A Novel Potentially Recombinant Rodent Coronavirus with a Polybasic Cleavage Site in the Spike Protein.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reignited global interest in animal coronaviruses and their potential for human transmission. While bats are thought to be the wildlife reservoir of SARS-CoV and SARS-CoV-2, the widespread human coronavirus OC43 is thought to have originated in rodents. Here, we sampled 297 rodents and shrews, representing eight species, from three municipalities of southern China. We report coronavirus prevalences of 23.3% and 0.7% in Guangzhou and Guilin, respectively, with samples from urban areas having significantly higher coronavirus prevalences than those from rural areas. We obtained three coronavirus genome sequences from Rattus norvegicus, including a Betacoronavirus (rat coronavirus [RCoV] GCCDC3), an Alphacoronavirus (RCoV-GCCDC5), and a novel Betacoronavirus (RCoV-GCCDC4). Recombination analysis suggests that there was a potential recombination event involving RCoV-GCCDC4, murine hepatitis virus (MHV), and Longquan Rl rat coronavirus (LRLV). Furthermore, we uncovered a polybasic cleavage site, RARR, in the spike (S) protein of RCoV-GCCDC4, which is dominant in RCoV. These findings provide further information on the potential for interspecies transmission of coronaviruses and demonstrate the value of a One Health approach to virus discovery. IMPORTANCE Surveillance of viruses among rodents in rural and urban areas of South China identified three rodent coronaviruses, RCoV-GCCDC3, RCoV-GCCDC4, and RCoV-GCCDC5, one of which was identified as a novel potentially recombinant coronavirus with a polybasic cleavage site in the spike (S) protein. Through reverse transcription-PCR (RT-PCR) screening of coronaviruses, we found that coronavirus prevalence in urban areas is much higher than that in rural areas. Subsequently, we obtained three coronavirus genome sequences by deep sequencing. After different method-based analyses, we found that RCoV-GCCDC4 was a novel potentially recombinant coronavirus with a polybasic cleavage site in the S protein, dominant in RCoV. This newly identified coronavirus RCoV-GCCDC4 with its potentially recombinant genome and polybasic cleavage site provides a new insight into the evolution of coronaviruses. Furthermore, our results provide further information on the potential for interspecies transmission of coronaviruses and demonstrate the necessity of a One Health approach for zoonotic disease surveillance.

Intrahost variation and evolutionary dynamics of adenoviruses correlate to neutrophils in infected patients.

With deep sequencing of virus genomes within the hosts, intrahost single nucleotide variations (iSNVs) have been used for analyses of virus genome variation and evolution, which is indicated to correlate with viral pathogenesis and disease severity. Little is known about the features of iSNVs among DNA viruses. We performed the epidemiological and laboratory investigation of one outbreak of adenovirus. The whole genomes of viruses in both original oral swabs and cell-cultured virus isolates were deeply sequenced. We identified 737 iSNVs in the viral genomes sequenced from original samples and 46 viral iSNVs in cell-cultured isolates, with 33 iSNVs shared by original samples and cultured isolates. Meanwhile, we found these 33 iSNVs were shared by different patients, among which, three hot spot areas 6367-6401, 9213-9247, and 10 584-10 606 within the functional genes of the adenovirus genome were found. Notably, the substitution rates of iSNVs were closely correlated with the clinical and immune indicators of the patients. Especially a positive correlation to neutrophils was found, indicating a predictable biomarker of iSNV dynamics. Our findings demonstrated the neutrophil-correlated dynamic evolution features of the iSNVs within adenoviruses, which indicates a virus-host interaction during human infection of a DNA virus.

Visual-spatial skills contribute to Chinese reading and arithmetic for different reasons: A three-wave longitudinal study.

Previous literature has revealed that visual-spatial processing is associated with both reading and arithmetic. Yet the strength of their relations and the reasons why visual-spatial processing contributes to reading and arithmetic remain ambiguous. The current study focused on two types of visual-spatial skills that recent evidence has suggested are crucial in children's early reading and arithmetic development: visual-perceptual and spatial visualization skills. With an interval of 6 months, we assessed 104 Hong Kong kindergarten children's visual-spatial skills, word reading, arithmetic performance, and vocabulary knowledge at Wave 1; orthographic awareness, basic number knowledge, and number line estimation at Wave 2; and Chinese word reading and arithmetic performance at Wave 3. Correlational analysis showed that both visual-perceptual and spatial visualization skills were associated with later Chinese word reading and arithmetic performance. Further mediation analyses revealed that spatial visualization skills, rather than visual-perceptual skills, contributed to Chinese word reading via orthographic awareness and also predicted arithmetic performance through basic number knowledge. However, number line estimation failed to mediate any relations of visual-spatial skills with children's arithmetic abilities. The results suggest the importance of visual-spatial processing in Chinese word reading and mathematics, with spatial visualization contributing to reading and mathematics for different reasons.

Comparisons of microbiological characteristics and antibiotic resistance of Klebsiella pneumoniae isolates from urban rodents, shrews, and healthy people.

BACKGROUND: The comparisons of molecular characterization and antibiotic resistance of Klebsiella pneumoniae (KP) isolates from humans and other animal hosts are not well studied. Our goal was to compare the molecular epidemiology of KP strains that were isolated from urban rodents, shrews, and healthy people. RESULTS: K. pneumoniae (KP) isolates were isolated from fecal samples of rodents, shrews and healthy adults in 2015 in southern China. In total, 465 fecal samples were collected, of which 85 from rodents, 105 from shrews, and 275 from healthy adults. Antimicrobial susceptibility and production of extended-spectrum ß-lactamases (ESBL) of the isolates were tested. PCR-based methods were used to detect specific genes, including ESBL genes (blaTEM, blaSHV, and blaCTX-M) in ESBL-producing isolates, capsular serotypes (K1, K2, K5, K20, K54, and K57) in hypervirulent KPs (hvKPs), and virulence genes (magA, wcaG, rmpA, uge, kfu, and aerobactin) in hvKP isolates. Multilocus sequence type (MLST) and pulsed-field gel electrophoresis (PFGE) were performed to exclude the homology of these isolates. The carriage rate of KP in urban rodents and shrews (78.42%) was higher than that in healthy adults (66.18%) (χ2 = 8.206, P = 0.004). The prevalence rates of ESBL-producing isolates among rodents, shrews, and humans were 7.94, 12.79, and 17.03%, respectively. The positive rates of CTX-M, TEM and SHV types in ESBL-producing isolates were 29.79, 27.66, and 17.02%, respectively. Serotype K1, K5, K20, and K57 were detected in both small mammals and humans. PFGE typing revealed thirty-six clusters. PFGE cluster A was clustered by samples of shrews and healthy adult, with a similarity of 88.4%. MLST typing revealed thirty-eight types. ST23 and ST35 were detected in samples of shrews and healthy adults. ST37 was detected in samples of 2 rodents and a healthy adult. CONCLUSIONS: Overlapping serotypes of hvKP were observed in both the animals and humans. The same PFGE or MLST types were also found in isolates derived humans, rodents and shrews. Therefore, urban rodents and shrews might play a certain role in the transmission of drug-resistant and hypervirulent KP.

Composition of gut and oropharynx bacterial communities in Rattus norvegicus and Suncus murinus in China.

BACKGROUND: Rattus norvegicus and Suncus murinus are important reservoirs of zoonotic bacterial diseases. An understanding of the composition of gut and oropharynx bacteria in these animals is important for monitoring and preventing such diseases. We therefore examined gut and oropharynx bacterial composition in these animals in China. RESULTS: Proteobacteria, Firmicutes and Bacteroidetes were the most abundant phyla in faecal and throat swab samples of both animals. However, the composition of the bacterial community differed significantly between sample types and animal species. Firmicutes exhibited the highest relative abundance in throat swab samples of R. norvegicus, followed by Proteobacteria and Bacteroidetes. In throat swab specimens of S. murinus, Proteobacteria was the predominant phylum, followed by Firmicutes and Bacteroidetes. Firmicutes showed the highest relative abundance in faecal specimens of R. norvegicus, followed by Bacteroidetes and Proteobacteria. Firmicutes and Proteobacteria had almost equal abundance in faecal specimens of S. murinus, with Bacteroidetes accounting for only 3.07%. The family Streptococcaceae was most common in throat swab samples of R. norvegicus, while Prevotellaceae was most common in its faecal samples. Pseudomonadaceae was the predominant family in throat swab samples of S. murinus, while Enterobacteriaceae was most common in faecal samples. We annotated 33.28% sequences from faecal samples of S. murinus as potential human pathogenic bacteria, approximately 3.06-fold those in R. norvegicus. Potential pathogenic bacteria annotated in throat swab samples of S. murinus were 1.35-fold those in R. norvegicus. CONCLUSIONS: Bacterial composition of throat swabs and faecal samples from R. norvegicus differed from those of S. murinus. Both species carried various pathogenic bacteria, therefore both should be closely monitored in the future, especially for S. murinus.

Methicillin-resistant Staphylococcus aureus among urban rodents, house shrews, and patients in Guangzhou, Southern China.

BACKGROUND: The transmission of methicillin-resistant Staphylococcus aureus (MRSA) between humans and animals has been identified in a number of countries. In this study, MRSA in urban rodents and shrews in a community was investigated. Further, comparisons of MRSA isolates from rodents, shrews, and humans were conducted to evaluate the relationships of these isolates from different origins. RESULTS: Between 2015 and 2016, 397 oropharynx samples from 212 rodents and 185 shrews, and 8 MRSA isolates from hospital patients were collected. Twelve MRSA were isolated from the small mammals (3.0, 95%CI: 1.3-4.7%), including 11 isolates from rodents and one from a shrew. Three MRSA isolates from Rattus norvegicus were PVL-positive, and seven isolates were IEC-negative (one from Suncus murinus, five from Rattus norvegicus, and one from a patient). The spa type, MLST, and antimicrobial resistance patterns showed that the MRSA retrieved from rodents and shrews are likely related to human strains. CONCLUSION: MRSA derived from rodent shares similar antimicrobial resistance and molecular characteristics to those from humans, suggesting that urban rodents may play as maintenance host or vectors for MRSA which is important to human health.

Young Chinese Children's Academic Skill Development: Identifying Child-, Family-, and School-Level Factors.

This chapter addresses how child-, family-, and school-level characteristics are associated with Chinese children's academic skill development during their preschool years. Academic skills are defined in terms of young children's emergent competencies in academic domains including literacy, mathematics, and science. First, we review the relations of young Chinese children's cognition (language, visuospatial, and executive functioning), behavior (social behavior and behavioral regulation), and affect (interest and attitude) to their performance in these academic domains. Second, we review the roles of familial variables, including family socioeconomic status and broad and specific aspects of parenting practices and parental involvement. Third, we review school- and classroom-level factors, with a special emphasis on preschool and classroom quality that is particularly relevant to young Chinese children's academic skills. We discuss the educational implications of these study findings and identify methodological limitations that may threaten their internal and external validity. Our aim is to bring attention to the growing body of research on young Chinese children's academic skill development and to highlight several areas that need further research.

Viral metagenomics of six bat species in close contact with humans in southern China.

Accumulating studies have shown that bats could harbor various important pathogenic viruses that could be transmitted to humans and other animals. Extensive metagenomic studies of different organs/tissues from bats have revealed a large number of novel or divergent viruses. To elucidate viral diversity and epidemiological and phylogenetic characteristics, six pooled fecal samples from bats were generated (based on bat species and geographic regions characteristic for virome analysis). These contained 500 fecal samples from six bat species, collected in four geographic regions. Metagenomic analysis revealed a plethora of divergent viruses originally found in bats. Multiple contigs from influenza A virus and coronaviruses in bats shared high identity with those from humans, suggesting possible cross-species transmission, whereas a number of contigs, whose sequences were taxonomically classifiable within Alphapapillomavirus, Betaretrovirus, Alpharetrovirus, Varicellovirus, Cyprinivirus, Chlorovirus and Cucumovirus had low identity to viruses in existing databases, which indicated possible evolution of novel viral species. None of the established caliciviruses and picornaviruses were found in the 500 fecal specimens. Papillomaviruses with high amino acid identity were found in Scotophilus kuhlii and Rhinolophus blythi, challenging the hypotheses regarding the strict host specificity and co-evolution of papillomaviruses. Phylogenetic analysis showed that four bat rotavirus A strains might be tentative G3 strains, according to the Rotavirus Classification Working Group classification.

The Risk of Human Papillomavirus Infection for Spontaneous Abortion, Spontaneous Preterm Birth, and Pregnancy Rate of Assisted Reproductive Technologies: A Systematic Review and Meta-Analysis.

AIMS: To estimate the impact of human papillomavirus (HPV) infection on spontaneous abortion, spontaneous preterm birth (sPTB), pregnancy rate of females undergoing assisted reproductive technologies (ART), and spontaneous abortion of ART pregnancy. METHODS: PubMed, Medline, Embase, and the Cochrane Library were searched until December 16, 2016. The OR or relative risk (RR) with its corresponding 95% CI were selected as effect size. Subgroup analysis of HPV genotype infection (high-risk HPV [HR-HPV] or indiscriminate genotype) was performed. Statistical analysis was conducted using STATA 12.0. RESULTS: Eighteen studies were included. Eight studies revealed no significant association between HPV infection and spontaneous abortion (OR 1.40, 95% CI 0.56-3.50). However, subgroup analysis showed indiscriminate genotype HPV infection increased the ratio of spontaneous abortion with OR of 2.24 (95% CI 1.37-3.65), while HR-HPV infection had no significant effect (OR 0.65, 95% CI 0.21-1.98). The results indicated that HR-HPV infection was a risk for sPTB with a pooled OR of 2.84 (95% CI 1.95-4.14). HPV infection was found to be independent of the ART-based clinical pregnancy rate (RR 1.04, 95% CI 0.64-1.70) and spontaneous abortion of ART pregnancy (RR 1.47, 95% CI 0.86-2.50). CONCLUSIONS: Indiscriminate HPV genotype infection can increase the risk of spontaneous abortion and HR-HPV infection was a risk factor for sPTB. However, there was not enough evidence to indicate the association between HPV infection and pregnancy rate of ART, and spontaneous abortion of ART pregnancy. Different genotypes of HPV infection may play a discrepant role in adverse pregnancy outcomes.

High prevalence and diversity of viruses of the subfamily Gammaherpesvirinae, family Herpesviridae, in fecal specimens from bats of different species in southern China.

Several studies have reported the detection of herpesviruses (HVs) in bats. However, the prevalence and phylogenetic characteristics of HVs in bats are still poorly understood. To elucidate the epidemiological characteristics of bat HVs in southern China, 520 fecal samples from eight bat species were collected in four geographic regions of southern China. Of these samples, 73 (14.0 %) tested positive for HVs using nested polymerase chain reaction assay. Phylogenetic analysis revealed a high degree of molecular diversity of HVs in bats of different species from different geographic regions. Our study provides evidence for co-evolution of bats and HVs.

Detection of novel adenoviruses in fecal specimens from rodents and shrews in southern China.

The prevalence and phylogenetic characteristics of AdVs in rodents and shrews in China are still unknown. To explore the epidemiological characteristics of rodent and shrew AdVs in southern China, 255 fecal samples derived from four rodent species and 90 from shrews were collected in Xiamen and Guangzhou city of southern China. Amplification of a 314-324-bp fragment from the DNA polymerase gene of AdVs was attempted by using a nested PCR. Twenty-nine (11.4 %) specimens from rodents and one (1.1 %) specimen from shrews were tested positive for AdVs. Phylogenetic analysis revealed that nine samples from Rattus norvegicus in Guangzhou city between 2012 and 2013 might be the genuine AdV of R. norvegicus. The same putative AdV sequences were derived from samples of different host species from different/same places. A novel adenovirus was detected in Suncus murinus Linnaeus (SML/14GDGZ72) for the first time. Our findings provide new data on the prevalence and diversity of AdVs in rodents and shrews.

Auditory detection of non-speech and speech stimuli in noise: Native speech advantage.

Detection thresholds of Chinese vowels, Korean vowels, and a complex tone, with harmonic and noise carriers were measured in noise for Mandarin Chinese-native listeners. The harmonic index was calculated as the difference between detection thresholds of the stimuli with harmonic carriers and those with noise carriers. The harmonic index for Chinese vowels was significantly greater than that for Korean vowels and the complex tone. Moreover, native speech sounds were rated significantly more native-like than non-native speech and non-speech sounds. The results indicate that native speech has an advantage over other sounds in simple auditory tasks like sound detection.

Development of EEG alpha and theta oscillations in the maintenance stage of working memory.

Several studies have shown developmental changes in EEG oscillations during working memory tasks. Although the load-modulated theta and alpha activities in adults are well-documented, the findings are inconsistent if children possess the adult-like brain oscillations that are similarly modulated by memory load. The present study compares children's and adults' true theta and alpha EEG oscillations, separated from aperiodic components, in the maintenance stage of working memory. The EEG was recorded in 25 Chinese-speaking children (14 male, Mage = 9.4 yrs) and 31 adults (19 male, Mage = 20.8 yrs) in Hong Kong while they performed an n-back task that included four conditions differing in load (1- vs. 2-back) and stimulus type (Chinese character vs. visual pattern). The results show that aperiodic activities (i.e., broadband power and slope) during the maintenance stage in the n-back task were significantly higher in children than adults. The periodic theta and alpha oscillations also changed with age. More importantly, adults showed significant periodic theta increase with memory load, whereas such an effect was absent in children. Regardless of age, there was a significant alpha power decrease with load increase, and a significant theta power enhancement when maintaining visual patterns than Chinese characters. In adults, load-modulated alpha peak shift (towards higher frequency) was linked to higher behavioral efficiency in the n-back task. In children, higher load-modulated theta enhancement was linked to better behavioral efficiency. The findings suggest that the load-modulated theta power during working memory maintenance matures from childhood to adulthood.

Neural specialization to English words in Chinese children: Joint contribution of age and English reading abilities.

N1 tuning to words, a neural marker of visual word recognition, develops by an interaction between age and ability. The development of N1 tuning to a second learnt print is unclear. The present study examined the joint contribution of age and English reading abilities to N1 amplitude and tuning to English print in Chinese children in Hong Kong. EEG signals were recorded from 179 children (six to nine years old) while they were performing a repetition detection task comprised of different print stimuli measuring three types of tuning, i.e., coarse tuning (real word versus false font), fine tuning (real versus nonword), and lexicality effect (real versus pseudo word). Children were assessed in English word reading accuracy (EWR) and English sub-lexical orthographic knowledge (EOK). Results indicated that coarse tuning decreased with age but increased with EWR and EOK. Fine tuning uniquely increased with EOK, and the lexicality effect increased with EWR. At last, higher EWR was linked to less right-lateralized coarse tuning in younger children. Taken together, the findings support the visual perceptual expertise account in the L2 context, in that N1 coarse tuning, fine tuning, and lexicality effect are driven by skill improvement.

The genomic characteristics and pathogenicity of a mammalian orthoreovirus within a new lineage from wild pika in plateau.

Emerging and re-emerging viruses from wild animals have seriously threatened the health of humans and domesticated animals in recent years. Herein, we isolated a new mammalian orthoreovirus (MRV), Pika/MRV/GCCDC7/2019 (PMRV-GCCDC7), in the Qinghai-Tibet Plateau wild pika (Ochotona curzoniae). Though the PMRV-GCCDC7 shows features of a typical reovirus with ten gene segments arranged in 3:3:4 in length, the virus belongs to an independent evolutionary branch compared to other MRVs based on phylogenetic tree analysis. The results of cellular susceptibility, species tropism, and replication kinetics of PMRV-GCCDC7 indicated the virus could infect four human cell lines (A549, Huh7, HCT, and LoVo) and six non-human cell lines, including Vero-E6, LLC-MK2, BHK-21, N2a, MDCK, and RfKT cell, derived from diverse mammals, i.e. monkey, mice, canine and bat, which revealed the potential of PMRV-GCCDC7 to infect a variety of hosts. Infection of BALB/c mice with PMRV-GCCDC7 via intranasal inoculation led to relative weight loss, lung tissue damage and inflammation with the increase of virus titer, but no serious respiratory symptoms and death occurred. The characterization of the new reovirus from a plateau-based wild animal has expanded our knowledge of the host range of MRV and provided insight into its risk of trans-species transmission and zoonotic diseases.

Children With Chinese Dyslexia Acquiring English Literacy: Interaction Between Cognitive Subtypes of Dyslexia and Orthographies.

This study investigated the impact of Chinese dyslexia subtypes on English literacy skills (i.e., reading fluency and dictation) in Hong Kong children. Eighty-four Cantonese-speaking children officially diagnosed with dyslexia (Mage = 103 months) and 48 age-matched typically developing (TD) children were tested. Cluster analysis with performances on Chinese syllable awareness (CSA), Chinese phonemic awareness (CPA), Chinese phonological memory (CPM), Chinese orthographic awareness (COA), and matrix reasoning (MR) yielded three cognitive subtypes: the phonological deficit (PD) subtype, the orthographic deficit (OD) subtype, and the global deficit (GD) subtype. After controlling for English language experience, age, and gender, all three dyslexia subtype groups performed significantly worse in English word reading fluency and dictation than the TD children. In addition, the PD group performed worse in English PA; the OD group performed worse in English OA; and the GD group performed worse in all English skills except English PM. We compared the level of impairment in literacy between languages and dyslexia subtypes. In word reading fluency, all subtype groups experienced less impairment in English than Chinese, while the OD group showed the largest English advantage. In dictation, only the OD group showed a significant language effect favoring English. The findings suggest that different subtypes of Chinese dyslexia bear different risks for difficulties in English literacy.

Behavioral and neurophysiological aspects of working memory impairment in children with dyslexia.

The present study aimed to identify behavioral and neurophysiological correlates of dyslexia which could potentially predict reading difficulty. One hundred and three Chinese children with and without dyslexia (Grade 2 or 3, 7- to 11-year-old) completed both verbal and visual working memory (n-back) tasks with concurrent EEG recording. Data of 74 children with sufficient usable EEG data are reported here. Overall, the typically developing control group (N = 28) responded significantly faster and more accurately than the group with dyslexia (N = 46), in both types of tasks. Group differences were also found in EEG band power in the retention phase of the tasks. Moreover, forward stepwise logistic regression demonstrated that both behavioral and neurophysiological measures predicted reading difficulty uniquely. Dyslexia was associated with higher frontal midline theta activity and reduced upper-alpha power in the posterior region. This finding is discussed in relation to the neural efficiency hypothesis. Whether these behavioral and neurophysiological patterns can longitudinally predict later reading development among preliterate children requires further investigation.

Spatial-temporal heterogeneity and determinants of HIV prevalence in the Mano River Union countries.

BACKGROUND: Utilizing population-based survey data in epidemiological research with a spatial perspective can integrate valuable context into the dynamics of HIV prevalence in West Africa. However, the situation in the Mano River Union (MRU) countries is largely unknown. This research aims to perform an ecological study to determine the HIV prevalence patterns in MRU. METHODS: We analyzed Demographic and Health Survey (DHS) and AIDS Indicator Survey (AIS) data on HIV prevalence in MRU from 2005 to 2020. We examined the country-specific, regional-specific and sex-specific ratios of respondents to profile the spatial-temporal heterogeneity of HIV prevalence and determine HIV hot spots. We employed Geodetector to measure the spatial stratified heterogeneity (SSH) of HIV prevalence for adult women and men. We assessed the comprehensive correct knowledge (CCK) about HIV/AIDS and HIV testing uptake by employing the Least Absolute Shrinkage and Selection Operator (LASSO) regression to predict which combinations of CCKs can scale up the ratio of HIV testing uptake with sex-specific needs. RESULTS: In our analysis, we leveraged data for 158,408 respondents from 11 surveys in the MRU. From 2005-2015, Cote d'Ivoire was the hot spot for HIV prevalence with a Gi_Bin score of 3, Z-Score 8.0-10.1 and P < 0.001. From 2016 to 2020, Guinea and Sierra Leone were hot spots for HIV prevalence with a Gi_Bin score of 2, Z-Score of 3.17 and P < 0.01. The SSH confirmed the significant differences in HIV prevalence at the national level strata, with a higher level for Cote d'Ivoire compared to other countries in both sexes with q-values of 0.61 and 0.40, respectively. Our LASSO model predicted different combinations of CCKs with sex-specific needs to improve HIV testing uptake. CONCLUSIONS: The spatial distribution of HIV prevalence in the MRU is skewed and the CCK about HIV/AIDS and HIV testing uptake are far below the threshold target set by UNAIDS for ending the epidemic in the sub-region. Geodetector detected statistically significant SSH within and between countries in the MRU. Our LASSO model predicted that different emphases should be implemented when popularizing the CCK about HIV/AIDS for adult women and men.

Development of two multiplex real-time PCR assays for simultaneous detection and differentiation of monkeypox virus IIa, IIb, and I clades and the B.1 lineage.

An ongoing multi-country outbreak of monkeypox was reported in May 2022 with several deaths, affecting 107 countries of all six World Health Organization (WHO) regions. The WHO has declared the current monkeypox outbreak to be a Public Health Emergency of International Concern. It is, thus, necessary to rapidly and accurately detect and distinguish different monkeypox virus (MPXV) clades. We designed primers and probes based on the alignment of 138 complete genomes of poxviruses. In Panel 1, we mixed one pair of primers and three probes to detect and differentiate the MPXV Western Africa (IIa, IIb clade) and Congo Basin (I clade) and other orthopoxviruses. In Panel 2, we mixed one pair of primers and two probes to detect the 2022 MPXV (B.1 lineage and its descendant lineages). In addition, we tested the specificity and sensitivity of the assay using real-time PCR. In Panel 1, the assay reproducibly identified various concentrations of two plasmids of the monkeypox virus, whereas other orthopoxviruses did not cross-react. In Panel 2, the probe annealed well to MPXV B.1 and showed the expected linearity. These two multiple real-time assays are inclusive and highly specific for identifying different clades of MPXV.