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Genomic surveillance is a critical tool for tracking emerging variants of SARS-CoV-2 (the virus that causes COVID-19), which can exhibit characteristics that potentially affect public health and clinical interventions, including increased transmissibility, illness severity, and capacity for immune escape. During June 2021-January 2022, CDC expanded genomic surveillance data sources to incorporate sequence data from public repositories to produce weighted estimates of variant proportions at the jurisdiction level and refined analytic methods to enhance the timeliness and accuracy of national and regional variant proportion estimates. These changes also allowed for more comprehensive variant proportion estimation at the jurisdictional level (i.e., U.S. state, district, territory, and freely associated state). The data in this report are a summary of findings of recent proportions of circulating variants that are updated weekly on CDC's COVID Data Tracker website to enable timely public health action.

Recombinant SHFV infectious cDNA clones expressing a foreign gene from an additional sg mRNA were constructed. Two 3′ genomic region sites, between ORF4′ and ORF2b and between ORF4 and ORF5, were utilized for insertion of the myxoma M013 gene with a C-terminal V5 tag followed by one of the three inserted transcription regulatory sequences (TRS), TRS2′, TRS4′ or TRS7. M013 insertion at the ORF4′/ORF2b site but not the ORF4/ORF5 site generated progeny virus but only the recombinant virus with an inserted TRS2′ retained the entire M013 gene through passage four. Insertion of an auto-fluorescent protein gene, iLOV, with an inserted TRS2′ at the ORF4′/ORF2b site, generated viable progeny virus. iLOV expression was maintained through passage eight. Although regulation of SHFV subgenomic RNA synthesis is complex, the ORF4′/ORF2b site, which is located between the two sets of minor structural proteins, is able to tolerate foreign gene insertion.

In mice, resistance to central nervous system (CNS) disease induced by members of the genus Flavivirus is conferred by an allele of the 2′-5′ oligoadenylate synthetase 1b gene that encodes the inactive full-length protein (Oas1b-FL). The susceptibility allele encodes a C-terminally truncated protein (Oas1b-tr). We show that the efficiency of neuron infection in the brains of resistant and susceptible mice is similar after an intracranial inoculation of two flaviviruses, but amplification of viral proteins and double-stranded RNA (dsRNA) is inhibited in infected neurons in resistant mouse brains at later times. Active OAS proteins detect cytoplasmic dsRNA and synthesize short 2′-5′-linked oligoadenylates (2′-5′A) that interact with the latent endonuclease RNase L, causing it to dimerize and cleave single-stranded RNAs. To evaluate the contribution of RNase L to the resistance phenotype in vivo, we created a line of resistant RNase L(−/−) mice. Evidence of RNase L activation in infected RNase L(+/+) mice was indicated by higher levels of viral RNA in the brains of infected RNase L(−/−) mice. Activation of type I interferon (IFN) signaling was detected in both resistant and susceptible brains, but Oas1a and Oas1b mRNA levels were lower in RNase L(+/+) mice of both types, suggesting that activated RNase L also has a proflaviviral effect. Inhibition of virus replication was robust in resistant RNase L(−/−) mice, indicating that activated RNase L is not a critical factor in mediating this phenotype. IMPORTANCE The mouse genome encodes a family of Oas proteins that synthesize 2′-5′A in response to dsRNA. 2′-5′A activates the endonuclease RNase L to cleave single-stranded viral and cellular RNAs. The inactive, full-length Oas1b protein confers flavivirus-specific disease resistance. Although similar numbers of neurons were infected in resistant and susceptible brains after an intracranial virus infection, viral components amplified only in susceptible brains at later times. A line of resistant RNase L(−/−) mice was used to evaluate the contribution of RNase L to the resistance phenotype in vivo. Activation of RNase L antiviral activity by flavivirus infection was indicated by increased viral RNA levels in the brains of RNase L(−/−) mice. Oas1a and Oas1b mRNA levels were higher in infected RNase L(−/−) mice, indicating that activated RNase L also have a proflaviviral affect. However, the resistance phenotype was equally robust in RNase L(−/−) and RNase L(+/+) mice.

Zika virus (ZIKV) infection attenuates the growth of human neural progenitor cells (hNPCs). As these hNPCs generate the cortical neurons during early brain development, the ZIKV-mediated growth retardation potentially contributes to the neurodevelopmental defects of the congenital Zika syndrome. Here, we investigate the mechanism by which ZIKV manipulates the cell cycle in hNPCs and the functional consequence of cell cycle perturbation on the replication of ZIKV and related flaviviruses. We demonstrate that ZIKV, but not dengue virus (DENV), induces DNA double-strand breaks (DSBs), triggering the DNA damage response through the ATM/Chk2 signaling pathway while suppressing the ATR/Chk1 signaling pathway. Furthermore, ZIKV infection impedes the progression of cells through S phase, thereby preventing the completion of host DNA replication. Recapitulation of the S-phase arrest state with inhibitors led to an increase in ZIKV replication, but not of West Nile virus or DENV. Our data identify ZIKV’s ability to induce DSBs and suppress host DNA replication, which results in a cellular environment favorable for its replication. IMPORTANCE Clinically, Zika virus (ZIKV) infection can lead to developmental defects in the cortex of the fetal brain. How ZIKV triggers this event in developing neural cells is not well understood at a molecular level and likely requires many contributing factors. ZIKV efficiently infects human neural progenitor cells (hNPCs) and leads to growth arrest of these cells, which are critical for brain development. Here, we demonstrate that infection with ZIKV, but not dengue virus, disrupts the cell cycle of hNPCs by halting DNA replication during S phase and inducing DNA damage. We further show that ZIKV infection activates the ATM/Chk2 checkpoint but prevents the activation of another checkpoint, the ATR/Chk1 pathway. These results unravel an intriguing mechanism by which an RNA virus interrupts host DNA replication. Finally, by mimicking virus-induced S-phase arrest, we show that ZIKV manipulates the cell cycle to benefit viral replication.

Members of the order Nidovirales express their structural protein ORFs from a nested set of 3′ subgenomic mRNAs (sg mRNAs), and for most of these ORFs, a single genomic transcription regulatory sequence (TRS) was identified. Nine TRSs were previously reported for the arterivirus Simian hemorrhagic fever virus (SHFV). In the present study, which was facilitated by next-generation sequencing, 96 SHFV body TRSs were identified that were functional in both infected MA104 cells and macaque macrophages. The abundance of sg mRNAs produced from individual TRSs was consistent over time in the two different cell types. Most of the TRSs are located in the genomic 3′ region, but some are in the 5′ ORF1a/1b region and provide alternative sources of nonstructural proteins. Multiple functional TRSs were identified for the majority of the SHFV 3′ ORFs, and four previously identified TRSs were found not to be the predominant ones used. A third of the TRSs generated sg mRNAs with variant leader–body junction sequences. Sg mRNAs encoding E′, GP2, or ORF5a as their 5′ ORF as well as sg mRNAs encoding six previously unreported alternative frame ORFs or 14 previously unreported C-terminal ORFs of known proteins were also identified. Mutation of the start codon of two C-terminal ORFs in an infectious clone reduced virus yield. Mass spectrometry detected one previously unreported protein and suggested translation of some of the C-terminal ORFs. The results reveal the complexity of the transcriptional regulatory mechanism and expanded coding capacity for SHFV, which may also be characteristic of other nidoviruses.