TASK DEFINITION: In this task, you're given a paragraph from the research paper and your task is to generate a suitable title for the research paper based on the given paper. Under 100 words is a good title length.
PROBLEM: The production of functional nidovirus replication-transcription complexes involves extensive proteolytic processing by virus-encoded proteases. In this study, we characterized the viral main protease (M pro ) of the type species, White bream virus (WBV), of the newly established genus Bafinivirus (order Nidovirales, family Coronaviridae, subfamily Torovirinae). Comparative sequence analysis and mutagenesis data confirmed that the WBV M pro is a picornavirus 3C-like serine protease that uses a Ser-His-Asp catalytic triad embedded in a predicted two-␤-barrel fold, which is extended by a third domain at its C terminus. Bacterially expressed WBV M pro autocatalytically released itself from flanking sequences and was able to mediate proteolytic processing in trans. Using N-terminal sequencing of autoproteolytic processing products we tentatively identified Gln2(Ala, Thr) as a substrate consensus sequence. Mutagenesis data provided evidence to suggest that two conserved His and Thr residues are part of the S1 subsite of the enzyme's substrate-binding pocket. Interestingly, we observed two N-proximal and two C-proximal autoprocessing sites in the bacterial expression system. The detection of two major forms of M pro , resulting from processing at two different N-proximal and one C-proximal site, in WBV-infected epithelioma papulosum cyprini cells confirmed the biological relevance of the biochemical data obtained in heterologous expression systems. To our knowledge, the use of alternative M pro autoprocessing sites has not been described previously for other nidovirus M pro domains. The data presented in this study lend further support to our previous conclusion that bafiniviruses represent a distinct group of viruses that significantly diverged from other phylogenetic clusters of the order Nidovirales. Cell culture and propagation of virus. Epithelioma papulosum cyprini (EPC) cells (RIE173) were obtained from the cell culture collection of the Friedrich-Loeffler-Institut (Greifswald-Insel Riems, Germany). They were grown in minimum essential medium with Earle's salts, 25 mM HEPES, GlutaMAX I (Invitrogen, catalog no. 42360-024) supplemented with 1 mM sodium pyruvate (Invitrogen, catalog no. 11360-039) and 10% fetal bovine serum (PAA Laboratories) at 27°C in a 5% CO 2 atmosphere. WBV isolate DF24/00 (23) was propagated on EPC cells at 20°C. Construction and mutagenesis of expression plasmids. Plasmids were generated by using standard cloning procedures. All PCR products were generated by using Pfu polymerase (Promega) according to the manufacturer's instructions. The correct sequences of the inserts were verified by sequencing using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems). To generate pMal-WBV-3424-3725-GST and pMal-WBV-3424-3725_S3589A-GST, the coding sequence for the WBV pp1a/pp1ab amino acid residues Gln-3424 to Met-3725 was amplified by using the primers MalE24 (5Ј-GGTCGTCAGACTGTCGATGAA GCC-3Ј) and M pro _BbsI (5Ј-AATAAGAAGACTGTTGTTAACTGGTGATG TGAGTGT-3Ј) and pMal-WBV-3CL_559-560 or pMal-WBV-3CL_S3589A (38) as a template, while the Schistosoma japonicum glutathione S-transferase (GST) coding sequence was amplified from pCITE-GST (44) by using the primers GSTup_BsaI (5Ј-AAAAGGTCTCTACAACATGAGCCCTATACTAGGTTA TTGG-3Ј) and GSTdn_BamHI (5Ј-AAAGGATCCTTAACGCGGAACCAGA TCCGATTTTGG-3Ј). The resulting PCR products were digested with BbsI and BsaI, respectively, and subsequently ligated by using T4 DNA ligase (New England Biolabs [NEB]). The resulting pp1a-3424-3725-GST fusion sequence was amplified by using MalE24 and GSTdn_BamHI, digested with SacI and BamHI, and ligated with SacI/BamHI-digested pMal-c2X plasmid DNA (NEB). To construct pMal-WBV-M pro -CHis, the coding region for pp1a/pp1ab amino acids Gln-3424 to Thr-3707 was amplified from pMal-pp1a-3424-3725-GST by using the primers JZ559 (5Ј-TCAGCATCAGAATGCATTCTGTAT-3Ј) and RU32 (5Ј-TTTGGATCCTTAGTGATGGTGATGGTGATGTGTTACTGGT GCTACTGGGAG-3Ј). The resulting PCR product was phosphorylated by using T4 polynucleotide kinase (PNK; NEB), digested with EcoRI and ligated with XmnI/EcoRI-digested pMal-c2X plasmid DNA (NEB). The inserts for plasmids pMal-WBV-N1-C1, pMal-WBV-N1-C2, pMal-WBV-N2-C1, and pMal-WBV-N2-C2 were amplified from pMal-WBV-3424-3725_S3589A-GST with the primers RU36 (5Ј-GCCCTCCAACGTATCAGAC AAGC-3Ј) in combination with RU177 (5Ј-ATAGAATTCTTATTGTCTGGT GATGGTGTTAC-3Ј), RU36 in combination with RU191 (5Ј-AAAGGATCCT TATTGTCCTACTGTGACAGATGT-3Ј), RU37 (5Ј-GCAGTCACAGTTGTT GTAACAC-3Ј) in combination with RU177, and RU37 in combination with RU191, respectively. The 5Ј termini of the resulting PCR products were phosphorylated by using PNK. The inserts for pMal-WBV-N1-C1 and pMal-WBV-N2-C1 were digested with EcoRI and then ligated with XmnI/EcoRI-digested pMal-c2X plasmid DNA. The inserts for pMal-WBV-N1-C2 and pMal-WBV-N2-C2 were digested with BamHI and ligated with XmnI/BamHI-digested pMal-c2X plasmid DNA. Site-directed mutagenesis of plasmid constructs was carried out by PCR-based methods essentially as described previously (28, 55) . Sequences of oligonucleotides used for mutagenesis are available on request. Expression of fusion proteins. Escherichia coli TB1 (NEB) cells transformed with pMal-pp1a-3424-3725-GST or pMal-pp1a-3424-3725_S3589A-GST were grown in Luria-Bertani (LB) medium containing ampicillin at a concentration of 100 g per ml until the culture reached an optical density at 600 nm (OD 600 ) of

SOLUTION: Characterization of Bafinivirus Main Protease Autoprocessing Activities ᰔ Downloaded from

PROBLEM: A new porcine coronavirus SADS-CoV was recently identified from suckling piglets 20 with severe diarrhea in southern China and its genome sequence is most identical (~95% 21 identity) to that of bat α-coronavirus HKU2. It again indicates bats are the natural 22 reservoir of many coronaviruses that have great potential for cross-species transmission 23 to animals and humans by recombination and/or mutation. Here we report the cryo-EM 24 structures of HKU2 and SADS-CoV spike glycoprotein trimers at 2.38 Å and 2.83 Å 25 resolution, respectively. HKU2 and SADS-CoV spikes exhibit very high structural 26 similarity, with subtle differences mainly distributed in the NTD and CTD of the S1 27 subunit responsible for cell attachment and receptor binding. We systematically 28 analyzed and compared the NTD, CTD, SD1 and SD2 domains of the S1 subunit and 29 the S2 subunit of HKU2 spike with those of α-, β-, γ-, and δ-coronavirus spikes. The 30 results show that the NTD and CTD of HKU2/SADS-CoV are probably the most 31 ancestral in the evolution of spike. Although the S2 subunit mediating membrane fusion 32 is highly conserved, the connecting region after fusion peptide in HKU2/SADS-CoV 33 S2 subunit also adopts a conformation distinct from other coronaviruses. These results 34 structurally demonstrate a close evolutionary relationship between HKU2 /SADS-CoV 35 and β-coronavirus spikes and provide new insights into the evolution and cross-species 36 transmission of coronaviruses. : bioRxiv preprint 39 Coronaviruses, categorized into the order Nidovirales family Coronaviridae and 40 subfamily Coronavirinae, are a large group of viral pathogens with a wide host range 1 . 41 Their infections in humans, other mammals and birds can cause respiratory, hepatic, 42 enteric and neurological diseases with varying severity 2 . The severe acute respiratory 43 syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus 44 (MERS-CoV) have posed severe threats to human health in the 21 st century 1,3 . In the 45 meantime, coronaviruses infecting domestic animals also bring substantial economic 46 losses 4 . For example, the swine acute diarrhea syndrome coronavirus (SADS-CoV) 47 (also known as SeACoV and PEAV) isolated in 2017 caused outbreaks of severe watery 48 diarrhea of suckling piglets with a mortality up to 90% in several commercial pig farms 49 in Guangdong Province of China 5-10 . SADS-CoV is an α-coronavirus and other 50 representative members in the α-genus are porcine epidemic diarrhea virus 51 (PEDV), porcine transmissible gastroenteritis coronavirus (TGEV), porcine respiratory 52 coronavirus (PRCV), human NL63 and 229E coronaviruses (HCoV-NL63 and HCoV-53 229E) 1 . Representative members in other three genera include mouse hepatitis 54 coronavirus (MHV), bovine coronavirus (BCoV), SARS-CoV, MERS-CoV, HCoV-55 OC43 and HCoV-HKU1 in the β-genus, avian infectious bronchitis virus (IBV) in the 56 γ-genus and porcine deltacoronavirus (PdCoV) in the δ-genus 1 . 57 Cross-species transmission promoted by genetic recombination and/or mutations 58 underlies the host range expansion of coronaviruses 3,11-13 . Bats are the natural reservoir 59 of more than 30 different α-and β-coronaviruses that have great potential for 60 interspecies transmission by recombination and/or mutation 12,14-16 . Data on genetic 61 evolution, receptor binding and pathogenesis have demonstrated that human SARS-62 CoV and MERS-CoV most likely originate from bats 1 . Palm civets and dromedary 63 camels are the intermediate hosts of SARS-CoV and MERS-CoV from bats to humans 1 , 64 respectively. The newly identified porcine SADS-CoV isolates are also found to share 65 ~95% sequence identity with Rhinolophus bat coronavirus HKU2, and this further 66 stressed the severe results of coronavirus spillover from bats to domestic animals 5-10 .

SOLUTION: Cryo-EM structures of HKU2 and SADS-CoV spike 1 glycoproteins and insights into coronavirus evolution 2 3 4

PROBLEM: A dense population, global connectivity and frequent human-animal interaction give southern China an important role in the spread and emergence of infectious disease. However, patterns of person-to-person contact relevant to the spread of directly transmitted infections such as influenza remain poorly quantified in the region. We conducted a household-based survey of travel and contact patterns among urban and rural populations of Guangdong, China. We measured the character and distance from home of social encounters made by 1821 individuals. Most individuals reported 5-10 h of contact with around 10 individuals each day; however, both distributions have long tails. The distribution of distance from home at which contacts were made is similar: most were within a kilometre of the participant's home, while some occurred further than 500 km away. Compared with younger individuals, older individuals made fewer contacts which tended to be closer to home. There was strong assortativity in age-based contact rates. We found no difference between the total number or duration of contacts between urban and rural participants, but urban participants tended to make contacts closer to home. These results can improve mathematical models of infectious disease emergence, spread and control in southern China and throughout the region.

SOLUTION:
Social mixing patterns in rural and urban areas of southern China