Over the past few decades, there has been a growing demand for genome analysis of ancient human remains. Destructive sampling is increasingly difficult to obtain for ethical reasons, and standard methods of breaking the skull to access the petrous bone or sampling remaining teeth are often forbidden for curatorial reasons. However, most ancient humans carried head lice and their eggs abound in historical hair specimens. Here we show that host DNA is protected by the cement that glues head lice nits to the hair of ancient Argentinian mummies, 1,500–2,000 years old. The genetic affinities deciphered from genome-wide analyses of this DNA inform that this population migrated from north-west Amazonia to the Andes of central-west Argentina; a result confirmed using the mitochondria of the host lice. The cement preserves ancient environmental DNA of the skin, including the earliest recorded case of Merkel cell polyomavirus. We found that the percentage of human DNA obtained from nit cement equals human DNA obtained from the tooth, yield 2-fold compared with a petrous bone, and 4-fold to a bloodmeal of adult lice a millennium younger. In metric studies of sheaths, the length of the cement negatively correlates with the age of the specimens, whereas hair linear distance between nit and scalp informs about the environmental conditions at the time before death. Ectoparasitic lice sheaths can offer an alternative, nondestructive source of high-quality ancient DNA from a variety of host taxa where bones and teeth are not available and reveal complementary details of their history. Over the last 30 years, ancient DNA (aDNA) has proven to be a key source of information about past organism assemblages, diversity, and evolution. To date, aDNA has been obtained from a variety of materials including bones and teeth , bird toAs the demand for human bones and teeth for destructive analyses have increased over the past 20 years, so have cases of unreported, indiscriminate exploitation of historical and unique specimens . ConcernPulex fleas from animal mummies (dogs and guinea pigs) belonging to the Chiribaya culture, South Peru . In addition, To date, parasites found in 19th century collections of natural history museums as well as archaeological sites have mainly been studied morphologically. Only a few, more recent studies have gathered short fragments of DNA from ancient parasites . DittmarPediculus humanus, is the most ubiquitous human ectoparasite. It is an obligatory bloodsucking parasite  to yield human DNA from host cells trapped inside their cement. This allowed us to explore the suitability of this DNA for genome-wide analysis, to compare with current sources of ancient human DNA , and to study aspects of preservation, genome coverage, haplotyping, sexing of individuals, and phylogeographic history. Eight ancient South American human remains were sampled for hair containing nits. The hair of two of these specimens, SJArg-1-Nit and SJArg-2-Nit, showed the presence of a few nits; a maximum of six from each individual were subjected to DNA extraction. For comparison of DNA preservation and coverage, we obtained one tooth (SJArg-1-Tooth) from the same sedimentary layer as SJArg-1-Nit, and a petrous bone (SJArg-3-Petrous) from an individual (SJArg-3) from the same culture as SJArg-1 and SJArg-2 but located at an upper layer. For comparison of host DNA from nits with host DNA from lice bloodmeal (inside their gut), two adult lice from the Denny’s historical Anoplura collection from the Oxford University Museum of Natural History were included. They were recovered ∼170 years ago (between 1849 and 1859) from a Dayak/Dyak/Dayuh native of Borneo (sample Dyak-Louse). Because lice are species specific parasites that parallel the history of their hosts, Pediculus genetic affinities were also investigated using mitochondrial genes. In addition to the genetic studies on nits, three of the nit samples were studied using cytogenetic methods to detect and validate DNA trapped in cement, and we compared these findings to the genomic data generated. Lastly, we investigated the level of infestation to gather information on the health status, carried out morphometric characterizations such as length of cement coverage to assess preservation, and nit/sheath distance to scalp to unravel environmental conditions such as variations in ambient temperature just before the death of these ancient individuals.In this work, we investigated the potential for ancient nit sheaths  Radiocarbon laboratory (SUERC-81423) to 1,461 cal. years BP . This is near to the end of Early Period in Argentina (500 BCE–650 CE) and is equivalent to the early intermediate period in the Central Andes (0–750 CE), which has one of the best constructed archaeological chronologies. It is also equivalent to Nazca and Tiwanaku cultures in Peru and Bolivia, respectively. This radiocarbon date is in good agreement with the previous age estimates  based on radiocarbon dating of associated bulk sediments. We therefore did not re-date the other three specimens used for DNA analysis, SJArg-1-Tooth, SJArg-2-Nit, and SJArg-3-Petrous, for which radiocarbon ages had also been obtained by bulk sediments and which were found to be 1,461 , 1,934 , and 1,018  cal. years BP with 95.4% probability. Radiocarbon ages of all eight mummies together with other age estimates based on cultural chronologies can be found in r = −0.73). The variations between samples of different ages were found significant , with the oldest samples from SJArg-2-Nit differing from most of the ages , whereas this distance in SJArg-4-Nit varied significantly . Nits carrying embryos are eggs laid shortly before the host’s death and were found at a shorter distance than the generally expected ∼7 mm from the root , occurring at high frequency at an unusually shorter distance of 2–4 mm  to root in three mummies , as the colder the air temperature the closer to the scalp the louse lays its eggs see  online. f 2–4 mm  online.For SJArg-1-Nit and SJArg-2-Nit, confocal microscopy analysis showed preservation of cuticle of hair with a similar quality to that of a fresh, healthy hair, when covered by cement. However, for SJArg-1-Nit, cuticle scales showed signs of deterioration, for example, being broken or detached in the cement-uncovered areas and showing rupture reaching the cortex or the medulla. Whereas SJArg-2-Nit showed much better preservation of the hair outside this segment, despite being about 500 years older. In addition, we noticed that the cuticle types of SJArg-1-Nit hair were different in shape, with large variation in the arrangement and size of the scales  online. Across all samples, we were unable to identify mitochondria in scans due to the limited magnification of confocal scanning microscopy, despite fully scanning the diameter of the hair and comparing between the sections covered by cement with the cement-free sections of the hair surface. None of the cement-free sections of the scanned shafts were positive for the fluorescent stains. Inside the cement, signals were observed from trapped material, such as dirt, likely bacterial, or fungal cells (based on shape). These particles, together with the shedding skin cells were collected or retained inside the liquid cement when it was secreted by the mother louse to glue the new egg to the hair, providing additional protection not only to the DNA of host cells, but to potentially also cells and extracellular DNA from other organisms.P. humanus corporis assembly GCA_000006295.1, in absence of a human head louse genome [We extracted and sequenced ∼285 million (raw) reads, which yielded 21 million reads upon trimming, duplicate removal, and quality filtering (e genome ). Louse The above results indicate that endogenous human DNA was preserved and extracted from all samples, however, we still sought to confirm its ancient origin by calculating the proportion of nucleotide misincorporations arising from DNA damage for aligning to both the human and louse genome using mapDamage  online. We used contamMix  to quanty) similar to males. We then calculated the fraction of Y chromosome in the extraction blank to ensure that the SJArg-1-Nit results were not due to background contamination and found the extraction blank to be female. Therefore, we concluded that the male proportion of the SJArg-1-Nit was not caused by contamination from laboratory reagents. The analysis also confirmed SJArg-3-Petrous to be XX and SJArg-2-Nit XY, and revealed Dyak-Louse host as XY.The fraction of Y chromosome alignments compared with the total number of alignments to the X and Y chromosome was used to determine the chromosomal sex of each sample  supplem online. We next sought to determine the Y chromosomal haplogroup. Although the coverage for Dyak-Louse and SJArg-1-Nit were found too low for calling the haplotypes, SJArg-2-Nit (Y-coverage 0.038×) was characterized as Q1b1a1a1 or Q-M3 (M848), which today is the largest Native American haplogroup  to investigate the broad population genetic affinities of the ancient individuals a. We merCandidatus Riesia (Enterobacteriaceae), were found with ∼79% and ∼28% of the reads classified, respectively. Ca. Riesia was not found in SJArg-1-Nit, instead, the bacterial class Actinobacteria was found to be most abundant  with more than 50% assigned at the family level Nocardiosaceae or lower; together with the intestinal associated Enterococcus where 5% of the reads were assigned. In the Dyak-Louse, we also identified ∼800 reads (0.7%) assigned to the fruit fly Rhagoletis sp. In the SJArg-2-Nit sample, we found 9% of the reads assigned to the commensal bacterial genus Proteus which holds several opportunistic pathogenic species including P. mirabilis. Interestingly, we also found 115 reads (0.5%) uniquely assigned to the cancerogenic Merkel cell polyomavirus MCPyV , hereby exposed at an ideal temperature for development . This isNotably, nit sample SJArg-2-Nit contains a similar proportion of host human DNA to the tooth, and more than two times what was found in the petrous bone, whereas the proportions of louse DNA were also found to be similar between both nit samples ∼22.5–26.5%. Differences in the total content of DNA between samples can arise from different rates of degradation due to environmental variability, such as temperature, UV light exposure, and humidity. In the four South American samples from Argentina, SJArg-1,2-Nit, SJArg-1-Tooth, and SJArg-3-Petrous, we observed a high variability between the amount of DNA damage , where dWe found the proportion of host DNA from the gut of the Dyak-Louse sample to be lower than that in nit cements. The DNA yield of host DNA from the adult lice was low, especially considering that the endogenous louse DNA content was high (65.5%). There can be multiple reasons for the poor DNA yield. The first reason might be that the bloodmeal is already at an advanced stage of physiological digestion. The second, the conditions in museums which often keep entomological collections at room temperature and occasionally use pesticides . The thiUsing the human mitochondrial information of the nits, we showed that haplotyping can be carried out from minute samples such as six nit sheaths. For both SJArg-1-Nit and SJArg-2-Nit, we found that they belong to the mitochondrial haplogroup A2 (+64), and the same haplogroup was assigned to the tooth and the petrous bone. This is one of the main founder haplogroups of the Americas , and hasIn the latest genetic analyses using current time populations from the same region of this study, Calingasta , b gene network constructed with the Dyak-Louse sample locates these lice as P.humanus capitis from South East Asia, within the haplogroup A, of worldwide distribution and highly prevalent in Asia and Africa. Remarkably, the network for the two ancient nit samples from Argentina, SJArg-1-Nit and SJArg-2-Nit, traces back their ancestry to P. humanus capitis from the Amazon lowlands, the same location found as for the human host DNA: the mitochondrial haplogroup analyses and MDS analysis for SJArg-2-Nit. A pre-Columbian origin of this human louse clade has been proposed and that it may be prevalent in the indigenous American lice populations  and SJArg-2-Nit , respectively.For whole genome-wide studies of the DNA trapped in sheaths’ cement, only six nits from each specimen SJArg-1-Nit and SJArg-2-Nit were available for this work, and the nits cut off the hair. The remaining few nits attached to the hair of the specimens were left untouched for future research. One tooth, SJArg-1-Tooth , originating from the same layer of the rock shelter than SJArg-1-Nit, and one petrous bone, SJArg-3-Petrous  from the same archaeological site were used for whole genome sequencing, and to validate the quality of the DNA obtained for sheath cement. Sample Dyak-Louse corresponds to two adult lice that were pooled for the DNA extraction, and the resulting genetic analyses were also used for comparison with the genome-wide data obtained from the cells trapped in sheath cement. We also checked for the presence of DNA in the fractions at both ends of the sheath attachments on shafts and did not detect any DNA in hair shafts. This was checked using DNA binding dyes and performing DNA extractions that resulted in no DNA. The lack of DNA in shafts can be also visualized in For morphological measures on hair with sheaths, we considered the following specimens  online: Detailed information on the data procedures and statistical analyses of morphometric data  are found in the Permits and clearance for conducting morphological and genetic studies on ancient remains for international transport and for destructive sampling (when/where required) were obtained before the start date  for 2 min whilst shaking in order to fenestrate the vitelline membrane of the nits. They were then washed with cold methanol twice and kept in it for 24 h at 4 °C for fixation. The methanol was replaced with chilled (−20 °C) acetone and left for 1 min. This was followed by double washing with room temperature PBS and incubation for 15 min in PBST (with 1% Triton X-100). PBST was replaced with PBS (3% BSA + 0.1% Tween) and incubated at room temperature for 1 h. Double washing with room temperature PBS was repeated followed by overnight staining with either propidium iodide or DAPI  (10 µg/ml). A final double wash with PBS was carried out followed by mounting in glycerol/PBS . The samples were observed with a Zeiss LSM 710 (inverted) confocal microscope using a 561-nm laser and filter for propidium iodide, and UV laser and filter for DAPI. All images were acquired and analyzed with the associated software Zen 3.1.aDNA work was performed in dedicated clean laboratory facilities at the Lundbeck Foundation Centre for GeoGenetics, GLOBE institute, University of Copenhagen.2, 50 mM DTT, 1% SDS, 0.1 mg Proteinase K), pH 8.0, at 37 °C  was sampled from a single well-preserved molar (SJArg-1-Tooth) and the inner dense material from the petrous bone (SJArg-3-Petrous) and extracted for aDNA following standard protocol . DNA libN” whenever a sampled base gave rise to a triallelic site.After sequencing, all reads were demultiplexed and trimmed using AdapterRemoval v2  and a toWe examined the fragment length distributions and the base substitution patterns using mapDamage  and estiThe broad relationships between ancient and present-day genomes were investigated using MDS applied to the identity-by-state-distance matrix (bammds: 10.1093/bioinformatics/btu410), and PCA, where ancient low-depth data were projected onto the reference data set .Haploid variants were called using the endoCaller program implemented in Schmutzi60 and only variants with a posterior probability exceeding 50 on the PHRED scale  were retained. We then used Haplogrep v2.261 to determine the mtDNA haplogroup, specifying PhyloTree (build 17) as the reference phylogeny. Y-chromosome variant calling and placement were performed following We next performed a metagenomic analysis by taxonomically classifying all reads following the Holi pipeline , excludib and COI from GenBank (accession numbers, We mapped reads that were classified as lice using bowtie2 v.2.3.2  to two cMolecular Biology and Evolution online.msab351_Supplementary_DataClick here for additional data file.


N/dS ratio accounting for the asymmetrical mutational spectrum also shows evidence of negative selection on viral genes, consistent with previous reports. Our findings provide deep insights into the mutational processes in SARS-CoV-2 viral infection and advance the understanding of the history and future evolution of the virus.Viruses accumulate mutations under the influence of natural selection and host–virus interactions. Through a systematic comparison of 351,525 full viral genome sequences collected during the recent COVID-19 pandemic, we reveal the spectrum of SARS-CoV-2 mutations. Unlike those of other viruses, the mutational spectrum of SARS-CoV-2 exhibits extreme asymmetry, with a much higher rate of C>U than U>C substitutions, as well as a higher rate of G>U than U>G substitutions. This suggests directional genome sequence evolution during transmission. The substantial asymmetry and directionality of the mutational spectrum enable pseudotemporal tracing of SARS-CoV-2 without prior information about the root sequence, collection time, and sampling region. This shows that the viral genome sequences collected in Asia are similar to the original genome sequence. Adjusted estimation of the d Sequencing the genetic material of >350,000 samples of the SARS-CoV-2 virus responsible for the COVID-19 pandemic has revealed details of the spectrum of mutations occurring in the recent viral transmission. The analysis was performed by researchers in South Korea and the UK, led by Young Seok Ju at the Korea Advanced Institute for Science and Technology, Daejeon. The results confirm that mutations in the SARS-CoV-2 genome are being shaped in a specific pattern: the nucleic acid bases cytosine and guanine have been replaced by uracil much more often than its reverse. Overall, the genome diversity of SARS-CoV-2 has not yet reached equilibrium, suggesting that the virus entered the human population very recently and is currently evolving rapidly. The findings enhance understanding of the history of the virus and help predict possibilities for its future evolution. Phylogenetically, SARS-CoV-2 belongs to the subgenus Sarbecovirus, a branch of Betacoronavirus in the Coronaviridae family3. Recent comparative studies proposed bats5 and pangolins8 as possible natural reservoirs of the virus.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading rapidly and globally9. Analogous to Darwinian evolution occurring in the origins of species, mutant viral strains gain a driving force when they acquire variations that increase infectivity and survival in the host environment. Therefore, understanding the genome changes of SARS-CoV-2 during the recent outbreak and their proper interpretations are critical for developing preventive, diagnostic, and therapeutic strategies against the virus. Although hundreds of thousands of SARS-CoV-2 genomes have been sequenced11, interpretations of the mutations as a whole have not been conducted.Acquisition of new mutations in viral genomes and natural selection acting on the resultant phenotypic diversity are the two constituent processes of viral evolution14. Unique patterns of mutations provide deep insights into the mechanistic sources of genomic mutations active in the somatic lineages of cancer cells. These approaches also suggest environmental exposure of the cellular lineage, as seen in transmissible cancers15. Here, we apply similar approaches to the catalog of mutations collected from a considerable number of sequenced SARS-CoV-2 genomes . Then, using the spectrum, we trace the origin of SARS-CoV-2 spreading in the human population and speculate on the molecular mechanisms that have shaped SARS-CoV-2 evolution in the recent outbreak and subsequent functional impacts of the mutations.Mutational signature analyses have been widely applied to cancer genomes to understand the mutational processes operative in human somatic cells10) (Supplementary Table Alphacoronavirus), as well as those of several bat coronaviruses, were downloaded from GenBank10 19 as a temporary root sequence, ancestral alleles in the internal nodes were predicted using the ‘ancestral.pml’ function with the ‘type = MPR’ option in the ‘phangorn’ R package. Mutations were called from the edges of the tree only when the mutated base could be unambiguously assigned to the conserved neighborhood, and the two upstream and two downstream bases from the mutated base had to be identical between the nodes being compared. Multiple base substitutions and short indels (<15 bases) were called in a similar way.The sequences of the viruses other than SARS-CoV-2 were aligned using Kalign 3.2.320.For a better understanding of the recurrent substitutions at position 11,083, we randomly selected and downloaded ~30 Illumina short-read sequencing datasets from the SRA database and reviewed them using the Integrative Genomic Viewer22 and represented as base frequency plots using the ‘ggseqlogo’ R package23.For mutational signature analysis, single-base substitutions were classified into 192 subclasses: 4 types of the original bases (4) mutated to the other bases (×3) in the context of immediate upstream (×4) and downstream (×4) bases. In the given set of substitutions, the mutational spectrum was defined by the numbers of each class of substitutions. We performed a subsampling analysis, which revealed that ~200 base substitutions are necessary to reconstruct a stable mutational spectrum . The conditional probability for each configuration of ancestral and descendant contexts was computed based on the substitution rate estimated from the data using the observed counts for each of the 192 contexts. The estimated substitution rates per class of substitutions were directly adopted from the observed mutational spectrum by scaling the spectral numbers to sum to 1. The likelihood of being the root of each node was calculated using the sum of the logarithmic value of substitution rates of all mutations, assuming the node is the root.25. We adopted the concept from previous work by Martincorena et al.26, an application of a 192-context model to the somatic evolution of cancer. We calculated ground neutral mutation rates for one base to another in a certain position by simply dividing the count for each substitution type by the number of corresponding contexts that appeared in the specified region. For a given region in the genome , the expected number of neutral synonymous mutations was calculated by the sum of the probabilities where a base change resulted in a silent mutation. The expected number of nonsynonymous mutations was calculated in a similar way. The dN and dS values were then calculated using the observed and expected numbers of nonsynonymous and silent mutations, respectively. To estimate the confidence interval of dN/dS for each region, bootstrapping was performed by randomly resampling the observed mutations in that region. Conventional maximum likelihood methods with different models and codon frequencies were tried to evaluate natural selection using IQ-TREE software (v2.1.1)27.Variations in the methods used to calculate dN/dS come from different assumptions regarding the expected density of neutral mutations10. These sequences were collected from 82 countries from December 24, 2019, to January 12, 2021  and its reverse F3606L  occurred 1505 times and 49 times, respectively . Interestingly, we found substantial asymmetry in base changes. For example, the rate of the C→U transition was much higher than that of its reverse U→C substitution . Likewise, the rate of G→U transversion was almost ten times higher than that of U→G substitution .CU, ACA, UCA, UCU, and GCU   Fig. .29, and (3) the selective advantage of possessing a certain context due to an underlying mechanism such as codon usage adaptation31. Of these, the RdRP error cannot explain the asymmetry of the mutational spectrum we observed, in particular the imbalance between substitution classes, because both positive- and negative-sense RNAs are equivalently replicated by the polymerase in infected cells  mistakes in viral genome replication by RNA-dependent RNA polymerase (known as RdRP), (2) active damaging processes in RNA molecules caused by host immune systems32. However, there are some missing links between RNA editing and our mutational spectrum. Although our mutational spectrum is quite similar to the pattern of mutations caused by APOBEC1 ([A/U]p[C→U]p[A/U]), the enzyme is exclusively expressed in the small and large intestine, which is not a predominant target organ for this viral infection. Moreover, APOBEC1-mediated RNA editing requires a mooring sequence downstream of the mutant site (WRAUYANUAU 3-10 bases downstream of the target cytosine site)33. However, in the mutations we profiled, no such sequence was found even after allowing two bases of mismatch. In addition, the mutational spectrum of APOBEC3A or APOBEC3B (Up[C→U]pN)35 was not very similar to our spectrum, although the enzymes are known to be expressed in the lung To tackle this problem, we used the directionality of the mutations to trace the root of SARS-CoV-2 without information about the sampling date. Our asymmetric mutational signature directly suggests that viral sequences harboring a higher proportion of C and G are likely to be more ancestral, that is, more similar to the original genomic sequence. To this end, we employed a likelihood approach to statistically evaluate the root from 351,525 sequences . We used a fixed substitution rate matrix proportional to the mutational spectrum, based on the observation that the spectrum (1) is robust to the position of the root .We investigated whether spectrum asymmetry is also present in long-term fixation in close relatives of SARS-CoV-2, such as bat coronaviruses (RaTG13 and RmYN02) and a pangolin coronavirus recently sequenced from Guangdong and Guanxi, Chinaons Fig. . Similarn = 12,202; 4.6% of all nonsynonymous mutations). Of these, 7252 (59.4%) coincided with the trinucleotide context signature, occurring by GCU→GUU codon change. Similarly, of the 17,138 threonine to isoleucine  changes, 8124 (47.4%) occur by ACU→AUU, congruent with the mutational signature.Of the 227,639 substitutions we identified, 162,221 (58.4%) induce amino acid alterations of the SARS-CoV-2 protein-coding genes. These nonsynonymous mutations were relatively evenly distributed in proportion to the length of the gene and had various ratios of C→U transitions . These models estimate the ratio of nonsynonymous to synonymous mutations under neutral selection by estimating transition and transversion rate parameters and codon frequencies in given sequences. However, similar to base substitution, when strong asymmetry exists in the mutation spectrum, those codon substitution models may not fully capture the underlying substitutional process. The mutational signature would result in bias in the set of codon changes, and the dN/dS calculation will be skewed unless this is corrected. Here, we calculated the dN/dS ratio, taking into account both the asymmetric mutational signature and codon usage (“Methods”47). The adjusted calculation showed that, on average, the overall dN/dS ratio was significantly < 1, indicating that the number of nonsynonymous mutations was lower than that expected under the assumption of neutrality . Seven nonstructural genes, ORF3a, ORF7a, ORF7b, ORF8, ORF9a, ORF9b, and ORF10, by contrast, showed higher dN/dS ratios than other genes, but these values were close to one, suggesting neutral evolution. Collectively, our results indicate that the vast majority of mutations accumulated in the pool of SARS-CoV-2 genomes during transmission would be functionally disadvantageous or neutral.The ratio of nonsynonymous to synonymous substitutions, the dN/dS ratio, is a key metric that has frequently been used for inference of natural selectionity Fig. . Compare51. Such rapid evolution of new viral strains appears to contradict more clock-like evolution, and this shift is caused by a variety of factors, such as host population changes, migration, and vaccination52. The SARS-CoV-2 data contain a full spectrum of mutations that were obtained after the virus was transmitted to its new host. However, many researchers use the most widely used general time-reversible (GTR) model to build phylogenetic trees and evaluate mutation rates and selection without considering whether the evolution of SARS-CoV-2 is clock-like or punctuated53. Our observation clearly shows that SARS-CoV-2 was introduced to the human population very recently and is not at evolutionary equilibrium.Punctuated viral evolution is a frequently proposed concept in which a virus evolves rapidly over short periods of time followed by long periods of no change54. Our results are well aligned with this observation. When zoonotic viruses invade human cells from nonhuman hosts, the human cells are not optimized to provide ideal growth conditions, and the virus base composition is not at equilibrium. In these circumstances, such directional changes in viral genome sequences are possible.The C→U and G→U asymmetry in the SARS-CoV-2 and MERS-CoV mutation spectra may be a characteristic of zoonotic RNA viruses recently introduced to human tissues. Previously, linear changes in the base composition over the time of spread were observed in Ebola and influenza viruses57 or to induce active translational selection itself31. Because viruses share a tRNA pool with their hosts, codon usage patterns can evolve to become similar to maximize translational efficiency30, or conversely, when the patterns are too similar, they can become dissimilar to avoid competition31. The codon usage profile of SARS-CoV-2 is known to be more similar to that of Bungarus multicinctus (snake) or Rhinolophus sinicus (bat) and not similar to that of humans, Manis javanica (pangolin)58, and Camelus dromedarius 59. The same mutational spectrum in the noncoding region strongly implies that the mutational spectrum overall is not due to selective pressure on codon usage.Codon composition is one of the extensively studied genomic properties of many viruses. The codon usage profile of a virus is thought to be passively formed by the mutational pressure exerted on the virus62) is a possible method for estimating the root based on the asymmetry of substitutions. However, the UNREST model assumes stationary homogeneous base composition over the time of evolution. This stationarity assumption is frequently reported to be violated in empirical observations, such as continuous decay of the CpG content due to spontaneous cytosine deamination64. There are Markov-based approaches that allow a nonstationary nonhomogeneous time-irreversible nature66. However, it would be extremely difficult to apply those methods to our data  because of the requirements for large memory and long computing time. Overall, our findings demonstrate the utility of the asymmetric mutational spectrum in searching for the root sequence without imposing information about the sampling date or region43.The Markov model-based unrestricted model (UNREST modelOur observation of U→C-dominant mutations in the evolutionary tree of SARS-CoV-2-related viruses suggests differences in the evolutionary characteristics of long-term fixation and those of accelerated (punctuated) evolution. In other words, the genetic diversity of human SARS-CoV-2 has a different mechanistic property from the high divergence of SARS-CoV-2-related viruses of natural origin. Although there is missing information between human SARS-CoV-2 and its natural relatives, making assertions difficult, the predominant C,G→U mutations in human SARS-CoV-2 infections are due to the recent host shift and act as a driving force of the punctuated equilibrium that accelerates viral evolution.67. Here, we account for the context of mutations to estimate dN/dS. Our approach has two assumptions. One is that the observed mutation count per context class is sufficiently robust to use the crude count ratio as the actual expected mutation ratio. The other assumption is that the context-specific mutational spectrum is mainly shaped by mutational pressure but not by selection. Further studies aiming to reveal the biochemical mechanism of the C→U- and G→U-dominant mutations in SARS-CoV-2 are required to resolve these assumptions, which will require further validation in functional studies. The context-adjusted estimate of dN/dS is generally in good agreement with previous reports. For example, negative selection is observed for most genes. The diversifying selection on ORF3 and ORF8 is more obvious with our context-adjusted method than with conventional models69. Among the nonstructural protein groups, Nsp7 showed the lowest dN/dS with the context-adjusted method. It seems to be under the strongest negative selection and thus could be used as a target for antiviral therapeutics70 or vaccine development74.Variations from estimating the dN/dS ratios have been developed to evaluate natural selection on coding sequences. These variations include different ways to estimate nonsynonymous/synonymous mutation counts under the neutral selection assumption. This depends on the belief of how mutations occur and how four bases occupy different codon positions in a given coding sequenceIn summary, we investigated the mutational signature during the global spread of SARS-CoV-2 using 351,525 genome sequences. A highly asymmetric spectrum was observed, with an exceptionally high proportion of C→U substitutions enriched in a few sequence contexts. We speculate that SARS-CoV-2 is currently in a rapid evolutionary stage prior to reaching static equilibrium. Tracing the outbreak’s origin by utilizing the directional substitution pattern indicates that the sequences collected early in Asia are more similar to the original sequence than other sequences are. Despite many nonsynonymous mutations, we observed that the viral genomes were under negative selection.Supplementary InformationSupplementary Tables 1-4


Vaccination is one of the most effective ways of controlling the COVID-19 pandemic. However, vaccine hesitancy is prevalent, and relatively few studies have explored how variables related to personal and external motives have affected the intention to vaccinate. The present study investigated the association between perceived personal benefits, variables reflecting external motives  and intention to receive COVID-19 vaccination among university students in China. The interaction between perceived personal benefits and the three factors reflecting external motives on intention to receive COVID-19 vaccination was also examined. A total of 6922 university students from five provinces of China completed a cross-sectional survey. Results showed that adjusting for significant background variables, perceived personal benefits, perceived social benefits, collectivism, and national pride were all significant factors of intention to receive COVID-19 vaccination. Results from interaction analyses also showed that the association between perceived personal benefits and COVID-19 vaccination intention was stronger among those with lower levels of national pride. Findings highlighted the important role of self-directed interest and external motives in promoting uptake of COVID-19 vaccination. COVID-19 has caused significant morbidity and severe threats to public health across the globe. As of 2 August 2021, 120,837 COVID-19 cases have been reported in China, resulting in 5637 deaths ,5. For iPrevious studies investigating factors of COVID-19 vaccination intention primarily focused on socio-demographics , cognitive factors  and psychosocial factors  ,9,10. AmIn addition to the self-directed motive of perceived personal benefit, this study examined the associations between some external factors and COVID-19 vaccination intention, including the others-directed motive of perceived social benefits, and two other socio-cultural factors related to social capital . COVID-19 vaccination can be viewed as a prosocial behavior, as it prevents transmissions to others and would contribute to herd immunity . While pCollectivism was also tested in the present study as a potential factor of COVID-19 vaccination intention. A previous study found that collective responsibility was positively associated with COVID-19 vaccination intention . In contThe relationships between national pride and COVID-19 vaccination intention and behavior were also tested in the present study for the first time. Pride is an important positive emotion that plays a critical role in psychosocial well-being . In genePerceived personal self-benefits and the three ‘external factors’  are thus potentially independent, yet inter-related driving forces behind COVID-19 vaccination. Their relative importance and emphasis have strong bearing on health promotion strategies. No study has looked at the inter-relationships among these variables and COVID-19 vaccination intention. It is hypothesized that the associations between perceived social benefits/collectivism/national pride and COVID-19 vaccination intention would be independent from that between perceived personal benefit and vaccination intention. Thus, the significance between the three external factors and vaccination intention would become non-significant after controlling for perceived personal benefit. It is further hypothesized that, although individuals are primarily driven by perceived personal benefit, perceived personal benefit would be moderated by the three ‘external variables’  in affecting vaccination intention. It is contended that the association between perceived personal benefits and vaccination intention might be weaker among those with high levels of perceived social benefits, collectivism, and national pride, as those people with high levels of ‘external factors’ might be less driven by self-interest when considering COVID-19 vaccination.Given the background, the present study investigated factors of COVID-19 vaccination intention  among university students in China. The studied factors included perceived personal benefits , and three ‘external factors’ related to others-directed motive and social capital . Second, it tested whether the three ‘external factors’ variables would be significantly associated with COVID-19 vaccination intention after adjusted for perceived personal benefit. Third, the interactions between perceived personal benefits and three ‘external motives’ variables were examined.An anonymous cross-sectional survey was conducted during 1 to 28 November 2020 among university students in five Chinese provinces (Zhejiang (East China), Yunnan (Southeast), Guangdong (South), Inner Mongolia (North), and Henan ) via an online survey link. A total of 165 classes of various grades  mainly within the faculties of arts, sciences, social sciences, economics or management, engineering and medicine or pharmacy of the 5 participating universities were selected by convenience sampling. The collaborating teachers and student helpers sent an invitation message, the online survey link, and several reminders to all the students in the selected classes via WeChat groups that were being used for class administration. The inclusion criteria of participants included being a full-time student at the selected universities and able to read and write Chinese. The questionnaire was self-administered and took about 10–15 min to complete. It was written on the invitation message and the online questionnaire that the participation was anonymous, voluntary and confidential, and the return of the completed questionnaire implied informed consent. Upon completion, the participants could join a lottery draw which offered eight prizes of 10–50 RMB (about 1.5–7.5 USD) and a symbolic “lucky money” of 1 RMB for half of the participants in each participating university. Ethical approval was obtained from the ethics committee of the corresponding author’s affiliated association.A total of 9593 invitations were sent out; 6940 students returned the completed questionnaires (a response rate of 72.3%), 18 of which were excluded due to incomplete data. Thus, 6922 participants were included in the final data analysis.Background information was collected, including the studied province, gender, ethnicity, faculty, and grade. Their perceived risk of COVID-19 was measured using a single item “If not taking up COVID-19 vaccination, what is the chance that you would contract COVID-19 in the future one year? (1 = extremely low to 5 = extremely high)”.Two items were used to examine behavioral intention of COVID-19 vaccination. Participants were asked to rate their chance of taking up free COVID-19 vaccination with common mild side effects and 80% and 50% efficacy in preventing COVID-19 infection, respectively, within the first six months upon the vaccines’ availability (likely/definitely yes versus likely/definitely not).Three items assessed the level of agreement with the following personal benefits in taking up COVID-19 vaccination: (i) self-protection, (ii) less frequent facemask wearing, and (iii) restoration of normal life. The items were rated on a 5-point Likert Scale from 1 = strongly disagree to 5 = strongly agree. The Cronbach’s alpha was 0.71 in this study.Two item assessed participants’ level of agreement with the following social benefits of taking up COVID-19 vaccination: (i) effectively protect others from contracting COVID-19 via contacting you, and (ii) help the country to contain the COVID-19 pandemic and prevent another round of COVID-19 outbreak in China. The items were rated on a 5-point Likert Scale from 1 = strongly disagree to 5 = strongly agree. The Cronbach’s alpha was 0.80 in this study.Collectivism was assessed by the collectivism subscale of the 26-item Five-Dimensional Scale of Individual Cultural Values, which has been validated in among U.S. and Korean university students . Two indNational pride was assessed by using the four items modified from the 2013 ISSP  National Identity III questionnaire, which has been used in 33 countries  and applp-value < 0.05.The four independent variables  were standardized to facilitate the comparisons of effect size in the subsequent analysis. Univariate and multivariate (with adjustment of background factors) logistic regression analysis was conducted to test the individual associations between the four independent variables and the two dependent variables . Crude odds ratios (ORc) and adjusted odds ratios (ORa) and their respective 95% confidence intervals (CIs) were derived. Moderation analyses were conducted to test the significance of the interaction effects of the three interaction terms  on the two outcomes, after adjusting for background factors. Data analysis was conducted by using SPSS 21.0 . Statistical significance was defined as two-tailed Of all the participants, about 40% were male (36.4%) and first-year students (43.2%). Half were studying medicine and health subjects (50.9%). The majority were Han ethnic (86.8%). About one-tenth (9.0%) perceived high risk of contracting COVID-19 in the future one year if not taking up COVID-19 vaccination see .Of the participants, 37.3% showed behavioral intention of receiving free COVID-19 vaccination of 80% efficacy and common mild effects within the first six months upon vaccines’ availability. The prevalence dropped to 19.8% if the efficacy were only 50% see .Those of two particular provinces , male sex, non-Han ethnicity, major in medicine and self-perceived high/extremely high risk of contracting COVID-19 were positively associated with behavioral intention of receiving free COVD-19 vaccination with common mild side effects and 80% and 50% efficacy. The associations between grade and the two outcomes were non-significant see .In In Model 1a,c,e of p < 0.001) than among those with higher levels .In Model 1b,d,f of To summarize, the study found prevalence of COVID-19 vaccination intention of about 37% if the efficacy of the vaccine were 80%, and as expected, dropped to about 19.8% if the efficacy was only 50%. The eventual vaccination rate may differ as health promotion, incentives, and policies would be introduced. As females, non-Hans (minorities), and those perceived lower risk of infection showed lower intention, health promotion may need to pay more attention to these subgroups. As seen from this study, the ‘external factors’ such as perceived social benefits, collectivism, and national pride were significantly associated with vaccination intention after adjusted for the background variables. Furthermore, the ‘external factors’ variables remained statistically significant in the 80% vaccine efficacy scenario after controlled for perceived personal benefits and the background variables. In addition, perceived personal benefits interacted significantly with national pride, i.e., the association between personal benefits and vaccination intention was stronger among those with lower national pride in the 80% efficacy scenario.In the present study, corroborating extant literature ,15, percNevertheless, at the time of a social crisis such as the COVID-19 pandemic, prosocial behaviors have emerged and short-term self-interest is often sacrificed for longer-term collective interests . Health This is the first study reporting a significant association between collectivism and COVID-19 vaccination. There are some plausible explanations. First, collectivists may tend to take the interest of others into account, they would be more likely to cooperate and show concerns with collective actions that would benefit to the group (COVID-19 vaccination in this case). Second, as they are more sensitive to others’ views, they are more likely to observe and adopt the social norm about a behavior, which is an important factor of behavioral intention and performance ,40. In tAnother novel finding was the association between national pride and COVID-19 vaccination intention after adjusting for the background factors and personal perceived benefits (given 80% vaccine efficacy). Dorfman et al.  found thInterestingly, the present study observes that the association between perceived personal benefits and COVID-19 vaccination intention would be stronger among those with low level of national pride. It seems that the decision to receive COVID-19 vaccination among those with lower national pride are more driven by the self-interest of perceived benefits of COVID-19 vaccination. However, the interactions between perceived personal benefits and perceived social benefits/collectivism were non-significant. The understanding of the inter-relationships between perceived personal benefits and the ‘external motives’ variables are thus only preliminary and requires future investigations.Our findings yield important theoretical and practical implications. First, it highlights the importance of emphasizing both personal and social benefits of COVID-19 vaccination when promoting COVID-19 vaccination intention. Messages that depict both the efficacy of vaccinations in protecting individuals from infection and promoting individual health and well-being and the benefits of herd immunity for other people  may be more effective than those mentioning personal benefits alone. In the current case of COVID-19 of which reaching herd immunity thresholds is crucial for controlling the pandemic, it may be particularly important to promote the awareness of the social benefits that vaccination is not only an individual but also a collective effort. Findings of the current study also provide promising evidence that collectivism contribute to higher intention to receive COVID-19 vaccination that may bring large benefits to the community. The current study extends previous work and highlights that collectivism would also contribute to intention to receive COVID-19 vaccination. To promote the uptake of COVID-19 vaccination, there is a need to promote a collectivistic orientation among the general population. Messages that emphasize social harmony, interpersonal relationship, and the importance of meeting social obligations and common goal can be useful techniques to encourage the population to receive COVID-19 vaccination. Future studies comparing levels of collectivism and national pride and COVID-19 vaccination coverage may test the contention that variations in such social capital would partially explain differences in national coverage rates.The study involved a large sample and was timely. It, however, had several limitations. First, the study was cross-sectional in nature so causality could not be assumed. Data was collected from five provinces, and the sample contained a relatively large number of first year students and those from the medicine major. Therefore, findings may not be representative to the whole university student population in China. Participants were self-selected so the data might have been subject to self-selection bias. As COVID-19 vaccination was not yet available during the time of the study, participants were asked to rate their intention to receive COVID-19 vaccination within six months upon its availability. The situations  keep changing so that the obtained information on vaccination intention might be different from those of actual performance of vaccination. University students may also be very different from the general population and generalization may be limited. Future studies should include those with lower socioeconomic status, such as older populations, those living in rural areas, or individuals with a lower level of education and test the applicability of perceived benefits, collectivism, and national pride in these populations. Furthermore, some scales were developed for this study as they were not available in the literature, and other potential factors, such as perceived susceptibility and perceived severity of COVID, and history of COVID-19 infection, have not been investigated. University students may have a lower level of perceived susceptibility and severity of COVID-19 due to their relatively young age. The role of these factors on their vaccination intention might be different compared to the older populations or those with chronic diseases. Last, clustering within the schools have not been taken into account; multi-level analysis or meta-analytic approach might improve accuracyIt is doubtless that the success in reducing COVID-19 transmissions relies on people voluntarily adopting vaccination. Identifying the factors that may potentially promote intention to receive COVID-19 would allow health care professionals to develop evidence-based strategies to increase national COVID-19 vaccination coverages. Overall, the present study provides encouraging insights that emphasizing both individual and social benefits, and collectivism and national pride may be effective at increasing COVID-19 vaccination, therefore reducing the burden of the disease. If confirmed by future studies, tailored messages should be designed based on individual’s perceived benefits in order to maximize the effects of health interventions. The understanding of the roles of self-directed interest versus others-directed interest, related personal traits and social capitals  is an important possible new direction for research and health promotion of COVID-19 vaccination.


There is limited knowledge on how the SARS-CoV-2 affects pregnancy outcomes. Studies investigating the impact of COVID-19 in early pregnancy are scarce and information on long-term follow-up is lacking.The purpose of this project is to study the impact of COVID-19 on pregnancy outcomes and long-term maternal and child health by: (1) establishing a database and biobank from pregnant women with COVID-19 and presumably non-infected women and their infants and (2) examining how women and their partners experience pregnancy, childbirth and early parenthood in the COVID-19 pandemic.This is a national, multicentre, prospective cohort study involving 27 Swedish maternity units accounting for over 86 000 deliveries/year. Pregnant women are included when they: (1) test positive for SARS-CoV-2 (COVID-19 group) or (2) are non-infected and seek healthcare at one of their routine antenatal visits (screening group). Blood, as well as other biological samples, are collected at different time points during and after pregnancy. Child health up to 4 years of age and parent experience of pregnancy, delivery, early parenthood, healthcare and society in general will be examined using web-based questionnaires based on validated instruments. Short- and long-term health outcomes will be collected from Swedish health registers and the parents’ experiences will be studied by performing qualitative interviews.Confidentiality aspects such as data encryption and storage comply with the General Data Protection Regulation and with ethical committee requirements. This study has been granted national ethical approval by the Swedish Ethical Review Authority  and national biobank approval by the Biobank Väst (dnr B2000526:970). Results from the project will be published in peer-reviewed journals.NCT04433364. The COVID-19 in Pregnancy and Early Childhood (COPE) study is a unique linkage between the Swedish Pregnancy Register, the Swedish Neonatal Quality Register, the Hospital Integrated Biobank Sweden and patient-reported outcomes through web-based questionnaires enabling both short-term and long-term follow-up of pregnant women, their partners and children during the COVID-19 pandemic.Prospective and automated collection of healthcare data in the comprehensive Swedish Pregnancy Register and Swedish Neonatal Quality Register covering more than 98% of all deliveries in Sweden ensures high-quality data.Logistics provided by Hospital Integrated Biobank Sweden enable high-quality biological sampling at several time points during pregnancy according to standardised protocols. However, due to resource limitations at the hospitals during the pandemic, some women will not have complete samples from all time points of interest.Based on validated instruments, child health and development during the first 4 years of life will be reported by parents along with comprehensive register-based long-term follow-up. There is a risk of selection bias regarding the follow-up questionnaires where we expect that a proportion of the study population will not answer the questionnaires.Other limitations include self-selection bias as women need to give consent to participate, and a prerequisite for participation in the interview part of the study is adequate Swedish or English language skills.The emergence of a new coronavirus was brought to WHO’s attention on 31 December 2019. Within weeks, a global health emergency ensued and COVID-19 was declared a pandemic by the WHO. There was an urgent need to identify and protect vulnerable populations within the society and from the knowledge gained from the previous human coronavirus outbreaks of SARS-CoV-1 and Middle East respiratory syndrome CoV, it was clear that pregnant women and their fetuses may be particularly at risk for poor outcomes.Recent reviews have found that pregnant women are more likely to need intensive care treatment related to COVID-19 as compared with non-pregnant women.et alet alTransplacental transmission of SARS-CoV-2 remains a topic of much debateCOVID-19 during pregnancy can have a profound impact on how a woman and her partner experience pregnancy, childbirth and early parenthood. The many unknowns connected to the virus have the potential to create anxiety in the pregnant population.Similarly, severe maternal morbidity may aggravate emotional distresshttp://www.biobanksverige.se/). Data retrievement and sampling by SIB and pre-established standardised registers with automatic transfer of data from medical records will not impact the workload of the ordinary hospital staff, and therefore, allow the study to proceed during the pandemic where there are already great constraints and limitations on healthcare resources. With almost universal smartphone usage in Sweden, patient-reported outcomes can be safely and efficiently collected using electronically distributed questionnaires.Antenatal care is offered free of cost for all women in Sweden and follows standardised guidelines.In summary, uniformity in laboratory testing, use of hospital integrated biobanks with standardised protocols, follow-up of child health and parent-reported outcomes through survey data and linkage of data from national registers will improve knowledge on how COVID-19 can affect the mother, partner, fetus and child. Sweden is one of the few countries with preconditions that can enable an almost population-based follow-up on parent and child health despite the considerable strains imposed by the COVID-19 pandemic.The overall aim of this project is to study the impact of SARS-CoV-2 infection on maternal, fetal and child health, as well as experience of pregnancy and parenthood during the COVID-19 pandemic by:Establishing a biobank with biosamples from both pregnant women with COVID-19 and presumably non-infected pregnant women and their infants.Establishing a database of survey-based data linked to Swedish quality and healthcare registers and information from electronic charts for both mother and child.Performing serological, viral and immunological analyses on biobank samples and linking these to maternal, fetal and child outcomes in order to assess short-term and long-term maternal and child health.Collecting prospective data on how women and their partners experience pregnancy, childbirth and early parenthood in the COVID-19 pandemic using validated questionnaires and qualitative interviews.https://www.snaks.se/). Data are collected in four different ways: (1) biosampling, (2) survey-based follow-up until 4 years after delivery, (3) linkage to Swedish health and quality registers enabling long-term follow-up and (4) interviews. All Swedish maternity units with their corresponding neonatal care units have been invited to participate in the study. Centres can participate in the biobank and/or the questionnaire part of the study. So far, 27 maternity units corresponding to more than 87 000 of the approximately 114 000 deliveries per year in Sweden are participating in the study (The COVID-19 in Pregnancy and Early Childhood (COPE) study is an ongoing Swedish multicentre study, facilitated by the Swedish network for national clinical studies in Obstetrics and Gynaecology . A prerequisite for participation in the interview part of the study is adequate Swedish or English language skills.www.copestudien.se), social media, interviews and articles available in mass media along with active recruitment by the local study research team.Participants have access to study information which is freely available in the waiting rooms of the antenatal care and maternity units involved in the study, the COPE study homepage  a ‘COVID-19 group’  and (2) This group includes women that (1) test-positive for SARS-CoV-2 during pregnancy or at delivery, (2) have a positive SARS-CoV-2 antibody test from infection during the current pregnancy or (3) have COVID-19 as a ‘clinical diagnose’ at the time point of delivery before test results are available.Before June 2020, there was limited testing capacity in Sweden and only symptomatic patients admitted to the hospital were tested. Since June–July 2020, testing for SARS-CoV-2 has become widely available, even outside hospitals, to all citizens in Sweden. In the beginning of 2021, SARS-CoV-2 screening was introduced for all patients admitted to Swedish hospitals including pregnant women on admission to maternity units. All adults, including pregnant women, are currently required to take a SARS-CoV-2 test in case of symptoms. Detailed data on the number of performed tests and test-positivity in different regions, age groups and over time are available at the homepage of the Public Health Agency of Sweden.This group consists of women without symptoms and/or with a negative test for SARS-CoV-2 during the current pregnancy. These women are recruited at participating centres during their antenatal care check-up or during their visit to the maternity unit.A woman in the screening group may be included into the COVID-19 group later on during the pregnancy if she contracts COVID-19. This may also be the case at the time of statistical analyses, in case the biobank specimens should indicate an asymptomatic SARS-CoV-2 infection or a positive test result is found registered in the Swedish Register for mandatory registration of notifiable infectious diseases (SmiNet).Samples will be analysed with real-time PCR (RT-PCR) for SARS-CoV-2, serology for SARS-CoV-2 along with other immunological analyses that will be specified according to up-to-date techniques pertinent to SARS-CoV-2 infection.https://biobanksverige.se/english/research/). For women and children included in the study, these samples may be analysed for serology for SARS-CoV-2 in order to define the time point for an asymptomatic SARS-CoV-2 infection. Similarly, in some centres in Sweden, blood samples from routine testing for immunisation at gestational week 28 are stored and may be analysed for serology for SARS-CoV-2. Further, we plan to combine already existing pregnancy biobanks that have biosamples from different gestational weeks , Uppsala/Örebro; GO PROVE, dnr 955-18, Gothenburg; UPMOST, dnr 2019-00309, Uppsala/Örebro and Biobank för gravida kvinnor, Uppsala, dnr 2007-181, Uppsala/Örebro) with the COPE biobank. These already existing biobanks will provide blood samples collected from February 2020 onwards.Pre-existing samples taken as part of routine antenatal care and screening will be obtained from already-existing biobanks along with the newly established COPE biobank. As part of routine antenatal care in Sweden, all women provide a blood sample that is used to screen for hepatitis B, syphilis, rubella and HIV infection in early pregnancy. These samples are stored in Hospital biobanks, according to the Swedish Biobanks Medical Care Act , Neonatal Quality Register (SNQ) and Intensive Care register), registers from the National Board of Health and Welfare, for example, data on long-term child health , Statistics Sweden, for example, data on education and income , Public Health Agency, data on time point for testing positive for SARS-CoV-2 , growth data during childhood  and medical records for additional data, for example, cardiotocography during delivery. The registers are described in detail in Box 1http://www.graviditetsregistret.se/).A certified national quality register initiated by the Swedish healthcare regions that combines prospectively collected data from the Swedish Maternal Health Care Register, the Swedish National Quality Register for Prenatal Diagnosis and data from electronic, standardised, prenatal delivery and neonatal records. The register includes more than 95% of all deliveries in Sweden and covers pregnancies from the first antenatal care visit until the follow-up visit at 8–12 weeks post partum. It contains information on maternal characteristics, medical and reproductive history, pregnancy examinations, delivery outcomes and follow-upExamples of variables that will be extracted: pregnancy loss after first visit to antenatal care, body mass index at booking visit, weight gain during pregnancy, gestational age at delivery, mode of delivery, postpartum blood loss, birth weight, Apgar score and pregnancy complications such as gestational hypertension, pre-eclampsia and gestational diabetes.www.snq.se).A national quality register for neonatal care. All neonatal departments in Sweden report standardised data on admitted infants including basic information about pregnancy and childbirth, as well as the condition, treatment and diagnoses of the infant according to the Swedish version of International Classification of Diseases 10th revision (ICD-10), as well as information from follow-up visits. During the COVID-19 pandemic, all children born to mothers testing positive for SARS-CoV-2 are registered in the Swedish Neonatal Quality RegisterExamples of variables that will be extracted: admission to neonatal intensive care unit (NICU) or neonatal special care (NSC), duration of NICU/NSC stay, mechanical ventilation, asphyxia related complications, hypothermia treatment.http://www.icuregswe.org/).A Swedish quality register on intensive care. Data regarding severity of disease and interventions will be retrieved from Swedish Intensive Care Register for women requiring care at an ICU .A mandatory health register including diagnoses on hospital admissions and outpatient visits in specialist care. Information will be retrieved on ICD-10 diagnoses and interventions for women during pregnancy and the postpartum period, chronic or previous disease in the mother as well long-term follow-up of their childrenExamples of variables that will be extracted: ICD-10 diagnosis of COVID-19 or diagnosis of thromboembolism for the mother during pregnancy as well as 3 months postpartum, diagnosis of neuropsychiatric disease during childhood.The national cause of death register is based on the obligatory death certificates that need to be signed by a medical doctor confirming the cause of death in the deceased. Both the date and cause of death are registered.https://www.socialstyrelsen.se/statistik-och-data/register/alla-register/dodsorsaksregistret/).COVID-19 is classified as a notifiable infectious disease. All positive SARS-CoV-2 tests are reported to SmiNet by the laboratories analysing the tests as well as the medical doctor responsible for sampling (Variables that will be extracted: Date for positive SARS-CoV-2 test or date of COVID-19 disease during pregnancy.http://bhvq.se/).A national quality register on child healthcare .Statistics Sweden is a government agency collecting data on education and income , abdominal, airway and other symptoms , visiting a doctor, prescription of antibiotics or whether the child has been admitted to hospital are noted. The questionnaire has previously been used in two other Swedish studies.At 4, 9 and 12 months (corrected age) along with 2, 3 and 4 years of age, parents report their infant’s development based on the validated Ages and Stages Questionnaire (ASQ)-version IIIInformants will be approximately 12–20 women who were diagnosed with COVID-19 during pregnancy and their partners, as well as 12–20 women and partners who were not diagnosed with COVID-19. Participants will be selected to ensure a broad range of views and experiences, for example, age, parity and socioeconomic background including severity of symptoms in the COVID-19 group. The women and their partners will be interviewed separately using face-to face interviews or by video link or telephone. Open-ended questions with follow-ups will be asked to deepen the understanding.The COPE study is collecting information for a database and biobank in order to study the association of COVID-19 during the pregnancy with a wide variety of different pregnancy, maternal and neonatal outcomes including the long-term follow-up of maternal and child health, as well as parental experience.Predefined research questions concern the incidence of infection and COVID-19 at different time points of the pandemic; the impact of COVID-19, gestational age at infection, severity of disease, viral load, presence of SARS-CoV-2 and/or antibodies against SARS-CoV-2 in different compartments of the mother and/or child, pregnancy outcome, maternal and child health; experience of childbirth and early parenthood during the pandemic  and non-infected women (no COVID-19 during pregnancy). In some analyses/subanalyses, gestational age at infection, severity of disease, viral load and immune response will be considered as additional exposure variables for the infected group.Pregnancy and neonatal outcomes will be retrieved from the SPR and SNQ, either registered as tick boxes, actual measures or International Classification of Diseases 10th revision (ICD-10) codes: Pre-eclampsia (ICD-10 O14), gestational age at birth, preterm delivery , birth weight, small for gestational age, birth asphyxia, and perinatal death.Thromboembolic event diagnosis will be retrieved from the SPR and the National Patient Register .et al.Vertical transmission as defined by Shah Child development and health: Developmental delays or potential delays as measured by ASQ up to 4 years of age. Neurological disorders diagnosed during the first 4 years of life (composite) retrieved from the National Patient Register; Any mental or behavioural disorder (ICD-10 F00–F99), impaired vision (H54), impaired hearing , cerebral palsy and other paralytic syndromes (G80–G83).Presence of antibodies in umbilical cord blood  and breast milk .Subanalyses will be performed to study the impact of country of birth, socioeconomic status or underlying disease in the mother .2 test or Fisher’s exact test. Correlations will be analysed by Pearson’s r or Spearman’s r as appropriate by distribution of the variable. Regression analyses, unadjusted and adjusted, will be performed to adjust for known confounding variables. Ten cases per variable at the lowest will be considered appropriate to avoid overfitting of the model.Demographics will be presented as numbers (percentage), medians or means as appropriate by distribution. Comparisons between groups will be analysed by Student’s t-test or Mann-Whitney U-test with means or medians and confidence intervals or IQR, as appropriate according to distribution of the variables. Categorical variables will be compared by χThis is an exploratory study where the initial sample size for recruitment is set to 200 women in the COVID-19 group and 1000 in the screening group . Sample size has been calculated with 80% power and a significance level of <0.05 based on the assumptions shown in The percentage of women with vertical transmission secondary to active COVID-19 at the time of delivery (14 days before to 2 days after delivery) will be calculated.Depending on the prevalence of malformations, the presence and type of malformations in case of infection during early pregnancy will be presented in a purely descriptive manner.The presence of antibodies in umbilical cord blood and breast milk will be studied in relation to gestational week of infection, severity of infection and maternal serum antibody levels. Antibody levels will be followed over time.Due to the unprecedented nature of the pandemic, it is difficult to perform formal power calculation with regard to parent health and mental well-being.A pregnant patient representative, her partner and a patient organisation namely, the Swedish Association for Premature Infants (Svenska prematurförbundet) were invited to participate in the early stages of planning the study. Several video meetings were held between a research group representative (KL) and the patient representative and her partner where the research questions and outcomes were discussed. Participant time investment in the study was discussed and adjustments in the questionnaires were made according to their feedback. The opinion and feedback obtained from the non-pregnant parent were also given special importance.www.copestudien.se) was designed in collaboration with the patient representative and provides a convenient platform for participants to connect with the research team. Communication with the wider pregnant population and the public through social and mass media has enabled uptake of patient-evoked research questions and led to appropriate modifications in the study protocol.The study website (www.medscinet.com), an international company specialising in web applications in the field of academic medicine. Biobank samples will be identified using the personal identification numbers of the patients included in the study and pseudonymised after identification in the biobank before laboratory analyses.COPE is a comprehensive cohort study involving the use of register based data, biological sampling, questionnaires and interviews. The study has received national ethical approval by the Ethics Review Board, Lund, Sweden  and national biobank approval at Biobank Väst (dnr B2000526:970). Confidentiality aspects such as data encryption and storage comply with the general data protection regulation. All data are stored in a secure online database provided by MedSciNet  while others are study specific . Sampling will be performed by experienced nurses and the application of a topical anaesthetic patch will help minimise pain for the child.Results from this study will be presented at different national and international conferences, in peer-reviewed journals and in mass media. Wide-spread public interest in COVID-19-related research will help facilitate study dissemination and participation from all major Swedish maternity units.Knowledge from previous coronavirus outbreaksLocal and regional differences in the testing and management of pregnant women with SARS-CoV-2, make the results of published studies difficult to interpret. Many studies do not report the gestational age at infection, do not include a control group, recruit women only when they seek healthcare for obstetric complications or on admission for delivery, do not specify the indication for an obstetric intervention (intervention with or due to COVID-19) or are not generalisable since they are based on a single site with a population having a certain risk profile regarding, for example, socioeconomic status or pregnancy complications.Few studies have focused on women’s experience of pregnancy, childbirth and early parenthood during the COVID-19 pandemic and the results are often context specific, making them difficult to generalise. Partner experience has been largely overlookedHowever, the study design has certain limitations. Although the majority of all maternity units are participating, the study will not be completely representative of the total population. Since women and partners need to actively consent to participate, there will be self-selection bias. Additionally, the questionnaire and interview parts of the study require adequate language skills as described earlier. During the 4-year follow-up period, a certain degree of ‘drop-out’ is expected in the questionnaire part of the study. With regard to the biobank part of the study, we expect maternity units to show considerable variation in retrieving a full list of samples as the healthcare sector is under enormous pressure due to the pandemic.To summarise, the COPE study will help lay the foundations for understanding the society’s ability to protect one of its most vulnerable groups, pregnant women and their children.


Large US colleges and universities that re-opened campuses in the fall of 2020 and the spring of 2021 experienced high per capita rates of COVID-19. Returns to campus were controversial because they posed a potential risk to surrounding communities. A large university in Pennsylvania that returned to in-person instruction for Fall 2020 and Spring 2021 semesters reported high incidence of COVID-19 among students. However, the co-located non-student resident population in the county experienced fewer COVID-19 cases per capita than reported in neighboring counties. Activity patterns from mobile devices indicate that the non-student resident population near the university restricted their movements during the pandemic more than residents of neighboring counties. Respiratory virus prevention and management in student and non-student populations requires different, specifically targeted strategies. To return to campus operations in the fall of 2020, some universities developed COVID-19 policies including frequent testing, mask mandates, distancing in classrooms, and hybrid online and in-person instruction plans3. Other universities pivoted to fully remote learning and did not return to campus4. The decision to re-open campuses was controversial because high density student populations would create high-risk transmission situations for students who returned to campus and have negative impacts on their health. Further, outbreaks in students populations had the potential to increase incidence in surrounding communities6. While individual students could make informed decisions about whether to return to campus based on their risk tolerance, town residents were forced to accept a changing level of local risk based on the decisions of others.Following the World Health Organization's (WHO) March 2020 official declaration of the COVID-19 pandemic, caused by the novel SARS-CoV-2 virus7. The return to campus quickly resulted in a large COVID-19 outbreak among students8. We assessed the COVID-19 outbreak in non-student residents in Centre County for comparison to the outbreaks in the surrounding counties, where large universities are not present. We show that that the timing of the increase in cases among non-student residents of Centre County during the Fall 2020 term lagged the increase in the student population and that the per capita cases among non-student residents of Centre County were lower than in surrounding counties.In Centre County, Pennsylvania, The Pennsylvania State University’s (PSU) University Park (UP) campus returned to campus operations in August 2020. The university offered students options for virtual, hybrid learning, and in-person classes with physical distancing in classrooms. Unofficially, the university estimates that about 30,000 undergraduate students returned to the county for the Fall 2020 term, down from approximately 40,000 in the Fall 2019 term due to the pandemic12. We compare movement and activity patterns derived from mobile devices for census block groups within Centre County where students make up more than half the residents, to non-student dominated census block groups in Centre County, and to the neighboring counties.In many settings, movement, as measured by mobile devices, is an early indicator of contacts that contribute to COVID-19 transmission and spatial spread; movement is a common target of behavioral interventionshttps://www.health.pa.gov/topics/disease/coronavirus/pages/cases.aspx)14 for Centre County and the six neighboring counties with which it shares borders: Blair, Clearfield, Clinton, Huntingdon, Mifflin, and Union  (https://virusinfo.psu.edu/covid-19-dashboard/)8. Two types of testing were conducted: students who were enrolled in on-campus classes were randomly selected for surveillance testing and all students could use on-demand testing. Through March 23, 2021, a total of 45,092 random tests were conducted for surveillance, of which 462, or 1.0%, were infected. Surveillance testing efforts ranged from 2440 to 4020 weekly tests through the Fall 2020 semester and were designed to consistently test approximately 1% of students throughout the school year.Within Centre County, PSU provided COVID-19 testing for UP students from August 7, 2020 onward and reported anonymized weekly (2020) and daily (2021) confirmed cases, negative test results, and total tests completed for each campus in a public dashboard Figs.   from the Centre County estimates provided by PA DOH. However, not all student tests were reported to DOH. A portion of the on-demand tests conducted for PSU UP students were completed by a third-party vendor, which required student registration. At the time of student registration, an estimated 0–25% of students registered with an address for a family home that did not reflect their residence in Centre County. Their test results were reported to the county of their registered address. This impacts a maximum of 1,166 positive student test results and 10,760 negative student tests.We conducted a sensitivity analysis to assess the uncertainty in reporting around the negative and positive students tests that may have been misallocated due to the reported residence of student tests. We have calculated the minimum and maximum number of affected positive and negative student tests. This uncertainty from student tests impacts non-student values, which are calculated by subtracting student values from county level reports. The calculations are based on a range of a possible 0–1166 positive student tests misallocated to other counties and up to 10,760 misallocated negative student tests. We have used the ranges of misallocated student tests to calculate, for non-student Centre County residents, the full possible range of (1) total cases, (2) reported cases per capita, and (3) tests per capita  for all seven previously mentioned counties in central PA15.We acquired county-level data on median household income, population size, and college enrollment status from the 2019 United States Census Bureau’s American Community Survey (ACS) 5-year data (We divide the census block groups (CBG) of Centre County into two categories. We first designated ‘student-dominated CBGs’ as CBGs where > 50% of ACS responses report enrollment as undergraduate students. We consider data from the 19 student-dominated CBGs in Centre County to be representative of the student population in Centre County. In addition to off-campus locations, the 19 student-dominated CBGs include all on-campus dorms. These 19 CBGs are either on or adjacent to PSU’s UP campus and occupy exactly 6 census tracts. The remaining 25 county census tracts were designated as non-student dominated areas.16 receives geolocation data from anonymized mobile devices collected from numerous applications. We analyzed SafeGraph’s mobile device-derived daily visit counts to points of interest (POI), which are fixed locations, such as businesses or attractions. SafeGraph data provide daily counts for total numbers of visits by mobile devices while using at least one application that provides geolocation data to SafeGraph. A “visit” indicates that the device entered the building or spatial perimeter designated as a POI. We acquired daily visit counts for POIs in the seven previously mentioned counties in central PA from January 1, 2019 forward . SafeGraph’s 2019 mean count of mobile devices residing in each county represents 4% (range 3.4–4.9%) of the 2019 census population of each county. The Pearson correlation between the mean count of devices residing in a county and 2019 ACS population size of the county was 0.99 for the seven counties . Nearly half of the reported cases from Centre County occurred in university students. The outbreak in students was asynchronous with the outbreak in non-students in Centre County and led the reported cases in the non-student residents of Centre County. Other studies of university populations also noted little genomic evidence for spread between student populations with high incidence and surrounding non-student communitiesTesting per capita was highest for university students, which may have contributed to higher case detection in students. PA DOH reports showed testing totals were also high for all of Centre County compared to neighboring counties. While we can’t precisely calculate the testing per capita for non-students in Centre County, the number of COVID-19 deaths per capita would not be affected by testing rates. Centre County non-students experienced 162.02 COVID-19 deaths per 100,000, which falls below the mean of 219.77 for these seven counties. This supports the assertion that Centre County non-student residents also experienced fewer cases per capita than the surrounding counties and the low number of cases per capita was not an artifact caused by low testing rates.23. The mobility data used here do not reflect differences in duration or strength of contacts in these settings . Mobile-device derived data from SafeGraph showed widespread declines in the number of visits to points of interest (POI) from 2019 to 2020. Centre County recorded greater declines in visit counts from 2019 to 2020 than the surrounding counties. During the pandemic, individuals residing in Centre County spent fewer minutes per day outside their homes during university academic terms than they did for the same time period prior to the pandemic. The decline in minutes spent outside the home in Centre County was greater than the decline in the neighboring counties during the pandemic. Relative to other counties, Centre County non-student residents showed greater declines in time spent outside the home than the number of daily trips to POI, although both decreased drastically when comparing pre-pandemic measures to post-SARS-CoV-2 emergence measures. This may indicate that short trips were less affected by pandemic restrictions while long trips outside the home were preferentially reduced, such as replacing day-long trips to a place of employment with working from home.Mobile device data measure the relative amount of movement between locations and serve as a proxy for contacts and interactions among individuals25. Slightly higher median household incomes may have contributed to Centre County residents’ abilities to stay at home during the pandemic more than the residents of neighboring counties26. Centre County residents may have also disproportionately adopted suggested behavioral interventions, including staying home, in reaction to the large outbreak in Centre County in university students. Ultimately, PSU’s return to campus operations did not cause excess COVID-19 cases per capita in non-student residents around the university but did create a high risk of infection for students who returned to campus.Despite high incidence of COVID-19 among students in Centre County, the delayed outbreak progression in non-student residents of Centre County is consistent with the outbreak timing in neighboring counties, which do not contain large universities. Self-imposed stay-at-home behaviors among Centre County residents may have limited transmission between students and non-student residents of Centre County and slowed transmission among non-student residents27. We show that on average, Centre County residents spent less time outside the home during the pandemic than the residents of the surrounding six counties. The proportion of Centre County non-student residents above 65 years of age, the most vulnerable group early in the outbreak, is lower than in the surrounding counties, but comparable    Table 15. Compa%) Table 15. SenioSupplementary Information.


Commercially available wearable (ambulatory) pulse oximeters have been recommended as a method for managing patients at risk of physiological deterioration, such as active patients with COVID-19 disease receiving care in hospital isolation rooms; however, their reliability in usual hospital settings is not known.2).We report the performance of wearable pulse oximeters in a simulated clinical setting when challenged by motion and low levels of arterial blood oxygen saturation  estimates were compared with simultaneous SaO2 samples to calculate the root-mean-square error (RMSE). The area under the receiver operating characteristic curve was used to analyze the detection of hypoxemia .The performance of 1 wrist-worn (Wavelet) and 3 finger-worn  wearable, wireless transmission–mode pulse oximeters was evaluated. For this, 7 motion tasks were performed: at rest, sit-to-stand, tapping, rubbing, drinking, turning pages, and using a tablet. Hypoxia exposure followed, in which inspired gases were adjusted to achieve decreasing SaO2 estimates matching 215 SaO2 samples in both study phases, from 33 participants, were analyzed. Tapping, rubbing, turning pages, and using a tablet degraded SpO2 estimation (RMSE>4% for at least 1 device). All finger-worn pulse oximeters detected hypoxemia, with an overall sensitivity of ≥0.87 and specificity of ≥0.80, comparable to that of the Philips MX450 pulse oximeter.SpO2 accuracy of wearable finger-worn pulse oximeters was within that required by the International Organization for Standardization guidelines. Performance was degraded by motion, but all pulse oximeters could detect hypoxemia. Our findings support the use of wearable, wireless transmission–mode pulse oximeters to detect the onset of clinical deterioration in hospital settings.The SpOISRCTN Registry 61535692; http://www.isrctn.com/ISRCTN61535692RR2-10.1136/bmjopen-2019-034404 The remaining sensitivity and specificity values were comparable.Several studies have been published on both the usefulness and the potential issues of pulse oximetry in the clinical setting using nonambulatory devices. In this study, we compared the performance of wearable pulse oximeters and 1 nonambulatory pulse oximeter using gold-standard arterial blood samples drawn from healthy adult participants. Availability of waveform data was a requirement that limited the selection of devices. Our provision of waveform data will allow clinical staff to assess the reliability of the signal. However, a risk with all continuous-monitoring systems is that they increase the burden on clinical teams by providing excess data. Further work is required to determine the usefulness of these systems in clinical practice and how continuous-monitoring data should be summarized in the electronic patient record.2  at the cost of a lower specificity value . This underestimation explains why recalibration by 2% achieves the optimal operating point. The remaining devices only required a change in the threshold by 1% at their optimal operating point.In tests of finger-based devices, WristOx2 3150 significantly underestimated SaOFrom the 7 motion tasks, tapping, rubbing, turning book pages, and using a tablet challenged the finger-based wearable devices the most , indicating that the SpO2 value might change between the time used to compute the SpO2 estimates and that of the ABG samples taken after the exercise. Our hypothesis was that the STS task would be the motion task with the greatest effect on the participants’ SaO2. However, the error in the SpO2 estimates from the wearable devices during motion was much larger, so this correction would not have changed our findings.We chose to sample ABGs at the end of each task to avoid accidental removal of the cannula. However, it became clear during our study that the ABGs could have been sampled while the motion task was occurring, perhaps better representing that interval reference SaO2 estimation by finger-worn pulse oximeters was within that required by the ISO 80601-2-61:2019 guideline (≤4%). The accuracy was degraded by motion but not more than that with usual-care bedside monitors. All finger-worn pulse oximeters were capable of detecting hypoxemia, their performance being comparable to that used in nonambulatory standard care.The accuracy of SpO2, especially values below 90%, may be helpful to manage the care of ambulatory in-hospital patients who have been infected with the SARS-CoV-2 virus [Our findings support the use of wearable, finger-based, wireless transmission–mode pulse oximeters to detect the onset of clinical deterioration in the hospital, possibly earlier than intermittent vital-sign measurements. The continuous assessment of SpO-2 virus . Further