Enterovirus C96 (EV-C96) is a novel human enterovirus associated with diseases such as hand, foot, and mouth disease and acute flaccid paralysis (AFP). In recent years, the global detection rate of EV-C96 has risen markedly, underscoring its epidemic potential. However, there is currently a lack of suitable animal models for studying its pathogenic mechanisms and antiviral research. This study utilized the clinical isolate AFP149/GD/CHN/2014 of EV-C96 and, through screening of infection doses, mouse strains, age, and inoculation routes, established the first EV-C96 suckling mouse infection model. The experimental results showed that intracranial injection of a viral dose of 103.425 TCID50 caused limb paralysis, weight loss, and 100% mortality in 1-day-old ICR suckling mice. Histopathological analysis indicated that the virus primarily targeted the central nervous system (brain and spinal cord) and myocardial tissue, a distribution characteristic that corresponds with the typical clinical manifestations of EV-C96 infections. Using this model to evaluate antiviral drugs, we found that treatment with IFN-α2a (1000 U) and IFN-γ (1000 U) increased the survival rate of infected suckling mice to 75% and 67%, respectively. In vaccine evaluation experiments, the survival rate of the EV-C96 inactivated vaccine immunization group was 100%. Pathological examinations showed that the myocardial and spinal cord tissue structures of the treatment and immunization groups were intact, and the degree of brain tissue lesions was significantly reduced. The EV-C96 suckling mouse model provides a platform for investigating viral pathogenesis and supports preclinical evaluation of antiviral drugs and vaccines.

Echovirus 30 (E30) is a significant pathogen associated with various illnesses such as viral meningitis, viral myocarditis. Currently, there are no specific drugs or vaccines targeting this virus. An appropriate animal model is imperative for assessing drug and vaccine efficacy. This investigation aimed to establish a neonatal mouse model using a clinical isolate E30/A538 and apply it to screen anti-E30 drugs. The study involved evaluating the susceptibility of different mouse strains to the isolate, determining the infectious dose, transmission route, and optimal age of the mice. This model was then used to assess antiviral efficacy. Neonatal ICR mice infected intracranially with 5LD50 of E30/A538 at one-day-old displayed clinical symptoms such as tremors, lethargy, limb paralysis, and mortality. Importantly, the E30/A538-infected mice exhibited brain neuron apoptosis and severe myocardial necrolysis, closely resembling human infections. Elevated levels of viral RNA and positive antigen presence were predominantly detected in the brains and hearts of infected mice. Using this model to assess antiviral efficacy, it was demonstrated that interferon-α2a inhibited E30/A538 replication in vivo, mitigated histopathological changes in the brain, spinal cord, and myocardium, and enhanced the survival rate of neonatal mice. In summary, this research established a wild neonatal mouse model of E30/A538 isolate infection that mirrors the characteristics of human infection. The model demonstrated the efficacy of interferon-α2a in combating E30. This model would serve as a foundation for investigating the pathogenesis of E30, as well as for assessing the efficacy of vaccines and other antiviral treatments against E30.

Developing both rapid- and long-acting antiviral drugs for single-dose administration can improve medication adherence and protect people at risk of infection. To provide proof of this concept, here, we designed multimerized form of viral receptor-binding domains (RBDs) to immediately occupy viral receptors to block infection and subsequently induce virus-specific protective immunity. We engineered SARS-CoV-2 RBD, enhancing its affinity to ACE2 and immunogenicity through multimerization and Fc modification. A single administration of 4RBD-Fc not only effectively blocked ACE2-dependent SARS-CoV-2 infections but also elicited robust virus-specific mucosal and systemic immunity in the absence of adjuvants, providing superior early and long-lasting protection compared to adjuvanted vaccines in mice. These findings demonstrate the feasibility and efficacy of engineered viral RBD as immediate-acting and long-lasting single-dose antiviral drugs through rapid receptor blocking and ensuing adaptive immunity induction.

The Oral Polio Vaccine (OPV) is genetically unstable and may mutate to form vaccinederived polioviruses (VDPVs). At present, the finding of VDPV mainly come from AFP surveillance and environmental sewage surveillance. In 2023, a vaccine-derived poliovirus (VDPV) strain type 3 isolated from a patient with hand, foot, and mouth disease (HFMD) in Guangdong Province, China, was sequenced and analyzed. The strain had reversion mutations at the known attenuation site 5’ non-coding region and recombined with Sabin1, Phylogenetic analysis of the VP1 coding region showed that the Guangdong type 3 VDPV evolved independently of other type 3 VDPVs found globally. It is meaningful to increase the sensitivity of surveillance of other enteroviral diseases for polioviruses.

Background: The Spike protein mutation severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to decreased protective effect of various vaccines and mAbs, suggesting that blocking SARS-CoV-2 infection by targeting host factors would make the therapy more resilient against virus mutations. Angiotensin-converting enzyme 2 (ACE2) is the host receptor of SARS-CoV-2 and its variants, as well as many other coronaviruses. Downregulation of ACE2 expression in the respiratory tract may prevent viral infection. Antisense oligonucleotides (ASOs) can be rationally designed on the basis of sequence data, require no delivery system, and can be administered locally. Objective: We sought to design ASOs that can block SARSCoV-2 by downregulating ACE2 in human airway. Methods: ACE2-targeting ASOs were designed using a bioinformatic method and screened in cell lines. Human primary nasal epithelial cells cultured at the air-liquid interface and humanized ACE2 mice were used to detect the ACE2 reduction levels and the safety of ASOs. ASO-pretreated nasal epithelial cells and mice were infected and then used to detect the viral infection levels. Results: ASOs reduced ACE2 expression on mRNA and protein level in cell lines and in human nasal epithelial cells. Furthermore, they efficiently suppressed virus replication of 3 different SARS-CoV-2 variants in human nasal epithelial cells. In vivo, , ASOs also downregulated human ACE2 in humanized ACE2 mice and thereby reduced viral load, histopathologic changes in lungs, and increased survival of mice. Conclusions: ACE2-targeting ASOs can effectively block SARSCoV-2 infection. Our study provides a new approach for blocking SARS-CoV-2 and other ACE2-targeting virus in high- risk populations. (J Allergy Clin Immunol 2024;154:1044-59.)

In 2019, we conducted a cross-sectional study for polio virus seroprevalence in Guangdong province, China. We assessed the positivity rates of poliomyelitis NA and GMT in serum across various demographic groups, and the current findings were compared with pre-switch data from 2014. Using multistage random sampling method, four counties/districts were randomly selected per city, and within each, one general hospital and two township hospitals were chosen. Healthy individuals coming for medical checkups or vaccination were invited. A total of 1318 individual samples were collected and tested. In non-newborn population, age-dependent positivity rates ranged from 77.8% to 100% for PV1 NA and 70.3% to 98.9% for PV3 NA (p < .01). The lowest GMT values for both types (17.03 and 8.46) occurred in the 20 to <30 years age group, while peak GMTs for PV1 and PV3 were observed in 1 to <2 (340.14) and 0 to <1-year (168.90) age groups, respectively. GMTs for PV1 (P = .002) and PV3 (P = .007) in Eastern Guangdong were lower than those in the other three regions. Male participants showed higher GMTs than females (P = .016 and .033, respectively). In newborn population, both males and females showed higher PV1 NA positivity rates and GMTs compared to PV3 (p < .05). Post-switch PV3 NA positivity rates were higher than pre-switch rates (p = .016). GMTs of both PV1 and PV3 were significantly higher post-switch (p < .001). The positivity rates of NAs and GMTs remain high level, which play an important role in resisting poliomyelitis infection. Effect of the converted immunization program was more pronounced than that before.

This study aimed to compare the sensitivity of two-phase separation and the filtration method using a mixed cellulose ester (MCE) membrane to detect enteroviruses in sewage samples. From December 2015 to July 2016, four domestic sewage samples (1 L/sample) were collected monthly from the Guangzhou Liede Sewage Treatment Plant, and each sewage sample was divided into two aliquots (500 mL). The sewage sample was concentrated using the two-phase separation and the filtration method using an MCE membrane, and the treated solutions were inoculated into cells for enterovirus isolation. Polymerase chain reaction amplification, VP1 sequencing, and enterovirus molecular typing were performed on the positive isolates. The detection rates of poliovirus (PV) and non-polio enterovirus (NPEV) obtained using the filtration method using an MCE membrane were higher than those using the two-phase separation method. McNemar's test showed that the detection rates of PV, NPEV, type 1 Sabin-like (SL1), type 2 Sabin-like (SL2), and type 3 Sabin-like (SL3) strain were not statistically significant (P > 0.05). In Guangdong Province, China, the detection rates for PV and NPEV were 53.13% and 62.50% (20/32), respectively. Twenty-seven PVs were isolated, three highly variable strains of the type 1 vaccine, with seven nucleotide substitutions in the VP1 region, compared with the type 1 Sabin strains. Eighty-seven strains of NPEV were isolated and nine serotypes were identified, among which coxsackievirus B3 (CVB3), echovirus 6(E6), and echovirus 11(E11) were the dominant strains. The filtration method using an MCE membrane is more sensitive than two-phase separation and can be used as a robust, sensitive, and cost-effective method to isolate enteroviruses from sewage.

Measles incidence reached a historic low in 2020-2021, and virological surveillance data showed that transmission of the domestic H1 genotype as well as multiple imported genotypes had been interrupted. This evidence confirms that China is currently approaching measles elimination. Background To provide useful insights into measles elimination progress in China, measles surveillance data were reviewed, and the transmission patterns of measles viruses circulating in China during 1993-2021 were analyzed. Methods Measles incidence data from the National Notifiable Disease Reporting System of the China Center for Disease Control and Prevention were analyzed. A total of 17 570 strains were obtained from 30 of 31 provinces in mainland China during 1993-2021. The recommended genotyping window was amplified. Genotyping analysis was conducted for comparison with the reference strains. Phylogenetic analyses were performed to identify genetic relationships among different lineages within the genotypes. Results With high coverage of routine immunization and intensive supplementary immunization activities, measles incidence has shown a downward trend since 1993, despite 2 resurgences, reaching a historic low level in 2020-2021 (average 0.5 per million). During 1993-2021, 9 genotypes including domestic genotype H1; imported genotypes B3, D4, D8, D9, D11, G3, and H2; and vaccine-associated genotype A were identified. Among them, the genotype H1 strain circulated endemically in China for more than 25 years; the last strain was detected in Yunnan Province in September 2019. Multiple imported genotypes have been identified since 2009 showing different transmission patterns. Since April 2020, no imported strains have been detected, while vaccine-associated genotype A continues to be detected. Conclusions The evidence of low incidence during 2020-2021 and virological surveillance data in this study confirm that China is currently approaching measles elimination.

Polio cases can be missed by acute flaccid paralysis (AFP) case surveillance alone, emphasizing the importance of environmental surveillance (ES). In this study, to investigate the serotype distribution and epidemiological trends of poliovirus (PV), we characterized PV isolated from domestic sewage in Guangzhou City, Guangdong Province, China from 2009 to 2021. A total of 624 sewage samples were collected from the Liede Sewage Treatment Plant, and the positive rates of PV and non-polio enteroviruses were 66.67% (416/624) and 78.37% (489/624), respectively. After sewage sample treatment, each sewage sample was inoculated in six replicate tubes of three cell lines, and 3370 viruses were isolated during the 13-year surveillance period. Among these, 1086 isolates were identified as PV, including type 1 PV (21.36%), type 2 PV (29.19%), and type 3 PV (49.48%). Based on VP1 sequences, 1057 strains were identified as Sabin-like, 21 strains were high-mutant vaccines, and eight strains were vaccine-derived poliovirus (VDPV). The numbers and serotypes of PV isolates in sewage were influenced by the vaccine switch strategy. After type 2 OPV was removed from the trivalent oral PV (OPV) vaccine and a bivalent OPV (bOPV) was adopted in May 2016, the last type 2 PV strain was isolated from sewage, with no detection thereafter. Type 3 PV isolates increased significantly and became the dominant serotype. Before and after the second vaccine switch in January 2020, that is, from the first dose of IPV and second-fourth doses of bOPV to the first two doses of IPV and third-fourth doses of bOPV, there was also a statistical difference in PV positivity rates in sewage samples. Seven type 2 VDPVs and one type 3 VDPV were identified in sewage samples in 2009-2021, and phylogenetic analysis indicated that all VDPVs isolated from ES in Guangdong are newly discovered VDPVs, different from VDPV previously discovered in China, and were classified as ambiguous VDPV. It is noteworthy that no VDPV cases were reported in AFP case surveillance in the same period. In conclusion, continued PV ES in Guangzhou since April 2008 has been a useful supplement to AFP case surveillance, providing an important basis for evaluating the effectiveness of vaccine immunization strategies. ES improves early detection, prevention, and control; accordingly, this strategy can curb the circulation of VDPVs and provide a strong laboratory basis for maintaining a polio-free status.

Objective:To analyze the evolutionary characteristics and variation of etiological agent in an acute hemorrhagic conjunctivitis (AHC) outbreak in a city of Guangdong province in May, so as to provide scientific basis for formulating a new round of measures for prevention and control of AHC epidemic.Methods:In this study, 20 conjunctival swabs were collected from AHC patients, and enterovirus, human enterovirus 70 (HEV70) and coxsackievirus A 24 variant (CVA24v) nucleic acids were detected by real-time fluorescence quantitative PCR. In addition, the VP1 and 3Cpro regions of the CVA24v positive samples were sequenced to analyze their evolutionary relationship with the CVA24v strains circulating in China and abroad.Results:All the 20 eye swab samples were EV-positive, and CVA24v-positive, with a positive rate of 100.00%, and all were HEV70-negative.The genomes of CVA24v in VP1 and 3Cpro regions of CVA24v in 5 and 7 samples were successfully sequenced. Based on molecular characterization analysis of VP1 and 3Cpro regions, it was found that the CVA24v isolated in this outbreak had the greatest nucleotide similarity with the CVA24v strains isolated in Thailand in 2014 and French Reunion Islands in 2015. The phylogenetic analysis of 3Cpro and VP1 regions showed that the CVA24v isolated in this outbreak is clustered together with the CVA24v that was prevalent in Thailand in 2014 and the French Reunion Islands in 2015, and have high affinity. Compared with CVA24v isolated in Guangdong in 2010, Thailand in 2014, and French Reunion Islands in 2015, CVA24v isolated in this outbreak was replaced at 4 amino acid sites in 3Cpro region and 1 amino acid site in VP1 region.Conclusions:The cause of this outbreak is enterovirus CVA24v, which has the highest similarity to CVA24v isolated in Thailand in 2014 and in the French Reunion Islands in 2015. There were new amino acid mutations in both 3Cpro and VP1 regions.

This study surveilled urban sewage samples from north China for 9 years to monitor enteroviruses. Samples were collected, processed, and viral identification and molecular typing were performed.

ObjectiveTo study the epidemic and genetic characteristics of coxsackievirus A8 (CVA8) in Guangdong province. MethodsFrom all specimens sampled in hand, foot, and mouth disease (HFMD) cases reported during 2010 – 2020 in Guangdong province, we collected those of other enterovirus (EV) – non-EV71/CVA16/CVA6. After amplified VP1 gene based-genotype identification, CVA8 strains were isolated from the specimens using rhabdomyosarcoma (RDa) cells. The entire VP1 region of the isolated CVA8 strains were amplified and sequenced and then phylogenetically analyzed using DNASTAR6.0 and MEGA6.06 software packages. ResultsOf totally 2650 other EV specimens collected during the period, 2.6% (69) were positive for CVA8 and the annual ratio of CVA8 positive specimens ranged from 0.5% to 12.0%, with the highest ratio in 2020. The CVA8 positive specimens were detected in 13 cities of the province and the highest clustering of positive specimens (28) was detected in Shantou city during July – November 2020. CVA8 strains were isolated from 73.9% (51) of the positive specimens. The phylogenetic analysis results indicated that CVA8 could be divided into five genotypes: A, B, C, D and E. The 44 CVA8 strains isolated in this study shared 77.8% – 100.0% homologies in nucleotide and 92.9% – 100.0% in amino acid, including genotype D (5 strains) and genotype E (39 strains), and the nucleotide homology of 28 CVA8 strains isolated in Shantou city during July – November 2020 was 99.3% – 100.0%. ConclusionCVA8 was prevalent sporadically in Guangdong province during 2010 – 2020 generally but highly prevalence among local HFMD cases in Shantou city during July – November 2020. CVA8 strains of genotype D and E were prevalent and strains of genotype E are currently prevalent in Guangdong province.

柯萨奇病毒B组3型（Coxsackievirus B3,CVB3）是肠道病毒中流行较为广泛的血清型之一,在全球多个国家和地区曾报道儿童急性心肌炎、无菌性脑膜炎、手足口病等的暴发流行,严重威胁儿童健康和公共卫生安全。本研究对广东省2020年手足口病患者标本中分离到的8株CVB3进行基因特征和进化分析,结果显示分离到的8株CVB3 VP1区核苷酸序列相似性为98.2%～99.5%,与原型株Nancy的核苷酸序列相似性在77.9%～78.5%之间,与其他CVB3中国大陆流行株的核苷酸序列相似性为79.6%～82.2%。8株CVB3均为E基因型,为广东省首次报道。8株CVB3分离株在进化树上聚集,提示病毒发生了局部传播。全基因组序列分析提示2株广东CVB3分离株在非结构蛋白区有重组现象的发生。本研究为E基因型CVB3在我国的流行传播和疾病防控提供基础资料。

柯萨奇病毒B组3型(Coxsackievirus B3,CVB3)是肠道病毒中流行较为广泛的血清型之一,在全球多个国家和地区曾报道儿童急性心肌炎、无菌性脑膜炎、手足口病等的暴发流行,严重威胁儿童健康和公共卫生安全.本研究对广东省2020年手足口病患者标本中分离到的8株CVB3进行基因特征和进化分析,结果显示分离到的8株CVB3 VP1区核苷酸序列相似性为98.2％～99.5％,与原型株Nancy的核苷酸序列相似性在77.9％～78.5％之间,与其他CVB3中国大陆流行株的核苷酸序列相似性为79.6％～82.2％.8株CVB3均为E基因型,为广东省首次报道.8株CVB3分离株在进化树上聚集,提示病毒发生了局部传播.全基因组序列分析提示2株广东CVB3分离株在非结构蛋白区有重组现象的发生.本研究为E基因型CVB3在我国的流行传播和疾病防控提供基础资料.

The coronavirus disease-19 (COVID-19) pandemic has been ongoing since December 2019, with more than 6.3 million deaths reported globally as of August 2022. Despite the success of several SARS-CoV-2 vaccines, the rise in variants, some of which are resistant to the effects of vaccination, highlights the need for a so-called pan-coronavirus (universal) vaccine. Here, we performed an immunogenicity comparison of prototype vaccines containing spike protein receptor-binding domain (RBD) residues 319–541, or spike protein regions S1, S2 and S fused to a histidine-tagged or human IgG1 Fc (hFC) fragment with either a longer (six residues) or shorter (three residues) linker. While all recombinant protein vaccines developed were effective in eliciting humoral immunity, the RBD-hFc vaccine was able to generate a potent neutralizing antibody response as well as a cellular immune response. We then compared the effects of recombinant protein length and linker size on immunogenicity in vivo. We found that a longer recombinant RBD protein (residues 319–583; RBD-Plus-hFc) containing a small alanine linker (AAA) was able to trigger long-lasting, high-titer neutralizing antibodies in mice. Finally, we evaluated cross-neutralization of wild-type and mutant RBD-Plus-hFc vaccines against wild-type, Alpha, Beta, Delta and Omicron SARS-CoV-2 variants. Significantly, at the same antigen dose, wild-type RBD-Plus-hFc immune sera induced broadly neutralizing antibodies against wild-type, Alpha, Beta, Delta and Omicron variants. Taken together, our findings provide valuable information for the continued development of recombinant protein-based SARS-CoV-2 vaccines and a basic foundation for booster vaccinations to avoid reinfection with SARS-CoV-2 variants.

The SARS-CoV-2 Delta variant has spread rapidly worldwide. To provide data on its virological profile, we here report the first local transmission of Delta in mainland China. All 167 infections could be traced back to the first index case. Daily sequential PCR testing of quarantined individuals indicated that the viral loads of Delta infections, when they first become PCR-positive, were on average ~1000 times greater compared to lineage A/B infections during the first epidemic wave in China in early 2020, suggesting potentially faster viral replication and greater infectiousness of Delta during early infection. The estimated transmission bottleneck size of the Delta variant was generally narrow, with 1-3 virions in 29 donor-recipient transmission pairs. However, the transmission of minor iSNVs resulted in at least 3 of the 34 substitutions that were identified in the outbreak, highlighting the contribution of intra-host variants to population-level viral diversity during rapid spread.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus driving the ongoing coronavirus disease 2019 (COVID-19) pandemic, continues to rapidly evolve. Because of the limited efficacy of vaccination in prevention of SARS-CoV-2 transmission and continuous emergence of variants of concern (VOCs), orally bioavailable and broadly efficacious antiviral drugs are urgently needed. Previously, we showed that the parent nucleoside of remdesivir, GS-441524, has potent anti–SARS-CoV-2 activity. Here, we report that esterification of the 5′-hydroxyl moieties of GS-441524 markedly improved antiviral potency. This 5′-hydroxyl-isobutyryl prodrug, ATV006, demonstrated excellent oral bioavailability in rats and cynomolgus monkeys and exhibited potent antiviral efficacy against different SARS-CoV-2 VOCs in vitro and in three mouse models. Oral administration of ATV006 reduced viral loads and alleviated lung damage when administered prophylactically and therapeutically to K18-hACE2 mice challenged with the Delta variant of SARS-CoV-2. These data indicate that ATV006 represents a promising oral antiviral drug candidate for SARS-CoV-2.