Genomic phylogenetic analyses of four major hand, foot and mouth disease-related enteroviruses.

Enteroviruses had diverged into many types, some of which cause hand, foot and mouth disease (HFMD) in children. The predominant enterovirus types associated with HFMD are EVA71, CVA16, CVA6 and CVA10. Four enterovirus types were classified into subtypes based on VP1 sequences. However, the phylogenetics of these enteroviruses is rarely concerned at the genomic level. In this study, we performed the phylogenetic analyses of the EVA71, CVA16, CVA6 and CVA10 using available full-length genomic sequences. We found that the topologies of phylogenetic trees of full-length genomic sequences and VP1 sequences were almost consistent, except few subtypes of EVA71 and CVA10. The mean genetic divergence was 15.8-27% between subtypes and less than 12% within subtypes/sub-subtypes at genomic level. Comparison of phylogenetic topologies between genomic and VP1 sequences helped us to identify two new EVA71 inter-subtype recombinants RF01_CC4 and RF02_CC4. Furthermore, EVA71 subtypes C1 and C2 and CVA10 subtype D were found to originate through inter-subtype recombination. The genomic reference sequences of these enteroviruses are provided here for subtyping. The results provide important insights into the understanding of the evolution and epidemiology of the four enteroviruses. Keywords: enterovirus; hand; foot and mouth disease; classification; genetic distance; recombination.

A rapid variant-tolerant reverse transcription loop-mediated isothermal amplification assay for the point of care detection of HIV-1.

Human immunodeficiency virus (HIV) continues to be a major burden on public health globally with on-going increases in the number of new infections each year. Rapid and sensitive point-of-care tests allow timely interventions and are essential to control the spread of the disease. However the highly variable nature of the virus, resulting in the evolution of many subtypes and inter-subtype recombinants, poses important challenges for its diagnosis. Here we describe a variant-tolerant reverse-transcription RT-LAMP amplification of the virus's INT gene, providing a simple to use, rapid (<30 min) in vitro point-of-care diagnostic test with a limit of detection <18 copies/reaction. The assay was first validated in clinical studies of patient samples, using both established RT-LAMP and RT-qPCR assays for reference, with results showing that this new variant-tolerant HIV-1 RT-LAMP diagnostic test is highly sensitive without compromising its high specificity for HIV-1 subtypes. The diagnostic test was subsequently configured within an easy-to-read paper microfluidic lateral flow test and was validated clinically using patient samples, demonstrating its future potential for use in timely, effective, low cost HIV diagnostics in global regions where healthcare resources may be limited.

Evolutionary dynamics of group A and B respiratory syncytial virus in China, 2009-2018.

Respiratory syncytial virus (RSV) is a major cause of acute respiratory tract infections in children and is a public health threat globally. To investigate the spatiotemporal dynamics of RSV evolution, we performed systematic phylogenetic analysis using all available sequences from the GenBank database, together with sequences from Shanghai, China. Both RSV-A and RSV-B appear to have originated in North America, with an inferred origin time of 1954.0 (1938.7-1967.6) and 1969.7 (1962.6-1975.5), respectively. BA-like strains of RSV-B, with a 60-nt insertion, and the ON1 strain of RSV-A, with a 72-nt insertion, emerged in 1997.6 (1996.2-1998.6) and 2010.1 (2009.1-2010.3), respectively. Since their origin, both genotypes have gradually replaced the former circulating genotypes to become the dominant strain. The population dynamic of RSV-A showed a seasonal epidemic pattern with obvious expansion in the periods of 2006-2007, 2010-2011, 2011-2012, and 2013-2014. Thirty fixed amino acid substitutions were identified during the divergence of NA4 from GA1 genotypes of RSV-A, and 13 were found during the divergence of SAB4 from GB1 of RSV-B. Importantly, ongoing evolution has occurred since the emergence of ON1, including four amino acid substitutions (I208L, E232G, T253K, and P314L). RSV-A genotypes GA5, NA4, NA1, and ON1 and RSV-B genotypes CB1, SAB4, BA-C, BA10, BA7, and BA9 were co-circulating in China from 2005 to 2015. In particular, RSV-A genotype ON1 was first detected in China in 2011, and it completely replaced GA2 to become the predominant strain after 2016. These data provide important insights into the evolution and epidemiology of RSV.

Single-tube detection of nine bacterial antibiotic-resistance genes by a 2-dimensional multiplex qPCR assay based on fluorescence and melting temperature.

Simple, multiplex qPCR methods are advantages for rapid molecular diagnosis of multiple antibiotics-resistant genes simultaneously. However, the number of genes can be detected in a single reaction tube is often limited by the fluorescence channels of a real-time PCR instrument. In this study, we developed a simple 2-D multiplex qPCR method by combining the probe colors and amplicon Tm values to overcome the mechanical limit of the machine. The principle of the novel assay was validated by detection of nine bacterial antibiotic-resistance genes (KPC, NDM, VIM, OXA-48, GES, CIT, EBC, ACC and DHA) in a single reaction tube. This assay is highly sensitive within a range of 30-3000 copies per reaction. The simplicity, rapidity, high sensitivity and specificity, and low cost of the novel method make it a promising tool for developing clinical diagnostic kits for monitoring resistance and other genetic determinants of infectious diseases.

A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2.

COVID-19 has become a major global public health burden, currently causing a rapidly growing number of infections and significant morbidity and mortality around the world. Early detection with fast and sensitive assays and timely intervention are crucial for interrupting the spread of the COVID-19 virus (SARS-CoV-2). Using a mismatch-tolerant amplification technique, we developed a simple, rapid, sensitive and visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection based on its N gene. The assay has a high specificity and sensitivity, and robust reproducibility, and its results can be monitored using a real-time PCR machine or visualized via colorimetric change from red to yellow. The limit of detection (LOD) of the assay is 118.6 copies of SARS-CoV-2 RNA per 25 µL reaction. The reaction can be completed within 30 min for real-time fluorescence monitoring, or 40 min for visual detection when the template input is more than 200 copies per 25 µL reaction. To evaluate the viability of the assay, a comparison between the RT-LAMP and a commercial RT-qPCR assay was made using 56 clinical samples. The SARS-CoV-2 RT-LAMP assay showed perfect agreement in detection with the RT-qPCR assay. The newly-developed SARS-CoV-2 RT-LAMP assay is a simple and rapid method for COVID-19 surveillance.

AMPH-1 is a tumor suppressor of lung cancer by inhibiting Ras-Raf-MEK-ERK signal pathway.

Amphiphysin 1 (AMPH-1) is a nerve terminal-enriched protein and it is a 128-kD protein with three identified functional domains. Some studies found that AMPH-1 was a dominant autoantigen associated with breast cancer and melanoma. However, its function in lung cancer is unknown. Here, we showed that AMPH-1 knockdown dramatically increased cell proliferation, attenuated cell apoptosis, and promoted cell cycle progression in human lung cancer cells. In vivo xenograft studies confirmed that the AMPH-1-knockdown cells were more tumorigenic than the controls. Moreover, we demonstrated that silencing AMPH-1 markedly activated Ras-Raf-MEK-ERK signal pathway. In summary, our results identified the anti-oncogenic function of AMPH-1 in lung cancer in vitro and in vivo. It is proposed that AMPH-1 may have potential as a new therapeutic target in human lung cancer treatment.

Subtype distribution of hepatitis C virus in Jiangsu, China.

Hepatitis C virus (HCV) genotype distribution varied by regions and transmission modes. In this study, we investigated HCV genotype distribution in five cities of Jiangsu, China, all of which are located in the Yangtze River Delta Region. A total of 363 samples were collected during 2011-2012. C/E2 and NS5B fragments of HCV were amplified using a multiple RT-nested PCR strategy and subjected to sequencing. Phylogenetic analysis was performed for HCV genotyping. Among 106 PCR positive cases, HCV subtypes 1a (0.9%), 1b (61.3%), 2a (15.1%), 3a (4.7%), 3b (9.4%), 6a (6.6%), and 6n (1.9%) were detected. Together with our previous data, we found that HCV subtypes were more among injection drug users (IDUs) (nine) than among general population (GP) (six), and the most common subtype among GP was 1b (73.9%), followed by 2a (14.5%), while the top four common subtypes among IDUs were 3a, 1b, 3b, and 6a, with similar prevalence rates (24.4%, 22.7%, 20.9%, and 17.4%, respectively). There were nine HCV subtypes prevalent among IDUs in Jiangsu, more than those in Xinjiang, Hubei, Yunnan, Guangxi, Guangdong, and Hong Kong. The top four common subtypes among IDUs in Jiangsu covered all the two most common HCV subtypes (except 6n subtype) observed in six targeted provinces/region. These results suggested that Jiangsu may be an important gathering place for various HCV subtypes and the gathering may be involved in the large scale of population migration from other regions of China to Eastern China.

A Melting Curve-Based Multiplex RT-qPCR Assay for Simultaneous Detection of Four Human Coronaviruses.

Human coronaviruses HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1 are common respiratory viruses associated with acute respiratory infection. They have a global distribution. Rapid and accurate diagnosis of HCoV infection is important for the management and treatment of hospitalized patients with HCoV infection. Here, we developed a melting curve-based multiplex RT-qPCR assay for simultaneous detection of the four HCoVs. In the assay, SYTO 9 was used to replace SYBR Green I as the fluorescent dye, and GC-modified primers were designed to improve the melting temperature (Tm) of the specific amplicon. The four HCoVs were clearly distinguished by characteristic melting peaks in melting curve analysis. The detection sensitivity of the assay was 3 × 10² copies for HCoV-OC43, and 3 × 10¹ copies for HCoV-NL63, HCoV-229E and HCoV-HKU1 per 30 µL reaction. Clinical evaluation and sequencing confirmation demonstrated that the assay was specific and reliable. The assay represents a sensitive and reliable method for diagnosis of HCoV infection in clinical samples.

How to report and make sense of a new HIV-1 circulating recombinant form?

Co-circulation of multiple HIV-1 subtypes in the same high-risk groups leads to the on-going generation of various inter-subtype recombinants, including unique (URFs) and circulating (CRFs) recombinant forms, which brings a new challenge for the prevention and eradication of HIV/AIDS. Identification and prompt reporting of new CRFs will provide not only new insights into the understanding of genetic diversity and evolution of HIV-1, but also an early warning of potential prevalence of these variants. Currently, 140 HIV-1 CRFs have been described; however, their prevalence and clinical importance are less concerned. Apart from the mosaic genomic maps, less other valuable information, including the clinical and demographic data, genomic sequence characteristics, origin and evolutionary dynamics, as well as representative genomic fragments for determining the variants, are available for most of these CRFs. Accompanied with the growing increase of HIV-1 full-length genomic sequences, more and more CRFs will be identified in the near future due to the high recombination potential of HIV-1. Here, we discuss the prevalence and clinical importance of various HIV-1 CRFs and propose how to report and make sense of a new HIV-1 CRF.

Genomic Characteristics of the New HIV-1 CRF07_BC K28E32 Variant.

Accompanied with the appearance and prevalence of the new K28E32 variant among men who have sex with men, HIV-1 circulating recombinant form 07_BC (CRF07_BC) was becoming the most predominant subtype circulating in China. The K28E32 variant with five specific mutations in reverse transcriptase coding region appears to have significantly higher in vitro HIV-1 replication ability than the wild-type strain. In this study, we characterized the special mutations/substitutions in the K28E32 variant at the genomic level. Ten specific mutations that rarely appeared in other six main HIV-1 subtypes/CRFs (A-D, CRF01_AE, and CRF02_AG) were identified in the coding genes/regions of the K28E32 variant, including S77L and a novel seven-amino acid detection (32DKELYPL38) (p6Δ7) in p6, I135L in integrase, T189S in Vif, H/Y15L/F in Vpr, I264V/A and LV/LI328-329VG in gp41, and H82C and S97P in Rev. The special locations of the novel p6Δ7, and gp41 mutations I264V/A and LV/LI328-329VG in crucial protein functional domains suggest that these mutations might be functionally important to the K28E32 variant. Furthermore, eight specific substitutions were identified in Rev responsive element (RRE) of the K28E32 variant, and were revealed to increase the stability of RRE structure with a lower minimum free energy. Whether these mutations/substitutions contribute to improved transmissibility of the CRF07_BC K28E32 variant needs to be further confirmed.

A novel extraction-free dual HiFi-LAMP assay for detection of methicillin-sensitive and methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is a leading cause of bacteremia and blood stream infections. Methicillin-resistant S. aureus (MRSA) that first appeared in 1961 often caused hospital-acquired infections (HAIs) and community-acquired infections (CAIs) and was associated with high mortality rate. Accurate and rapid point-of-care testing (POCT) of MRSA is crucial for clinical management and treatment of MRSA infections, as well as the prevention and control of HAIs and CAIs. Here, we reported a novel extraction-free dual HiFi-LAMP assay for discriminative detection of methicillin-susceptible S. aureus and MRSA. The dual HiFi-LAMP assay can detect 30 copies/reaction of nuc and mecA genes with detection limits of 147 and 158 copies per 25 µL reaction, respectively. A retrospective clinical evaluation with 107 clinical S. aureus isolates showed both sensitivity and specificity of 100%. A prospective clinical evaluation with 35 clinical samples revealed a specificity of 100% and a sensitivity of 92.3%. The dual HiFi-LAMP assay can detect almost all S. aureus samples (141/142; 99.3%) within 20 min, implying that the entire HiFi-LAMP assay (including sample process) can be completed within 40 min, extremely significantly shorter than 3-5 days by the traditional clinical microbial culture and antibiotic susceptibility testing. The novel extraction-free dual HiFi-LAMP assay can be used as a robust POCT tool to promote precise diagnosis and treatment of MRSA infections in hospitals and to facilitate surveillance of MRSA at hospital and community settings.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) was associated with high mortality rate and listed as a "priority pathogen" by the World Health Organization. Accurate and rapid point-of-care testing (POCT) of MRSA is critically required for clinical management and treatment of MRSA infections. Some previous LAMP-based POCT assays for MRSA might be questionable due to their low specificity and the lack of appropriate evaluation directly using clinical samples. Furthermore, they are relatively tedious and time-consuming because they require DNA extraction and lack multiplex detection capacity. Here, we reported a novel extraction-free dual HiFi-LAMP assay for discriminative detection of MRSA and methicillin-susceptible S. aureus. The assay has high specificity and sensitivity and can be completed within 40 min. Clinical evaluation with real clinical samples and clinical isolates showed excellent performance with 100% specificity and 92.3%-100% sensitivity. The novel extraction-free assay may be a robust POCT tool to promote precise diagnosis of MRSA infections and facilitate surveillance of MRSA at hospital and community settings.

Longitudinal anellome dynamics in the upper respiratory tract of children with acute respiratory tract infections.

Anelloviruses (AVs) are ubiquitous in humans and are the most abundant components of the commensal virome. Previous studies on the diversity, transmission, and persistence of AVs mainly focused on the blood or transplanted tissues from adults; however, the profile of the anellome in the respiratory tract in children are barely known. We investigated the anellome profile and their dynamics in the upper respiratory tract from a cohort of children with acute respiratory tract infections (ARTIs). Different to that in adult, betatorquevirus is the most abundant genus, followed by alphatorquevirus. We found that the relative abundance of betatorquevirus was higher in earlier time points, and in contrast, the abundance of alphatorquevirus was higher in later time points; these results might suggest that betatorquevirus decreased with age and alphatorquevirus increased with age in childhood. No difference regarding the diversity and abundance of anellome was found between single and multiple ARTIs, consistent with the idea that AV is not associated with certain disease. Most AVs are transient, and a small proportion (8 per cent) of them were found to be possibly persistent, with persistence time ranging from 1 month to as long as 56 months. Furthermore, the individual respiratory anellome appeared to be unique and dynamic, and the replacement of existing AVs with new ones are common over different time points. These findings demonstrate that betatorquevirus may be the early colonizer in children, and the individual respiratory anellome is unique, which are featured by both chronic infections and AV community replacement.

Rapid detection of human influenza A viruses by HFman probe-based loop-mediated isothermal amplification assays.

Since China abandoned the zero-COVID policy at the end of 2022, a wave of severe Flu pandemic emerged in China. Rapid and accurate diagnosis of Influenza A virus (IAV) is critical for clinical management and therapeutic decision-making of patients with fever. Here, we reported a novel IAV HF-LAMP assay, which can be performed with purified RNA or directly using clinical samples. The assays with purified RNA and clinical samples have high sensitivity with limit of detection (LOD) of 9.6 copies/reaction, 9900 copies/mL, and short sample-to-answer times of 36 and 50 min, respectively. Both assays showed high specificity and significantly higher IAV detection rate than the rapid antigen detection (RAD) assays. Furthermore, we found the vast majority (91.2 %) of children with fever during the pandemic were infected by IAV, and current IAV infection has a very narrow detectable window. The novel IVA HF-LAMP assays will provide robust tools to facilitate early diagnosis of IAV infection in current and future seasonal influenza epidemics.

Review of human pegivirus: Prevalence, transmission, pathogenesis, and clinical implication.

Human pegivirus (HPgV-1), previously known as GB virus C (GBV-C) or hepatitis G virus (HGV), is a single-stranded positive RNA virus belonging to the genus Pegivirus of the Flaviviridae family. It is transmitted by percutaneous injuries (PIs), contaminated blood and/or blood products, sexual contact, and vertical mother-to-child transmission. It is widely prevalent in general population, especially in high-risk groups. HPgV-1 viremia is typically cleared within the first 1-2 years of infection in most healthy individuals, but may persist for longer periods of time in immunocompromised individuals and/or those co-infected by other viruses. A large body of evidences indicate that HPgV-1 persistent infection has a beneficial clinical effect on many infectious diseases, such as acquired immunodeficiency syndrome (AIDS) and hepatitis C. The beneficial effects seem to be related to a significant reduction of immune activation, and/or the inhabitation of co-infected viruses (e.g. HIV-1). HPgV-1 has a broad cellular tropism for lymphoid and myeloid cells, and preferentially replicates in bone marrow and spleen without cytopathic effect, implying a therapeutic potential. The paper aims to summarize the natural history, prevalence and distribution characteristics, and pathogenesis of HPgV-1, and discuss its association with other human viral diseases, and potential use in therapy as a biovaccine or viral vector.

COVID-19 public health measures reduce the incidence of respiratory infectious diseases.

BACKGROUND: Children and the elderly are two special subpopulations for coronavirus disease 2019 (COVID-19) and respiratory tract infections (RTIs). The study aimed to evaluate the effect of COVID-19 public health measures on the burden of RTIs in China by performing a two-center investigation. METHODS: The electronic medical records of all inpatients in departments of pediatrics and respiratory medicine of Taizhou Fourth People's Hospital (Taizhou, China) and Shaanxi Provincial People's Hospital (Xi'an, China) during January 1, 2019 to June 30, 2021 were analyzed. A total of 18,084 child inpatients and 14,802 adult inpatients were included. RESULTS: The vast majority (88.3%-90.6%) of the adult inpatients were the elderly, aged over 50 years. The numbers of child and adult (elderly) inpatients, and the proportions of RTI-associated diseases substantially decreased during COVID-19 pandemic (2020-2021) compared to that before the pandemic (2019) in Taizhou and Xi'an. A significantly higher proportion of LRTI-associated diseases was observed in elderly female inpatients (53.4-55.6%) than elderly male inpatients (34.3-41.5%) (p < 0.001) in spite of more male inpatients than female inpatients (1.94-1.95:1). CONCLUSIONS: COVID-19-related interventions provide an additional beneficial effect on reduction of RTI-associated diseases in both children and the elderly.

Multiplex, Real-Time, Point-of-care RT-LAMP for SARS-CoV-2 Detection Using the HFman Probe.

Viral evolution impacts diagnostic test performance through the emergence of variants with sequences affecting the efficiency of primer binding. Such variants that evade detection by nucleic acid-based tests are subject to selective pressure, enabling them to spread more efficiently. Here, we report a variant-tolerant diagnostic test for SARS-CoV-2 using a loop-mediated isothermal nucleic acid-based amplification (LAMP) assay containing high-fidelity DNA polymerase and a high-fidelity DNA polymerase-medicated probe (HFman probe). In addition to demonstrating a high tolerance to variable SARS-CoV-2 viral sequences, the mechanism also overcomes frequently observed limitations of LAMP assays arising from non-specific amplification within multiplexed reactions performed in a single "pot". Results showed excellent clinical performance (sensitivity 94.5%, specificity 100%, n = 190) when compared directly to a commercial gold standard reverse transcription quantitative polymerase chain reaction assay for the extracted RNA from nasopharyngeal samples and the capability of detecting a wide range of sequences containing at least alpha and delta variants. To further validate the test with no sample processing, directly from nasopharyngeal swabs, we also detected SARS-CoV-2 in positive clinical samples (n = 49), opening up the possibility for the assay's use in decentralized testing.

Comparative evaluation of 19 reverse transcription loop-mediated isothermal amplification assays for detection of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has caused a global pandemics. To facilitate the detection of SARS-CoV-2 infection, various RT-LAMP assays using 19 sets of primers had been developed, but never been compared. We performed comparative evaluation of the 19 sets of primers using 4 RNA standards and 29 clinical samples from COVID-19 patients. Six of 15 sets of primers were firstly identified to have faster amplification when tested with four RNA standards, and were further subjected to parallel comparison with the remaining four primer sets using 29 clinical samples. Among these 10 primer sets, Set-4 had the highest positive detection rate of SARS-CoV-2 (82.8%), followed by Set-10, Set-11, and Set-13 and Set-17 (75.9%). Set-14 showed the fastest amplification speed (Tt value < 8.5 min), followed by Set-17 (Tt value < 12.5 min). Based on the overall detection performance, Set-4, Set-10, Set-11, Set-13, Set-14 and Set-17 that target Nsp3, S, S, E, N and N gene regions of SARS-CoV-2, respectively, were determined to be better than the other primer sets. Two RT-LAMP assays with the Set-4 primers in combination with any one of four other primer sets (Set-14, Set-10, Set-11, and Set-13) were recommended to be used in the COVID-19 surveillance.

A Novel 2-dimensional Multiplex qPCR Assay for Single-Tube Detection of Nine Human Herpesviruses.

Human herpesviruses are double-stranded DNA viruses that are classified into nine species. More than 90% of adults are ever infected with one or more herpesviruses. The symptoms of infection with different herpesviruses are diverse ranging from mild or asymptomatic infections to deadly diseases such as aggressive lymphomas and sarcomas. Timely and accurate detection of herpesvirus infection is critical for clinical management and treatment. In this study, we established a single-tube nonuple qPCR assay for detection of all nine herpesviruses using a 2-D multiplex qPCR method with a house-keeping gene as the internal control. The novel assay can detect and distinguish different herpesviruses with 30 to 300 copies per 25 µL single-tube reaction, and does not cross-react with 20 other human viruses, including DNA and RNA viruses. The robustness of the novel assay was evaluated using 170 clinical samples. The novel assay showed a high consistency (100%) with the single qPCR assay for HHVs detection. The features of simple, rapid, high sensitivity, specificity, and low cost make this assay a high potential to be widely used in clinical diagnosis and patient treatment.

Temporal association between human upper respiratory and gut bacterial microbiomes during the course of COVID-19 in adults.

SARS-CoV-2 is the cause of COVID-19. It infects multiple organs including the respiratory tract and gut. Dynamic changes of regional microbiomes in infected adults are largely unknown. Here, we performed longitudinal analyses of throat and anal swabs from 35 COVID-19 and 19 healthy adult controls, as well as 10 non-COVID-19 patients with other diseases, by 16 S rRNA gene sequencing. The results showed a partitioning of the patients into 3-4 categories based on microbial community types (I-IV) in both sites. The bacterial diversity was lower in COVID-19 patients than healthy controls and decreased gradually from community type I to III/IV. Although the dynamic change of microbiome was complex during COVID-19, a synchronous restoration of both the upper respiratory and gut microbiomes from early dysbiosis towards late more diverse status was observed in 6/8 mild COVID-19 adult patients. These findings reveal previously unknown interactions between upper respiratory and gut microbiomes during COVID-19.