key: cord-0832719-mlpnbm76
authors: Gao, Ge; Hu, Xue; Zhou, Yiwu; Rao, Juhong; Zhang, Xiaoyu; Peng, Yun; Zhao, Jiaxuan; Yao, Yanfeng; Liu, Kunpeng; Liang, Mengying; Liu, Hang; Deng, Fei; Xia, Han; Shan, Chao; Yuan, Zhiming
title: Infection and pathogenesis of the Delta variant of SARS-CoV-2 in Rhesus macaque
date: 2022-02-10
journal: Virol Sin
DOI: 10.1016/j.virs.2022.02.001
sha: ae172a9e17d861db9dfa5f9263aaff22f6a502b5
doc_id: 832719
cord_uid: mlpnbm76

Delta variant B.1.167.2 is becoming the dominant SARS-CoV-2 lineage worldwide and is designated as a variant of concern (VOC) by the World Health Organization (WHO). The infectivity and pathogenicity of the delta variant were evaluated in the Rhesus macaque model and compared with the early strains that caused the first wave of COVID-19 in Wuhan, China. Viral load and lung disease were studied. There was no significant difference in the virus shedding pattern, viral load and the severity of disease, but the delta variant reached the growth peak faster even using a lower infection dose than the early strains, which supported the attributed rapid disease spread of the variant.

to the surveillance data (WHO, 2021b) . 57

The Delta variant was firstly identified in India in Dec 2020 and the World Health Organization (WHO) 58 declared it a "variant of concern (VOC)", since it was shown to be more contagious, more deadly and more 59 resistant to the current vaccines and treatments (WHO, 2021b) . During May to the beginning of December, 60 2021, Delta variant was the dominant circulating strain in most regions of the world, with around 60% greater 61 transmissibility than the Alpha variant, 1,000 times higher viral load than the strains in the initial epidemic 62 wave of 2020, an increased 2.26-fold risk of hospitalization compared with Alpha and was moderately 63 resistant at a degree of around 30.7% to vaccines BNT162b2 or ChAdOx1 nCoV-19 particularly in people 64 with a single dose (Bian et al., 2021; Callaway, 2021; Planas et al., 2021; Shi and Dong, 2021 Previous studies have shown that SARS-CoV-2 replicates to a high viral load in the respiratory tract of 68 Rhesus macaques and causes pneumonia utilized for studying pathology, immune response, and multiple 69 preclinical drug and vaccine evaluations (Chandrashekar et al., 2021 (Chandrashekar et al., , 2020 Deng et al., 2020; Feng et al., 70 2020; Mercado et al., 2020; Shan et al., 2020) . This model is important for generating information on disease 71 characteristics and replicative fitness and for evaluating the pharmaceutical treatment approaches against the 72 new variant. 73

In the present study, pathogenicity and virus shedding of the Delta variant in rhesus macaques were 74 assessed. The shedding pattern and pathogenesis changes were like that observed in our prior studies, but with 75 rapid and strong replication in macaques when compared to the early SARS-CoV-2 strains during the first 76 epidemic that occurred in humans, in Wuhan, China in late 2019 (Shan et al., 2020) . 77

Three adult rhesus macaques aged from six to eleven years, were inoculated with a total of 1 × 10 6 as mild weight loss in the high-dose group. Fever, respiratory distress and mortality were not observed. 82 RNA extraction and RT-qPCR detection for SARS-CoV-2 from swabs, blood, and tissues as described 83

previously (Shan et al., 2020) . Elevated levels of viral RNA in the throat were observed with a median peak of 84 6.7 (range 6.0 to 7.3) log10 and 7.6 (range 3.4 to 7.9) log10 RNA copies/swab in HD and LD groups, 85 respectively (Fig. 1B) . No significant difference was found regardless of dose. Much lower virus load was 86 found in the nasal specimens. No viral RNA was detected in blood and anal samples (Fig. 1B) . Viral RNA in 87 the throat increased in all animals in HD and LD groups from days one to two, suggesting viral replication. 88

All macaques in both groups were euthanized and necropsied at 7 days post inoculation (dpi). Different 89 lobes of lung, trachea, and bronchus were collected and viral RNA was detected in all tissues. Much higher 90 viral load was presented in the trachea and bronchus when compared to lung, with a median value of 8.2 91 (range 6.8 to 8.6) log10 and 8.2 (range 4.1 to 8.6) log10 RNA copies/g in trachea and a median value of 8.1 92 (range 5.2 to 8.6) log10 and 8.1 (range 3.8 to 8.6) log10 RNA copies/g in the bronchus (Fig. 1C) . 93

The anterior-posterior thoracic X-rays of macaques from both of the HD and LD groups showed intense 94 patchy opacity in the middle and lower parts of lung lobes at 7 dpi (Fig. 1D) . For the histopathological 95 analysis and SARS-CoV-2 antigen detection, tissues from lung, bronchus, and trachea were fixed, then 96 hematoxylin-eosin (HE) stain, immunological fluorescence assay (IFA), and Immunohistochemical (IHC) 97 were conducted separately as described previously (Shan et al., 2020) . Microscopically, lesions were in the 98 lungs where the interstitial pneumonia present was severe. Pneumonia from LD macaques was characterized 99 macaques which showed as strongly positive in trachea and bronchus (Fig. 1E ). IHC analysis of HD and LD 111 macaques by SARS-CoV-2 NP antigen revealed that the positivity was distributed in pulmonary macrophages 112 around the alveolar wall, bronchial mucosa epithelium, goblet cells and pseudostratified ciliated epithelial 113 cells of the tracheal mucosa epithelium, lymphocytes of the mucosal lamina propria and tracheal submucosal 114 glands ( Supplementary Fig. S1) . 115

Even the infection dose for Delta variant in this experiment was 1 × 10 6 TCID50 or 1 × 10 5 TCID50 per 116 animal, which was 7 or 70 folds lower than the early strain IVCAS 6.7512 (at 7 × 10 6 TCID50 per animal) used 117 in our previous study, high viral load in throat and severe pathogenic changes in lung were still be observed 118 (Shan et al., 2020) . The viral RNA load in throat of infected animal with Delta variant took 2-3 days to reach 119 peak while the early strain took around 5 days (Shan et al., 2020) , supporting the attributed disease rapid 120 spread of the Delta variant. In addition, whether infected with early strain or Delta variant, no significant 121 changes in the body temperature, weight loss, blood cell counts (Supplementary Fig. S2 ) and X-ray imaging 122 were detected, which might be due to the virus could only cause asymptomatic or mild clinical symptoms in 123 macaque (Shan et al., 2020) . This suggests that the viral load in the throat and the pathogenic changes of lung 124 can be used as the two main parameters for evaluating the SARS-CoV-2 infection in the rhesus macaque Callaway, E., 2021. Delta coronavirus variant: scientists brace for impact. Nature. Fig. 1 Experimental scheme, viral RNA load, thoracic X-rays, histopathological and Immunological

The SARS-CoV-2 B

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WHO, 2021b. Tracking SARS-CoV-2 variants

B Viral load in swabs and blood of the Delta variant infected rhesus macaque. Viral 222 load (mean ± SEM) of anal, blood, nasal and throat specimens collected from the inoculated macaques (HD

= 3) on 1 to 7 dpi. C Viral loads in lung of the Delta variant infected rhesus macaque. Varied 224 samples including lobe of lung tissue

Anterior-posterior thoracic X-rays of infected rhesus macaque. The images have been 226 taken at 0 and 7 dpi of HD and LD macaques. Areas of interstitial infiltration, indicative of pneumonia are 227 highlighted with red ellipse. E Histopathological and immunological fluorescence assay (IFA) observation of 228

Yellow arrows indicate alveolar wall thickening, black arrows 229 indicate the epithelial cells shedding, red arrows indicate the inflammation cells in HE staining. Red color 230 indicates nucleocapsid protein (NP), and blue color indicated the cell nuclei in IFA. Lung, bronchus, and 231 trachea tissue were collected at 7 dpi from HD and LD macaques

SEM, standard error of mean