Evaluations of modes of pooling specimens for COVID-19 screened by quantitative PCR and droplet digital PCR.

Though pooling samples for SARS-CoV-2 detection has effectively met the need for rapid diagnostic and screening tests, many factors can influence the sensitivity of a pooled test. In this study, we conducted a simulation experiment to evaluate modes of pooling specimens and aimed at formulating an optimal pooling strategy. We focussed on the type of swab, their solvent adsorption ability, pool size, pooling volume, and different factors affecting the quality of preserving RNA by different virus solutions. Both quantitative PCR and digital PCR were used to evaluate the sampling performance. In addition, we determined the detection limit by sampling which is simulated from the virus of different titers and evaluated the effect of sample-storage conditions by determining the viral load after storage. We found that flocked swabs were better than fibre swabs. The RNA-preserving ability of the non-inactivating virus solution was slightly better than that of the inactivating virus solution. The optimal pooling strategy was a pool size of 10 samples in a total volume of 9 mL. Storing the collected samples at 4 °C or 25 °C for up to 48 h had little effect on the detection sensitivity. Further, we observed that our optimal pooling strategy performed equally well as the single-tube test did. In clinical applications, we recommend adopting this pooling strategy for low-risk populations to improve screening efficiency and shape future strategies for detecting and managing other respiratory pathogens, thus contributing to preparedness for future public health challenges.

Severe pigeon paramyxovirus 1 infection in a human case with probable post-COVID-19 condition.

Pigeon paramyxovirus 1 (PPMV-1) is an antigenic host variant of avian paramyxovirus 1. Sporadic outbreaks of PPMV-1 infection have occurred in pigeons in China; however, few cases of human PPMV-1 infection have been reported. The purpose of this article is to report a case of severe human PPMV-1 infection in an individual with probable post-COVID-19 syndrome (long COVID) who presented with rapidly progressing pulmonary infection. The patient was a 66-year-old man who was admitted to the intensive care unit 11 days after onset of pneumonia and recovered 64 days after onset. PPMV-1 was isolated from the patient's sputum and in cloacal smear samples from domesticated pigeons belonging to the patient's neighbour. Residual severe acute respiratory syndrome coronavirus 2 was detected in respiratory and anal swab samples from the patient. Sequencing analyses revealed that the PPMV-1 genome belonged to genotype VI.2.1.1.2.2 and had the 112RRQKRF117 motif in the cleavage site of the fusion protein, which is indicative of high virulence. This case of cross-species transmission of PPMV-1 from a pigeon to a human highlights the risk of severe PPMV-1 infection in immunocompromised patients, especially those with long COVID. Enhanced surveillance for increased risk of severe viral infection is warranted in this population.

A Comparison of Clinical Characteristics of Infections with SARS-CoV-2 Omicron Subvariants BF.7.14 and BA.5.2.48 - China, October-December 2022.

What is already known about this topic?: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, the clinical manifestations resulting from different SARS-CoV-2 variants may demonstrate significant variation. What is added by this report?: We conducted a comparative analysis of the clinical features associated with SARS-CoV-2 Omicron subvariants BF.7.14 and BA.5.2.48 infections. The results of our study indicate that there are no substantial differences in clinical manifestations, duration of illness, healthcare-seeking behaviors, or treatment between these two subvariants. What are the implications for public health practice?: Timely identification of alterations in the clinical spectrum is crucial for researchers and healthcare practitioners in order to enhance their comprehension of clinical manifestations, as well as the progression of SARS-CoV-2. Furthermore, this information is beneficial for policymakers in the process of revising and implementing appropriate countermeasures.

Epidemiological features, genomic characteristics, and origin tracing of the COVID-19 outbreaks in Beijing from January to September 2022.

Different variants of severe acute respiratory syndrome coronavirus 2 have been discovered globally. At present, the Omicron variant has been extensively circulated worldwide. There have been several outbreaks of the Omicron variant in China. Here, we investigated the epidemiologic, genetic characteristics, and origin-tracing data of the outbreaks of COVID-19 in Beijing from January to September 2022. During this time, 19 outbreaks occurred in Beijing, with the infected cases ranging from 2 to 2230. Two concern variants were detected, with eight genotypes. Based on origin tracing analysis, two outbreaks were from the cold-chain transmission and three from items contaminated by humans. Imported cases have caused other outbreaks. Our study provided a detailed analysis of Beijing's outbreaks and valuable information to control the outbreak's spread.

Screening and Analysis of Serum Protein Biomarkers Infected by Coronavirus Disease 2019 (COVID-19).

Coronavirus disease 2019 (COVID-19) has spread widely around the world, and in-depth research on COVID-19 is necessary for biomarkers and target drug discovery. This analysis collected serum from six COVID-19-infected patients and six healthy people. The protein changes in the infected and healthy control serum samples were evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high-performance liquid chromatography (HPLC). The differential protein signature in both groups was retrieved and analyzed by the Kyoto Encyclopedia of Gene and Genomes (KEGG), Gene ontology, COG/KOG, protein-protein interaction, and protein domain interactions tools. We shortlisted 24 differentially expressed proteins between both groups. Ten genes were significantly up-regulated in the infection group, and fourteen genes were significantly down-regulated. The GO and KEGG pathway enrichment analysis suggested that the chromosomal part and chromosome were the most enriched items. The oxytocin signaling pathway was the most enriched item of KEGG analysis. The netrin module (non-TIMP type) was the most enriched protein domain in this study. Functional analysis of S100A9, PIGR, C4B, IL-6R, IGLV3-19, IGLV3-1, and IGLV5-45 revealed that SARS-CoV-2 was closely related to immune response.

Detection by environmental surveillance and genomic characterization of H5N8 highly pathogenic avian influenza virus from a poultry meat market in Beijing, China, 2021-22.

Since 2010 the year when it was first reported in domestic ducks in China, highly pathogenic avian influenza (HPAI) H5N8 has caused several outbreaks in different countries. The first outbreak wave was documented in South Korea and Japan in 2014 and the second wave was reported in Asian and European countries in 2016. More importantly, zoonotic infection was first reported in poultry workers in Russia in 2021. Therefore, active surveillance on H5N8 is highly needed. Surveillance on live birds instead of environmental samples is commonly reported. In the present study, we reported detection and genomic characterization of an environmental H5N8 strain in environmental samples of Tongzhou poultry meat markets in Beijing on a monthly basis from March 2021 to February 2022. Among 600 samples screened, a total of 27 samples were positive for influenza A virus with 4 typed as H5N8, 10 H7N9, and 13 H9N2. Whole genome sequencing and analysis of one duck neck with a higher virus load showed that A/Environment sample/Beijing/TZ001/20 21 (H5N8) clade 2.3.4.4b had the highest identities (over 99%) in all eight segments with H5N8 isolates from wild birds swan and tern in Hubei and had polybasic cleavage site PLREKRRKR/G, characteristic of a HPAI virus. Overall, our data indicate that HPAI H5N8 virus is still circulating in domestic ducks in China in the study period and continued surveillance in domestic and wild birds is needed to control H5N8.

An Updated Review on SARS-CoV-2 Infection in Animals.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has lasted for two years and caused millions of infections and deaths in humans. Although the origin of SARS-CoV-2 infection in humans remains unknown, infection in animals has been frequently reported in varieties of animals all over the world. Both experimental and natural infections of SARS-CoV-2 in different animal species provide useful information on viral host range and pathogenicity. As the pandemic continues to evolve, SARS-CoV-2 infection in animals will be expanding. In this review, we summarized SARS-CoV-2 testing and infection in animals as well as SARS-CoV-2 strains and transmission in animals. Current data showed that at least 18 different animal species tested positive for SARS-CoV-2. These 18 animal species belong to pet, captive, farmed, and wild animals. Fifteen of the eighteen animal species were known to be positive for the Delta variant and ten animal species were infected with two different types of variants. Human-to-animal, animal-to-animal, and animal-to-human transmission events were suggested in different outbreaks involved in animal infection with SARS-CoV-2. Continued testing, immunization, and surveillance are warranted.

Genomic characteristics of SARS-CoV-2 in Beijing, China, 2021.

At present, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread worldwide, which has emerged multiple variants and brought a threat to global public health. To analyze the genomic characteristics and variations of SARS-CoV-2 imported into Beijing, we collected the respiratory tract specimens of 112 cases of coronavirus disease 2019 (COVID-19) from January to September 2021 in Beijing, China, including 40 local cases and 72 imported cases. The whole-genome sequences of the viruses were sequenced by the next-generation sequencing method. Variant markers and phylogenic features of SARS-CoV-2 were analyzed. Our results showed that in all 112 sequences, the mutations were concentrated in spike protein. D614G was found in all sequences, and mutations including L452R, T478K, P681R/H, and D950N in some cases. Furthermore, 112 sequences belonged to 23 lineages by phylogenetic analysis. B.1.1.7 (Alpha) and B.1.617.2 (Delta) lineages were dominant. Our study drew a variation image of SARS-CoV-2 and could help evaluate the potential risk of COVID-19 for pandemic preparedness and response.

Importation of SARS-CoV-2 Omicron variant in Beijing, China.

Omicron (B.1.1.529), the fifth variant of concern (VOC) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was firstly identified in November 2021 in South Africa. Omicron contains far more genome mutations than any other VOCs ever found, raising significant concerns about its increased transmissibility and immune evasion. Here, we report the importation of the Omicron variant into Beijing, China, in December 2021. Full-length genome sequences of five imported strains were obtained, with their genetic features characterized. Each strain contained 57 to 61 nucleotide substitutions, 39 deletions, and 9 insertions in the genome. Thirty to thirty-two amino acid changes were found in the spike proteins of the five strains. The phylogenetic tree constructed by the maximum likelihood method showed that all five imported genomes belonged to Omicron (BA.1) (alias of B.1.1.529.1), which is leading to the current surge of coronavirus disease 2019 (COVID-19) cases worldwide. The globally increased COVID-19 cases driven by the Omicron variant pose a significant challenge to disease prevention and control in China. Continuous viral genetic surveillance and increased testing among international travellers are required to contain this highly contagious variant.

Comparison of four commercial RT-PCR diagnostic kits for COVID-19 in China.

We compared the sensitivity and specificity of four commercial coronavirus disease (COVID-19) diagnostic kits using real-time reverse transcription-polymerase chain reaction (RT-PCR). Kits I-IV approved by the State Drug Administration of China were selected, and the detection targets were ORF1ab gene and N gene. Specificity was evaluated by detecting other respiratory viruses. The sensitivity and batch effect of each kit were evaluated by testing 10-fold dilutions of RNA. Clinical application was verified by testing nasopharyngeal swab and sputum specimens from COVID-19 patients. Among the 78 cases infected by other respiratory viruses, no amplification curve was observed using these four COVID-19 RT-PCR kits. The minimum detection limits of kits I-IV were 10-6 , 10-5 , 10-5 , and 10-6 dilutions, respectively, and concentrations were 10 copies/mL (10-5 dilution) and 1 copies/mL (10-6 dilution). The sensitivities of kits I-IV detected using 142 nasopharyngeal swab specimens from COVID-19 patients were 91.55%, 81.69%, 80.28%, and 90.85%, respectively, while they were 92.68%, 85.37%, 82.93%, and 93.90%, respectively, for the 82 sputum samples. The specificity of each kit was 100.00% (77/77). The total expected detection rate using sputum samples was 88.59% (691/780) higher than 86.15% (672/780) of nasopharyngeal swabs. Comparison of nasopharyngeal swab and sputum samples from the same COVID-19 patient led to the detection of ORF1ab and N genes in 16 (100%) sputum samples; only ORF1ab and N genes were detected in 12 (75%) and 14 (87.5%) nasopharyngeal swab specimens, respectively. In conclusion, comparison of commercial COVID-19 RT-PCR kits should be performed before using a new batch of such kits in routine diagnostics.

Potential False-Negative Nucleic Acid Testing Results for Severe Acute Respiratory Syndrome Coronavirus 2 from Thermal Inactivation of Samples with Low Viral Loads.

BACKGROUND: Coronavirus disease-2019 (COVID-19) has spread widely throughout the world since the end of 2019. Nucleic acid testing (NAT) has played an important role in patient diagnosis and management of COVID-19. In some circumstances, thermal inactivation at 56°C has been recommended to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) before NAT. However, this procedure could theoretically disrupt nucleic acid integrity of this single-stranded RNA virus and cause false negatives in real-time polymerase chain reaction (RT-PCR) tests. METHODS: We investigated whether thermal inactivation could affect the results of viral NAT. We examined the effects of thermal inactivation on the quantitative RT-PCR results of SARS-CoV-2, particularly with regard to the rates of false-negative results for specimens carrying low viral loads. We additionally investigated the effects of different specimen types, sample preservation times, and a chemical inactivation approach on NAT. RESULTS: Our study showed increased Ct values in specimens from diagnosed COVID-19 patients in RT-PCR tests after thermal incubation. Moreover, about half of the weak-positive samples (7 of 15 samples, 46.7%) were RT-PCR negative after heat inactivation in at least one parallel testing. The use of guanidinium-based lysis for preservation of these specimens had a smaller impact on RT-PCR results with fewer false negatives (2 of 15 samples, 13.3%) and significantly less increase in Ct values than heat inactivation. CONCLUSION: Thermal inactivation adversely affected the efficiency of RT-PCR for SARS-CoV-2 detection. Given the limited applicability associated with chemical inactivators, other approaches to ensure the overall protection of laboratory personnel need consideration.

Enterovirus D68 in a 6-year-old acute flaccid myelitis case in China, 2018: a case report.

BACKGROUND: Acute flaccid myelitis (AFM) are reported to be associated with enterovirus D68 infection. Though an increasing number of AFM cases were reported with EV-D68 infection in the US, few such cases have been found in China. CASE PRESENTATION: A 6-year-old boy presented with acute flaccid myelitis (AFM) involving left arm after fever and respiratory symptoms for 6 days. Computed Tomography (CT) revealed inflammation in both lungs and magnetic resonance imaging (MRI) of the brain and spine showed swelling in the left frontal lobe and brain stem. The patient was diagnosed with meningomyelitis. EV-D68 was detected from pharyngeal samples 36 days after the onset of the disease. CONCLUSION: We report the first EV-D68 infection in case of AFM in mainland China. AFM surveillance systems is recommended to be established in China to guide diagnosis, case reporting, and specimen collection and testing for better understanding its etiologies.

The effectiveness of influenza vaccination in preventing hospitalizations in elderly in Beijing, 2016-18.

BACKGROUND: Influenza vaccinations play an important role in preventing influenza related hospitalizations. The objective of this study was to estimate the effectiveness of vaccination in protecting Beijing residents aged ≥60 years from influenza related hospitalizations during the 2016/17 and 2017/18 influenza seasons. METHODS: Patients who met the definition of severe acute respiratory infection (SARI) and were hospitalized in the nine sentinel hospitals in Beijing during the 2016/17 and 2017/18 influenza seasons were identified as the study population. The vaccination status of patients was obtained from a vaccination registry. Real-time reversetranscription polymerasechainreaction (RT-PCR) experiments were conducted to test pharyngeal or lower respiratory tract samples collected from SARI patients for influenza A and B viruses. Vaccine effectiveness (VE) was examined using a test-negative design that compare the odds of vaccination among influenza positives and negatives, adjusting for calendar week of illness onset, age, and underlying medical conditions. RESULTS: We identified 50,364 patients in the study, in which there were 145 influenza cases and 528 influenza-negative controls aged ≥60 years in 2016/17 season and 149 cases and 358 controls aged ≥60 years in 2017/18 season. The most commonly identified subtype among participants was influenza A(H3N2) in 2016/17 and 2017/18 season (78.5% and 70.6%). Among the adults aged ≥60 years, the adjusted VE of vaccination against any influenza virus for serious acute respiratory infection (SARI) patients was 32.8% (95% confidence interval [CI]: -22.0 to 63.0%) in 2016/17 season. While the adjusted VE in 2017/18 season were 4.6% (95% CI: -72.4 to 47.2%) against any types of influenza, 29.2% (95% CI: -92.9 to 74%) against influenza A(H1N1)pdm09, -37.7% (95% CI: -293.8; 51.9%) against influenza A(H3N2) viruses, and 3.6% (95% CI: -113.8 to 56.5%) against influenza B. CONCLUSION: The influenza vaccine provided moderate protection in 2016/17 season and mild protection in 2017/18 season for influenza related inpatients of adults aged ≥60 years in Beijing.

Avian influenza A (H9N2) virus infections among poultry workers, swine workers, and the general population in Beijing, China, 2013-2016: A serological cohort study.

BACKGROUND: Few studies have reported on the seroprevalence of antibodies against avian influenza A (H9N2) virus and the incidence of these infections in the northern China and among swine workers. METHODS: We conducted a serological cohort study among people working with poultry or swine or the general population in Beijing, China. It comprised four cross-sectional serological surveys in November 2013, April 2014, April 2015, and April 2016. Blood samples collected from the participants were tested for anti-H9N2 antibodies using a hemagglutination-inhibition (HI) assay. Multivariable Poisson regression model was then used to compare the person-month incidence rates for H9N2 viral infections among the three groups, assessed by incidence rate ratio (IRR). RESULTS: In the four cross-sectional surveys, the highest seroprevalence of anti-H9N2 antibodies (HI titer ≥ 80) was recorded in the poultry workers (2.77%, 19/685) in April 2016, while the lowest was recorded in the general population (0.09%, 1/1135) in April 2015. The highest incidence density rate for H9N2 infections across the whole study period was recorded among the poultry workers (3.75/1000 person-months), followed by the swine workers (1.94/1000 person-months) and the general population (1.78/1000 person-months). Multivariable analysis showed that the poultry workers were at higher risk (IRR: 2.42, 95% CI: 1.07-5.48; P = 0.034) of contracting H9N2 virus than the general population. CONCLUSIONS: Although the seroprevalence of H9N2 antibodies was low in Beijing, the poultry workers were at higher risk of contracting H9N2 viral infections than the general population. Closer monitoring and strengthened protection measures for poultry workers are warranted.

Fungal pathogens pose a potential threat to animal and plant health in desertified and pika-burrowed alpine meadows on the Tibetan Plateau.

Intact Tibetan meadows provide significant defense against soil-borne pathogen dispersal. However, dramatic meadow degradation has been observed due to climate change and pika damage, but their impacts on soil-borne pathogens are still unclear. With approximately 40% of the world's population living in Tibetan Plateau and its downstream watersheds, this lack of knowledge should be of great concern. Here, we used Illumina amplicon sequencing to characterize the changes in potential human, domestic animal, plant, and zoonotic bacterial and fungal pathogens in nondegraded, desertified, and pika-burrowed meadows. The relative abundance of bacterial domestic animal pathogens and zoonotic pathogens were significantly increased by desertification. Pika burrowing significantly increased the relative abundance of bacterial human pathogens and zoonotic pathogens. The species richness and relative abundance of fungal pathogens was significantly increased by desertification and pika burrowing. Accordingly, fungal plant and animal pathogens categorized by FUNGuid significantly increased in desertified and pika-burrowed meadows. Soil chemical and plant properties explained 38% and 64% of the bacterial and fungal pathogen community variance, respectively. Therefore, our study indicates for the first time that both alpine meadow desertification and pika burrowing could potentially increase infectious disease risks in the alpine ecosystem, especially for fungal diseases.

Responses of biotic interactions of dominant and subordinate species to decadal warming and simulated rotational grazing in Tibetan alpine meadow.

Warming increases competition among plant species in alpine communities by ameliorating harsh environmental conditions, such as low temperatures. Grazing, as the main human activity, may mitigate the effect of warming, as previously reported. However, it is critical to refine the effects of warming on biotic interactions among species, for example, by taking the competitive ability of species into consideration. Based on a 10-year warming and grazing experiment in a Tibetan alpine meadow, we evaluated interspecific biotic interactions of dominant and subordinate species, using the approach of interspecific spatial associations. Warming significantly increased competition between subordinate and dominant species as well as among subordinate species, but not among dominant species. Moreover, facilitation of dominant-subordinate species also increased under warming. Simulated rotational grazing had similar effects to warming, with increasing interspecific competition. Our results show that, when studying the effects of warming on biotic interactions among species, it is necessary to characterize different species pairs relative to their competitive ability, and that simulated rotational grazing does not mitigate the effects of warming in the long term. Our results also provide evidence that the spatial pattern of species is a critical mechanism in species coexistence.

Human parainfluenza virus infection in severe acute respiratory infection cases in Beijing, 2014-2016: A molecular epidemiological study.

BACKGROUND: Severe acute respiratory infection (SARI) threatens human health and even survival, causing a huge number of hospitalized patients every year. However, as one of the most common respiratory viruses circulated worldwide, the epidemiological and phylogenetic characteristics of human parainfluenza virus (HPIV) in these cases were not well known. OBJECTIVES: To reveal the epidemiological features of HPIV infection in SARIs in Beijing area from September 2014 to August 2016. METHODS: A total of 1229 SARI cases in Beijing area were enrolled, investigated, sampled, and tested by multiplex real-time PCR to identify HPIVs and other common respiratory viruses. Eighteen HPIV-3 viruses isolated from all HPIV-positive samples in these SARI cases were sequenced and analyzed. RESULTS: Among all enrolled cases, 0.81%, 0.73%, 4.48%, and 0.57% were positive for HPIV-1 to HPIV-4, respectively. The highest yield rate of HPIV infection occurred in children under 5 years old (9.07%), followed by the patients over 60 years old (6.02%). The phylogenetic information of HPIV-3 showed that all viruses belonged to Cluster C3a. CONCLUSIONS: Besides the young children, the elders older than 60 years also showed a relatively high infection rate of HPIVs, which should be given comparable attentions. Moreover, the HPIV-3 circulating in China undergoes continued evolution, suggesting the potential risk of evolved HPIV infection should not be overlooked.

IFITM3 Rs12252-C Variant Increases Potential Risk for Severe Influenza Virus Infection in Chinese Population.

Background: Interferon Inducible Transmembrane 3 (IFITM3) is a key factor in interferon pathway and it involves host's immune response against multiple viruses. IFITM3 rs12252-C was associated with severe influenza virus infection in several studies, however whether this association is universal to all types of influenza virus or diverse ethnic populations remain controversial. Method: A case-control genetic association study was performed from September 2013 to April 2014 and September 2014 to April 2015. All samples were tested for influenza using RT-PCR, and genotyped by High Resolution Melting assay. Results: A total of 65 healthy people, 165 mild influenza-like illness (ILI) cases and 315 severe acute respiratory infection (SARI) cases were enrolled in this study. The frequency of CC genotype was much higher in SARI cases with IVI than that in ILI cases with IVI (61.59 vs. 27.16%), leading a 4.67-fold greater risk for severe IVI than other two genotypes. Moreover, the risk of IFITM3 rs12252-C variant for severe IVI was specific for both influenza A and influenza B. Conclusion:IFITM3 rs12252 CC genotype was associated with severity rather than susceptibility of IVI in Chinese population, and this strong effect was observed in all subtypes of seasonal influenza infection.

Detection of yellow fever virus genomes from four imported cases in China.

Yellow fever virus (YFV), as the first proven human-pathogenic virus, is still a major public health problem with a dramatic upsurge in recent years. This is a report on four imported cases of yellow fever virus into China identified by whole genome sequencing. Phylogenetic analysis was performed and the results showed that these four viruses were highly homologous with Angola 71 strains (AY968064). In addition, effective mutations of amino acids were not observed in the E protein domain of four viruses, thus confirming the effectiveness of the YFV-17D vaccine (X03700). Although there is low risk of local transmission in most part of China, the increasing public health risk of YF caused by international exchange should not be ignored.