key: cord-1008432-9k0haiw0
authors: Yadav, Pragya D.; Mohandas, Sreelekshmy; Shete, Anita M; Nyayanit, Dimpal A; Gupta, Nivedita; Patil, Deepak Y.; Sapkal, Gajanan N.; Potdar, Varsha; Kadam, Manoj; Kumar, Abhimanyu; Kumar, Sanjay; Suryavanshi, Deepak; Mote, Chandrashekhar S.; Abraham, Priya; Panda, Samiran; Bhargava, Balram
title: SARS CoV-2 variant B.1.617.1 is highly pathogenic in hamsters than B.1 variant
date: 2021-05-05
journal: bioRxiv
DOI: 10.1101/2021.05.05.442760
sha: 65defa346322442549b93d0bde51916d71f57c00
doc_id: 1008432
cord_uid: 9k0haiw0

Background The recent emergence of new SARS-CoV-2 lineage B.1.617 in India has been associated with a surge in the number of daily infections. This variant has combination of specific mutations L452R, E484Q and P681R reported to possibly enhance the transmissibility with likelihood of escaping the immunity. We investigated the viral load and pathogenic potential of B.1.617.1 in Syrian golden hamsters. Methods Two groups of Syrian golden hamsters (9 each) were inoculated intranasally with SARS CoV-2 isolates, B.1 (D614G) and B.1.617.1 respectively. The animals were monitored daily for the clinical signs and body weight. The necropsy of three hamsters each was performed on 3, 5- and 7-days post-infection (DPI). Throat swab (TS), nasal wash (NW) and organ samples (lungs, nasal turbinate, trachea) were collected and screened using SARS-CoV-2 specific Real-time RT-PCR. Results The hamsters infected with B.1.617.1 demonstrated increased body weight loss compared to B.1 variant. The highest viral load was observed in nasal turbinate and lung specimens of animals infected with B.1.167.1 on 3 DPI. Neutralizing antibody (NAb) and IgG response in hamsters of both the groups were observed from 5 and 7 DPI respectively. However, higher neutralizing antibody titers were observed against B.1.167.1. Gross pathology showed pronounced lung lesions and hemorrhage with B.1.671 compared to B.1. Conclusions B.1617.1 and B.1 variant varied greatly in their infectiousness, pathogenesis in hamster model. This study demonstrates higher pathogenicity in hamsters evident with reduced body weight, higher viral load in lungs and pronounced lung lesions as compared to B.1 variant. Summary B.1.617.1 is the new SARS-CoV-2 lineage that emerged in India. Maximal body weight loss and higher viral load in hamsters infected with B.1.617.1. It caused pronounced lung lesions in hamsters compared to B.1 variant which demonstrates the pathogenic potential of B.1.617.1.

Since, the first report of severe acute respiratory syndrome coronavirus-2 (SARS CoV -2) in Wuhan, China in 2019, the virus has constantly evolved leading to the emergence of new variants [1, 2] . The first variant of SARS CoV-2, D614G (B.1 lineage) became dominant and is prevalent worldwide since March 2020. Later mutations at different amino acid positions were reported with regional dominance [3] and were designated as variants under investigation (VUI).

The variant of concerns (VOC) has also been identified in many countries with increased transmissibility, pathogenicity and phenomenon of immune escape. [7] . The state of Maharashtra shares 21% of the active cases (0.67 million) from among the reported cases of 3.17 million from the country [8] . The [6, 9] . The origin of this variant is still 5 unknown and as mentioned above, the presence of this variant has been identified not only in India but from 21 different countries [10]. Our recent study has shown that Covaxin (a whole virion inactivated vaccine) works against B.1.617 variant [6] . However, answering the question of this variant is the main cause of the surge of the second wave in India remains a challenge.

In an earlier study, we investigated the pathogenicity of the SARS CoV-2 B.1 and B.1.1.7 lineage in the Syrian hamster model [11] . It was observed that B.1 lineage of SARS CoV-2 variant produced interstitial pneumonia with marked alveolar damage and type-II pneumocyte hyperplasia in hamsters [11, 12] . In the present study, we investigated the viral load and 

The animal experiments were performed with the approval of the Institutional Animal lineages propagated at ICMR-NIV, Pune as described earlier under isoflurane anesthesia [9, 13] .

Clinical signs and body weight of animals were observed daily and three hamsters each were euthanized on day 3, 5-and 7-days post-infection (DPI) to perform a necropsy. Throat swab 6 (TS), nasal wash (NW) and organ samples (lungs, nasal turbinate, trachea) were collected following the necropsy.

Nasal wash and TS collected in 1ml viral transport medium and weighed organ samples (lungs, nasal turbinate and trachea) triturated in 1 ml media were used for RNA extraction using MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit as per the manufacturer's instructions.

Real-time RT-PCR was performed for the E gene for SARS-CoV-2 as well as for detection of sgRNA of the E gene using published primers [14, 15] .

Lungs samples collected during necropsy were fixed in 10% neutral buffered formalin.

The tissues were processed by routine histopathological techniques for hematoxylin and eosin staining. The lung lesions were graded from 0 to +4 as nil, minimal, mild, moderate and severe based on the vascular changes, inflammatory changes and bronchial/ alveolar damage.

The assay was performed against both the isolates as described earlier [16] . Four-fold serial dilution of hamster serum samples mixed with an equal amount of virus suspension and incubated at 37°C for 1 hour. Further 0.1 ml of the mixture was inoculated in a 24-well tissue culture plate containing a confluent monolayer of Vero CCL-81 cells and was incubated at 37°C for 60 min and overlay medium was added to the cell monolayer, which was further incubated at 37°C in 5% CO2 incubator for 4-5 days and PRNT50 titres were calculated.

The lungs and nasal turbinate samples were used for virus titration in Vero CCL-81 cells.

A hundred microliter of the sample was added onto a 24-well plate with Vero CCL-81 monolayers and incubated for one hour at 37°C. The media was removed and the cell monolayer 7 was washed with PBS and was incubated with maintenance media with 2% FBS in a CO2

incubator. The plate was examined daily for any cytopathic effects (CPE) and the culture supernatant from the wells showing CPE was confirmed by real-time RT-PCR.

Graphpad Prism version 8.4.3 software was used to assess the statistical significance. A non-parametric two-tailed Mann-Whitney test was used to compare between two groups and pvalues of < 0.05 were considered to be statistically significant. (Table 1 ). In the case of the B.1 variant, the pneumonic changes observed were minimal to mild which included inflammatory cell infiltration, focal consolidation and mild congestion (Fig 4A-4C) . The pronounced changes (moderate to severe) with mononuclear infiltration in the alveolar interstitial space, interstitial septal thickening, consolidation and pneumocyte hyperplasia were observed with B.1.617.1 variant consistently till 7DPI (Fig 4D-4F ). The protective efficacy of the immunity generated from an initial SARS-CoV-2 infection against subsequent reinfection is puzzling. Studies in animal models have shown that prior infection results in protective immunity [22] . In the present study, we have evaluated the neutralization efficacy of the sera of hamsters infected with B.1.617 against B.1 variant and vice versa and found robust neutralizing immune response in both conditions. Re-infection studies need to be performed to understand the extent of the protection by these neutralizing antibodies in decreasing the viral load and severity of the disease. We recently reported that sera from BBV152 vaccine recipients and recovered cases of COVID-19 produced robust neutralizing antibodies against B.1.617 variant [6] . This means that the immunization program has great relevance against the different variants circuiting in India and other country and vaccines will have the potential to protect the recipient against severe morbidity following infection. 

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