Development and evaluation of a quadruple real-time fluorescence-based quantitative reverse transcription polymerase chain reaction assay for detecting Langya, Mojiang, Nipah, and Cedar viruses

The emerging viruses within the genus Henipavirus in the family Paramyxoviridae pose a great threat to public biosafety.To develop a quadruple real-time fluorescence-based quantitative reverse transcription polymerase chain reaction(qRT-PCR)assay is pivotal for the early warning of the potential of zoonotic infectious diseases.Specific primers and probes were designed for the relatively conserved regions based on whole genome sequences of Langya virus(LayV),Mojiang virus(MojV),Nipah virus(NiV),and Cedar virus(CedV),followed by the establishment of a quadruple real-time fluorescence-based qRT-PCR detection method.No cross-reactivity was observed with other viral nucleic acids.The optimal linear detection range for LayV,MojV,NiV,and CedV was 10^(1)-10^(8)copies/μL,and the lower limit of detection was 10 copies/μL.Three different DNA concentrations of LayV,MojV,NiV,and CedV(10^(4),10^(5),and 10^(6)copies/μL)were tested 14 times,achieving good repeatability.The standard deviation of the cycle threshold values for each concentration was<0.5 and the coefficient of variation was<3%.Furthermore,the amplification efficiency of quadruple real-time fluorescence-based qRT-PCR was>90%,and the correlation coefficient was>0.99.The established quadru-ple real-time fluorescence-based qRT-PCR assay for the detection of LayV,MojV,NiV,and CedV exhibits good sensitivity,specificity,and repeatability.Therefore,it can be used to detect Henipavirus and other related clinical specimens.

Enhanced potency of an IgM-like nanobody targeting conserved epitope in SARS-CoV-2 spike N-terminal domain

Almost all the neutralizing antibodies targeting the receptor-binding domain(RBD)of spike(S)protein show weakened or lost efficacy against severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)emerged or emerging variants,such as Omicron and its sub-variants.This suggests that highly conserved epitopes are crucial for the development of neutralizing antibodies.Here,we present one nanobody,N235,displaying broad neutralization against the SARS-CoV-2 prototype and multiple variants,including the newly emerged Omicron and its sub-variants.Cryo-electron microscopy demonstrates N235 binds a novel,conserved,cryptic epitope in the N-terminal domain(NTD)of the S protein,which interferes with the RBD in the neighboring S protein.The neutralization mechanism interpreted via flow cytometry and Western blot shows that N235 appears to induce the S1 subunit shedding from the trimeric S complex.Furthermore,a nano-IgM construct(MN235),engineered by fusing N235 with the human IgM Fc region,displays prevention via inducing S1 shedding and cross-linking virus particles.Compared to N235,MN235 exhibits varied enhancement in neutralization against pseudotyped and authentic viruses in vitro.The intranasal administration of MN235 in low doses can effectively prevent the infection of Omicron sub-variant BA.1 and XBB in vivo,suggesting that it can be developed as a promising prophylactic antibody to cope with the ongoing and future infection.

Efficient inhibition of SARS-CoV-2 emerging EG.5,EG.5.1 and BA.2.86 variants by fusion inhibitor HY3000 peptide

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)continues to pose a significant threat to the world,as it continually evolves and gives rise to multiple variants and sub-variants.Recently,the Omicron EG.5 linage,which was first detected in Indonesia on 17 February 2023,has raised concerns due to its increased prevalence and extended immune escape properties,according to the risk analysis by the World Health Organization(WHO)[1].As of 3 October 2023,EG.5 and its sub-linages have been reported in 83 countries with shared 49,008 genome sequences in GISAID database(https://gisaid.org/hcov19-variants/).EG.5 has become the dominant strain in the United States according to Centers for Disease Control and Prevention(CDC),accounting for 29.4%of SARS-CoV-2 infections(https://covid.cdc.gov/covid-data-tracker/#variant-proportions).

ZIKA virus isolated from mosquitoes:a field and laboratory investigation in China,2016

A field investigation of arboviruses was conducted in Dejiang, Guizhou Province in the summer of 2016. A total of 8,795 mosquitoes, belonging to four species of three genera, and 1,300 midges were collected. The mosquito samples were identified on site according to their morphology, and the pooled samples were ground and centrifuged in the laboratory. The supernatant was incubated with mosquito tissue culture cells(C6/36) and mammalian cells(BHK-21) for virus isolation. The results indicated that 40%(3,540/8,795) were Anopheles sinensis, 30%(2,700/8,795) were Culex pipiens quinquefasciatus, and 29%(2,530/8,795)were Armigeres subbalbeatus. Furthermore, a total of eight virus isolates were obtained, and genome sequencing revealed two Zika viruses(ZIKVs) isolated from Culex pipiens quinquefasciatus and Armigeres subbalbeatus, respectively; three Japanese encephalitis viruses(JEVs) isolated from Culex pipiens quinquefasciatus; two Banna viruses(BAVs) isolated from Culex pipiens quinquefasciatus and Anopheles sinensis, respectively; and one densovirus(DNV) isolated from Culex pipiens quinquefasciatus.The ZIKVs isolated from the Culex pipiens quinquefasciatus and Armigeres subbalbeatus mosquitoes represent the first ZIKV isolates in China's Mainland. This discovery presents new challenges for the prevention and control of ZIKV in China, and prompts international cooperation on this global issue.

Genome sequencing and phylogenetic analysis of Banna virus(genus Seadornavirus,family Reoviridae)isolated from Culicoides

In an investigation of blood-sucking insects and arboviruses, a virus(YN12243) was isolated from Culicoides samples collected in the Sino-Burmese border region of Yunnan Province, China. The virus caused cytopathic effect(CPE) in C6/36 cells and passaged stably. Polyacrylamide gel analysis showed that the genome of YN12243 was composed of 12 segments of double-stranded RNA(dsRNA), with a distribution pattern of 6-6. The nucleotide and amino acid sequences of the coding region(1.12 segments) were17,803 bp and 5,925 amino acids in length, respectively. The phylogenetic analysis of VP1 protein(RdRp) revealed that YN12243 belonged to genus Seadornavirus of family Reoviridae, and further analysis indicated that YN12243 belongs to the Banna virus(BAV) genotype A2. Additionally, YN12243 was located in the same evolutionary cluster as BAV strains isolated from different mosquito species, suggesting that the BAV isolated from Culicoides does not have species barriers. These results indicate that Culicoides can also be a vector for BAV. In view of the hematophagous habits of Culicoides on cattle, horses, deer, and other large animals, as well as the possibility of spreading and causing a variety of animal arboviral diseases, it is important to improve infection detection and monitor the BAV in large livestock.

Cold-chain transportation in the frozen food industry may have caused a recurrence of COVID-19 cases in destination:Successful isolation of SARS-CoV-2 virus from the imported frozen cod package surface

Coronavirus disease 2019(COVID-19)pandemic has spread in 220 countries/regions towreak havoc to human beings around theworld.At present,the second wave of COVID-19 has begun in many European countries.The complete control of COVID-19 is very urgent.Although China quickly brought the virus under control,there have been eight sporadic outbreaks in China since then.Both in Xinfadi of Beijing and Dalian outbreak of COVID-19,environmental swab samples related to imported cold chain food were tested nucleic acid positive for SARS-CoV-2.In this outbreak in Qingdao,we directly isolated SARS-CoV-2 from the cod outer package's surface swab samples.This is the first time worldwide,SARS-CoV-2 were isolated from the imported frozen cod outer package's surface,which showed that imported frozen food industry could import SARS-CoV-2 virus.

Pigsties near dwellings as a potential risk factor for the prevalence of Japanese encephalitis virus in adult in Shanxi,China

Background:The increasing trend of adult cases of Japanese encephalitis(JE)in China,particularly in northern China,has become an important public health issue.We conducted an epidemiological investigation in the south of Shanxi Province to examine the relationships between mosquitoes,Japanese encephalitis virus(JEV),and adult JE cases.Methods:Mosquito specimens were collected from the courtyards of farmers’households and pig farms in Shanxi Province.Mosquitoes were pooled,homogenized,and centrifuged.Reverse transcription-polymerase chain reaction(RT-PCR)was used to detect mosquito-borne arbovirus genes in homogenates.Specimens positive for these genes were inoculated into the baby hamster kidney cell line(BHK-21)to isolate virus.Minimum infection rate was calculated and phylogenetic analyses were performed.Results:A total of 7943 mosquitoes belonging to six species in four genera were collected;Culex tritaeniorhynchus accounted for 73.08%(5805/7943),C.pipiens pallens for 24.75%(1966/7943),and the remaining 3%(104/7943)consisted of Anopheles sinensis,Aedes vexans,Ae.dorsalis,and Armigeres subalbatus.Sixteen pools were positive for JEV based on RT-PCR using JEV pre-membrane gene nested primers.Phylogenetic analyses showed that all JEVs belonged to genotype I;two pools were positive using Getah Virus(GETV)gene primers.In addition,one JEV strain(SXYC1523)was isolated from C.pipiens pallens specimens.These results indicate that the minimum infection rate of JEV in mosquito specimens collected from the courtyards of farmers’households with pigsties was 7.39/1000;the rate for pig farms was 2.68/1000;and the rate for farmers’courtyards without pigsties was zero.Conclusions:The high-prevalence regions of adult JE investigated in this study are still the natural epidemic focus of JEV.Having pigsties near dwellings is a potential risk factor contributing to the prevalence of adult JE.To prevent the occurrence of local adult JE cases,a recommendation was raised that,besides continuing to implement the Expanded Program on Immunization for children,the government should urge local farmers to cease raising pigs in their own courtyards to reduce the probability of infection with JEV.

Emergence of Zika Virus in Culex tritaeniorhynchus and Anopheles sinensis Mosquitoes in China

Zika virus(ZIKV)has been isolated from mosquitoes such as Aedes,Mansonia uniformis,and Culex perfuscus;However,the isolation of ZIKV from Anopheles sinensis and Culex tritaeniorhynchus has not yet been reported.In June and July2018,22,985 mosquitoes and 57,500 midges were collected in Jiangxi Province in southeastern China.Among them,six strains of ZIKV were isolated from mosquitoes:four from An.sinensis and two from Cx.tritaeniorhynchus.Molecular genetic analysis showed that the ZIKV isolated from An.sinensis and Cx.tritaeniorhynchus belonged to genotype 2 in the Asian evolutionary branch of ZIKV.In addition,the ZIKV strains isolated from An.sinensis and Cx.tritaeniorhynchus had amino acid substitutions identical to ZIKV strains prevalent in South America since 2015.This study is the first to isolate ZIKV from mosquito specimens collected in the wild of Jiangxi Province,China;This is also the first time that ZIKV has been isolated from An.sinensis and Cx.tritaeniorhynchus.Given that An.sinensis and Cx.tritaeniorhynchus have a very wide geographical distribution in China and even in eastern and southern Asia,the isolation of several strains of ZIKV from these two mosquitoes poses new challenges for the prevention and control of ZIKV infection in the mainland of China and countries and regions with the same distribution of mosquitoes.

A pan-coronavirus peptide inhibitor prevents SARS-CoV-2 infection in mice by intranasal delivery

Coronaviruses(Co Vs)have brought serious threats to humans,particularly severe acute respiratory syndrome coronavirus 2(SARS-Co V-2),which continually evolves into multiple variants.These variants,especially Omicron,reportedly escape therapeutic antibodies and vaccines,indicating an urgent need for new antivirals with pan-SARS-Co V-2 inhibitory activity.We previously reported that a peptide fusion inhibitor,P3,targeting heptad repeated-1(HR1)of SARS-Co V-2 spike(S)protein,could inhibit viral infections.Here,we further designed multiple derivatives of the P3 based on structural analysis and found that one derivative,the P315V3,showed the most efficient antiviral activity against SARS-Co V-2 variants and several other sarbecoviruses,as well as other human-Co Vs(HCo Vs).P315V3 also exhibited effective prophylactic efficacy against the SARS-Co V-2 Delta and Omicron variants in mice via intranasal administration.These results suggest that P315V3,which is in PhaseⅡclinical trial,is promising for further development as a nasal pan-SARS-Co V-2 or pan-Co Vs inhibitor to prevent or treat CoV diseases.

一株靶向沙贝病毒高度保守的S2亚基的广谱中和纳米抗体

新型冠状病毒感染给人类健康和全球公共安全带来了巨大威胁,但是病毒仍在持续变异产生新的变异毒株,尤其OmicronBQ.1.1和XBB变异株,完全逃逸现有抗体药物,疫苗保护效力也严重降低。此外,多种动物源新冠相关冠状病毒(沙贝病毒)具有感染人的危险。

Low levels of neutralizing antibodies against XBB Omicron subvariants after BA.5 infection

The COVID-19 response strategies in Chinese mainland were recently adjusted due to the reduced pathogenicity and enhanced infectivity of Omicron subvariants.In Chengdu,China,an infection wave was predominantly induced by the BA.5 subvariant.It is crucial to determine whether the hybrid anti-SARS-CoV-2 immunity following BA.5 infection.

Characterization of viromes within mosquito species in China

Mosquitoes are vectors of multiple disease-causing agents that cause worldwide outbreaks of serious infectious diseases in both humans and animals. These arboviral agents include Dengue virus (DENV), Zika virus (ZIKV), chikungunya virus, yellow fever virus, Japanese encephalitis virus (JEV),and Rift Valley fever virus. Thus, mosquitoes are key to the study of viral origin, spread, and the epidemic tendency of these infectious diseases (Hall and Macdonald, 2016).

Immunogenicity and safety of NVSI-06-07 as a heterologous booster after priming with BBIBP-CorV:a phase 2 trial

The increased coronavirus disease 2019(COVID-19)breakthrough cases pose the need of booster vaccination.We conducted a randomised,double-blinded,controlled,phase 2 trial to assess the immunogenicity and safety of the heterologous prime-boost vaccination with an inactivated COVID-19 vaccine(BBIBP-CorV)followed by a recombinant protein-based vaccine(NVSI-06-07),using homologous boost with BBIBP-CorV as control.Three groups of healthy adults(600 individuals per group)who had completed two-dose BBIBP-CorV vaccinations 1–3 months,4–6 months and≥6 months earlier,respectively,were randomly assigned in a 1:1 ratio to receive either NVSI-06-07 or BBIBP-CorV boost.Immunogenicity assays showed that in NVSI-06-07 groups,neutralizing antibody geometric mean titers(GMTs)against the prototype SARS-CoV-2 increased by 21.01–63.85 folds on day 28 after vaccination,whereas only 4.20–16.78 folds of increases were observed in control groups.For Omicron variant,the neutralizing antibody GMT elicited by homologous boost was 37.91 on day 14,however,a significantly higher neutralizing GMT of 292.53 was induced by heterologous booster.Similar results were obtained for other SARS-CoV-2 variants of concerns(VOCs),including Alpha,Beta and Delta.Both heterologous and homologous boosters have a good safety profile.Local and systemic adverse reactions were absent,mild or moderate in most participants,and the overall safety was quite similar between two booster schemes.Our findings indicated that NVSI-06-07 is safe and immunogenic as a heterologous booster in BBIBP-CorV recipients and was immunogenically superior to the homologous booster against not only SARS-CoV-2 prototype strain but also VOCs,including Omicron.

Identification of Histoplasma causing an unexplained disease cluster in Matthews Ridge, Guyana

Here,we report the identification of Histoplasma causing an unexplained disease cluster in Matthews Ridge,Guyana.In March 2019,14 employees of Chongqing Bosai Mining Company,China,working in a manganese mining of Guyana,had unexplained fever,and two of them died.We obtained lung and brain tissues as well as the blood samples from the two deceased cases(patient No.1 and 2),and bronchoscopy lavages and cerebrospinal fluid samples from one severe case(patient No.3),respectively.All samples were tested by pathological examination,high-throughput sequencing,and real-time PCR.Pathological detection showed the presence of spore-like structures in the lung tissue of patient No.1,indicating a fungal infection in this patient.Nanopore sequencing identified the existing of H.capsulatum in the lung tissue sample within 13 h.Next-generation sequencing identified specific fragments of H.capsulatum in all of the samples tested(lung,brain and blood serum from the deceased cases,and plasma from the severe case).Real-time PCR assays did not reveal any viral infection related to transmission from bat feces.We conclude that H.capsulatum was the causative pathogen of this disease cluster based on epidemiologic,clinical,pathological and nucleic acid evidence.

Safety and immunogenicity of a mosaic vaccine booster against Omicron and other SARS-CoV-2 variants:a randomized phase 2 trial

An ongoing randomized,double-blind,controlled phase 2 trial was conducted to evaluate the safety and immunogenicity of a mosaic-type recombinant vaccine candidate,named NVSI-06-09,as a booster dose in subjects aged 18 years and older from the United Arab Emirates(UAE),who had administered two or three doses of inactivated vaccine BBIBP-CorV at least 6 months prior to enrollment.The participants were randomly assigned with 1:1 to receive a booster dose of NVSI-06-09 or BBIBP-CorV.The primary outcomes were immunogenicity and safety against severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)Omicron variant,and the exploratory outcome was cross-immunogenicity against other circulating strains.Between May 25 and 30,2022,516 adults received booster vaccination with 260 in NVSI-06-09 group and 256 in BBIBP-CorV group.Interim results showed a similar safety profile between two booster groups,with low incidence of adverse reactions of grade 1 or 2.For immunogenicity,by day 14 post-booster,the fold rises in neutralizing antibody geometric mean titers(GMTs)from baseline elicited by NVSI-06-09 were remarkably higher than those by BBIBP-CorV against the prototype strain(19.67 vs 4.47-fold),Omicron BA.1.1(42.35 vs 3.78-fold),BA.2(25.09 vs 2.91-fold),BA.4(22.42 vs 2.69-fold),and BA.5 variants(27.06 vs 4.73-fold).Similarly,the neutralizing GMTs boosted by NVSI-06-09 against Beta and Delta variants were also 6.60-fold and 7.17-fold higher than those by BBIBP-CorV.Our findings indicated that a booster dose of NVSI-06-09 was well-tolerated and elicited broad-spectrum neutralizing responses against divergent SARS-CoV-2 variants,including Omicron and its sub-lineages.

Back to Science in Searching for SARS-CoV-2 Origins

In recent decades,emerging and re-emerging human-infecting pathogens have been represented as huge threats to public health and have become a global concern(1).After outbreaks of two coronaviruses(CoVs),severe acute respiratory syndrome coronavirus(SARS-CoV)and Middle East respiratory syndrome coronavirus(MERS-CoV),severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)became the first-known pandemic hastening CoV with tremendous wrecking to the world(2).The origin tracing of these emerging pathogens is of great significance in infectious disease prevention and control(3–4).The origin of SARS-CoV-2 remains elusive after the more than 3-year pandemic,though scientists around the world are making great efforts.From the experience of studying many other infectious pathogens,origin tracing is systematic and time-consuming work.The supposed origins of many infectious pathogens are still in debate,including SARS-CoV and human immunodeficiency virus,etc(5).

Papua New Guinea Under the COVID-19 Pandemic and Public Health Support from the World Health Organization

On March 20,2020,the first positive case of coronavirus disease 2019(COVID-19)was detected in Papua New Guinea(PNG).That same day,a state of emergency was declared in the country,thereby halting all incoming and domestic flights and limiting travel between provinces.Meanwhile,the PNG government established the National Control Centre(NCC)to specifically respond to the pandemic.