Sequence Analysis Of Hypervariable Region Research Articles

Novel recombinant avian infectious bronchitis viruses from chickens in Korea, 2019-2021.

Infectious bronchitis virus (IBV) has evolved through various mutation mechanisms, including antigenic drift and recombination. Four genotypic lineages of IBVs including GI-15, GI-16, GI-19, and GVI-1 have been reported in Korea. In this study, we isolated two IBVs from chicken farms, designated IBV/Korea/289/2019 (K289/19) and IBV/Korea/163/2021 (K163/21), which are two distinct natural recombinant viruses most likely produced by genetic reassortment between the S1 gene of K40/09 strain (GI-19 lineage) and IBV/Korea/48/2020 (GI-15 lineage) in co-infected commercial chickens. Comparative sequence analysis of hypervariable regions (HVRs) revealed that the K289/19 virus had similar HVR I and II with the K40/09 virus (100% and 99.2% nucleotide sequence identity, respectively), and HVR III with the IBV/Korea/48/2020 virus (100% nucleotide sequence identity). In contrast, the K163/21 virus had HVR I and II similar to the IBV/Korea/48/2020 virus (99.1% and 99.3% nucleotide sequence identity, respectively), and HVR III to the K40/09 virus (96.6% nucleotide sequence identity). The K289/19 virus exhibited similar histopathologic lesions, tissue tropism in trachea and kidney, and antigenicity with the parental K40/09 virus. The K163/21 exhibited similar pathogenicity and tissue tropism with the K40/09 virus, which were similar results with the isolate K289/19. However, it showed a lower antigenic relatedness with both parental strains, exhibiting R-value of 25 and 42, respectively. The continued emergence of the novel reassortant IBVs suggests that multiple recombination events have occurred between different genotypes within Korea. These results suggest that antigenic profiles could be altered through natural recombination in the field, complicating the antigenic match of vaccine strains to field strains. Enhanced surveillance and research into the characteristics of newly emerging IBVs should be carried out to establish effective countermeasures.

Recolonizing lost habitat—how European beavers (Castor fiber) return to south-western Germany

For the last decades, the European beaver (Castor fiber) has been recolonizing its original habitats. Reintroductions of beavers from different relict populations into southern Germany have resulted in several admixed populations, which are spreading out along various river systems. The eastern part of the German state of Baden-Wurttemberg is a melting pot of colonization waves originating from various introduced populations. The aim of this study was to exemplify origins and dispersal behaviour of beavers in this region using genetic fingerprint methods. Sequence analysis of hypervariable region 1 (HV1) of the mitochondrial control region and fragment length analyses at 11 microsatellite loci resulted in genetic profiles for 84 samples. The study region is being populated from three different local origins of beavers: the north of the Main-Tauber district, the Neckar River near the city of Mosbach and the Danube tributaries in the east. Main-Tauber samples were most diverse, including microsatellite alleles and HV1 haplotypes specific to C. f. albicus (from the German Elbe relict population). In view of the geographical proximity of this region to a release site of C. f. albicus in the Spessart area, this finding strongly suggests gene flow between beaver populations of different provenience. Two remaining local origins at the Neckar and Danube tributaries are closely related to each other, thus possibly descending from the same original (admixed) population. This study is intended to serve as a starting point for follow-up fine-scale research into dispersal behaviour of European beavers currently recolonizing their original habitats.

Sequence analysis of mitochondrial DNA hypervariable region I in Thai individuals

Nucleotide sequence analysis of hypervariable region I (HVRI) in human mitochondrial DNA (mtDNA) was investigated in 100 unrelated Thai individuals. A total of 85 variable sites and 423 base substitutions, which consisted of 390 nucleotide transitions and 33 nucleotide transversions were found. The following nucleotide substitutions were found: 48% at 16,223, 31% at 16,304, 30% at 16,332, and 26% at 16,129, respectively. Transition from T to C (43.7%) was the most frequent substitution. The nucleotide insertions were found at two sites with T at position 16,188 and C at position 16,194. Eighty-two haplotypes were investigated of which 72 haplotypes were unique. The most frequent haplotypes (16,108T-16,129A-16,162G-16,172C-16,304C and 16,260T-16,298C-16,355T-16,362C) were observed. From position 16,180 to 16,193, thirteen patterns of polycytosine or C-stretch were observed, whereas 68 Thai individuals were found to be similar to the references. The genetic diversity, random match probability, and discrimination power were estimated to be 0.9943, 0.0156, and 0.9844, respectively.

Sequencing human mitochondrial hypervariable region II as a molecular fingerprint for environmental waters.

To protect environmental water from human fecal contamination, authorities must be able to unambiguously identify the source of the contamination. Current identification methods focus on tracking fecal bacteria associated with the human gut, but many of these bacterial indicators also thrive in the environment and in other mammalian hosts. Mitochondrial DNA could solve this problem by serving as a human-specific marker for fecal contamination. Here we show that the human mitochondrial hypervariable region II can function as a molecular fingerprint for human contamination in an urban watershed impacted by combined sewer overflows. We present high-throughput sequencing analysis of hypervariable region II for spatial resolution of the contaminated sites and assessment of the population diversity of the impacting regions. We propose that human mitochondrial DNA from public waste streams may serve as a tool for identifying waste sources definitively, analyzing population diversity, and conducting other anthropological investigations.

Genetic characterization of VP1 gene of seven Sacbrood virus isolated from three provinces in northern China during the years 2008–2012

The genetic diversity of seven northern China isolated Sacbrood virus strains (SBV) has been analyzed, and hypervariable regions of the VP1 gene of 7 SBV were sequenced and characterized, in order to obtain epidemiological and immunological information, and to suggest typing criteria for SBV. Sequence analysis of hypervariable regions of the VP1 gene in the genome of these isolates revealed a sequence homology of 91.0–99.3% among all seven local SBV isolates from Apis cerana from China, with a similarity of 93.3–100.0% in deduced amino acid sequences. These local isolates shared 87.4–92.8% sequence homology with six SBV reference strains in GenBank (including two SBV reference strains from Apis cerana from China), which represents a 91.8–97.6% similarity in deduced amino acid sequences. Genetic analysis also showed that five SBV strains from Apis cerana from China had a 13-amino-acid deletion at amino acid positions 287–299, and two SBV strains infecting the Korean honeybee had a 17-amino-acid deletion at amino acid positions 284–300 in comparison with other SBV. Phylogenetic analysis revealed two major groups (AC genotype SBV infecting Apis cerana and AM genotype SBV infecting Apis mellifera). The AC genotype could be further divided into subgroups. Based on the results of phylogenetic analysis, a similarity scan of SBV nucleotide sequences was carried out by using Simplot software and results in similar results. Our results suggest possible typing criteria for SBV based on the phylogenetic tree and sequence homology, and also that the virus has host specificity and regional variations.

Molecular epidemiology of infectious bursal disease viruses isolated from Southern China during the years 2000–2010

We performed a molecular epidemiology study of infectious bursal disease virus (IBDV) from six provinces in southern China by analyzing IBDVs isolated during the years 2000-2010. Sequence analysis of hypervariable regions of the VP2 gene (vVP2) in the genome of these isolates revealed that the majority of these viruses (45/59) were characterized as vv (very virulent) IBDV genotype, 12 out of 59 isolates were avirulent IBDV genotype and two from Guangxi were intermediate IBDV genotype. Phylogenetic analysis revealed that 45 vvIBDV genotype isolates have divided into five groups, all displaying strong divergence from the currently used vaccine strains. In all isolates, 14 non-critical amino acid substitutions were found in vVP2. The isolates from 2006 to 2010 had more substitutions (11 sites) than the isolates from 2000 to 2005 (7 sites). This study demonstrates that there were different genotypes of IBDV prevailing in six provinces of southern China. The mutations in vVP2 were common, which might be one of the reasons for the evolution of the IBDVs. Therefore, in regards to IBDV prevention, it is vital to have continuous monitoring of the genetic variability (long-term tracking of viral evolution) to provide optimal protection against IBDV.

Biometrical and genetical characterization of large Babesia Ovis in Iran

One species of Babesia was identified on the blood smear of 20 different naturally infected sheep in the Northwest of Iran. It was polymorphic, including double pyriform with acute or obtuse angle, single pyriform, and ring form. The size of typical paired pyriforms with acute angle was 2.7 x 0.4 microm (n=10) and with obtuse angle was 3.5 x 0.6 microm (n=10). Although the morphological and biometrical parameters resembled the Babesia motasi, the results of seminested polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism using primers specific for small subunit of 18S rRNA confirmed this species as Babesia ovis. Furthermore, the sequence analysis of hypervariable region of small subunit of 18S rRNA revealed the corresponding sequences for B. ovis as well. Experimental infection of healthy lambs with the morphological larger B. ovis showed a milder clinical signs compared to the small one.

Early Evolution of Hepatitis C Virus (HCV) Quasispecies after Liver Transplant for HCV‐Related Disease

End-stage liver disease as a result of chronic hepatitis C virus (HCV) infection is the main indication for liver transplant (LT), but allografts are systematically infected with HCV soon after transplant. Viral quasispecies are poorly described during the early posttransplant period. For 17 patients who received an LT for HCV disease, plasma viral quasispecies evolution was determined by sequence analysis of hypervariable region 1 of the E2 envelope gene before transplant (BT), after 7 days (D7), and after 1 month (M1). T helper (Th)1/Th2 cytokine levels were determined concomitantly. HCV quasispecies showed a significant decrease in amino acid diversity at D7 and M1, compared with BT (P<.05). A correlation was observed between low plasma tumor necrosis factor-alpha levels at D7 and decreased quasispecies amino acid complexity at the same date. Nucleic acid diversity was lower for genotype 1 than for genotype 3 infection (P<.05). The complexity and diversity of amino acids were lower in patients with hepatocellular carcinoma (HCC) BT than in those without HCC (P<.05). Conserved amino acid residues within quasispecies were shared by the whole cohort before and after LT. Viral structural and/or host immunological features could favor the emergence of fitter HCV strains after LT.

Molecular analysis of measles virus genome derived from SSPE and acute measles patients in Papua, New Guinea

A very high annual incidence of 56 per million population below the age of 20 years for subacute sclerosing panencephalitis (SSPE) has been reported from Papua New Guinea (PNG). In a more recent study, we have confirmed this unusual high incidence for Eastern Highlands Province (EHP) of PNG. In the study, it was observed that the vaccination rate among SSPE patients registered at Goroka Base General Hospital (GBGH) in EHP was higher than that of other infants in the province in recent years. To identify the measles virus (MV) responsible for SSPE in EHP, sequence analysis of hypervariable region of the N gene was performed from 13 MV genomes: 2 amplified from clinical specimens of SSPE patients and 11 from acute measles patients. In 2 cases among the 11 with acute measles, nucleotide sequence of the entire H gene derived from isolated viruses was determined. Both nucleotide sequence and phylogenetic tree analyses showed that the amplified MV cDNAs were closely related to one another and belonged to the D3 genotype though they were different from any previously reported MV sequences. No genome sequences of vaccine strains were detected. These findings suggest that the MV strains prevailing in the highlands of PNG belong to genotype D3 of the MV and this wild-type MV rather than the vaccine strains was likely to be responsible for SSPE in these patients.

Sequence analysis of hypervariable region of hepatitis C virus (HCV) associated with immune complex in patients with chronic HCV infection.

Immune complex (IC) frequently exists in sera of patients with hepatitis C virus (HCV) infection. When circulating HCV particles are fractionated by differential flotation ultracentrifugation, HCV in the bottom fraction appears to be in the form of IC. Flotation ultracentrifugation of serum was done in 7 patients positive for serum anti-C3d-binding circulating IC and in 7 negative patients. In all IC-positive patients, HCV RNA was detected in the bottom fraction but not in the top fraction of 3 patients. Amino acid sequences of hypervariable region (HVR) of HCV in the bottom fraction of IC-positive patients were almost the same as those of whole serum, while those of the top fraction considerably differed from those of whole serum except for 1 case. These data suggest that the major population of HCV forms IC in IC-positive patients, and antibody response to the HVR of HCV is responsible for IC formation.

Susceptibility of Human T-Lymphotropic Virus Type I-Infected Cell Line MT-2 to Hepatitis C Virus Infection

To obtain a hepatitis C virus (HCV) proliferation system, we examined the susceptibility of various cultured cell lines to HCV infection. We found that a human T-lymphotropic virus type I infected cell Line MT-2 was fairly sensitive to HCV infection. Using the polymerase chain reaction, intracellular positive-stranded HCV RNA was detected until at least 15 days postinoculation (p.i.). Intracellular negative-stranded HCV RNA was also detected at 10 days p.i., although not at 7 days p.i., suggesting that HCV is replicating in MT-2 cells 10 days p.i. Sequence analysis of hypervariable region 1 (HVR1) revealed that HVR1 sequences from cells 10 days p.i. had become homogeneous, although HVR1 sequences from the inoculum showed the typical quasi-species. We also found a lack of anti-HVR1 antibody against the HVR1 species which became homogeneous at 10 days p.i., although we easily detected antibody against the other HVR1 species obtained from the inoculum. These findings suggest that MT-2 cells are susceptible to HCV infection and are capable of supporting HCV replication.