The spike protein of SARS-CoV-2 has been undergoing mutations and is highly glycosylated. It is critically important to investigate the biological significance of these mutations. Here, we investigated 80 variants and 26 glycosylation site modifications for the infectivity and reactivity to a panel of neutralizing antibodies and sera from convalescent patients. D614G, along with several variants containing both D614G and another amino acid change, were significantly more infectious. Most variants with amino acid change at receptor binding domain were less infectious, but variants including A475V, L452R, V483A, and F490L became resistant to some neutralizing antibodies. Moreover, the majority of glycosylation deletions were less infectious, whereas deletion of both N331 and N343 glycosylation drastically reduced infectivity, revealing the importance of glycosylation for viral infectivity. Interestingly, N234Q was markedly resistant to neutralizing antibodies, whereas N165Q became more sensitive. These findings could be of value in the development of vaccine and therapeutic antibodies.

Pseudoviruses are useful virological tools because of their safety and versatility, especially for emerging and re-emerging viruses. Due to its high pathogenicity and infectivity and the lack of effective vaccines and therapeutics, live SARS-CoV-2 has to be handled under biosafety level 3 conditions, which has hindered the development of vaccines and therapeutics. Based on a VSV pseudovirus production system, a pseudovirus-based neutralization assay has been developed for evaluating neutralizing antibodies against SARS-CoV-2 in biosafety level 2 facilities. The key parameters for this assay were optimized, including cell types, cell numbers, virus inoculum. When tested against the SARS-CoV-2 pseudovirus, SARS-CoV-2 convalescent patient sera showed high neutralizing potency, which underscore its potential as therapeutics. The limit of detection for this assay was determined as 22.1 and 43.2 for human and mouse serum samples respectively using a panel of 120 negative samples. The cutoff values were set as 30 and 50 for human and mouse serum samples, respectively. This assay showed relatively low coefficient of variations with 15.9% and 16.2% for the intra-and inter-assay analyses respectively. Taken together, we established a robust pseudovirus-based neutralization assay for SARS-CoV-2 and are glad to share pseudoviruses and related protocols with the developers of vaccines or therapeutics to fight against this lethal virus.

The 501Y.V2 variants of SARS-CoV-2 containing multiple mutations in Spike are now dominant in South Africa and are rapidly spreading to other countries. Here, experiments with 18 pseudotyped viruses showed that the 501Y.V2 variants do not confer increased infectivity in multiple cell types except for murine ACE2-overexpressing cells, where a substantial increase in infectivity was observed. Notably, the susceptibility of the 501Y.V2 variants to 12 of 17 neutralizing monoclonal antibodies was substantially diminished, and the neutralization ability of the sera from convalescent patients and immunized mice was also reduced for these variants. The neutralization resistance was mainly caused by E484K and N501Y mutations in the receptor-binding domain of Spike. The enhanced infectivity in murine ACE2-overexpressing cells suggests the possibility of spillover of the 501Y.V2 variants to mice. Moreover, the neutralization resistance we detected for the 501Y.V2 variants suggests the potential for compromised efficacy of monoclonal antibodies and vaccines.

Pseudotyped viruses are useful virological tools because of their safety and versatility. On the basis of a vesicular stomatitis virus (VSV) pseudotyped virus production system, we developed a pseudotyped virus-based neutralization assay against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in biosafety level 2 facilities. Compared with the binding antibody test, the neutralization assay could discriminate the protective agents from the antibody family. This protocol includes production and titration of the SARS-CoV-2 S pseudotyped virus and the neutralization assay based on it. Various types of samples targeting virus attachment and entry could be evaluated for their potency, including serum samples derived from animals and humans, monoclonal antibodies and fusion inhibitors (peptides or small molecules). If the pseudotyped virus stock has been prepared in advance, it will take 2 days to get the potency data for the candidate samples. Experience in handling cells is needed before implementing this protocol.

In total, 335 serum samples were collected from rabbits from two farms in Gansu province, China, and tested for anti-hepatitis E virus (HEV) antibody using EIA and for HEV RNA using nested RT- PCR with ORF2 primers. The overall prevalence of anti-HEV antibody and HEV RNA was 57.0% (191/335) and 7.5% (25/335), respectively. The positivity rate of HEV RNA in the anti-HEV antibody negative group (7.6% (11/144)) did not differ significantly from that in the positive group (7.3% (14/191)). The concordance between HEV RNA and anti-HEV antibody was 43.3% with no significant correlation (P < 0.05). All 25 amplicons from the ORF2 region were cloned and sequenced. On the basis of nucleotide sequence comparison, they had 84-99% identity to each other and 73-77%, 70-76%, 75-82%, 71-77%, and 53-65% with the corresponding regions of genotypes 1, 2, 3, 4, and avian HEV, respectively. Samples that were positive with the ORF2 primers were amplified using ORF1 region primers; 17 were positive and shared 71-78%, 73-76%, 74-82%, 72-78%, and 39-58% identity with the corresponding regions of genotypes 1, 2, 3, 4, and avian HEV, respectively, at the nucleotide level. Two representative full-length sequences were determined. These two sequences shared 85% identity with each other and had 74%, 73%, 78-79%, 74-75%, and 46-47% identity to full-length genotypes 1, 2, 3, 4, and avian HEV, respectively. Thus, the sequences isolated from the rabbits represent a novel genotype of HEV. This study provides novel information about HEV genotypes infecting rabbits as well as evidence of a new mammalian genotype of HEV.