Assessment of a multiplex PCR and Nanopore-based method for dengue virus sequencing in Indonesia.

BACKGROUND: Dengue virus (DENV) infects hundreds of thousands of people annually in Indonesia. However, DENV sequence data from the country are limited, as samples from outbreaks must be shipped across long-distances to suitably equipped laboratories to be sequenced. This approach is time-consuming, expensive, and frequently results in failure due to low viral load or degradation of the RNA genome. METHODS: We evaluated a method designed to address this challenge, using the 'Primal Scheme' multiplex PCR tiling approach to rapidly generate short, overlapping amplicons covering the complete DENV coding-region, and sequencing the amplicons on the portable Nanopore MinION device. The resulting sequence data was assessed in terms of genome coverage, consensus sequence accuracy and by phylogenetic analysis. RESULTS: The multiplex approach proved capable of producing near complete coding-region coverage from all samples tested ([Formula: see text] = 99.96%, n = 18), 61% of which could not be fully amplified using the current, long-amplicon PCR, approach. Nanopore-generated consensus sequences were found to be between 99.17-99.92% identical to those produced by high-coverage Illumina sequencing. Consensus accuracy could be improved by masking regions below 20X coverage depth (99.69-99.92%). However, coding-region coverage was reduced at this depth ([Formula: see text] = 93.48%). Nanopore and Illumina consensus sequences generated from the same samples formed monophyletic clades on phylogenetic analysis, and Indonesian consensus sequences accurately clustered by geographical origin. CONCLUSION: The multiplex, short-amplicon approach proved superior for amplifying DENV genomes from clinical samples, particularly when the virus was present at low concentrations. The accuracy of Nanopore-generated consensus sequences from these amplicons was sufficient for identifying the geographic origin of the samples, demonstrating that the approach can be a useful tool for identifying and monitoring DENV clades circulating in low-resource settings across Indonesia. However, the inaccuracies in Nanopore-generated consensus sequences mean that the approach may not be appropriate for higher resolution transmission studies, particularly when more accurate sequencing technologies are available.

Full genomic characterization of a porcine rotavirus strain detected in an asymptomatic piglet in Accra, Ghana.

BACKGROUND: The introduction of rotavirus A vaccination across the developing world has not proved to be as efficacious as first hoped. One cause of vaccine failure may be infection by zoonotic rotaviruses that are very variable antigenically from the vaccine strain. However, there is a lack of genomic information about the circulating rotavirus A strains in farm animals in the developing world that may be a source of infection for humans. We therefore screened farms close to Accra, Ghana for animals sub-clinically infected with rotavirus A and then sequenced the virus found in one of these samples. RESULTS: 6.1% of clinically normal cows and pigs tested were found to be Rotavirus A virus antigen positive in the faeces. A subset of these (33.3%) were also positive for virus RNA. The most consistently positive pig sample was taken forward for metagenomic sequencing. This gave full sequence for all open reading frames except segment 5 (NSP1), which is missing a single base at the 5' end. The virus infecting this pig had genome constellation G5-P[7]-I5-R1-C1-M1-A8-N1-T7-E1-H1, a known porcine genotype constellation. CONCLUSIONS: Farm animals carry rotavirus A infection sub-clinically at low frequency. Although the rotavirus A genotype discovered here has a pig-like genome constellation, a number of the segments most closely resembled those isolated from humans in suspected cases of zoonotic transmission. Therefore, such viruses may be a source of variable gene segments for re-assortment with other viruses to cause vaccine breakdown. It is recommended that further human and pig strains are characterized in West Africa, to better understand this dynamic.

Phylogenetic Characterization of Crimean-Congo Hemorrhagic Fever Virus Detected in African Blue Ticks Feeding on Cattle in a Ugandan Abattoir.

Crimean-Congo hemorrhagic fever virus (CCHFV) is the most geographically widespread of the tick-borne viruses. However, African strains of CCHFV are poorly represented in sequence databases. In addition, almost all sequence data collected to date have been obtained from cases of human disease, while information regarding the circulation of the virus in tick and animal reservoirs is severely lacking. Here, we characterize the complete coding region of a novel CCHFV strain, detected in African blue ticks (Rhipicephalus (Boophilus) decoloratus) feeding on cattle in an abattoir in Kampala, Uganda. These cattle originated from a farm in Mbarara, a major cattle-trading hub for much of Uganda. Phylogenetic analysis indicates that the newly sequenced strain belongs to the African genotype II clade, which predominantly contains the sequences of strains isolated from West Africa in the 1950s, and South Africa in the 1980s. Whilst the viral S (nucleoprotein) and L (RNA polymerase) genome segments shared >90% nucleotide similarity with previously reported genotype II strains, the glycoprotein-coding M segment shared only 80% nucleotide similarity with the next most closely related strains, which were derived from ticks in Western India and Northern China. This genome segment also displayed a large number of non-synonymous mutations previously unreported in the genotype II strains. Characterization of this novel strain adds to our limited understanding of the natural diversity of CCHFV circulating in both ticks and in Africa. Such data can be used to inform the design of vaccines and diagnostics, as well as studies exploring the epidemiology and evolution of the virus for the establishment of future CCHFV control strategies.

Molecular epidemiology of dengue in a setting of low reported endemicity: Kupang, East Nusa Tenggara province, Indonesia.

BACKGROUND: Most regions in Indonesia experience annual dengue epidemics. However, the province of East Nusa Tenggara has consistently reported low incidence. We conducted a dengue molecular epidemiology study in Kupang, the capital of the province. METHODS: Dengue patients were recruited from May 2016 to September 2017. Dengue virus (DENV) screening was performed using NS1 and immunoglobulin G (IgG)/IgM detection. Serotype was determined using reverse transcription polymerase chain reaction and the envelope genes were sequenced to infer the genetic identity and phylogeny. RESULTS: From 119 patients, dengue was confirmed in 62 (52%). Compared with official data, underreporting of dengue incidence was observed. The majority (36%) of patients were children <10 y of age. Most patients (80%) experienced mild fever. All serotypes were detected, with DENV-3 as the predominant (57%). Kupang DENV-1 isolate was classified as genotype IV, an old and endemic strain, DENV-2 as cosmopolitan, DENV-3 as genotype I and DENV-4 as genotype II. Most isolates showed relatively low evolutionary rates and are closely related with strains from Bali and Timor Leste. CONCLUSIONS: The low dengue incidence was most likely caused by sustained local circulation of endemic viruses. This study provides information on the epidemiology of dengue in a low-endemicity setting that should help future mitigation and disease management.

An investig-ation into the epidemiology of chikungunya virus across neglected regions of Indonesia.

BACKGROUND: Chikungunya virus (CHIKV) is an important emerging and re-emerging public health problem worldwide. In Indonesia, where the virus is endemic, epidemiological information from outside of the main islands of Java and Bali is limited. METHODOLOGY/PRINCIPAL FINDINGS: Four hundred and seventy nine acutely febrile patients presenting between September 2017-2019 were recruited from three city hospitals situated in Ambon, Maluku; Banjarmasin, Kalimantan; and Batam, Batam Island as part of a multi-site observational study. CHIKV RNA was detected in a single serum sample while a separate sample was IgM positive. IgG seroprevalence was also low across all three sites, ranging from 1.4-3.2%. The single RT-PCR positive sample from this study and 24 archived samples collected during other recent outbreaks throughout Indonesia were subjected to complete coding region sequencing to assess the genetic diversity of Indonesian strains. Phylogenetic analysis revealed all to be of a single clade, which was distinct from CHIKV strains recently reported from neighbouring regions including the Philippines and the Pacific Islands. CONCLUSIONS/SIGNIFICANCE: Chikungunya virus strains from recent outbreaks across Indonesia all belong to a single clade. However, low-level seroprevalence and molecular detection of CHIKV across the three study sites appears to contrast with the generally high seroprevalences that have been reported for non-outbreak settings in Java and Bali, and may account for the relative lack of CHIKV epidemiological data from other regions of Indonesia.

Distinct Dengue Disease Epidemiology, Clinical, and Diagnosis Features in Western, Central, and Eastern Regions of Indonesia, 2017-2019.

The people of Indonesia have been afflicted by dengue, a mosquito-borne viral disease, for over 5 decades. The country is the world's largest archipelago with diverse geographic, climatic, and demographic conditions that may impact the dynamics of disease transmissions. A dengue epidemiology study was launched by us to compare and understand the dynamics of dengue and other arboviral diseases in three cities representing western, central, and eastern Indonesia, namely, Batam, Banjarmasin, and Ambon, respectively. A total of 732 febrile patients were recruited with dengue-like illness during September 2017-2019 and an analysis of their demographic, clinical, and virological features was performed. The seasonal patterns of dengue-like illness were found to be different in the three regions. Among all patients, 271 (37.0%) were virologically confirmed dengue, while 152 (20.8%) patients were diagnosed with probable dengue, giving a total number of 423 (57.8%) dengue patients. Patients' age and clinical manifestations also differed between cities. Mostly, mild dengue fever was observed in Batam, while more severe cases were prominent in Ambon. While all dengue virus (DENV) serotypes were detected, distinct serotypes dominated in different locations: DENV-1 in Batam and Ambon, and DENV-3 in Banjarmasin. We also assessed the diagnostic features in the study sites, which revealed different patterns of diagnostic agreements, particularly in Ambon. To detect the possibility of infection with other arboviruses, further testing on 461 DENV RT-PCR-negative samples was performed using pan-flavivirus and -alphavirus RT-PCRs; however, only one chikungunya infection was detected in Ambon. A diverse dengue epidemiology in western, central, and eastern Indonesia was observed, which is likely to be influenced by local geographic, climatic, and demographic conditions, as well as differences in the quality of healthcare providers and facilities. Our study adds a new understanding on dengue epidemiology in Indonesia.