Genotype Shift Research Articles

Comparative phylogenetic analysis and transcriptomic profiling of Dengue (DENV-3 genotype I) outbreak in 2021 in Bangladesh

BackgroundThe next-generation sequencing (NGS) technology facilitates in-depth study of host-pathogen metatranscriptome. We, therefore, implicated phylodynamic and transcriptomic approaches through NGS technology to know/understand the dengue virus (DENV) origin and host response with dengue fever.MethodsIn this study, blood serum RNA was extracted from 21 dengue patients and 3 healthy individuals. Total transcriptomic data were analyzed for phylogenetic, phylodynamic, differential express gene (DEG), and gene ontology (GO) using respective bioinformatics tools.ResultsThe viral genome sequence revealed dengue viral genome size ranges 10647 to 10707 nucleotide. Phylogenetic and phylodynamic analysis showed that the 2021 epidemic isolates were DENV-3 genotype-I and maintained as a new clade in compared to 2019 epidemic. Transcriptome analysis showed a total of 2686 genes were DEG in dengue patients compared to control with a q-value < 0.05. DESeq2 plot counts function of the top 24 genes with the smallest q-values of differential gene expression of RNA-seq data showed that 11 genes were upregulated, whereas 13 genes were downregulated. GO analysis showed a significant upregulation (p = < 0.001) in a process of multicellular organismal, nervous system, sensory perception of chemical stimulus, and G protein-coupled receptor signaling pathways in the dengue patients. However, there were a significant downregulation (p = < 0.001) of intracellular component, cellular anatomical entity, and protein-containing complex in dengue patients. Most importantly, there was a significant increase of a class of immunoregulatory proteins in dengue patients in compared to the controls, with increased GO of immune system process. In addition, upregulation of toll receptor (TLR) signaling pathways were found in dengue patients. These TLR pathways were particularly involved for the activation of innate system coupled with adaptive immune system that probably involved the rapid elimination of dengue virus infected cells. These differentially expressed genes could be further investigated for target based prophylactic interventions for dengue.ConclusionThis is a first report describing DENV complete genomic features and differentially expressed genes in patients in Bangladesh. These genes may have diagnostic and therapeutic values for dengue infection. Continual genomic surveillance is required to further investigate the shift in dominant genotypes in relation to viral pathogenesis.

Epidemiological and genomic analysis of dengue cases in Guangzhou, China, from 2010 to 2019

With a long epidemic history and a large number of dengue cases, Guangzhou is a key city for controlling dengue in China. The demographic information regarding dengue cases, and the genomic characteristics of the envelope gene of dengue viruses, as well as the associations between these factors were investigated from 2010 to 2019, to improve the understanding of the epidemiology of dengue in Guangzhou. Demographic data on 44,385 dengue cases reported to the Notifiable Infectious Disease Report System were analyzed using IBM SPSS Statistics v. 20. Dengue virus isolates from patient sera were sequenced, and phylogenetic trees were constructed using PhyML 3.1. There was no statistical difference in the risk of dengue infection between males and females. Unlike other areas in which dengue is endemic, the infection risk in Guangzhou increased with age. Surveillance identified four serotypes responsible for dengue infections in Guangzhou. Serotype 1 remained prevalent for most of the study period, whereas serotypes 3 and 4 were prevalent in 2012 and 2010, respectively. Different serotypes underwent genotype and sublineage shifts. The epidemiological characteristics and phylogeny of dengue in Guangzhou suggested that although it has circulated in Guangzhou for decades, it has not been endemic in Guangzhou. Meanwhile, shifts in genotypes, rather than in serotypes, might have caused dengue epidemics in Guangzhou.

Streptococcus dysgalactiae Bloodstream Infections, Norway, 1999-2021.

Streptococcus dysgalactiae increasingly is recognized as a pathogen of concern for human health. However, longitudinal surveillance data describing temporal trends of S. dysgalactiae are scarce. We retrospectively identified all β-hemolytic streptococcal bloodstream infections reported in Bergen, in western Norway, during 1999-2021. To explore S. dysgalactiae disease burden in a broader context, we mapped the incidence of all microbial species causing bloodstream infections during 2012-2021. We found S. dysgalactiae incidence rates substantially increased during the study period; by 2021, S. dysgalactiae was the fifth most common pathogen causing bloodstream infections in our region. We noted genotypic shifts and found that the rising trend was related in part to the introduction and expansion of the stG62647 emm-type. S. dysgalactiae is among the most common causes of bloodstream infections in western Norway, and increased surveillance and unambiguous species identification are needed to monitor the disease burden attributable to this pathogen.

Novel insights on the genetic population structure of human-infecting Cyclospora spp. and evidence for rapid subtype selection among isolates from the USA

Human-infecting Cyclospora was recently characterized as three species, two of which (C. cayetanensis and C. ashfordi) are currently responsible for all known human infections in the USA, yet much remains unknown about the genetic structure within these two species. Here, we investigate Cyclospora genotyping data from 2018 through 2022 to ascertain if there are temporal patterns in the genetic structure of Cyclospora parasites that cause infections in US residents from year to year. First, we investigate three levels of genetic characterization: species, subpopulation, and strain, to elucidate annual trends in Cyclospora infections. Next, we determine if shifts in genetic diversity can be linked to any of the eight loci used in our Cyclospora genotyping approach. We observed fluctuations in the abundance of Cyclospora types at the species and subpopulation levels, but no significant temporal trends were identified; however, we found recurrent and sporadic strains within both C. ashfordi and C. cayetanensis. We also uncovered major shifts in the mitochondrial genotypes in both species, where there was a universal increase in abundance of a specific mitochondrial genotype that was relatively abundant in 2018 but reached near fixation (was observed in over 96% of isolates) in C. ashfordi by 2022. Similarly, this allele jumped from 29% to 82% relative abundance of isolates belonging to C. cayetanensis. Overall, our analysis uncovers previously unknown temporal-genetic patterns in US Cyclospora types from 2018 through 2022 and is an important step to presenting a clearer picture of the factors influencing cyclosporiasis outbreaks in the USA.

Global and Local Evolutionary Dynamics of Dengue Virus Serotype 1, 3 and 4.

Evolutionary studies on Dengue virus (DENV) in endemic regions are necessary since naturally occurring mutations may lead to genotypic variations or shifts in serotypes, which may lead to future outbreaks. Our study comprehends the evolutionary dynamics of DENV, using phylogenetic, molecular clock, skyline plots, network, selection pressure, and entropy analyses based on partial CprM gene sequences. We have collected 250 samples, 161 in 2017 and 89 in 2018. Details for the 2017 samples were published in our previous article and that of 2018 are presented in this study. Further evolutionary analysis was carried out using 800 sequences, which incorporate the study and global sequences from GenBank: DENV-1 (n=240), DENV-3 (n=374), and DENV-4 (n=186), identified during 1944-2020, 1956-2020, and 1956-2021, respectively. Genotypes V, III, and I were identified as the predominant genotypes of the DENV-1, DENV-3, and DENV-4 serotypes, respectively. The rate of nucleotide substitution was found highest in DENV-3 (7.90×10-4s/s/y), followed by DENV-4 (6.23×10-4s/s/y) and DENV-1 (5.99×10-4s/s/y). The Bayesian skyline plots of the Indian strains revealed dissimilar patterns amongst the population size of the three serotypes. Network analyses showed the presence of different clusters within the prevalent genotypes. The data presented in this study will assist in supplementing the measures for vaccine development against DENV.

Introducing ROTAVAC® to the occupied Palestinian Territories: Impact on diarrhea incidence, rotavirus prevalence and genotype composition.

Rotavirus infection remains an important cause of morbidity and mortality in children. The introduction of vaccination programs in more than 100 countries has contributed to a decrease in hospitalizations and mortality. This study investigates the epidemiological impact of the rotavirus vaccine ROTAVAC® in the Palestinian Territories, the first country to switch from ROTARIX® to this new vaccine. Clinical surveillance data was collected fromchildren younger than 5attendingoutpatient clinics throughout Gaza withdiarrhea between 2015 and 2020. The incidence of all-cause diarrhea was assessed using an interrupted time-series approach. Rotavirus prevalence was determined at the Caritas Baby Hospital in the West Bank usingELISA on stool specimen of children younger than 5with diarrhea. Genotyping was performed on 325 randomly selected rotavirus-positive samples from January 2015 through December 2020 using multiplex PCR analysis. Average monthly diarrhea casesdropped by 16.7% annually fromintroduction of rotavirus vaccination in May 2016 to the beginning of the SARS-CoV-2 epidemic in March 2020 for a total of 53%. Case count declines were maintained afterthe switchto ROTAVAC® in October 2018. Rotavirus positivity in stool samples declined by 67.1% over the same period without change followingthe switch to ROTAVAC®. The distribution of predominant genotypes in rotavirus-positive stool samples changed from a pre-vaccination G1P [8] to G9P[8] and G12P[8] during the ROTARIX® period and G2P[4] after the introduction of ROTAVAC®. ROTAVAC® has shown epidemiological impact on par with ROTARIX® after its introduction to the national immunization schedule in the Palestinian Territories. A molecular genotype shift from a pre-vaccination predominance of G1P[8] to a current predominance of G2P[4] requires more long-term surveillance.

The Epidemiological surveillance of Human Papillomavirus (HPV) genotypes in oral and genital tracts and the impact of HPV vaccination on male university students from Rio de Janeiro state, Brazil

Introduction: Human Papillomavirus (HPV) infections are of significant concern in men, given its potential impact on their health and the risk of transmission to partners. Understanding and addressing this infection in men is crucial to evaluate the effectiveness of vaccination in reducing HPV-related diseases. Objective: To assess the impact of HPV vaccination, potential genotype shifts, and adverse effects, through a prospective study conducted with male university students. Methods:The study involved 286 volunteers who were examined at Sexually Transmitted Disease Clinics at the Universidade Federal Fluminense in Niterói, Rio de Janeiro, Brazil. The HPV prevalence was evaluated using generic PCR, genotyped by DNA microarray and monitored adverse effects. Results: The findings of this study revealed the absence of moderate or severe adverse effects. Genetic shifts were observed, including the disappearance of oncogenic HPV types 16 and 18. Surprisingly, even after completing the full vaccine regimen, students still harbored HPV11 in the oral tract. Furthermore, persistent HPV 6 and 11 infections were identified in three students, who had pre-existing infections prior to vaccination, at the follow-up visit. Multivariate analysis uncovered independent associations, notably an increased risk of HPV infection in the oral tract among men who have sex with men. HPV prevalence rates remained low both before and after the vaccination scheme (T0: 14.7%, T1: 8.7%). Even after the full vaccination scheme, the prevalence remained similar at T2 (14.6%), with no statistically significant differences recorded. HPV11 emerged as the most prevalent type throughout the study, followed by HPV6. Vaccine genotypes were detected in a significant proportion of samples at T0 (85.4%), T1 (89.5%), and T2 (100%). Conclusion: Overall, this study suggests that vaccination may represent a promising approach to reducing HPV-related health risks. These findings shed light on the potential benefits and challenges of HPV vaccination, emphasizing the need for continued monitoring and vaccination efforts.

Detection and Complete Genome Analysis of Porcine Circovirus 2 (PCV2) and an Unclassified CRESS DNA Virus from Diarrheic Pigs in the Dominican Republic: First Evidence for Predominance of PCV2d from the Caribbean Region.

We report here high rates (47.5%, 48/101) of detection of porcine circovirus 2 (PCV2) in diarrheic pigs from three pig farms in the Dominican Republic. Seventeen of the PCV2 positive samples, representing the three pig farms, different age groups and sampling periods (2020–2021), were amplified for the complete PCV2 genome. Based on analysis of open reading frame 2 and complete genome sequences, the 17 PCV2 strains were assigned to the PCV2d genotype. Significant differences were observed in PCV2 detection rates between the vaccinated (20% (10/50)) and unvaccinated (62.5% (10/16) and 80% (28/35)) farms, corroborating previous observations that PCV2a-based vaccines confer protection against heterologous PCV2 genotypes. The present study is the first to report detection and molecular characterization of PCV2 from the Dominican Republic, warranting large-scale molecular epidemiological studies on PCV2 in pig farms and backyard systems across the country. For the first time, PCV2d was identified as the predominant PCV2 genotype in a study from the Caribbean region, suggesting that a genotype shift from PCV2b to PCV2d might be happening in the Caribbean region, which mirrored the current PCV2 genotype scenario in many other parts of the world. Besides PCV2, we also identified a pigeon circovirus-like virus, and a circular Replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA virus, which was characterized for the complete genome. The CRESS DNA virus shared a similar genomic organization and was related to unclassified CRESSV2 DNA viruses (belonging to the Order Cirlivirales) from porcine feces in Hungary, indicating that related unclassified CRESS DNA viruses are circulating among pigs in different geographical regions, warranting further studies on the epidemiology and biology of these novel viruses.

Genotype Shift of Malaysian Porcine Circovirus 2 (PCV2) from PCV2b to PCV2d within a Decade.

Simple SummaryThis study aims to provide an updated Malaysian porcine circovirus 2 (PCV2) situation after a knowledge gap of one decade. Molecular detection rates of 83.78% and 83.54% at farm and sample population level were reported, close to previous publication. However, an obvious genotype shift from genotype PCV2b to PCV2d was revealed. Substitution rate for PCV2 cap gene sequences in this study was estimated at 1.102 × 10−3 substitutions/site/year, in agreement with the high substitution rate expected from PCV2 strains. Phylogenetic clustering pattern according to the year of sample origin was observed, suggesting possible nucleotide mutation occurring over time. Concurrent circulation of different PCV2 strains within one farm and within a single individual were also observed. This study also reports detection of PCV2 antigen across all production age groups from fetuses to sows; in abattoir lung samples from clinically healthy finishers; and in the wild boar population roaming Peninsular Malaysia. These observations of high molecular detection rates in farms, clinically healthy abattoir samples and in the wild boar population; and most importantly, a new wave of genotype shift from PCV2b to PCV2d—warrant further attention on the Malaysian PCV2 situation pertinent to the control and management strategy applicable to local swine farming.This paper aims to update the molecular status of porcine circovirus 2 (PCV2) in Malaysia. Firstly, the molecular detection rate of PCV2 in farm and sampled pig population were reported to be 83.78% (31/37 farms) and 83.54% (66/79 pigs) positive for PCV2, respectively. PCV2 was detected across all age groups, from fetuses, porkers to sows. Co-detection of PCV2 and PCV3 antigens was also reported at a rate of 28.77% (21/73). Secondly, PCV2 antigen was also detected in Malaysian abattoir lung samples: 18 out of 19 (94.74%) samples originating from clinically healthy finishers were tested positive. Further, this is the first study to confirm the circulation of PCV2 in the wild boar population roaming Peninsular Malaysia, where 28 out of 28 (100%) wild boar lung samples were found positive. One decade earlier, only genotype PCV2b was reported in Malaysia. This most recent update revealed that genotypes PCV2a, PCV2b and PCV2d were present, with PCV2d being the predominant circulating genotype. PCV2 cap gene nucleotide sequences in this study were found to be under negative selection pressure, with an estimated substitution rate of 1.102 × 10−3 substitutions/site/year (ssy).

Oocyst Shedding Dynamics in Children with Cryptosporidiosis: a Prospective Clinical Case Series in Ethiopia.

ABSTRACTKnowledge on the duration of Cryptosporidium oocyst shedding, and how shedding may be affected by subtypes and clinical parameters, is limited. Reduced transmission may be a secondary benefit of cryptosporidiosis treatment in high-prevalence areas. We conducted a prospective clinical case series in children of <5 years presenting with diarrhea to a health center and a hospital in Ethiopia over an 18-month period. Stool samples were collected repeatedly from children diagnosed with cryptosporidiosis for up to 60 days. Samples were examined, and Cryptosporidium shedding was quantified, using auramine phenol, immunofluorescent antibody staining, and quantitative PCR (qPCR). In addition, species determination and subtyping were used to attempt to distinguish between new infections and ongoing shedding. Duration and quantity of shedding over time were estimated by time-to-event and quantitative models (sex- and age-adjusted). We also explored how diarrheal severity, acute malnutrition, and Cryptosporidium subtypes correlated with temporal shedding patterns. From 53 confirmed cryptosporidiosis cases, a median of 4 (range 1 to 5) follow-up stool samples were collected and tested for Cryptosporidium. The median duration of oocyst shedding was 31 days (95% confidence interval [CI], 26 to 36 days) after onset of diarrhea, with similar estimates from the quantitative models (31 days, 95% CI 27 to 37 days). Genotype shift occurred in 5 cases (9%). A 10-fold drop in quantity occurred per week for the first 4 weeks. Prolonged oocyst shedding is common in a pediatric clinical population with cryptosporidiosis. We suggest that future intervention trials should evaluate both clinical efficacy and total parasite shedding duration as trial endpoints.IMPORTANCE Cryptosporidiosis is an important cause of diarrhea, malnutrition, and deaths in young children in low-income countries. The infection spreads from person to person. After infection, prolonged release of the Cryptosporidium parasite in stool (shedding) may contribute to further spread of the disease. If diagnosis and treatment are made available, diarrhea will be treated and deaths will be reduced. An added benefit may be to reduce transmission to others. However, shedding duration and its characteristics in children is not well known. We therefore investigated the duration of shedding in a group of young children who sought health care for diarrhea in a hospital and health center in Ethiopia. The study followed 53 children with cryptosporidiosis for 2 months. We found that, on average, children released the parasite for 31 days after the diarrhea episode started. Point-of-care treatment of cryptosporidiosis may therefore reduce onward spread of the Cryptosporidium parasite within communities and households.

Japanese encephalitis virus genotype III from mosquitoes in Tarlac, Philippines

ObjectivesThe aim of this study was to investigate the presence of Japanese encephalitis virus (JEV) in a rice-farming community in the Philippines and to determine its implications regarding the epidemiology of viral encephalitides in the Asia-Pacific Region. MethodsMosquitoes were collected monthly from animal-baited traps close to flooded rice fields in two barangays (villages) in the Municipality of San Jose, Tarlac Province in Luzon, from May 2009 to July 2010. Virus was detected by nested reverse transcription PCR. Phylogenetic analysis of the amplified virus envelope gene was done using the maximum-likelihood method. ResultsA total of 28 700 known vector mosquitoes were collected, namely Culex vishnui, Culex fuscocephala, Culex tritaeniorhynchus, and Culex gelidus. JEV genotype III was detected in C. tritaeniorhynchus, belonging to the same genotype but form a different clade from those reported in the 1980s and in 2020 in this country. ConclusionsJapanese encephalitis is associated with rice cultivation and the presence of infected mosquitoes in Tarlac, Philippines. It remains to be seen whether the observed genetic shift of genotype III to genotype I in Asia will in time have an impact on the epidemiology of Japanese encephalitis in the Philippines. For long-term disease control, regular surveillance and Japanese encephalitis immunization in children and travelers in high risk areas are recommended.

Circulation of genotypes of dengue virus serotype 2 in Guangzhou over a period of 20\xa0years

BackgroundThe dengue epidemic in Guangzhou has imposed a rising burden on society and health infrastructure. Here, we present the genotype data for dengue virus serotype 2 (DENV-2) to improve understanding of this dengue epidemic.MethodsWe sequenced the envelope gene of DENV-2 obtained from patient serum samples and subsequently performed maximum-likelihood phylogenetic analysis using PhyMLv3.1, maximum clade credibility analysis using BEAST v.1.10.4, and selection pressure analysis using Datamonkey 2.0.ResultsThe prevalent DENV-2 strains identified in Guangzhou region are related to those in Southeast Asian countries. In particular, the Malaysia/Indian subcontinent genotype is prevailing in Guangzhou with no apparent genotype shift having occurred over the past 20 years. However, episodic positive selection was detected at one site.ConclusionsLocal control of the DENV-2 epidemic in Guangzhou requires effective measures to prevent and monitor imported cases. Moreover, the shift between the Malaysia/Indian subcontinent genotype lineages, which originated at different time points, may account for the rise in DENV-2 cases in Guangzhou. Meanwhile, the low rate of dengue haemorrhagic fever in Guangzhou may be explained by the dominance of the less virulent Malaysia/Indian subcontinent genotype.

Higher sensitivity to hydrogen peroxide and light stress conditions of the microcystin producer Microcystis aeruginosa sp PCC7806 compared to non-producer strains.

The increasing incidence of cyanobacterial blooms with their associated production of cyanotoxins lead managers of aquatics systems to control their biomass to limit the health risk. Among the variety of existing treatment approaches, hydrogen peroxide (H2O2) shows increasing use but the effects of environmental parameters on its effectiveness are still not completely known. With the aim to assess the efficiency of H2O2 treatments in the control of cyanobacterial blooms and decrease toxic risk, we tested three Microcystis strains according to their ability to produce cyanotoxins (a microcystin-producing, non-microcystin-producing and mcyB-knockout mutant). Photochemical efficiency, percentage of living cells and microcystin cell content were compared under various hydrogen peroxide concentrations coupled with stress conditions encountered during the life cycle of cyanobacteria as darkness and high light. The microcystin-producing strain appeared the more sensitive to hydrogen peroxide treatment and to light condition, probably due to a lower rate of repair of Photo System II (PSII). We also highlighted various responses of PSII activity according to Microcystis strains which could partly explain the shift of dominant genotypes often occurring during a bloom event. Our results confirm the link between light and microcystin content and variations of microcystin contents appear as a consequence of photosynthetic activity. These findings could be of particular interest regarding water quality management, especially the use of H2O2 as a potential algaecide which seems to be more effective to use during periods of high light.

748. The Changing Epidemiology of Clostridioides difficile Infection and the NAP1/027 Strain in Two Quebec Hospitals

BackgroundIn 2003, many hospitals in Québec, Canada experienced an increase in the incidence of healthcare-associated C. difficile infection (HA-CDI) associated with increased morbidity and mortality. This increase was associated with the dissemination of the NAP1/027 strain. The objective of this study was to describe the epidemiology of HA-CDI in two tertiary care hospitals based in Montréal from 2003 to 2019.MethodsSurveillance for HA-CDI was performed using standard definitions from 2003 to 2019 at the Montreal General Hospital (MGH) and Royal Victoria Hospital (RVH), in Montréal, Québec. C. difficile was isolated from stool specimens using standard methods. Pulsed field gel electrophoresis and ribotyping were performed to determine genotype. Antibiotic utilization and infection control interventions implemented over the same time period were reviewed. ResultsA total of 4314 cases of CDAD were identified during the study period: 2295 at the RVH and 2019 at the MGH. The incidence decreased from 29.5 to 5.9 cases per 10,000 patient-days between 2003 and 2019 at the RVH and from 23.8 to 3.9 cases per 10,000 patient-days at the MGH. Of the 124 isolates available for genotyping in 2003, 112 were NAP1 (90.3%) compared to 5 out of 53 (9.4%) in 2019. Fluoroquinolone utilization decreased from 230 to 139 DDDs per 1,000 patient-days between 2003 and 2019, whereas total antibiotic utilization increased from 1296 to 1550 DDDs per 1,000 patient-days. Infection Control interventions included empirically placing patients with diarrhea on precautions, intensified cleaning measures, formal antibiotic stewardship, introduction of a real-time PCR C. difficile test in June 2010, and a move to a facility with only single rooms at the RVH in April 2015. Incidence of HA-CDI at the RVH and MGH and antibiotic utilization between 2003 and 2019 ConclusionAn important change in HA-CDI epidemiology was observed in two Canadian tertiary care hospitals based in Montréal between 2003 and 2019. There was a significant decrease in incidence of HA-CDI and a genotype shift from a predominance of NAP1 strains to non-NAP1 strains. Utilization of fluoroquinolones, to which the NAP1 strain is resistant, concurrently decreased. Infection control interventions targeting isolation, diagnosis, disinfection, and antibiotic stewardship have contributed to the major observed reduction in HA-CDI incidence.Disclosures All Authors: No reported disclosures

A vanA vancomycin-resistant Enterococcus faecium ST80 outbreak resulting from a single importation event.

A marked genotype shift among vancomycin-resistant Enterococcus faecium (VREfm) from vanB to vanA in Australia between 2011 and 2015 is a well-known phenomenon. It is hypothesized that this was caused by multiple independent clones emerging simultaneously in different settings and/or regions. To gain insights into the circumstances surrounding the shift from vanB to vanA VREfm in one Australian hospital. The genomes of 69 vanA VREfm isolates from St George Hospital collected between 2009 and 2018 were studied. An expansion of ST80 vanA VREfm was noted following a single introduction. ST80 isolates were thus further characterized using hybrid sequencing and contextualized through comparisons with other published Australian ST80 isolates. Phylogenies were constructed with plasmid sequences compared with the index isolate. The 2011 expansion of ST80 vanA VREfm isolates in our institution originated from the 2009 index isolate, from a patient transferred from overseas. Phylogenetic analysis with other Australian ST80 vanA VREfm isolates showed that the 2011 expansion event was unique, with limited spread to adjacent local health districts. Plasmid analysis showed multiple variants, which can also be traced back to the 2009 isolate, consistent with ongoing plasmid adaptation over time. These findings confirm an expansion event following a VREfm introduction event leading to a sustained clonal and plasmid outbreak over several years. Moreover, it demonstrates the complexity of countrywide replacement events. This study also highlights the use of hybrid sequencing in establishing an epidemiological relationship to the index isolate that was initially inapparent.

Occurrence of Colibacillosis in Broilers and Its Relationship With Avian Pathogenic Escherichia coli (APEC) Population Structure and Molecular Characteristics.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis, the disease with the highest economic loss for the broiler industry. However, studies focusing on the prevalence and population structure of APEC in the broiler production pyramid are scarce. Here, we used genotyping and serotyping data to elucidate the APEC population structure and its changes in different broiler production stages along with whole-genome sequencing (WGS) in a subset of APEC isolates to determine transmission patterns amongst dominant APEC sequence types (STs) and characterize them in detail. Comparison of genotypes encountered in both APEC and avian fecal E. coli (AFEC) provided further insights. Overall, APEC-related mortality, as the proportion of the total sampled mortality in the broiler production, was high (35%), while phylogroup C and serogroup O78 were predominant amongst APEC isolates. We found a low (34.0%) and high (53.3%) incidence of colibacillosis in chicks and end-cycle broilers, respectively, which may be related to a shift in APEC genotypes, suggesting a trend from commensalism to pathogenicity across different broiler production stages. Despite considerable APEC genotypic diversity, there was substantial genotype overlap (40.9%, overall) over the production stages and convergence of STs to the four clusters. Within these clusters, WGS data provided evidence of clonal transmission events and revealed an enriched virulence and resistance APEC repertoire. More specifically, sequenced APEC were assigned to defined pathotypes based on their virulence gene content while the majority (86%) was genotypically multi-drug resistant. Interestingly, WGS-based phylogeny showed that a subset of APEC, which are cephalosporin-resistant, may originate directly from cephalosporin-resistant AFEC. Finally, exploration of the APEC plasmidome indicated that the small fraction of the APEC virulome carried by IncF plasmids is pivotal for the manifestation of the APEC pathotype; thus, plasmid exchange can promote pathogenicity in strains that are at the edge of the commensal and pathogenic states.

A five-year retrospective study on the epidemiology of hand, foot and mouth disease in Sabah, Malaysia

Hand, foot, and mouth disease (HFMD) is endemic in Malaysia, with the number of cases increasing. Sabah has experienced several HFMD outbreaks, but information on the epidemiology and molecular characteristics of responsible viruses is scarce. In this study, data of 17,574 reports of HFMD cases in Sabah from 2015 to 2019 were extracted from a public health disease surveillance system and analyzed. Twenty-one swab samples from 13 children were collected from Beaufort, Sabah, during an outbreak in August 2018 for detection and serotyping of causative viruses by semi-nested reverse transcription-polymerase chain reaction (snRT-PCR) of the VP4–VP2 region and consensus degenerate hybrid oligonucleotide primer PCR of the VP1 region, respectively. Nucleotide sequencing and phylogenetic analysis were conducted by the neighbor-joining method. The average annual incidence of HFMD was 94.3 per 100,000 people, with the greatest yearly increase between 2017 and 2018. Swabs from six children were tested positive for enterovirus, of which five were positive for CVA16 and one for EV71. All CVA16 strains belonged to sub-genotype B1a, and the EV71 strain belonged to sub-genotype B5. Phylogenetic analyses indicate that enterovirus genotype shift might be responsible for the increasing trend of HFMD in Sabah, however, further study is needed.

3′UTR SL-IV and DB1 Regions Contribute to Japanese Encephalitis Virus Replication and Pathogenicity

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes fatal neurological disease in humans, is one of the most important emerging pathogens of public health significance. JEV is maintained in an enzootic cycle and causes reproductive failure in pigs. Notably, the shift in JEV genotypes is not fully protected by existing vaccines, so the development of a candidate vaccine is urgently needed. In this study, we compared pathogenicity between Japanese encephalitis virus SA14 and BJB (isolated from humans in the 1970s) strains. We found that the BJB strain was attenuated in mice and that there was no case fatality rate. The growth rate of BJB was higher than SA14 virus in BHK-21 cells. Based on the sequence alignment of the viral genome between the SA14 and BJB virus strains, some mutations at sites 248, 254, 258, and 307 were observed in the 3′ untranslated region (3′UTR). The 3′UTR of JEV plays a very important role in the viral life cycle. Furthermore, using a reverse genetic system, we conducted and rescued the parental JEV strain SA14 (T248, A254, and A258) and the mutant virus rSA14-3′UTRmut (T248C, A254G, A258G, and 307G). Through an analysis of the RNA secondary structure model of the 3′UTR, we discovered that the mutations of T248C, A254G, and A258G reduced the apiculus ring and increased the lateral ring significantly in the stem-loop structures IV (SL-IV) structure region of 3′UTR. Moreover, the insertion of 307G added a ring to the dumbbell structure 1 (DB1) structure region. Strikingly, these RNA secondary structure changes in 3′UTR of rSA14-3′UTRmut increased viral negative chain RNA production and enhanced the replication ability of the virus in BHK-21 cells. However, in vivo mouse experiments illustrated that the rSA14-3′UTRmut virus significantly decreased the neurovirulence of JEV. These results affirmed that the JEV SL-IV and DB1 regions play an important role in viral proliferation and pathogenicity. Taken together, we complement the study of RNA element function in the 3′UTR region of JEV by providing a new target for the rational design of live attenuated candidate vaccines and the increase of virus production.

Emergence of novel Porcine circovirus 2 genotypes in Southern India.

A total of 200 samples from Porcine circovirus 2 suspected (n = 112) and healthy (n = 88) swine populations collected from different districts of Tamil Nadu, south India were used in this study. The samples comprising of serum (n = 124), swabs from natural orifices (n = 52), and postmortem tissues (n = 24). All the samples were processed and subjected to the screening and detection of the PCV2 genome by a specific PCR assay. PCV2 genomes from positive samples were further subjected to genotyping with specifically designed primers for the full-length amplification of the ORF2 gene which codes for capsid protein (Cp) and serves as an epidemiological marker. Randomly, 13 amplified ORF2 genes were sequenced and the aligned sequences were subjected to signature motif analysis and phylogeny in MEGA X. The molecular prevalence of PCV2 infection in Tamil Nadu is 10.5% (n = 21). Signature motif and phylogenetic studies of 13 samples revealed 38.5% (n = 5) presence of each PCV2b intermediate 1(IM1) and PCV2b genotypes, followed by 15.4% (n = 2) PCV2d-2 and 7.7% (n = 1) PCV2d genotypes. The PCV2b-IM1 genotype has a 99.43% sequence homology with Vietnam isolate (JX506730). PCV2b genotypes showed 99.72% sequence identity with Chinese isolate (KX068219). PCV2d-2 genotypes reported in this study have 100% sequence identity with Taiwan isolate (MF169721). PCV2d genotype showed 97.87% sequence identity with Thailand isolate (MF314293). Amino acid analysis of all the 13 full-length ORF2 gene sequences revealed specific mutations in the immune reactive domains of A, B, C, and D. Capsid protein of three PCV2b and five PCV2b IM1 isolates had extra amino acid residue lysine (K) at 234 position of ORF2 similar to PCV2d. For the first time in South India, PCV2b IM1 and PCV2d-2 genotypes are reported. This study evidences the genetic shifts of PCV2 isolates in India and it is analogous to that of global genotypic shift.

Spatial autocorrelation of dengue cases and molecular biological characteristics of envelope gene of dengue virus in Guangzhou, 2019

Objective: Analyze epidemiological situation of dengue fever,and survey impacts of four serotypes dengue viruses in Guangzhou, 2019. Methods: Information of patients was collected in Notifiable Infectious Disease Report System (NIDRS). Spatial autocorrelation of dengue cases was evaluated using ArcGIS version 10.2. Serum samples were tested by real-time PCR. Virus strains were isolated from positive sera. Then E gene was sequenced. Phylogenetic trees were including PhyMLsoftwarev 3.1. Results: A total of 1 655 dengue cases, consisted of 1 382 local cases and 273 imported cases, was confirmed in 2019. The incidence was 11.10 per 100 000 dengue cases were autocorrelated in Guangzhou. There were 18 high-high clusters. Most of the imported dengue cases were imported from Southeast Asian countries (86.08%,235/273) and African countries (2.56%,7/273). Of 749 serum samples detected by real-time PCR, the positive rate was 93.06% (697/749). Four hundred and sixty-four dengue virus strains had been isolated in 2019. Compared with data from the genotype tree of the former years, no genotype shift was discovered. Serotype 1 was still predominant. Serotype 2 was the significant strain in Baiyun district and Liwan district. Conclusions: Dengue fever was spreading all over Guangzhou in 2019. The suburban areas, which played a more critical role in causing the spread and outbreak of dengue fever, should be given more prominence. Inspection at ports should be enforced to prevent importing cases from African countries and Southeast Asian countries. The risk of serotype 2 cannot be overlooked. Four serotypes dengue viruses prevailed simultaneously in Guangzhou, which warns us to take precaution of severe dengue outbreaks.