Mitophylogeny of Pangasiid Catfishes and its Taxonomic Implications for Pangasiidae and the Suborder Siluroidei.

Pangasiidae (catfish order: Siluriformes) comprises 30 valid catfish species in four genera: Pangasius, Pangasianodon, Helicophagus, and Pseudolais. Their systematics are frequently revised due to the addition of newly described species. Although Pangasiidae is known to be a monophyletic family, the generic and phylogenetic relationships among the taxa are poorly resolved. This study characterized three newly obtained complete mitogenomes of Mekong River catfishes from Vietnam (Pangasius mekongensis, Pangasius krempfi, and Pangasianodon hypophthalmus), as well as the inter-and intrafamilial relationships of the Pangasiidae and catfish families in Siluroidei. The genomic features of their mitogenomes were similar to those of previously reported pangasiids, including all regulatory elements, extended terminal associated sequences (ETAS), and conserved sequence blocks (CSBs) (CSB-1, CSB-2, CSB-3, and CSBs, A to F) in the control region. A comprehensive phylogeny constructed from datasets of multiple 13 PCG sequences from 117 complete mitogenomes of 32 recognized siluriform families established Pangasiidae as monophyletic and a sister group of Austroglanididae. The [Pangasiidae + Austroglanididae] + (Ictaluridae + Cranoglanididae) + Ariidae] clade is a sister to the "Big Africa" major clade of Siluriformes. Furthermore, both phylogenies constructed from the single barcodes (83 partial cox1 and 80 partial cytB, respectively) clearly indicate genus relationships within Pangasiidae. Pangasianodon was monophyletic and a sister to the (Pangasius + Helicophagus + Pseudolais) group. Within the genus Pangasius, P. mekongensis was placed as a sister taxon to P. pangasius. Pangasius sanitwongsei was found to be related to and grouped with Pangasianodon, but in single-gene phylogenies, it was assigned to the Pangasius + Helicophagus + Pseudolais group. The datasets in this study are useful for studying pangasiid systematics, taxonomy and evolution.

Phylogenotyping of infectious bursal disease virus in Vietnam according to the newly unified genotypic classification scheme.

Since 1987, infectious bursal disease virus (IBDV) has circulated and evolved in Vietnam, but little is known about the genotypes present. IBDV samples were collected in 1987, 2001-2006, 2008, 2011, 2015-2019, and 2021 in 18 provinces. We conducted phylogenotyping analysis based on an alignment of 143 VP2-HVR (hypervariable region) sequences from 64 Vietnamese isolates (26 previous and 38 additional sequences and two vaccines, and alignment of 82 VP1 B-marker sequences, including one vaccine and four Vietnamese field strains. The analysis identified three A-genotypes, A1, A3, and A7, and two B-genotypes, B1 and B3, among the Vietnamese IBDV isolates. The lowest average evolutionary distance (8.6%) was seen between the A1 and A3 genotypes, and the highest (21.7%) was between A5 and A7, while there was a distance of 14% between B1 and B3 and 17% between B3 and B2. Unique signature residues were observed for genotypes A2, A3, A5, A6, and A8, which could be used for genotypic discrimination. A timeline statistical summary revealed that the A3-genotype predominated (79.8% presence) in Vietnam from 1987 to 2021 and that it remained the dominant IBDV genotype over the last five years (2016-2021). The current study contributes to a better understanding of the circulating genotypes and evolution of IBDV in Vietnam and worldwide.

Mitogenomic and nuclear ribosomal transcription unit datasets support the synonymy of Paragonimus iloktsuenensis and P. ohirai (Paragonimidae: Platyhelminthes).

The complete mitogenome (mtDNA) of nominal Paragonimus iloktsuenensis (Paragonimidae: Trematoda) and the nuclear ribosomal transcription unit (rTU) coding region (rTU*: from 5'-terminus of 18S to 3'-terminus of 28S rRNA gene, excluding the external spacer region) of this species and of P. ohirai were obtained and used to further support the previously suggested synonymy of these taxa in the P. ohirai complex. The complete mitogenome of P. iloktsuenensis was 14,827 bp long (GenBank: ON961029) and nearly identical to that of P. ohirai (14,818 bp; KX765277), with a 99.12% nucleotide identity. The rTU* was 7543 bp and 6932 bp in these two taxa, respectively. All genes and spacers in the rTU were identical in length, with exception of the first internal transcribed spacer, which contained multiple tandem repeat units (6.7 for P. iloktsuenensis and 5.7 for P. ohirai). There was near 100% identity for the rTU genes. The phylogenetic topology inferred from the mtDNA and from individual gene regions (partial cox1 of 387 bp and the ITS-2 of 282 bp - 285 bp) indicated a very close relationship consistent with synonymy of P. iloktsuenensis and P. ohirai. The datasets provided here will be useful for taxonomic reappraisal as well as studies of evolutionary and population genetics of the genus Paragonimus and family Paragonimidae.

Molecular genotypic analysis of porcine circovirus type 2 reveals the predominance of PCV2d in Vietnam (2018-2020) and the association between PCV2h, the recombinant forms, and Vietnamese vaccines.

We conducted nucleotide and amino acid sequence alignment and phylogenetic analysis of porcine circovirus ORF2 (Cap protein) from 17 PCV2-positive clinical samples from nine different northern Vietnamese provinces (Mar 2018-Nov 2020), four local vaccines, and 77 reference strains. We identified one PCV2a (1/17 = 5.9%), five PCV2b (5/17 = 29.9%), and 11 PCV2d (11/17 = 64.7%) isolates, while only PCV2d was detected in 2020. Timeline analysis indicated an increasing predominance of PCV2d nationwide (2018-2020). With strong nodal support (98% for nucleotides and 74% for amino acids), the phylogenetic tree topology revealed a distinct PCV2h clade including recombinant/intermediate strains and local vaccines. The Cap protein sequences from 11 PCV2d field strains had the 2d-genotype-typical motif 86SNPLSV91 in loop CD, the motif TGID in loop GH-HI, and the motif 230PLNPK234 in loop CT. The PCV2h isolates (and vaccines) had the 86SNPLSV91, SAID, and 230L(N/H)PK234 motifs. Selection pressure analysis indicated positive selection at seven sites: A68N in immunoreactive region (IRR)-A; 119G and 130V in IRR-B; and 167L, T190(A/S), 194D and 202F in IRR-C. We identified PCV2h as the genotype of the recombinant strains, which resulted from intergenotype recombination of PCV2a, PCV2b, and PCV2d. The current data provide new information about the diversity, distribution, and dominance of the PCV2 genotype in Vietnam.

Canine parvovirus type 2c in Vietnam continues to produce distinct descendants with new mutations restricted to Vietnamese variants.

Viral protein 2 (VP2) of canine parvovirus (CPV) exhibits a high degree of genetic and antigenic diversity. We analyzed 88 Vietnamese CPV-VP2 sequences (1755 bp), 34 from this study and 54 from previous studies, and discovered a new sublineage, "new var.", within the lineage CPV-2c-"new", characterized by the mutation 5G/447M, which is restricted to the Vietnamese isolates. These new mutants appear to have emerged in recent years, accounting for 65.5% of the total. With strong nodal support (98%), the distinct Vietnamese 2c-"new-var." sublineage (5G/426E/447M) was found to be separate from the 2c-"new" sublineage (5G/426E/447I) within the 2c-(Asia)/Asia-2c lineage. Amino acid changes in epitopes of VP2 might have led to the generation of subvariants and affected the antigenicity, immunogenicity, or virulence of the virus, resulting in vaccine failure worldwide.

Comparative mitogenomics of the zoonotic parasite Echinostoma revolutum resolves taxonomic relationships within the 'E. revolutum' species group and the Echinostomata (Platyhelminthes: Digenea).

The complete mitochondrial sequence of 17,030 bp was obtained from Echinostoma revolutum and characterized with those of previously reported members of the superfamily Echinostomatoidea, i.e. six echinostomatids, one echinochasmid, five fasciolids, one himasthlid, and two cyclocoelids. Relationship within suborders and between superfamilies, such as Echinostomata, Pronocephalata, Troglotremata, Opisthorchiata, and Xiphiditata, are also considered. It contained 12 protein-coding, two ribosomal RNA, 22 transfer RNA genes and a tandem repetitive consisting non-coding region (NCR). The gene order, one way-positive transcription, the absence of atp8 and the overlapped region by 40 bp between nad4L and nad4 genes were similar as in common trematodes. The NCR located between tRNAGlu (trnE) and cox3 contained 11 long (LRUs) and short repeat units (SRUs) (seven LRUs of 317 bp, four SRUs of 207 bp each), and an internal spacer sequence between LRU7 and SRU4 specifying high-level polymorphism. Special DHU-arm missing tRNAs for Serine were found for both tRNAS1(AGN) and tRNAS2(UCN). Echinostoma revolutum indicated the lowest divergence rate to E. miyagawai and the highest to Tracheophilus cymbius and Echinochasmus japonicus. The usage of ATG/GTG start and TAG/TAA stop codons, the AT composition bias, the negative AT-skewness, and the most for Phe/Leu/Val and the least for Arg/Asn/Asp codons were noted. Topology indicated the monophyletic position of E. revolutum to E. miyagawai. Monophyly of Echinostomatidae and Fasciolidae was clearly solved with respect to Echinochasmidae, Himasthlidae, and Cyclocoelidae which were rendered paraphyletic in the suborder Echinostomata.

Molecular characterization of field isolates of infectious bursal disease virus from three decades, 1987-2018, reveals a distinct genotypic subgroup in Vietnam.

The complete nucleotide sequence of the viral protein 2 (VP2) ORF was determined for 26 Vietnamese infectious bursal disease isolates collected from clinical outbreaks in vaccinated flocks from 1987 to 2018 and two commercial vaccine specimens. These sequences were compared for molecular classification with 42 reference strains representing all four main classes of serotype 1, including very virulent (vvIBDV), classical (cvIBDV), antigenic variant (avIBDV) and attenuated (atIBDV) strains, and serotype 2 strains. Amino acids at nine key positions in the VP2-HVR in 20 Vietnamese isolates, A222, I242, Q253, I256, D279, A284, I294, S299, A329, which are typical of the vvIBDV class, were found to be identical in all of the isolates. Eighteen of these isolates had a unique change at residue 212 (D212N) located in the PAB loop. Phylogenetic analysis revealed a distinct lineage/subclade with strong nodal support (96%) that included recent Chinese IBDV strains that were distinct from typical vvIBDVs. Six isolates contained the amino acid substitutions P222, V242, Q253, V256, D279, A284, I294, N299, A329, which are present in two vaccine strains derived from strain 2512 and these isolates were also closely related to the classical virulent STC strain. Data from this study show that there is considerable genetic diversity among vvIBDVs, which vary according to geographic region. Antigenic drift and differences in genetic characteristics between virulent strains and IBDV vaccine strains may be the cause of vaccine failure. Better antigenic matching of vaccines to the strains circulating in Vietnam is therefore required.

Molecular analysis of Newcastle disease virus isolates reveals a novel XIId subgenotype in Vietnam.

Eight Vietnamese Newcastle disease virus field isolates from 2008-2015 and 3 vaccine specimens were genotyped based on their full F gene sequences and compared to 80 reference strains representing all 18 genotypes. Three isolates formed a novel subgenotype XIId, identified for the first time in Vietnam; while the others clustered as follows: four in subgenotypes VIId and VIIh; two in Genotype I; and two in Genotype II. Evolutionary distance calculations confirmed the Vietnamese XIId isolates were distinct from XIIa and XIIb by 0.062-0.070; and from other genotypes by 0.089-0.245. This data demonstrated that a novel XIId subgenotype emerged in Vietnam indicating considerable genetic diversity, thus highlighting the need to implement antigenic matching during vaccination against NDVs.

Molecular genotyping of duck hepatitis A viruses (DHAV) in Vietnam.

INTRODUCTION: The aim of this study was to identify the genetic characteristics and molecular genotyping of duck hepatitis A virus (DHAV) isolated in Vietnam during 2009-2013. METHODOLOGY: Thirty duckling livers from outbreaks between 2009 and 2013 in seven provinces were collected and identified by polymerase chain reaction (PCR). Then, VP1 genes of eleven positive samples and two attenuated vaccine strains were sequenced and analyzed. RESULTS: Genotypic and phylogenetic analyses indicated that the 13 Vietnamese isolates were classified into two genotypes, DHAV-1 and DHAV-3. The rate of identity and homology was 91%-100% between the 10 Vietnamese and 26 global strains of DHAV-3, and 92%-100% between 3 Vietnamese and 16 strains of DHAV-1. Between the DHAV-3 and DHAV-1 strains, the divergence reached 30%. At the C-terminal of VP1 for the different strains, a hypervariable region was observed, and notably, six of the Vietnamese DHAV-3 strains in this study showed four consistent differences (at positions T184M, Q200H, K207N, and K214R) within this group that were distinct from all other DHAV-3 strains. CONCLUSIONS: This is the first report of molecular characterization of DHAVs in Vietnam. At least two genotypes were identified, DHAV-1 and DHAV-3, with diversified clades within and between genotypes. DHAV-3 seemed to be dominant in Vietnam.

Development and evaluation of a single-step duplex PCR for simultaneous detection of Fasciola hepatica and Fasciola gigantica (family Fasciolidae, class Trematoda, phylum Platyhelminthes).

A single-step multiplex PCR (here referred to as a duplex PCR) has been developed for simultaneous detection and diagnosis of Fasciola hepatica and F. gigantica. These species overlap in distribution in many countries of North and East Africa and Central and Southeast Asia and are similar in egg morphology, making identification from fecal samples difficult. Based on a comparative alignment of mitochondrial DNA (mtDNA) spanning the region of cox1-trnT-rrnL, two species-specific forward primers were designed, FHF (for F. hepatica) and FGF (for F. gigantica), and a single reverse primer, FHGR (common for both species). Conventional PCR followed by sequencing was applied using species-specific primer pairs to verify the specificity of primers and the identity of Fasciola DNA templates. Duplex PCR (using three primers) was used for testing with the DNA extracted from adult worms, miracidia, and eggs, producing amplicons of 1,031 bp for F. hepatica and 615 bp for F. gigantica. The duplex PCR failed to amplify from DNA of other common liver and intestinal trematodes, including two opisthorchiids, three heterophyids, an echinostomid, another fasciolid, and a taeniid cestode. The sensitivity assay showed that the duplex PCR limit of detection for each Fasciola species was between 0.012 ng and 0.006 ng DNA. Evaluation using DNA templates from 32 Fasciola samples (28 adults and 4 eggs) and from 25 field-collected stools of ruminants and humans revealed specific bands of the correct size and the presence of Fasciola species. This novel mtDNA duplex PCR is a sensitive and fast tool for accurate identification of Fasciola species in areas of distributional and zonal overlap.