First detection of Tetraparvovirus ungulate 1 in diseased cattle (Chinese Simmental) from Hunan province, China.

Tetraparvovirus is an emerging parvovirus infecting a variety of mammals and humans, and associated with human diseases including severe acute respiratory infection and acute encephalitis syndrome. In the present study, a Tetraparvovirus ungulate 1 (formerly known as bovine hokovirus) strain HNU-CBY-2023 was identified and characterized from diseased Chinese Simmental from Hunan province, China. The nearly complete genome of HNU-CBY-2023 is 5346 nt in size and showed genomic identities of 85-95.5% to the known Tetraparvovirus ungulate 1 strains from GenBank, indicating a rather genetic variation. Phylogenetic and genetic divergence analyses indicated that Tetraparvovirus ungulate 1 could be divided into two genotypes (I and II), and HNU-CBY-2023 was clustered into genotype II. This study, for the first time, identified Tetraparvovirus ungulate 1 from domestic cattle from mainland China, which will be helpful to understand the prevalence and genetic diversity of Tetraparvovirus ungulate 1.

Lineage classification and selective site identification of Orthoebolavirus zairense.

As the high pathogenic species of Filoviridae virus family, Orthoebolavirus zairense (EBOV) shows frequent outbreaks in human in recently years since its first emerging in 1976 in Democratic Republic of the Congo (COD), bringing ongoing risks and burden on public health safety. Here, the phylogenetic relationship among major outbreaks was analyzed. The results showed that EBOV isolates could be divided into four lineages according to spatial and temporal epidemics. Then, the positive selection sites (PSSs) were detected on all proteins of the EBOV, exhibiting lineage characteristic. Particularly, sites in GP and VP24 were identified to be significantly under positive selection, and partial of which were maintained in the latest isolates in 2021. GP and L were found to have high variability between lineages. Substitutions including F443L and F443S in GP, as well as F1610L and I1951V in L could be characteristic of the two large outbreaks in COD (2018) and West Africa (2014), respectively. Further, substitutions of significant PSSs in VP24 and L proteins were visualized for analysis of structural changes, which may affect EBOV pathogenesis. In summary, our results gains insights in genetic characteristic and adaptive evolution of EBOV, which could facilitate gene functional research against EBOV.

Key mutations in the spike protein of SARS-CoV-2 affecting neutralization resistance and viral internalization.

To control the ongoing COVID-19 pandemic, a variety of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines have been developed. However, the rapid mutations of SARS-CoV-2 spike (S) protein may reduce the protective efficacy of the existing vaccines which is mainly determined by the level of neutralizing antibodies targeting S. In this study, we screened prevalent S mutations and constructed 124 pseudotyped lentiviral particles carrying these mutants. We challenged these pseudoviruses with sera vaccinated by Sinovac CoronaVac and ZF2001 vaccines, two popular vaccines designed for the initial strain of SARS-CoV-2, and then systematically assessed the susceptivity of these SARS-CoV-2 variants to the immune sera of vaccines. As a result, 14 S mutants (H146Y, V320I + S477N, V382L, K444R, L455F + S477N, L452M + F486L, F486L, Y508H, P521R, A626S, S477N + S698L, A701V, S477N + T778I, E1144Q) were found to be significantly resistant to neutralization, indicating reduced protective efficacy of the vaccines against these SARS-CoV-2 variants. In addition, F486L and Y508H significantly enhanced the utilization of human angiotensin-converting enzyme 2, suggesting a potentially elevated infectivity of these two mutants. In conclusion, our results show that some prevalent S mutations of SARS-CoV-2 reduced the protective efficacy of current vaccines and enhance the infectivity of the virus, indicating the necessity of vaccine renewal and providing direction for the development of new vaccines.

VirusRecom: an information-theory-based method for recombination detection of viral lineages and its application on SARS-CoV-2.

Genomic recombination is an important driving force for viral evolution, and recombination events have been reported for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the Coronavirus Disease 2019 pandemic, which significantly alter viral infectivity and transmissibility. However, it is difficult to identify viral recombination, especially for low-divergence viruses such as SARS-CoV-2, since it is hard to distinguish recombination from in situ mutation. Herein, we applied information theory to viral recombination analysis and developed VirusRecom, a program for efficiently screening recombination events on viral genome. In principle, we considered a recombination event as a transmission process of ``information'' and introduced weighted information content (WIC) to quantify the contribution of recombination to a certain region on viral genome; then, we identified the recombination regions by comparing WICs of different regions. In the benchmark using simulated data, VirusRecom showed a good balance between precision and recall compared to two competing tools, RDP5 and 3SEQ. In the detection of SARS-CoV-2 XE, XD and XF recombinants, VirusRecom providing more accurate positions of recombination regions than RDP5 and 3SEQ. In addition, we encapsulated the VirusRecom program into a command-line-interface software for convenient operation by users. In summary, we developed a novel approach based on information theory to identify viral recombination within highly similar sequences, providing a useful tool for monitoring viral evolution and epidemic control.

Key mutations on spike protein altering ACE2 receptor utilization and potentially expanding host range of emerging SARS-CoV-2 variants.

Increasing evidence supports inter-species transmission of SARS-CoV-2 variants from humans to domestic or wild animals during the ongoing COVID-19 pandemic, which is posing great challenges to epidemic control. Clarifying the host range of emerging SARS-CoV-2 variants will provide instructive information for the containment of viral spillover. The spike protein (S) of SARS-CoV-2 is the key determinant of receptor utilization, and therefore amino acid mutations on S will probably alter viral host range. Here, to evaluate the impact of S mutations, we tested 27 pseudoviruses of SARS-CoV-2 carrying different spike mutants by infecting Hela cells expressing different angiotensin-converting enzyme 2 (ACE2) orthologs from 20 animals. Of these 27 pseudoviruses, 20 bear single mutation and the other 7 were cloned from emerging SARS-CoV-2 variants, including D614G, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (B.1.429), and Mu (B.1.621). Using pseudoviral reporter assay, we identified that the substitutions of T478I and N501Y enabled the pseudovirus to utilize chicken ACE2, indicating potential infectivity to avian species. Furthermore, the S mutants of real SARS-CoV-2 variants comprising N501Y showed significantly acquired abilities to infect cells expressing mouse ACE2, indicating a critical role of N501Y in expanding SARS-CoV-2 host range. In addition, A262S and T478I significantly enhanced the utilization of various mammal ACE2. In summary, our results indicated that T478I and N501Y substitutions were two S mutations important for receptor adaption of SARS-CoV-2, potentially contributing to the spillover of the virus to many other animal hosts. Therefore, more attention should be paid to SARS-CoV-2 variants with these two mutations.

Characterization of Deltacoronavirus in Black-Headed Gulls (Chroicocephalus ridibundus) in South China Indicating Frequent Interspecies Transmission of the Virus in Birds.

Deltacoronavirus (DCoV) is a genus of coronavirus (CoV) commonly found in avian and swine, but some DCoVs are capable of infecting humans, which causes the concern about interspecies transmission of DCoVs. Thus, monitoring the existence of DCoVs in animals near communities is of great importance for epidemic prevention. Black-headed gulls (Chroicocephalus ridibundus) are common migratory birds inhabiting in most urban and rural wetlands of Yunnan Province, China, which is a typical habitat for black-headed gulls to overwinter. Whether Yunnan black-headed gulls carry CoV has never been determined. In this study, we identified three strains of DCoVs in fecal samples of Yunnan black-headed gulls by reverse-transcriptional PCR and sequenced their whole genomes. Genomic analysis revealed that these three strains shared genomic identity of more than 99%, thus named DCoV HNU4-1, HNU4-2, and HNU4-3; their NSP12 showed high similarity of amino acid sequence to the homologs of falcon coronavirus UAE-HKU27 (HKU27), houbara coronavirus UAE-HKU28 (HKU28), and pigeon coronavirus UAE-HKU29 (HKU29). Since both HKU28 and HKU29 were found in Dubai, there might be cross-border transmission of these avian DCoVs through specific routes. Further coevolutionary analysis supported this speculation that HNU4 (or its ancestors) in black-headed gulls originated from HKU28 (or its homologous strain) in houbara, which was interspecies transmission between two different avian orders. In addition, interspecies transmission of DCoV, from houbara to falcon, pigeon and white-eye, from sparrow to common-magpie, and quail and mammal including porcine and Asian leopard cat, from munia to magpie-robin, was predicted. This is the first report of black-headed gull DCoV in Asia which was highly homolog to other avian DCoVs, and the very "active" host-switching events in DCoV were predicted, which provides important reference for the study of spread and transmission of DCoVs.

Epidemiology and evolution of novel deltacoronaviruses in birds in central China.

The variety and widespread of coronavirus in natural reservoir animals is likely to cause epidemics via interspecific transmission, which has attracted much attention due to frequent coronavirus epidemics in recent decades. Birds are natural reservoir of various viruses, but the existence of coronaviruses in wild birds in central China has been barely studied. Some bird coronaviruses belong to the genus of Deltacoronavirus. To explore the diversity of bird deltacoronaviruses in central China, we tested faecal samples from 415 wild birds in Hunan Province, China. By RT-PCR detection, we identified eight samples positive for deltacoronaviruses which were all from common magpies, and in four of them, we successfully amplified complete deltacoronavirus genomes distinct from currently known deltacoronavirus, indicating four novel deltacoronavirus stains (HNU1-1, HNU1-2, HNU2 and HNU3). Comparative analysis on the four genomic sequences showed that these novel magpie deltacoronaviruses shared three different S genes among which the S genes of HNU1-1 and HNU1-2 showed 93.8% amino acid (aa) identity to that of thrush coronavirus HKU12, HNU2 S showed 71.9% aa identity to that of White-eye coronavirus HKU16, and HNU3 S showed 72.4% aa identity to that of sparrow coronavirus HKU17. Recombination analysis showed that frequent recombination events of the S genes occurred among these deltacoronavirus strains. Two novel putative cleavage sites separating the non-structural proteins in the HNU coronaviruses were found. Bayesian phylogeographic analysis showed that the south coast of China might be a potential origin of bird deltacoronaviruses existing in inland China. In summary, these results suggest that common magpie in China carries diverse deltacoronaviruses with novel genomic features, indicating an important source of environmental coronaviruses closed to human communities, which may provide key information for prevention and control of future coronavirus epidemics.

Innate Immunity Evasion Strategies of Highly Pathogenic Coronaviruses: SARS-CoV, MERS-CoV, and SARS-CoV-2.

In the past two decades, coronavirus (CoV) has emerged frequently in the population. Three CoVs (SARS-CoV, MERS-CoV, SARS-CoV-2) have been identified as highly pathogenic human coronaviruses (HP-hCoVs). Particularly, the ongoing COVID-19 pandemic caused by SARS-CoV-2 warns that HP-hCoVs present a high risk to human health. Like other viruses, HP-hCoVs interact with their host cells in sophisticated manners for infection and pathogenesis. Here, we reviewed the current knowledge about the interference of HP-hCoVs in multiple cellular processes and their impacts on viral infection. HP-hCoVs employed various strategies to suppress and evade from immune response, including shielding viral RNA from recognition by pattern recognition receptors (PRRs), impairing IFN-I production, blocking the downstream pathways of IFN-I, and other evasion strategies. This summary provides a comprehensive view of the interplay between HP-hCoVs and the host cells, which is helpful to understand the mechanism of viral pathogenesis and develop antiviral therapies.

Genome Characterization of Bird-Related Rhabdoviruses Circulating in Africa.

Rhabdoviridae is the most diverse family of the negative, single-stranded RNA viruses, which includes 40 ecologically different genera that infect plants, insects, reptiles, fishes, and mammals, including humans, and birds. To date, only a few bird-related rhabdoviruses among the genera Sunrhavirus, Hapavirus, and Tupavirus have been described and analyzed at the molecular level. In this study, we characterized seven additional and previously unclassified rhabdoviruses, which were isolated from various bird species collected in Africa during the 1960s and 1970s. Based on the analysis of their genome sequences obtained by next generation sequencing, we observed a classical genomic structure, with the presence of the five canonical rhabdovirus genes, i.e., nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and polymerase (L). In addition, different additional open reading frames which code putative proteins of unknown function were identified, with the common presence of the C and the SH proteins, within the P gene and between the M and G genes, respectively. Genetic comparisons and phylogenetic analysis demonstrated that these seven bird-related rhabdoviruses could be considered as putative new species within the genus Sunrhavirus, where they clustered into a single group (named Clade III), a companion to two other groups that encompass mainly insect-related viruses. The results of this study shed light on the high diversity of the rhabdoviruses circulating in birds, mainly in Africa. Their close relationship with other insect-related sunrhaviruses raise questions about their potential role and impact as arboviruses that affect bird communities.

Receptor utilization of angiotensin-converting enzyme 2 (ACE2) indicates a narrower host range of SARS-CoV-2 than that of SARS-CoV.

Coronavirus (CoV) pandemics have become a huge threat to the public health worldwide in the recent decades. Typically, severe acute respiratory syndrome CoV (SARS-CoV) caused SARS pandemic in 2003 and SARS-CoV-2 caused the ongoing COVID-19 pandemic. Both viruses are most likely originated from bats. Thus, direct or indirect inter-species transmission from bats to humans is required for the viruses to cause pandemics. Receptor utilization is a key factor determining the host range of viruses which is critical to the inter-species transmission. Angiotensin-converting enzyme 2 (ACE2) is the receptor of both SARS-CoV and SARS-CoV-2, but only ACE2s of certain animals can be utilized by the viruses. Here, we employed pseudovirus cell-entry assay to evaluate the receptor-utilizing capability of ACE2s of 20 animals by the two viruses and found that SARS-CoV-2 utilized less ACE2s than SARS-CoV, indicating a narrower host range of SARS-CoV-2. Especially, SARS-CoV-2 tended not to use murine or non-mammal ACE2s. Meanwhile, pangolin-CoV, another SARS-related coronavirus highly homologous to SARS-CoV-2 in its genome, yet showed similar ACE2 utilization profile with SARS-CoV rather than SARS-CoV-2. Nevertheless, the actual susceptibility of these animals to the coronaviruses should be further verified by in vivo studies. To clarify the mechanism underlying the receptor utilization, we compared the amino acid sequences of the 20 ACE2s and found 5 amino acid residues potentially critical for ACE2 utilization, including the N-terminal 20th and 42nd amino acid residues that might determine the different receptor utilization of SARS-CoV, SARS-CoV-2 and pangolin-CoV. Our studies enhance the understanding of receptor utilization of pandemic coronaviruses, potentially contributing to the virus tracing, intermediate host screening and epidemic prevention for pathogenic coronaviruses.

BioAider: An efficient tool for viral genome analysis and its application in tracing SARS-CoV-2 transmission.

The novel human coronavirus (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) pandemic worldwide. Control of COVID-19 pandemic is vital for public health and is the prerequisite to maintain social stability. However, the origin and transmission route of SARS-CoV-2 is unclear, bringing huge difficult to virus control. Monitoring viral variation and screening functional mutation sites are crucial for prevention and control of infectious diseases. In this study, we developed a user-friendly software, named BioAider, for quick sequence annotation and mutation analysis on large-scale genome-sequencing data. Herein, we detected 14 substitution hotspots within 3,240 SARS-CoV-2 genome sequences, including 3 groups of potentially linked substitution. NSP13-Y541C was crucial substitution which might affect the unwinding activity of the viral helicase. In particular, we discovered a SR-rich region of SARS-CoV-2 distinct from SARS-CoV, indicating more complex replication mechanism and unique N-M interaction of SARS-CoV-2. Interestingly, the quantity of SSRX repeat fragments in SARS-CoV-2 provided further evidence of its animal origin. Overall, we developed an efficient tool for rapid identification of viral genome mutations which could facilitate viral genomic studies. Using this tool, we have found critical clues for the transmission route of SARS-CoV-2 which would provide theoretical support for the epidemic control of pathogenic coronaviruses.

Predicting the angiotensin converting enzyme 2 (ACE2) utilizing capability as the receptor of SARS-CoV-2.

SARS-CoV-2, the newly identified human coronavirus causing severe pneumonia pandemic, was probably originated from Chinese horseshoe bats. However, direct transmission of the virus from bats to humans is unlikely due to lack of direct contact, implying the existence of unknown intermediate hosts. Angiotensin converting enzyme 2 (ACE2) is the receptor of SARS-CoV-2, but only ACE2s of certain species can be utilized by SARS-CoV-2. Here, we evaluated and ranked the receptor-utilizing capability of ACE2s from various species by phylogenetic clustering and sequence alignment with the currently known ACE2s utilized by SARS-CoV-2. As a result, we predicted that SARS-CoV-2 tends to utilize ACE2s of various mammals, except murines, and some birds, such as pigeon. This prediction may help to screen the intermediate hosts of SARS-CoV-2.

The epidemic of 2019-novel-coronavirus (2019-nCoV) pneumonia and insights for emerging infectious diseases in the future.

At the end of December 2019, a novel coronavirus, 2019-nCoV, caused an outbreak of pneumonia spreading from Wuhan, Hubei province, to the whole country of China, which has posed great threats to public health and attracted enormous attention around the world. To date, there are no clinically approved vaccines or antiviral drugs available for these human coronavirus infections. Intensive research on the novel emerging human infectious coronaviruses is urgently needed to elucidate their route of transmission and pathogenic mechanisms, and to identify potential drug targets, which would promote the development of effective preventive and therapeutic countermeasures. Herein, we describe the epidemic and etiological characteristics of 2019-nCoV, discuss its essential biological features, including tropism and receptor usage, summarize approaches for disease prevention and treatment, and speculate on the transmission route of 2019-nCoV.

Association between fentanyl test results and rescue morphine requirements in children after adenotonsillectomy.

PURPOSE: Preoperative sleep study helps to predict post-adenotonsillectomy morphine requirements. However, in some institutions, many suspected children with obstructive sleep apnoea syndrome have an adenotonsillectomy without polysomnography assessments. This study investigated the relationship between the results of a fentanyl test performed before extubation and the postoperative morphine requirements in children after adenotonsillectomy. METHODS: Intravenous fentanyl (1 µg/kg) was given as a test before extubation when spontaneous ventilation was restored in 80 children aged 3-7 years who underwent adenotonsillectomy. The result was considered positive if the patient's respiratory rate decreased >50% after the test. In the recovery room, pain was assessed every 10 min using the Children's Hospital of Eastern Ontario Pain Scale. Rescue morphine (10 µg/kg) was given when the score was ≥6. RESULTS: The median [IQR (range)] cumulative morphine consumption rates for children with a positive result (n = 25) and a negative result (n = 52) were 30 (20, 40) and 50 (40, 50) µg/kg, respectively (P = 0.002). Eighty-eight percent of the positive-result patients and 48% of the negative-result patients were light consumers of morphine (cumulative dose <50 µg/kg) (P = 0.001). CONCLUSIONS: We conclude that children with a positive result after a fentanyl test require less morphine to achieve comfort than those with a negative result. CLINICALTRIALS. GOV ID: NCT02484222.

The characteristics of the staircase phenomenon during the period of twitch stabilization in infants in TOF mode.

BACKGROUND: Acceleromyography used to monitor the neuromuscular transmission function is available in infants and children. However, information on the so-called staircase phenomenon during the baseline stabilization period in this population is limited. Our objective was to assess the characteristics of such phenomenon in infants in acceleromyography. METHODS: Thirty infants were divided into three groups: group A (aged 1-5 months, n = 10), group B (aged 6-11 months, n = 10), and group C (aged 12-24 months, n = 10). Anesthesia was induced and maintained without neuromuscular blocking agents. Patients received a supramaximal stimulus followed by TOF measurements every 15 s over the course of 30 min with a TOF-Watch(®) SX. All data were collected in a notebook computer using the TOF-Watch(®) SX Monitor software through optical fibers. RESULTS: The evoked T1 responses in younger infants (group A) were significantly lower than those in older infants (groups B and C) from 0-20 min. In group A, the signals increased to the maximum value of 121 ± 15% that of the control at 7.25 min and decreased toward a plateau of 113 ± 18% at 30 min. In group B, the signal heights increased to the maximum value of 143 ± 14% at 9.5 min, then decreased slowly to 136 ± 10% at 19.5 min, finally reaching 116 ± 12% at 30 min. In group C, the mean values of T1 reached the maximum of 139 ± 19% that of control at 9.5 min and finally reached 126 ± 22% of control at 30 min. CONCLUSIONS: The staircase effect presents in a shorter time course and at lower degrees in smaller infants. However, in older infants, staircase effect still presents in a long period and may influence the onset time and duration of twitch depression after muscle relaxants administration.

Serum microRNAs are promising novel biomarkers for diffuse large B cell lymphoma.

MicroRNAs (miRNAs) are regulatory RNA molecules that are deregulated in many disease types, including cancer. Recently, miRNAs have shown promise as markers for cancer diagnosis. The aim of this study was to investigate whether serum miRNAs can be used as biomarkers for the detection of diffuse large B cell lymphoma (DLBCL). We measured the levels of miRNAs (miR-15a, miR-16-1, miR-21, miR-29c, miR-34a, miR-155, and miR-223) in serum samples from patients with DLBCL and healthy controls using real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR). We show here that miRNAs are present in human serum in a remarkably stable form. Four of miRNAs (miR-15a, miR-16-1, miR-29c, and miR-155) were significantly elevated in DLBCL serum when compared with normal controls (P < 0.05), while miR-34a was downregulated in DLBCL serum when compared with controls (P < 0.05). Receiver operating characteristic analyses reflects strong discriminating DLBCL from controls, with area under the curves of 0.7722, 0.7002, 0.6672, 0.8538, and 0.7157 for miR-15a, miR-16-1, miR-29c, miR-34a, and miR-155, respectively. At the cut-off value of 0.0006 for miR-15a, the sensitivity was 80% and the specificity was 76%; at the cut-off value of 0.0886 for miR-16-1, the sensitivity was 94% and the specificity was 51%; at the cut-off value of 1.395 for miR-34a, the sensitivity was 100% and the specificity was 70%; at the cut-off value of 0.0022 for miR-155, the sensitivity was 83% and the specificity was 65%. In conclusion, these data suggest that serum miRNAs are potentially useful tools as novel noninvasive biomarker for the diagnosis of DLBCL.