory cells.

　　In areas where viral antigens are detected 2 and 5 days after virus inoculation, monocyte infiltration can be observed. The immunohistochemistry of the N protein of SARS-CoV-2 showed that two days after virus inoculation, there were viral antigens in the bronchial epithelial cells of golden hamsters.

　　5 days after virus inoculation, it developed into lung cells.

　　7 days after virus inoculation, 30-60% of the lungs are consolidated. However, no viral antigens were detected, and type 2 lung cell proliferation was obvious.

　　CD3-positive T lymphocytes were detected in the peribronchial area 5 days after virus inoculation, which may help to quickly clear infected cells.

　　There is inflammatory cell infiltration in the turbinate. Viral antigens were detected in nasal epithelial cells and olfactory sensory neurons of nasal mucosa.

　　"The infection in olfactory neurons was further confirmed in cells expressing the N protein of SARS-CoV and neuron-specific β-III tubulin.

　　Compared with the control group, the number of olfactory neurons in the nasal mucosa of golden hamsters decreased 2 days after virus inoculation, as shown in the figure below

　　7 days after virus inoculation, nasal epithelial cells attenuate significantly. Then tissue repair can be observed 14 days after virus inoculation. Although there was no inflammation in the duodenal epithelial cells, viral antigens were detected in the duodenal epithelial cells 2 days after virus inoculation. Five days after virus inoculation, no obvious histopathological changes were observed in the brain, heart, liver and kidney.