Amino acid substitutions in NSP6 and NSP13 of SARS-CoV-2 contribute to superior virus growth at low temperatures

In general, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates well at 37 degrees C, which is the temperature of the human lower respiratory tract, but it poorly at 30 degrees C-32 degrees C, which is the temperature of the human upper respiratory tract. The replication efficiency of SARS-CoV-2 in the upper respiratory tract may directly affect its transmissibility. In this study, an XBB.1.5 isolate showed superior replicative ability at 32 degrees C and 30 degrees C, whereas most other Omicron sub-variant isolates showed limited growth. Deep sequencing analysis demonstrated that the frequencies of viruses possessing the NSP6-S163P and NSP13-P238S substitutions increased to more than 97% during propagation of the XBB.1.5 isolate at 32 degrees C but did not reach 55% at 37 degrees C. Reverse genetics revealed that these substitutions contributed to superior virus growth in vitro at these low temperatures by improving virus genome replication. Mutant virus possessing both substitutions showed slightly higher virus titers in the upper respiratory tract of hamsters compared to the parental virus; however, transmissibility between hamsters was similar for the mutant and parental viruses. Taken together, our findings indicate that NSP6-S163P and NSP13-P238S contribute to superior virus growth at low temperatures in vitro and in the upper respiratory tract of hamsters. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates efficiently at 37 degrees C. However, the temperature of the human upper airway is 30 degrees C-32 degrees C. Therefore, the replicative ability of SARS-CoV-2 at low temperatures could influence virus replication in the upper airway and transmissibility. In this study, we assessed the growth of Omicron sub-variants at low temperatures and found that an XBB.1.5 isolate showed increased replicative ability. By deep sequencing analysis and reverse genetics, we found that amino acid changes in NSP6 and NSP13 contribute to the low-temperature growth; these changes improved RNA polymerase activity at low temperatures and enhanced virus replication in the upper airway of hamsters. Although these substitutions alone did not drastically affect virus transmissibility, in combination with other substitutions, they could affect virus replication in humans. Furthermore, since these substitutions enhance virus replication in cultured cells, they could be used to improve the production of inactivated or live attenuated vaccine virus.