Phylogeny and molecular evolution of the first local monkeypox virus cluster in Guangdong Province, China.

The first local mpox outbreak in Guangdong Province, China occurred in June 2023. However, epidemiological data have failed to quickly identify the source and transmission of the outbreak. Here, phylogeny and molecular evolution of 10 monkeypox virus (MPXV) genome sequences from the Guangdong outbreak were characterized, revealing local silent transmissions that may have occurred in Guangdong whose mpox outbreaks suggested a molecular epidemiological correlation with Portugal and several regions of China during the same period. The lineage IIb C.1, which includes all 10 MPXV from Guangdong, shows consistent temporal continuity in both phylogenetic characteristics and unique molecular evolutionary mutation spectrum, reflected in the continuous increase of single nucleotide polymorphisms (SNPs) and shared mutations over time. Compared with the Japan MPXV, the Guangdong MPXV showed higher genomic nucleotide differences and separated 14 shared mutations from the B.1 lineage, comprising 6 non-synonymous mutations in genes linked to host regulation, virus infection, and virus life cycle. The unique mutation spectrum with temporal continuity in IIb C.1, related to apolipoprotein B mRNA-editing catalytic polypeptide-like 3, promotes rapid viral evolution and diversification. The findings contribute to understanding the ongoing mpox outbreak in China and offer insights for developing joint prevention and control strategies.

Evaluation of a multiplex PCR assay for detection of respiratory viruses and Mycoplasma pneumoniae in oropharyngeal swab samples from outpatients.

BACKGROUND: Respiratory viruses, such as influenza viruses, initially infect the upper airways but can manifest as severe lower respiratory tract infections in high-risk patients with significant morbidity and mortality. For syndromic diagnosis, several multiplex nucleic acid amplification tests have been developed for clinics, of which SureX 13 Respiratory Pathogen Multiplex Kit (ResP) can simultaneously detect 13 pathogens directly from airway secretion specimens. The organisms identified are influenza virus A, influenza virus A pdmH1N1 (2009), influenza virus A H3N2, influenza virus B, adenovirus, boca virus, rhinovirus, parainfluenza virus, coronavirus, respiratory syncytial virus, human metapneumovirus, Mycoplasma pneumoniae, and Chlamydia. METHODS: This study provides performance evaluation data of this assay by comparing with pathogen-specific PCRs from oropharyngeal swab samples. RESULTS: Ten pathogens were detected in this assay, of which rhinovirus, adenovirus, and influenza virus A pdmH1N1 (2009) were the most common. The overall agreement between the ResP and the comparator tests was 93.8%. The ResP demonstrated 86.5% agreement for positive results and 97.8% agreement for negative results. CONCLUSION: The ResP assay demonstrated a highly concordant performance comparing with pathogen-specific PCRs for detection of respiratory pathogens in oropharyngeal swabs from outpatients and could aid in the diagnosis of respiratory infections in a variety of clinical scenarios.

The dengue preface to endemic in mainland China: the historical largest outbreak by Aedes albopictus in Guangzhou, 2014.

BACKGROUND: Dengue was regarded as a mild epidemic in mainland China transmitted by Aedes albopictus. However, the 2014 record-breaking outbreak in Guangzhou could change the situation. In order to provide an early warning of epidemic trends and provide evidence for prevention and control strategies, we seek to characterize the 2014 outbreak through application of detailed cases and entomological data, as well as phylogenetic analysis of viral envelope (E) gene. METHODS: We used case survey data identified through the Notifiable Infectious Disease Report System, entomological surveillance and population serosurvey, along with laboratory testing for IgM/IgG, NS1, and isolation of viral samples followed by E gene sequencing and phylogenetic analysis to examine the epidemiological and molecular characteristics of the outbreak. RESULTS: The 2014 dengue outbreak in Guangzhou accounted for nearly 80% of total reported cases that year in mainland China; a total of 37,376 cases including 37,340 indigenous cases with incidence rate 2908.3 per million and 36 imported cases were reported in Guangzhou, with 14,055 hospitalized and 5 deaths. The epidemic lasted for 193 days from June 11 to December 21, with the highest incidence observed in domestic workers, the unemployed and retirees. The inapparent infection rate was 18.00% (135/750). In total, 96 dengue virus 1 (DENV-1) and 11 dengue virus 2 (DENV-2) strains were isolated. Phylogenetic analysis indicated that the DENV-1 strains were divided into genotype I and V, similar to the strains isolated in Guangzhou and Dongguan in 2013. The DENV-2 strains isolated were similar to those imported from Thailand on May 11 in 2014 and that imported from Indonesia in 2012. CONCLUSIONS: The 2014 dengue epidemic was confirmed to be the first co-circulation of DENV-1 and DENV-2 in Guangzhou. The DENV-1 strain was endemic, while the DENV-2 strain was imported, being efficiently transmitted by the Aedes albopictus vector species at levels as high as Aedes aegypti.

Functional properties of DENV EDIII­reactive antibodies in human DENV­1­infected sera and rabbit antiserum to EDIII.

The envelope domain III (EDIII) of the dengue virus (DENV) has been confirmed to be involved in receptor binding. It is the target of specific neutralizing antibodies, and is considered to be a promising subunit dengue vaccine candidate. However, several recent studies have shown that anti­EDIII antibodies contribute little to the neutralizing or enhancing ability of human DENV­infected serum. The present study involved an analysis of the neutralization and antibody­dependent enhancement (ADE) activities of EDIII­reactive antibodies in human convalescent sera from patients with primary DENV­1 infection and rabbit antiserum immunized with recombinant DENV­1 EDIII protein. The results indicated that serum neutralization was not associated with titres of EDIII­binding antibodies in the human DENV­1­infected sera. The depletion of anti­EDIII antibodies from these serum samples revealed that the anti­EDIII antibodies of the patients contributed little to neutralization and ADE. However, the EDIII­reactive antibodies from the rabbit antiserum exhibited protective abilities of neutralization at a high dilution (~1:50,000) and ADE at a low dilution (~1:5,000) for the homotypic DENV infection. Notably, the rabbit antiserum displayed ADE activity only at a dilution of 1:40 for the heterotypic virus infection, which suggests that EDIII­reactive antibodies may be safe in secondary infection with heterotypic viruses. These results suggest that DENV EDIII is not the predominant antigen of the DENV infection process; however, purified or recombinant DENV EDIII may be used as a subunit vaccine to provoke an effective and safe antibody response.

[Mapping of the B Cell Neutralizing Epitopes on ED III of Envelope Protein from Dengue Virus].

Dengue virus (DENV) envelope [E] protein is the major surface protein of the virions that indued neutralizing antibodies. The domain III of envelope protein (EDIII) is an immunogenic region that holds potential for the development of vaccines; however, the epitopes of DENV EDIII, especially neutralizing B-cell linear epitopes, have not been comprehensively mapped. We mapped neutralizing B-cell linear epitopes on DENV-1 EDIII using 27 monoclonal antibodies against DENV-1 EDIII proteins from mice immunized with the DENV-1 EDIII. Epitope recognition analysis was performed using two set of sequential overlapping peptides (16m and 12m) that spanned the entire EDIII protein from DENV-1, respectively. This strategy identified a DENV-1 type- specific and a group-specific neutralizing epitope, which were highly conserved among isolates of DENV-1 and the four DENV serotypes and located at two regions from DENV-1 E, namely amino acid residues 309-320 and 381-392(aa 309-320 and 381-392), respectively. aa310 -319(310KEVAETQHGT319)was similar among the four DENV serotypes and contact residues on aa 309 -320 from E protein were defined and found that substitution of residues E309 , V312, A313 and V320 in DENV-2, -3, -4 isolates were antigenically silent. We also identified a DENV-1 type-specific strain-restricted neutralizing epitope, which was located at the region from DENV-1 E, namely amino acid residues 329-348 . These novel type- and group-specific B-cell epitopes of DENV EDIII may aid help us elucidate the dengue pathogenesis and accelerate vaccine design.

[Establishment and preliminary application of dengue virus envelope domain III IgG antibody capture enzyme-linked immuno-absorbent assay].

OBJECTIVE: To establish a highly sensitive and specific assay to detect dengue virus (DENV) envelope protein domain III (EDIII) IgG antibody, and to explore its value in the diagnosis and seroepidemiological survey of dengue. METHODS: The DENV EDIII IgG antibody capture ELISA was developed using the recombinant full-length DENV EDIII, which was prepared by Pichia yeast expression system as the capture antigen. The serum samples were collected from the same group of 35 DENV-1 patients of primary infection during disease period in 2006 and their follow-up phase in 2010; and the sensitivity of the assay was compared to that of the commercial Panbio DENV IgG ELISA. RESULTS: The sensitivity of DENV EDIII IgG ELISA in detecting the serum samples from disease period and follow-up phase was 87% (20/23) and 94% (33/35), respectively; whereas the sensitivity of Panbio DENV IgG ELISA was 71% (25/35) and 0, respectively. The sensitivity of DENV EDIII IgG ELISA in detecting the serum samples from both periods was similar, without statistical significance (χ(2) = 0.946, P = 0.331). For serum samples from disease period, the sensitivity of DENV EDIII IgG ELISA was comparable with that of Panbio DENV IgG ELISA (χ(2) = 1.924, P = 0.165). However, DENV EDIII IgG ELISA demonstrated a significantly higher sensitivity than Panbio DENV IgG ELISA in detecting the serum samples from follow-up phase (χ(2) = 62.432, P = 0.000). CONCLUSION: DENV EDIII IgG capture ELISA is highly sensitive in detecting IgG in the serum samples from either disease period or follow-up phase. This method might be a promising alternative for diagnosis and seroepidemiologic survey of dengue.

Molecular characterization of the envelope gene of dengue virus type 3 newly isolated in Guangzhou, China, during 2009-2010.

BACKGROUND: After an absence of 29 years, dengue virus type 3 (DENV-3) re-emerged in Guangzhou in 2009 and again in 2010. However, the geographical route by which the virus entered the city, and how it has changed genetically, remain unclear. Therefore, we carried out a comprehensive investigation into the molecular characteristics of the DENV-3 involved. METHODS: The envelope (E) genes of viruses isolated from dengue patients during the 2009-2010 epidemics were sequenced and compared with previously published E gene sequences of global representative DENV-3 strains available in GenBank, including isolates circulating in other provinces of China. RESULTS: A total of 13 isolates (seven from 2009 and six from 2010) were obtained from human serum samples. Phylogenetic analysis revealed that the isolates were grouped into three genotypes (I, III, and V) and then two clades within genotype III (genotype I from Indonesia, genotype III clade A from Côte d'Ivoire, genotype III clade B from Tanzania, and genotype V from Philippines). In addition, there were 1.3-9.0% and 0.5-3.9% differences in the nucleic and deduced amino acid sequences between the 2009 and 2010 strains, respectively. CONCLUSIONS: The DENV-3 viruses from the period 2009-2010 were not from the continuous spread of an epidemic strain or the re-emergence of the 2009 strains in the 2-year period. The introduction of different DENV-3 genotypes following more than one geographical route was an important contributing factor to the 2009-2010 dengue epidemics in Guangzhou.

Novel AAV-based genetic vaccines encoding truncated dengue virus envelope proteins elicit humoral immune responses in mice.

The envelope protein of dengue virus is involved in host cell attachment for entry and induction of protective immunity. Current efforts are focused on producing a tetravalent vaccine by mixing four monovalent vaccine components. In this work, we developed a genetic vaccine based on a novel adeno-associated viral (AAV) vector expressing the carboxy-terminal truncated envelope protein (79E) of dengue virus. The expression of the recombinant 79E protein in HEK 293 cells was confirmed by Western blot. Vectors packaged with novel AAV capsids (AAV2/8 or AAV2/rh32.33) were injected into C57BL/6 mice intramuscularly. Dengue virus antigen was produced in the mice and induced long-lasting antibody responses against the dengue virus still detectable 20 weeks after immunization. AAV2/8 vaccine induced higher anti-dengue virus antibody levels than AAV2/rh32.33 vaccine or AAV plasmid. Furthermore, the anti-dengue antibodies could neutralize homogeneous dengue virus. These results demonstrated that the AAV vaccines possessed appropriate immunogenicity and could be used for the development of an effective dengue vaccine.

[Epidemiological situation and the E gene evolution of dengue virus in Guangzhou, 2011].

OBJECTIVE: To investigate the epidemiological characteristics of Dengue and the E gene of the new isolated strains. METHODS: Epidemiological data and serum samples were collected. Serotypes were detected by real-time PCR and virus was isolated in C6/36. E gene of the new isolated strains were sequenced and analyzed by Mega 4.0. RESULTS: The cases of Dengue reached at the peak during September and November, with Serotype 1, 2 and 4 were involved. Five strains of serotype l were isolated, with 4 of them fell into the clad of Asia genotype, and 1 belonged to America/Africa genotype. CONCLUSION: The strains isolated in Guangzhou showed a high identity to the Southeast Asian strains. There seemed high risk of outbreak of Dengue in this area, However, the Dengue virus might have already been localized.

[Production and characterization of the monoclonal antibodies against nonstructural 1 protein of DENV-4].

AIM: To produce monoclonal antibodies (mAbs) against nonstructural 1 protein (NS1) of DENV-4 and characterization. METHODS: BALB/c mice were immunized with recombinant DENV4-NS1 protein and inactive DENV-4. Splenocytes of immunized mice were fused with myeloma cells (NS-1) to produce hybridoma cell lines, secreting anti-DENV4-NS1 protein mAbs. ELISA was used to identify the mAbs against NS1of DENV-4. Immnofluorescence assay (IFA) and Western blot analysis were applied to identify the specificity of mAbs. RESULTS: fifteen mAbs recognizing two different antigenic epitopes on NS1of DENV-4 were obtained. These mAbs had characteristics of specific binding to recombinant NS1 of DENV-4. The IFA showed that all of the mAbs strongly reacted to DENV-4, while had no cross-reactivity to the other three serotypes of dengue virus. CONCLUSION: mAbs against NS1 of DENV-4 with high specificity has been successfully established. These results lay the foundation value for study on the structural and functional of DENV4-NS1 protein and early diagnosis of DENV-4 infection.

[Molecular epidemiologic analysis on new emerged type 3 dengue virus in Guangzhou in 2009].

OBJECTIVE: To analyze and trace the infection source the envelope (E) gene of the new emerged type 3 dengue virus in Guangzhou in 2009. METHODS: Sera were collected from patients infected with local dengue fever. Dengue virus was cultured and isolated by C6/36 cells. The whole length E gene was amplified from the positive specimen by RT-PCR, thereby sequenced and phylogenetic tree drawn by neighbor-joining method. Both data on epidemiologic and molecular studies were processed and analysed. RESULTS: 7 strains of type 3 dengue virus were isolated from samples of the 19 patients. E gene of these strains was amplified. The complete E genes of 7 strains belonged to 1479 nucleotides in length, encoding a polyprotein of 493 amino acids. Data from the phylogenetic analysis showed that 09/GZ/1081, 09/GZ/1483 and 09/GZ/10806 strains fell within the Southeast Asia/South Pacific group. 09/GZ/10616, 09/GZ/11144, 09/GZ/11194 while 09/GZ/13105 strains fell within the India group. CONCLUSION: The type 3 dengue virus identified in Guangzhou area in 2009 was imported and could be decided into two genotypes.

[To evaluate the neutralizing abilities of anti-dengue virus antibodies with nonstructural protein 1 antigen capture enzyme-linked immunosorbent assay].

OBJECTIVE: To produce neutralizing antibodies against envelope protein domain III (EDIII) of dengue virus serotype I (DENV-1) and evaluate the nonstructural protein 1 (NS1) antigen capture enzyme-linked immunosorbent assay (ELISA) for identification of antibody neutralizing abilities. METHODS: Five BALB/c mice and one New Zealand Rabbit were immunized with recombinant EDIII protein of DENV-1 for the production of hybridomas and hyperimmune sera. Indirect ELISA, immunofluorescence assay (IFA) and Western Blot analyses were applied to identify specificity of antibodies. Comparing to plaque reduction neutralization test (PRNT), the new established DENV-1 specific NS1 antigen capture ELISA was used for detecting the neutralizing abilities of these antibody. RESULTS: Four strains of monoclonal antibodies (mAbs) named 1A1, 1B3, 3D3 and 9D6 and one hyperimmune serum of rabbit were obtained, all of which were approved to have neutralizing abilities to DENV-1 with the PRNT titer of 1:1024, 1:512, 1:256, 1:4096 and 1:4096. MAb 3D3 with the lowest neutralization titer in PRNT had not shown neutralizing ability to DENV-1 in NS1 antigen capture ELISA, while MAbs 1A1, 1B3 and 9D6 and the rabbit hyperimmune serum could protect the C6/36 from being infected by DENV-1 with the neutralization titer of 1:32, 1:32, 1:128 and 1:128 in this assay. CONCLUSION: NS1 antigen capture ELISA could be used to identify antibody neutralizing abilities to DENV, it was a faster and more convenient way to screen antibodies with high neutralization titer and might also be used as one of the methods to evaluate the effects of vaccines.

Cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human.

The genomic sequences of severe acute respiratory syndrome coronaviruses from human and palm civet of the 2003/2004 outbreak in the city of Guangzhou, China, were nearly identical. Phylogenetic analysis suggested an independent viral invasion from animal to human in this new episode. Combining all existing data but excluding singletons, we identified 202 single-nucleotide variations. Among them, 17 are polymorphic in palm civets only. The ratio of nonsynonymous/synonymous nucleotide substitution in palm civets collected 1 yr apart from different geographic locations is very high, suggesting a rapid evolving process of viral proteins in civet as well, much like their adaptation in the human host in the early 2002-2003 epidemic. Major genetic variations in some critical genes, particularly the Spike gene, seemed essential for the transition from animal-to-human transmission to human-to-human transmission, which eventually caused the first severe acute respiratory syndrome outbreak of 2002/2003.