A case of human rabies with a long incubation period in Wuhan.

Rabies remains endemic in China and continues to pose a major threat to public health with a nearly 100 % case fatality rate in humans. We confirmed a case of human rabies in Wuhan, in May 2018. The patient had got a dog bite wound 3 years before symptoms of confusion, hydrophobia, and photophobia onset. On May 14, our laboratory confirmed that the patient was infected with a rabies virus that circulates in dogs in China and died on May 24, two weeks later after admission. Complete glycoprotein gene sequences determined for this isolate indicated the source of a RABV infection was dog-related RABV variants.

Genetic characteristics of Echovirus 11 circulating in Xiangyang, Hubei Province between 2016 and 2017 / 中华微生物学和免疫学杂志

Objective:To analyze the molecular characteristics of Echovirus 11 (Echo11) strains isolated in Xiangyang, Hubei Province from 2016 to 2017 based on the sequences of VP1 gene.Methods:Rectal and throat swab specimens were collected from children with hand, foot and mouth disease (HFMD) in Xiangyang from 2016 to 2017. Echo11 strains were detected by real-time reverse transcriptase PCR (RT-PCR) and isolated after cultured in human rhabdosarcoma (RD) cells. The VP1 regions of Echo11 strains isolated from RD cells and the whole genomes of three representative Echo11 strains were amplified by conventional RT-PCR and the sequences were analyzed. DNAStar7.0 (MegAlign) and MEGA6.0 (Data) were used to analyze the homology and mutation sites in nucleotide and amino acid sequences. Neighbor-joining method was used to construct phylogenetic trees. Recombination analysis was performed with SimPlot software (BootScanning).Results:A total of 11 Echo11 strains were isolated from 3 494 HFMD cases, accounting for 0.31%. They were highly homologous in the VP1 gene. These strains shared 98.4%-100.0% homology in nucleotide sequences and 98.3%-100.0% homology in amino acid sequences. The homology between the 11 Echo11 strains and the prototype strain (Echo11/Gregory, X80059) was 73.9%-74.8% in nucleotide sequences and 87.7%-88.7% in amino acid sequences. All of the Echo11 strains circulating in Xiangyang were classified into lineage D, having a similarity to the strains circulating in some regions of mainland China since 2013. In multiple regions of the genome, the Echo11 strains isolated in Xiangyang were highly similar to the Henan Echo1 strains in 2010 and the Hubei Echo6 strains in 2015, suggesting there was recombination within the genome of Echo11 strains in Xiangyang.Conclusions:The Echo11 strains circulating in Xiangyang from 2016 to 2017 belonged to lineage D and were recombinant strains.

The feasibility of convalescent plasma therapy in severe COVID-19 patients: a pilot study

Currently, there are no approved specific antiviral agents for 2019 novel coronavirus disease (COVID-19). In this study, ten severe patients confirmed by real-time viral RNA test were enrolled prospectively. One dose of 200 mL convalescent plasma (CP) derived from recently recovered donors with the neutralizing antibody titers above 1:640 was transfused to the patients as an addition to maximal supportive care and antiviral agents. The primary endpoint was the safety of CP transfusion. The second endpoints were the improvement of clinical symptoms and laboratory parameters within 3 days after CP transfusion. The median time from onset of illness to CP transfusion was 16.5 days. After CP transfusion, the level of neutralizing antibody increased rapidly up to 1:640 in five cases, while that of the other four cases maintained at a high level (1:640). The clinical symptoms were significantly improved along with increase of oxyhemoglobin saturation within 3 days. Several parameters tended to improve as compared to pre-transfusion, including increased lymphocyte counts (0.65x109/L vs. 0.76x109/L) and decreased C-reactive protein (55.98 mg/L vs. 18.13 mg/L). Radiological examinations showed varying degrees of absorption of lung lesionswithin 7 days. The viral load was undetectable after transfusion in seven patients who had previous viremia. No severe adverse effects were observed. This study showed CP therapy was welltolerated and could potentially improve the clinical outcomes through neutralizing viremia in severe COVID-19 cases. The optimal dose and time point, as well as the clinical benefit of CP therapy, needs further investigation in larger well-controlled trials. Significance StatementCOVID-19 is currently a big threat to global health. However, no specific antiviral agents are available for its treatment. In this work, we explored the feasibility of convalescent plasma (CP) transfusion to rescue severe patients. The results from 10 severe adult cases showed that one dose (200 mL) of CP was welltolerated and could significantly increase or maintain the neutralizing antibodies at a high level, leading to disappearance of viremia in 7 days. Meanwhile, clinical symptoms and paraclinical criteria rapidly improved within 3 days. Radiological examination showed varying degrees of absorption of lung lesions within 7 days. These results indicate that CP can serve as a promising rescue option for severe COVID-19 while the randomized trial is warranted.

Molecular detection and sequence analysis of enteroviruses isolated from a suspected outbreak of aseptic meningitis / 中华微生物学和免疫学杂志

Objective To detect and analyze enteroviruses causing suspected aseptic meningitis in a kindergarten in Jinhua City, Zhejiang Province. Methods Viral RNA was extracted from samples and cDNA was prepared by reverse transcription. PCR was performed to amplify the partial sequences of the 5′-untranslated region ( UTR) and VP1 gene of enteroviruses. Serotypes of the viruses were determined by com-paring the homology between the partial sequences of VP1 gene. Phylogenetic tree of the partial VP1 se-quences was constructed using MEGA6. Results This study included seven patients and twenty-six asymp-tomatic students. Coxsakievirus A10 (CV-A10) was detected in 48. 5％ of the students and echovirus 6 (Echo 6) in 21. 2％. Besides, 12. 1％ of the students might be co-infected by the two viruses. Among the seven patients, six were infected by CV-A10 and the other one might have co-infection. According to the phylogenetic analysis, CV-A10 strains detected in this study were closely related to those isolated in China in recent years, including the strains isolated in Xiamen in 2015 and Yunnan in 2017, while the Echo 6 strains were phylogenetically related to those isolated in Yunnan, Guangzhou and Shandong in 2014. Conclusions CV-A10 and Echo 6 were detected in the cases with suspected aseptic meningitis and had close phylogenetic relationships to the strains appeared in China in recent years.

Prokaryotic expression of coxsackie virus A6 genes and preparation of polyclonal antibody against the VP1 and VP2 protein / 中华实验和临床病毒学杂志

Objective@#To construct the prokaryotic plasmid expressing the recombinant protein Coxsackie virus A6 VP1 or VP2 and prepare the antiserum of rabbit anti-CVA6 VP1 or anti-CVA6 VP2.@*Methods@#CVA6 VP1and VP2 gene fragments were amplified by reverse transcription PCR and inserted into the prokaryotic expression vectors. The recombinant plasmids were expressed in E. coli BL21 (DE3). After induction with Isopropyl β-D-Thiogalactoside (IPTG), the fusion proteins were obtained, and then purified by SDS-PAGE electrophoresis and gel extraction. The polyclonal antibodies were prepared by immunizing rabbits with the fusion proteins and analyzed with Western blot(WB) and indirect immunofluorescence assay(IFA).@*Results@#The prokaryotic expression vector of CVA6 VP1 or VP2 was confirmed by PCR, double enzyme digestion and sequencing. CVA6 VP1 and VP2 fusion proteins with high purity were obtained. WB and IFA were used to identify polyclonal antibodies.@*Conclusions@#The CVA6 VP1 and VP2 prokaryotic expression vectors were successfully constructed, and the recombinant CVA6 VP1 and VP2 proteins and their corresponding polyclonal antibodies were obtained.

Sequencing and analysis of N gene of street rabies virus isolated from different hosts in Zhejiang province / 中华流行病学杂志

<p><b>OBJECTIVE</b>To elucidate the characteristics of genetic variability and its relationship with prevalence, through sequencing and analysis of N gene among street rabies virus isolated from different hosts (homo sapiens, ferret badger, dog) in Zhejiang province.</p><p><b>METHODS</b>Samples were screened and confirmed by direct fluorescence assay and reverse transcript PCR. Sequences were analyzed using bio-information software.</p><p><b>RESULTS</b>Eighteen street rabies virus strains were identified, including 2 from homo sapiens, 5 from ferret badger, and 11 from dog. Similarities of N gene and N protein were calculated to be 89.7%-100.0% and 98.4%-100.0% respectively. Mutations occurred in N gene were almost non-sense mutations. In addition,Data from phylogenetic analysis showed that all these strains could be classified into traditional genotype 1.</p><p><b>CONCLUSION</b>The prevalence of rabies viruses among different hosts in Zhejiang province had certain regional properties. Rabies viruses isolated from the same kind of host or from the same/adjacent county/counties had the closest relationship. However, the characteristics of rabies virus prevalent in homo sapiens were somewhat complicated. In summary, the transmission of street rabies virus in Zhejiang province was from dogs to ferret badgers and homo sapiens, and the virus could circulate and cross-regional transmit among dogs and ferret badgers.</p>