Evaluation of SARS-CoV-2 ORF7a Deletions from COVID-19-Positive Individuals and Its Impact on Virus Spread in Cell Culture.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the COVID-19 outbreak, posed a primary concern of public health worldwide. The most common changes in SARS-CoV-2 are single nucleotide substitutions, also reported insertions and deletions. This work investigates the presence of SARS-CoV-2 ORF7a deletions identified in COVID-19-positive individuals. Sequencing of SARS-CoV-2 complete genomes showed three different ORF7a size deletions (190-nt, 339-nt and 365-nt). Deletions were confirmed through Sanger sequencing. The ORF7a∆190 was detected in a group of five relatives with mild symptoms of COVID-19, and the ORF7a∆339 and ORF7a∆365 in a couple of co-workers. These deletions did not affect subgenomic RNAs (sgRNA) production downstream of ORF7a. Still, fragments associated with sgRNA of genes upstream of ORF7a showed a decrease in size when corresponding to samples with deletions. In silico analysis suggests that the deletions impair protein proper function; however, isolated viruses with partial deletion of ORF7a can replicate in culture cells similarly to wild-type viruses at 24 hpi, but with less infectious particles after 48 hpi. These findings on deleted ORF7a accessory protein gene, contribute to understanding SARS-CoV-2 phenotypes such as replication, immune evasion and evolutionary fitness as well insights into the role of SARS-CoV-2_ORF7a in the mechanism of virus-host interactions.

Hydroxymethylbilane synthase gene mutations and polymorphisms in Brazilian families with acute intermittent porphyria.

Acute intermittent porphyria (AIP), an autosomal dominant disorder, is caused by a deficiency of hydroxymethylbilane synthase (HMBS). In the present study, we sought to establish a correlation between HMBS activity with the presence of mutations and polymorphisms. Enzyme activity was measured in red blood cells of four Brazilian unrelated AIP families (n = 124) and in blood donors (n = 80). The HMBS mutations in AIP family members were studied by PCR-SSCP followed by direct sequencing. Six intragenic SNPs (1345 G>A, 1500 T>C, 2377 C>A, 2478 A>G, 3581 A>G, and 7064 C>A) were determined by PCR-RFLP. Abnormal SSCP patterns in exons 7, 9, 12, and 15 were observed. DNA sequencing analysis revealed one nonsense mutation, R149X, two missense mutations, G111R and L338P, and one deletion, CT 730-731. All mutation carriers had lower enzyme activity. All polymorphisms, except 2377 C>A and 7064 C>A, showed no significant differences compared with previous reports. Mutation screening allowed the detection of the missense mutation, L338P, and the 730_731delCT deletion, two as yet unreported mutations in Brazilian AIP patients. Our findings also showed a high frequency of 2478 A>G and 3581 A>G polymorphism combinations suggesting that these polymorphisms contributed to enzymatic activity reduction in our study population.