Genetic Diversity and Spatiotemporal Dynamics of Chikungunya Infections in Mexico during the Outbreak of 2014–2016

Chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes mosquitoes, which causes Chikungunya fever. Three CHIKV genotypes have been identified: West African, East-Central-South African and Asian. In 2014, CHIKV was detected for the first time in Mexico, accumulating 13,569 confirmed cases in the following three years. Studies on the molecular diversification of CHIKV in Mexico focused on limited geographic regions or investigated only one structural gene of the virus. To describe the dynamics of this outbreak, we analyzed 309 serum samples from CHIKV acute clinical cases from 15 Mexican states. Partial NSP3, E1, and E2 genes were sequenced, mutations were identified, and their genetic variability was estimated. The evolutionary relationship with CHIKV sequences sampled globally were analyzed. Our sequences grouped with the Asian genotype within the Caribbean lineage, suggesting that the Asian was the only circulating genotype during the outbreak. Three non-synonymous mutations (E2 S248F and NSP3 A437T and L451F) were present in our sequences, which were also identified in sequences of the Caribbean lineage and in one Philippine sequence. Based on the phylogeographic analysis, the viral spread was reconstructed, suggesting that after the introduction through the Mexican southern border (Chiapas), CHIKV dispersed to neighboring states before reaching the center and north of the country through the Pacific Ocean states and Quintana Roo. This is the first viral phylogeographic reconstruction in Mexico characterizing the CHIKV outbreak across the country.     

2015-2016年深圳市登革Ⅲ、Ⅳ型分离病毒株E/NS1基因序列特征

目的 了解2015-2016年深圳市分离的登革Ⅲ、Ⅳ型病毒E/NS1基因序列特征及登革Ⅲ、Ⅳ型病毒流行规律,探究其可能的传播来源。方法 收集2015-2016年深圳市登革热患者病例资料及急性期血清,用C6/36细胞培养分离登革病毒,用FQ-PCR对其进行血清分型,分离成功的病毒株用反转录-聚合酶链反应(PCR)方法扩增E基因和NS1基因,进行序列分析,绘制系统进化树,氨基酸比对分析4个不同血清型NS1蛋白。结果 从5份Ⅲ型的登革病毒标本中成功分离4份病毒株,3份Ⅳ型登革病毒标本中成功分离2份登革病毒;BLAST分析保守E基因结果表明,与DENV-3分离株同源性最高(99%)的毒株主要是印度尼西亚2010和2015年分离株、菲律宾2015年分离株。与DENV-4分离株同源性最高(99%)的毒株主要是菲律宾2013年分离株、印度尼西亚2010分离株、意大利2009年分离株。NS1基因序列分析显示所选4株不同血清型的病毒株氨基酸相似性为77.69%,4个不同血清型的登革热NS1抗原存在5-7个氨基酸保守区域。结论 2015-2016年深圳市输入的登革Ⅲ、Ⅳ型病毒可能来自印度尼西亚和菲律宾,应加强出入境人员的监测工作,避免输入引起本地感染的风险。     

Hyperendemic Dengue and Possible Zika Circulation in the Westernmost Region of the Indonesian Archipelago

The transmission of dengue and other medically important mosquito-borne viruses in the westernmost region of Indonesia is not well described. We assessed dengue and Zika virus seroprevalence in Aceh province, the westernmost area of the Indonesian archipelago. Serum samples collected from 199 randomly sampled healthy residents of Aceh Jaya in 2017 were analyzed for neutralizing antibodies by plaque reduction neutralization test (PRNT). Almost all study participants (198/199; 99.5%) presented with multitypic profiles of neutralizing antibodies to two or more DENV serotypes, indicating transmission of multiple DENV in the region prior to 2017. All residents were exposed to one or more DENV serotypes by the age of 30 years. The highest geometric mean titers were measured for DENV-4, followed by DENV-1, DENV-2 and DENV-3. Among a subset of 116 sera, 27 neutralized ZIKV with a high stringency (20 with PRNT₉₀ > 10 and 7 with PRNT₉₀ > 40). This study showed that DENV is hyperendemic in the westernmost region of the Indonesian archipelago and suggested that ZIKV may have circulated prior to 2017.     

In Vitro Inhibition of Replication of Dengue Virus Serotypes 1–4 by siRNAs Bound to Non-Toxic Liposomes

Dengue virus is a ssRNA+ flavivirus, which produces the dengue disease in humans. Currently, no specific treatment exists. siRNAs regulate gene expression and have been used systematically to silence viral genomes; however, they require controlled release. Liposomes show favorable results encapsulating siRNA for gene silencing. The objective herein was to design and evaluate in vitro siRNAs bound to liposomes that inhibit DENV replication. siRNAs were designed against DENV1–4 from conserved regions using siDirect2.0 and Web-BLOCK-iT™ RNAiDesigner; the initial in vitro evaluation was carried out through transfection into HepG2 cells. siRNA with silencing capacity was encapsulated in liposomes composed of D-Lin-MC3-DMA, DSPC, Chol. Cytotoxicity, hemolysis, pro-inflammatory cytokine release and antiviral activity were evaluated using plaque assay and RT-qPCR. A working concentration of siRNA was established at 40 nM. siRNA1, siRNA2, siRNA3.1, and siRNA4 were encapsulated in liposomes, and their siRNA delivery through liposomes led to a statistically significant decrease in viral titers, yielded no cytotoxicity or hemolysis and did not stimulate release of pro-inflammatory cytokines. Finally, liposomes were designed with siRNA against DENV, which proved to be safe in vitro.     

Comparison of Hepatitis E Virus Sequences from Humans and Swine,The Netherlands, 1998–2015

Pigs are suspected to be a major source of zoonotic hepatitis E virus (HEV) infection in industrialized countries, but the transmission route(s) from pigs to humans are ill-defined. Sequence comparison of HEV isolates from pigs with those from blood donors and patients in 372 samples collected in The Netherlands in 1998 and 1999 and between 2008 and 2015 showed that all sequences were genotype 3 except for six patients (with travel history). Subgenotype 3c (gt3c) was the most common subtype. While the proportion of gt3c increased significantly between 1998 and 2008, it remained constant between 2008 and 2015. Among the few circulating HEV subtypes, there was no difference observed between the human and the pig isolates. Hepatitis E viruses in humans are very likely to originate from pigs, but it is unclear why HEV gt3c has become the predominant subtype in The Netherlands.     

A Review on Chikungunya Virus Epidemiology,Pathogenesis and Current Vaccine Development

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that recently re-emerged in many parts of the world causing large-scale outbreaks. CHIKV infection presents as a febrile illness known as chikungunya fever (CHIKF). Infection is self-limited and characterized mainly by severe joint pain and myalgia that can last for weeks or months; however, severe disease presentation can also occur in a minor proportion of infections. Among the atypical CHIKV manifestations that have been described, severe arthralgia and neurological complications, such as encephalitis, meningitis, and Guillain–Barré Syndrome, are now reported in many outbreaks. Moreover, death cases were also reported, placing CHIKV as a relevant public health disease. Virus evolution, globalization, and climate change may have contributed to CHIKV spread. In addition to this, the lack of preventive vaccines and approved antiviral treatments is turning CHIKV into a major global health threat. In this review, we discuss the current knowledge about CHIKV pathogenesis, with a focus on atypical disease manifestations, such as persistent arthralgia and neurologic disease presentation. We also bring an up-to-date review of the current CHIKV vaccine development. Altogether, these topics highlight some of the most recent advances in our understanding of CHIKV pathogenesis and also provide important insights into the current development and clinical trials of CHIKV potential vaccine candidates.     

Origin and Spread of the Dengue Virus Type 1, Genotype V in Senegal, 2015–2019

Dengue virus (DENV) is the most widespread arthropod-borne virus, with the number and severity of outbreaks increasing worldwide in recent decades. Dengue is caused by genetically distinct serotypes, DENV-1–4. Here, we present data on DENV-1, isolated from patients with dengue fever during an outbreak in Senegal and Mali (Western Africa) in 2015–2019, that were analyzed by sequencing the envelope (E) gene. The emergence and the dynamics of DENV-1 in Western Africa were inferred by using maximum likelihood and Bayesian methods. The DENV-1 grouped into a monophyletic cluster that was closely related to those from Southeast Asia. The virus appears to have been introduced directly into Medina Gounass (Suburb of Dakar), Senegal (location probability = 0.301, posterior = 0.76). The introduction of the virus in Senegal occurred around 2014 (95% HPD = 2012.88–2014.84), and subsequently, the virus moved to regions within Senegal (e.g., Louga and Fatick), causing intense outbreaks in the subsequent years. The virus appears to have been introduced in Mali (a neighboring country) after its introduction in Senegal. In conclusion, we present evidence that the outbreak caused by DENV-1 in urban environments in Senegal and Mali after 2015 was caused by a single viral introduction from Asia.     

Prevalence of Human Coronaviruses in Children and Phylogenetic Analysis of HCoV-OC43 during 2016–2022 in Riyadh,Saudi Arabia

With the emergence of SARS-CoV-2, routine surveillance combined with sequence and phylogenetic analysis of coronaviruses is urgently required. In the current study, the four common human coronaviruses (HCoVs), OC43, NL63, HKU1, and 229E, were screened in 361 clinical samples collected from hospitalized children with respiratory symptoms during four winter seasons. RT-PCR-based detection and typing revealed different prevalence rates of HCoVs across the four seasons. Interestingly, none of the four HCoVs were detected in the samples (n = 100) collected during the winter season of the COVID-19 pandemic. HCoV-OC43 (4.15%) was the most frequently detected, followed by 229E (1.1%). Partial sequences of S and N genes of OC43 from the winter seasons of 2015/2016 and 2021/2022 were used for sequence and phylogenetic analysis. Multiple sequence alignment of the two Saudi OC43s strains with international strains revealed the presence of sequence deletions and several mutations, of which some changed their corresponding amino acids. Glycosylation profiles revealed a number of O-and N-glycosylation sites in both genes. Based on phylogenetic analysis, four genotypes were observed with Riyadh strains grouped into the genotype C. Further long-term surveillance with a large number of clinical samples and sequences is necessary to resolve the circulation patterns and evolutionary kinetics of OC43 in Saudi Arabia.     

Surveillance of Influenza in Indonesia, 2003–2007

Background Longitudinal data are limited about the circulating strains of influenza viruses and their public health impact in Indonesia. We conducted influenza surveillance among outpatients and hospitalized patients with influenza‐like illness (ILI) across the Indonesian archipelago from 2003 through 2007. Methodology Demographic, clinical data, and respiratory specimens were collected for 4236 ILI patients tested for influenza virus infection by RT‐PCR and viral culture. Principal Findings Influenza A and B viruses co‐circulated year‐round with seasonal peaks in influenza A virus activity during the rainy season (December–January). During 2003–2007, influenza viruses were identified in 20·1% (4236/21 030) of ILI patients, including 20·1% (4015/20 012) of outpatients, and 21·7% (221/1018) of inpatients. One H5N1 case was identified retrospectively in an outpatient with ILI. Antigenic drift in circulating influenza A and B virus strains was detected during the surveillance period in Indonesia. In a few instances, antigenically drifted viruses similar to the World Health Organization (WHO) vaccine strains were detected earlier than the date of their designation by WHO. Conclusions Influenza A and B virus infections are an important cause of influenza‐like illness among outpatients and hospitalized patients in Indonesia. While year‐round circulation of influenza viruses occurs, prevention and control strategies should be focused upon the seasonal peak during rainy season months. Ongoing virologic surveillance and influenza disease burden studies in Indonesia are important priorities to better understand the public health impact of influenza in South‐East Asia and the implications of influenza viral evolution and global spread.     

High Incidence of Zika or Chikungunya Infection among Pregnant Women Hospitalized Due to Obstetrical Complications in Northeastern Brazil—Implications for Laboratory Screening in Arbovirus Endemic Area

The diagnostic of arbovirus-related obstetric complications in high-risk pregnancy and childbirth care is challenging, especially in endemic areas. We conducted a prospective study to track active or recent Zika (ZIKV), dengue (DENV), or chikungunya (CHIKV) virus infection among hospitalized pregnant women (PW) with obstetric complications in a hospital at the epicenter of Zika outbreak and ZIKV-related microcephaly in Brazil. Clinical data and blood samples were collected at enrollment and 10 days after the admission of study participants, between October 2018 and May 2019. Further clinical data were extracted from medical records. Samples were screened by molecular and serological tests. Out of 780 participants, 93.1% (95% CI: 91.1–94.7%) presented previous DENV exposure (IgG). ZIKV, CHIKV, and/or DENV laboratory markers of recent or active infection were detected in 130 PW, yielding a prevalence of 16.6% (95% CI: 14.2–19.5%); 9.4% (95% CI: 7.4–11.7%), 7.4% (95% CI: 5.7–9.7%), and 0.38% (95% CI: 0.1–1.2%) of CHIKV, ZIKV, and DENV infections, respectively. Most ZIKV infections were detected by molecular assays (89.6%), while CHIKV infections were detected by serology (95.9%). Our findings highlight the need for arbovirus infections screening in PW with obstetrical complications, potentially associated to these infections in endemic areas regardless of the signs or symptoms suggestive of arboviral disease.     

Molecular Transmission Dynamics of Primary HIV Infections in Lazio Region,Years 2013–2020

Molecular investigation of primary HIV infections (PHI) is crucial to describe current dynamics of HIV transmission. Aim of the study was to investigate HIV transmission clusters (TC) in PHI referred during the years 2013–2020 to the National Institute for Infectious Diseases in Rome (INMI), that is the Lazio regional AIDS reference centre, and factors possibly associated with inclusion in TC. These were identified by phylogenetic analysis, based on population sequencing of pol; a more in depth analysis was performed on TC of B subtype, using ultra-deep sequencing (UDS) of env. Of 270 patients diagnosed with PHI during the study period, 229 were enrolled (median follow-up 168 (IQR 96–232) weeks). Median age: 39 (IQR 32–48) years; 94.8% males, 86.5% Italians, 83.4% MSM, 56.8% carrying HIV-1 subtype B. Of them, 92.6% started early treatment within a median of 4 (IQR 2–7) days after diagnosis; median time to sustained suppression was 20 (IQR 8–32) weeks. Twenty TC (median size 3, range 2–9 individuals), including 68 patients, were identified. A diagnosis prior to 2015 was the unique factor associated with inclusion in a TC. Added value of UDS was the identification of shared quasispecies components in transmission pairs within TC.     

Whole Genome Sequencing and Phylogenetic Analysis of Rabies Viruses from Bats in Connecticut,USA, 2018–2019

We performed whole genome sequencing and genetic characterization of rabies viruses (RABV) detected in bats submitted to the Connecticut Veterinary Medical Diagnostic Laboratory (CVMDL) during 2018–2019. Among 88 bats submitted to CVMDL, six brain samples (6.8%, 95% confidence interval: 1.6% to 12.1%) tested positive by direct fluorescent antibody test. RABVs were detected in big brown bats (Eptesicus fuscus, n = 4), a hoary bat (Lasiurus cinereus, n = 1), and an unidentified bat species (n = 1). Complete coding sequences of four out of six detected RABVs were obtained. In phylogenetic analysis, the RABVs (18-62, 18-4347, and 19-2274) from big brown bats belong to the bats EF-E1 clade, clustering with RABVs detected from the same bat species in Pennsylvania and New Jersey. The bat RABV (19-2898) detected from the migratory hoary bat belongs to the bats LC clade, clustering with the eleven viruses detected from the same species in Arizona, Washington, Idaho, and Tennessee. The approach used in this study generated novel data regarding genetic relationships of RABV variants, including their reservoirs, and their spatial origin and it would be useful as reference data for future investigations on RABV in North America. Continued surveillance and genome sequencing of bat RABV would be needed to monitor virus evolution and transmission, and to assess the emergence of genetic mutations that may be relevant for public health.     

Typhoid fever in Ujung Pandang, Indonesia – high-risk groups and high-risk behaviours

We performed a hospital-based case-control study to identify high-risk groups and routes of transmission of typhoid fever in the city of Ujung Pandang on the island of Sulawesi, Indonesia. The annual incidence of this disease in southern Sulawesi is estimated at 3.1/1000 and the case fatality at 5.1% Cases were 50 patients over 13 years of age admitted to Stella Maris Hospital with a diagnosis of typhoid fever between June and September 1991. Diagnosis was made on clinical grounds and in 90% of cases confirmed by a Widal test.
Controls were 42 patients admitted for non-infectious disorders during the same period and individually matched by age and sex. Controls did not have a history of typhoid fever. Interviews took place in hospital. Analysis was by unconditional logistic regression. High-risk groups consisted of those who were single, unemployed and those who had a university education. Median age of cases was 22 years. Consumption of food from warungs (food stalls in the street) was strongly associated with risk (OR = 45). Both cases and controls washed hands after use of the toilet and before meals, but cases used soap significantly less often (OR = 30). The results of this study can be used to take preventive measures against this severe disease of educated and single young adults by targetting them for IEC-activities emphasizing the importance of thorough hand-washing and the need to take care in the selection of street-foods.     

Influenza clinical testing and oseltamivir treatment in hospitalized children with acute respiratory illness, 2015–2016

BackgroundAntiviral treatment is recommended for all hospitalized children with suspected or confirmed influenza, regardless of their risk profile. Few data exist on adherence to these recommendations, so we sought to determine factors associated with influenza testing and antiviral treatment in children.MethodsHospitalized children <18 years of age with acute respiratory illness (ARI) were enrolled through active surveillance at pediatric medical centers in seven cities between 11/1/2015 and 6/30/2016; clinical information was obtained from parent interview and chart review. We used generalized linear mixed‐effects models to identify factors associated with influenza testing and antiviral treatment.ResultsOf the 2299 hospitalized children with ARI enrolled during one influenza season, 51% (n = 1183) were tested clinically for influenza. Clinicians provided antiviral treatment for 61 of 117 (52%) patients with a positive influenza test versus 66 of 1066 (6%) with a negative or unknown test result. In multivariable analyses, factors associated with testing included neuromuscular disease (aOR = 5.35, 95% CI [3.58–8.01]), immunocompromised status (aOR = 2.88, 95% CI [1.66–5.01]), age (aOR = 0.93, 95% CI [0.91–0.96]), private only versus public only insurance (aOR = 0.78, 95% CI [0.63–0.98]), and chronic lung disease (aOR = 0.64, 95% CI [0.51–0.81]). Factors associated with antiviral treatment included neuromuscular disease (aOR = 1.86, 95% CI [1.04, 3.31]), immunocompromised state (aOR = 2.63, 95% CI [1.38, 4.99]), duration of illness (aOR = 0.92, 95% CI [0.84, 0.99]), and chronic lung disease (aOR = 0.60, 95% CI [0.38, 0.95]).ConclusionApproximately half of children hospitalized with influenza during the 2015–2016 influenza season were treated with antivirals. Because antiviral treatment for influenza is associated with better health outcomes, further studies of subsequent seasons would help evaluate current use of antivirals among children and better understand barriers for treatment.     

2001-2016年广州市登革3型病毒E基因序列及系统进化树分析

目的 了解2001-2016年广州市登革3型病毒的流行情况,掌握毒株的进化情况和趋势。方法 将登革热确诊病例的血清用荧光PCR检测,阳性血清用C6/36细胞进行病毒分离,测定分离毒株的E基因序列,与NCBI的毒株序列相比较,利用Mega 4.0软件构建系统进化树。结果2001-2016年共分离到登革3型病毒24株,从基因型上分类属于基因亚型I、II、III和V型,基因亚型III在流行年份和分离毒株数上稍占优势。流行病学调查和序列分析均显示与东南亚国家流行的登革热相关度较高。结论 广州市登革3型病毒以输入为主,随着输入压力增大、基因亚型增多和转换,可能会使广州市登革热流行传播更为复杂,流行风险进一步增高。     

Molecular Analysis of Caprine Enterovirus Circulating in China during 2016–2021: Evolutionary Significance

Here, we report the characterization of 13 novel caprine/ovine enterovirus strains isolated from different regions in China during 2016–2021. Immunoperoxidase monolayer assay showed that these viral strains shared strong cross-reaction with the previously reported caprine enterovirus CEV-JL14. Alignment analysis of the complete nucleotide sequences revealed 79.2%–87.8% and 75.0%–76.7% sequence identity of these novel caprine enterovirus strains to CEV-JL14 and TB4-OEV, respectively. Phylogenetic analyses clustered these novel strains to EV-G based on the amino acid sequences of P1 and 2C+3CD. Moreover, phylogenetic analysis of these caprine enterovirus strains identified three new EV-G types using VP1 sequences. These results demonstrate the genetic variations and the evolution of caprine enterovirus.