Nucleotide Sequence Analysis Research Articles

Emerging hantavirus infection in wild rodents captured in suburbs of Gwangju Metropolitan City, South Korea.

BackgroundHemorrhagic fever with renal syndrome (HFRS) caused by hantaviruses is a frequently reported acute hemorrhagic fever in South Korea. These viruses are transmitted by various rodent species such as Apodemus agrarius.Methodology/Principal findingsTo investigate hantavirus infection and seroprevalence in rodents, wild rodents were captured from two districts in the suburbs of Gwangju Metropolitan City from January 2016 to December 2018. Nested reverse-transcription polymerase chain reaction (RT-PCR) targeting the hantavirus-specific L segment and indirect immunofluorescence antibody (IFA) assay using Hantaan virus antigen slides were performed. A total of 585 wild rodents were captured—512 A. agrarius, 49 Crocidura lasiura, and 24 Myodes regulus. Nested RT-PCR was performed to examine the rate of hantavirus infection in wild rodents, and 1.88% (11/585) of all rodents, 1.17% (6/512) of A. agrarius, 6.12% (3/49) of C. lasiura, and 8.33% (2/24) of M. regulus tested positive. The nucleotide sequence analysis of the eleven PCR-positive products revealed that six PCR products showed over 85% sequence similarity with the Jeju virus, four showed over 99.7% similarity with the Hantaan virus, and one showed over 95.3% homology with the Imjin virus. Moreover, IgG antibodies against the Hantaan virus were detected in 6.15% (36/585) of all rodents, 6.8% (35/512) of A. agrarius, and 4.17% (1/24) of M. regulus. IgG antibodies were not detected in C. lasiura.Conclusions/SignificanceHantaviruses were detected in all three wild rodent species of A. agrarius, C. lasiura, and M. regulus captured in the suburbs of Gwangju Metropolitan City, South Korea, and it was demonstrated that they were various strains of hantaviruses such as the Hantaan, Jeju, and Imjin viruses.

Detection, characterization and virulence analysis of nucleopolyhedrovirus isolated from the cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae)

BackgroundIn the present study, detection, characterization and virulence analysis of a field collected nucleopolyhedrovirus isolated from the cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) was carried out. The obtained isolate, named SpliNPV-YW, was collected from diseased S. littoralis larvae in El-Menoufia governorate, Egypt.ResultsTransmission electron microscopy showed the presence of typical occlusion bodies with average size of (1.06 × 1.19 µm). Upon digestion using two different endonucleases, PstI and ScaI, no clear difference was detected in the collected isolate (SpliNPV-YW) DNA genome pattern compared to the reference strain SpliNPV-AN1956. The evolutionary analysis of the polyhedrin gene's partial nucleotide sequence revealed that SpliNPV-YW isolate was closed and had a genetic origin with the NPV isolate SpliMNPV-A26-5 that belongs to group II NPVs with identity of 99.7%. The median lethal concentration (LC50) and the median lethal time (LT50) values were estimated for second and fourth larval instars of S. littoralis. The LC50 values were 2.8 × 104 OB/ml for second larval instar and 5.2 × 105 OB/ml for fourth larval instar after 10 days of treatment. Regarding the speed of killing of the viral isolate, the results showed that the LT50 value for the second instar larvae (LT50 = 5.5 days) was lower than that of the fourth instar larvae (LT50 = 6.2 days) at concentrations of 4.3 × 1010 (ob/ml) and 1.2 × 1011 (ob/ml) for second and fourth instar larvae, respectively.ConclusionsHost specificity and virulence characteristics make SpliNPV-YW isolate a good potential to be utilized as a candidate biopesticide for the control of S. littoralis population in Egypt.

The Mitochondrial Genome of Cylicocyclus elongatus (Strongylida: Strongylidae) and Its Comparative Analysis with Other Cylicocyclus Species.

Cylicocyclus elongatus (C. elongatus) is one of the species in Cylicocyclus, subfamily Cyathostominae, but its taxonomic status in Cylicocyclus is controversial. Mitochondrial (mt) genome is an excellent gene marker which could be used to address the taxonomy controversy. In the present study, the complete mt genome of C. elongatus was determined, and sequence and phylogenetic analyses were performed based on mtDNA data to determine the classification of C. elongatus. The circular complete mt genome of C.elongatus was 13875 bp in size, containing 12 protein-coding genes (12 PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and 2 non-coding regions (NCRs). The A + T content of C. elongatus complete mt genome was 76.64%. There were 19 intergenic spacers with lengths of 2-53 bp and 2 overlaps with lengths of 1-2 bp in the impact complete mt genome. ATT and TAA were the most common start and termination codons of 12 PCGs, respectively. Comparative analyses of mt genomes nucleotide sequence and amino acid sequence showed that there were higher identities between C. elongatus and five other Cylicocyclus, rather than with P. imparidentatum. Phylogenetic analyses based on concatenated nucleotide sequences of 12 PCGs of 23 species in the family Strongylidae showed that C. elongatus was closely related to Cylicocyclus species, rather than P. imparidentatum. We concluded that C. elongatus was a member in Cylicocyclus based on comparative and phylogenetic analyses of mt genome sequences. The data of the complete mt genome sequence of C. elongatus provide a new and useful genetic marker for further research on Cyathostominae nematodes.

Epidemiological Interface of Sylvatic and Dog Rabies in the North West Province of South Africa.

Rabies is a viral zoonosis that causes an estimated 59,000 preventable human fatalities every year. While more than 120 countries remain endemic for dog-mediated rabies, the burden is the highest in Africa and Asia where 99% of human rabies cases are caused by domestic dogs. One such rabies-endemic country is South Africa where an estimated 42 preventable human deaths occur every year. Although canine rabies had been well described for most of the provinces in South Africa, the epidemiology of rabies within the North West Province had not been well defined prior to this investigation. As such, the aim of this study was to use nucleotide sequence analyses to characterise the extant molecular epidemiology of rabies in the North West Province of South Africa—with specific focus on the interface between dogs and sylvatic species. To this end, Rabies lyssavirus isolates originating from the North West Province were subjected to molecular epidemiological analyses relying on the Bayesian Markov Chain Monte Carlo methodology on two distinct gene regions, viz. the G-L intergenic region and partial nucleoprotein gene. Our results provided strong evidence in support of an endemic cycle of canine rabies in the East of the province, and three independent endemic cycles of sylvatic rabies spread throughout the province. Furthermore, evidence of specific events of virus spill-over between co-habiting sylvatic species and domestic dogs was found. These results suggest that the elimination of canine-mediated rabies from the province will rely not only on eliminating the disease from the dog populations, but also from the co-habiting sylvatic populations using oral rabies vaccination campaigns.

Spread of Human T-Lymphotropic Virus 1 and 2 Among Relatives of People Who Use Illicit Drugs in Northern Brazil.

The human T-lymphotropic virus 1 (HTLV-1) and 2 (HTLV-2) can be transmitted between humans by mechanisms associated with horizontal and vertical routes. Recently, high prevalence rates and levels of genetic diversity for HTLV-1 and HTLV-2 were detected among people who use illicit drugs (PWUDs) in the Brazilian state of Pará. None of the PWUDs with HTLV-1 or HTLV-2 were aware of their carrier condition of the retrovirus, and they ability to spread it to their family group, sexual partners, and other contacts. Thus, this study evaluated the presence of HTLV-1 and HTLV-2 in families of PWUDs in the state of Pará, in Northern Brazil. This descriptive study used convenience sampling and accessed 37 PWUDs and their respective families (n = 97) in 18 municipalities in the state of Pará, northern Brazil. All participants provided personal data and were tested for the presence of HTLV-1 and HTLV-2 using enzyme-linked immunosorbent assay and western blotting. HTLV positive samples were selected for Nested-PCR, and viral genotyping by nucleotide sequencing and phylogenetic analysis. HTLV-1 or HTLV-2 infections were detected in 15 families of PWUDs: 27 family members of PWUDs were infected with HTLV-1 (27.8%) and another 20 of them with HTLV-2 (20.6%). Subtypes 1a [subgroup A (54.5%)], 2b (20.5%), and 2c (25.0%) were detected. High horizontal (76.9%) and vertical (61.4%) transmission rates of HTLV were ascertained. Factors that facilitate the acquisition and transmission of HTLV-1 and HTLV-2 were reported by the participants, such as long-term relationships, unprotected sex, breastfeeding, and lack of knowledge about the condition of being a carrier of the retrovirus. Evidence indicates intrafamilial transmission of HTLV from PWUDs to members of their respective families. Key interventions should urgently be employed for the control and prevention of HTLV-1 and HTLV-2 to reduce the spread of this retrovirus in PWUDs and the general population in Northern Brazil and elsewhere.

Detection of multidrug resistance and associated genes among <em>Salmonella</em> species from enteric fever cases

Background: Salmonella spp. has rapidly gained resistance to ampicillin, chloramphenicol, co-trimoxazole, and tetracycline (ACCoT) which necessitated the use of fluoroquinolones and cephalosporins. However, there are reports on emergence of fluoroquinolone-resistant Salmonella isolates in various parts of Asia and low resistance to third-generation cephalosporins. The incidence of multi-drug resistant (MDR) Salmonella enterica serovar Typhi (S. Typhi) in India increased in the 1990s. After a decade, studies showed reduced MDR percentage. Hence this study was performed to detect antibiotic resistance patterns and associated genes among clinical isolates of Salmonella. Methods: A total of 171 clinical isolates of Salmonella isolates collected between 2011 to 2016 from a tertiary care hospital in Chennai, India were included. Antibiotic susceptibility testing and screening for extended-spectrum β-lactamase production was performed. Genes encoding resistance to β-lactam antibiotics (blaTEM, blaSHV, and blaCTX-M), chloramphenicol (cat, cmlA, and floR), co-trimoxazole (sul1, sul2, sul3, and dfr), and tetracycline (tet(A), tet(B), tet(C), tet(D), tet(E), and tet(G)) were detected using PCR and nucleotide sequencing analysis. Results: The majority of the isolates were susceptible to ACCoT antibiotics, cephalosporins, and carbapenems. Most of the isolates were resistant to nalidixic acid and 1.7% were sensitive to ciprofloxacin. All the isolates (n=171) were negative for ESBL. Multidrug resistance was seen in 4.1% isolates and all the MDR Salmonella isolates were found to contain blaTEM-1(TEM-1-type β-lactamase), cat, dfrA17, sul1, and tet(B) genes. Conclusion: We have observed a low MDR prevalence and reduced susceptibility to the fluoroquinolone, ciprofloxacin. This changing susceptibility pattern of Salmonella species over time warrants continuous monitoring and judicious use of antibiotics to prevent the emergence of resistant strains.

Identification of two novel ephemeroviruses in pigs infected by classical swine fever virus.

Ephemeroviruses are arthropod-borne rhabdoviruses within Ephemerovirus genus and have been isolated exclusively from cattle and haematophagous arthropods (mosquitoes and biting midges) without any member detected or isolated up to date from pigs, although some serological surveys have indicated that pigs may be a silent host for ephemerovirus infection. Here, many viral reads annotated to, but genetically distinct from, the existing members within the Ephemerovirus genus have been identified in the meta-transcriptomic data of two clinical classical swine fever virus (CSFV)-infected samples (HeN10 and GDMM7). The nearly complete genome sequences of the two novel ephemeroviruses have been obtained through contig assembly, specific RT-PCR and sequencing, therefore named as porcine ephemeroviruses (PoEVs). Genome nucleotide sequence analysis showed that PoEV strains HeN10 and GDMM7 have similar genome organization and 66.5% genomic identity to each other, but both are genetically distant from all members of the Ephemerovirus genus with identity being only 51.1-59.6%. Furthermore, comparison of the most conserved ephemeroviral proteins N and L indicated that PoEV strains HeN10 and GDMM7 share a high sequence identity to each other (N: 78.1%; L: 70.7%), but are diverged from the known ephemeroviruses (N: 43.4-60.7%; L: 47.6-58.5%). The genetic distance is significantly beyond the criteria for demarcation of viruses assigned to different ephemerovirus species. Thereby, two novel viruses named as PoEV1 (strain HeN10) and PoEV2 (strain GDMM7) are identified and these appear to represent two new species within the Ephemerovirus genus. The present study showed the first genome evidence of pig ephemeroviruses, likely expanding the known host range of ephemerovirus.

Molecular characterization of virus species causing yellow mosaic disease of redgram in Andhra Pradesh

Redgram is animportant drought tolerant leguminous crop in the semi-arid subtropics. Many fungal, bacterialand viral diseases are major constraints for redgram production, apart from all these diseases, yellowmosaic disease (YMD) is observed in redgram crop in few localities of Chittoor district, Andhra Pradeshduring 2017. The present study was aimed to find out associated begomo virus species causing YMD inredgram.The total genomic DNA was isolated from YMV infected redgram leaves by modified CTAB method.The rolling circle amplification based full length clones were constructed in pUC18 vector. The two constructed clones (DNA-A and DNA-B) were confirmed by using gene specific primers and weresequenced.The coat protein gene andcomplete nucleotide sequence analysis was carried out with the knownbegomoviruses sequences obtained from Genbank database.The phylogenetic analysis of coat protein geneof YMV-AP-Red gram isolate shared 94.3-98.1% nucleotide identity with HgYMV isolates, followed byMYMV (81.2-85.2%), MYMIV (78.1-80.2%) and DoYMV(72.2-74%). Hence it was confirmed that viral speciescausing YMD in redgram from Tirupati, AndhraPradesh is a Horsegram yellow mosaic virus and it is avariant of HgYMV as it shared>94% similarity with other HgYMV isolates at nucleotide level.

Molecular identification, whole-cell protein profiling, antibiotics sensitivity and pathogenicity of Chryseobacterium gleum recovered from diseased fishes of freshwater wetlands in India

Globally, bacterial gill disease is one of the most prominent bacterial diseases affecting both marine and freshwater fishes. This study aimed to investigate diseased Labeo rohita, Labeo catla and Oreochromis niloticus with gill discoloration, tail rot and skin ulcer that resembles the typical sign of bacterial gill disease from East Kolkata Wetlands, India. The results revealed that four isolates of Chryseobacterium gleum were the responsible pathogens. The isolates CIFRI-RRM3, CIFRI-BFG2, CIFRI-SRM4 and CIFRI-TRM5 were identified using bacterial colony morphology, biochemical test, whole-cell protein profiling, 16S rRNA gene amplification and nucleotide sequence analysis. The antibiotic susceptibility assay showed that all the isolates were completely resistant to ampicillin, amoxyclav, cefalexin, cephalothin, ceftriaxone, ceftazidime, cefotaxime, cefuroxime, cefadroxil, chloramphenicol, kanamycin, netilmicin, penicillin-G, ticarcillin, tobramycin, erythromycin and tetracycline. A challenge experiment was also conducted for four isolates with intraperitoneal injection at a dose of 2.5 × 1010 CFU mL−1 to 4.2 × 1010 CFU mL−1in respective fish hosts. Only three isolates (CIFRI-BFG2, CIFRI-RRM3 and CIFRI-TRM5) proved their virulence by producing diseases with mild symptoms of skin lesions, gill disease and cumulative mortality of 10–20%. To the best of our knowledge, this is the first description of the phenotypic, molecular and whole-cell protein profiling of C. gleum derived from freshwater fishes in India.

Garden Lupin (Lupinus polyphyllus) Is a New Natural Host of Lily Symptomless Virus in Korea.

Lupinus polyphyllus (Fabaceae), commonly known as garden lupin, is cultivated for commercial, private, and gardening purposes in many countries in Europe, North America, and Asia (Ramula and Kalske 2020). No previous studies have reported viruses infecting L. polyphyllus in the Republic of Korea. In May 2021, virus-like symptoms such as stunting, chlorotic striping, and mosaic were observed on the leaves of L. polyphyllus at the Suncheonman National Garden in Suncheon, South Korea; the disease incidence was estimated to be between 10 and 20%. These symptoms were similar to those observed in lily plants infected by lily symptomless virus (LSV) (Singh et al. 2005, Flores et al. 2019). Several different species of Lilium, including lilies, were grown in the same garden with L. polyphyllus on the adjacent block. Only carlavirus-like filamentous particles (approximately 610-700 nm in length and 12-15 nm in diameter) were observed using transmission electron microscopy in negatively stained extracts from five symptomatic samples. To further investigate the causative virus, a total of 20 symptomatic leaves were collected from 20 different L. polyphyllus plants and tested by DAS-ELISA using polyclonal antibodies against viruses infecting plants from the Lilium genus, including arabis mosaic virus, cucumber mosaic virus, LSV, lily mottle virus, and plantago asiatica mosaic virus (Agdia, Elkhart, IN, USA). Commercial positive and negative controls and extract from healthy L. polyphyllus tissue were included in each ELISA. The serological analyses showed that all symptomatic samples were positive only for LSV, but not for other viruses. To further confirm LSV infection, total RNA was extracted from 10 ELISA-positive plants and two healthy L. polyphyllus plants (negative control) using Clear-S Total RNA extraction kit (InVirusTech Co., Gwangju, Korea). RT-PCRs were performed using LSV coat protein (CP) specific primers, LSV1 (5'-CACATATGCAATCAAGACCAGCAC-3') and LSV2 (5'-GAGGATCCTCATCCATTATTTGCGTATC-3'), designed to amplify an 876-nt fragment of the CP gene (Lim et al. 2009). A DNA fragment of the expected size was amplified from only the ten symptomatic leaves. Three amplified RT-PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI) and sequenced. The sequences obtained from each amplicon showed 100% sequence identity; a single representative sequence was deposited to GenBank (LC649240). Pairwise nucleotide sequence analysis revealed that this LSV isolate LP2 had 99% nucleotide and 100% amino acid identity with Lilium longiflorum LSV isolate from South Korea (JX962776). To fulfill Koch's postulates, sap from an LSV-positive L. polyphyllus plant was inoculated to 10 young healthy L. polyphyllus plants. At 14 days post-inoculation, all the inoculated plants showed typical symptoms of LSV infection, such as mild vein-clearing and mosaic (Asjes 2000). The LSV infection in the inoculated plants was confirmed by RT-PCR using the aforementioned primers and sequencing of the 876-nt amplicons. To the best of our knowledge, this is the first report of LSV naturally infecting L. polyphyllus in the Republic of Korea. LSV could negatively impact the quality of L. polyphyllus and trade. Thus, it is important to investigate the extent of its distribution in South Korea and how it can be effectively managed.

Diversity and antimicrobial susceptibility profiles of Aeromonas spp. isolated from diseased freshwater fishes in Thailand.

Motile Aeromonas septicemia (MAS), a disease caused by Aeromonas spp., is recognized as a major disease in freshwater aquaculture. This study aimed to investigate the distribution and diversity of Aeromonas spp. and their antimicrobial susceptibility patterns. A total of 86 isolates of Aeromonas spp. were recovered from diseased freshwater fishes from 13 farms in Thailand. All isolates were identified using biochemical characteristics, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), polymerase chain reaction assays, and the gyrB gene sequence analysis. The result of MALDI-TOF MS showed 100% (86 isolates) accuracy at genus-level identification, and 88.4% (76 isolates) accuracy at species-level identification. Six species of Aeromonas were confirmed through nucleotide sequencing and phylogenetic analysis of the gyrB gene Aeromonas veronii (72.1%), Aeromonas jandaei (11.6%), Aeromonas schubertii (9.3%), Aeromonas diversa (3.5%), Aeromonas hydrophila (2.3%), and Aeromonas punctata (1.2%). Antimicrobial susceptibility tests for all isolatesrevealed resistance against amoxicillin (99%), ampicillin (98%), oxolinic acid (81.4%), oxytetracycline (77%), trimethoprim-sulfamethoxazole (24%), and enrofloxacin (21%). The multiple antibiotic resistance (MAR) index varied between 0.14 and 0.86, with MAR values more than 0.2 in 99% of isolates. Furthermore, four diverse multidrug-resistant (MDR) patterns were found among Aeromonas isolates. Our finding show that A.veronii is the most abundant species in Thai cultured freshwater fish with the highest MDR patterns.

The GP-45 Protein, a Highly Variable Antigen from Babesia bigemina, Contains Conserved B-Cell Epitopes in Geographically Distant Isolates.

In B. bigemina, the 45 kilodaltons glycoprotein (GP-45) is the most studied. GP-45 is exposed on the surface of the B. bigemina merozoite, it is believed to play a role in the invasion of erythrocytes, and it is characterized by a high genetic and antigenic polymorphism. The objective of this study was to determine if GP-45 contains conserved B-cell epitopes, and if they would induce neutralizing antibodies. The comparative analysis of nucleotide and amino acids sequences revealed a high percentage of similarity between field isolates. Antibodies against peptides containing conserved B-cell epitopes of GP-45 were generated. Antibodies present in the sera of mice immunized with GP-45 peptides specifically recognize B. bigemina by the IFAT. More than 95% of cattle naturally infected with B. bigemina contained antibodies against conserved GP-45 peptides tested by ELISA. Finally, sera from rabbits immunized with GP-45 peptides were evaluated in vitro neutralization tests and it was shown that they reduced the percentage of parasitemia compared to sera from rabbits immunized with adjuvant. GP-45 from geographically distant isolates of B. bigemina contains conserved B-cell epitopes that induce neutralizing antibodies suggesting that this gene and its product play a critical role in the survival of the parasite under field conditions.

Prevalence of hepatitis B and D viruses in HIV-infected persons in the Socialist Republic of Vietnam

The aim of this study was to estimate the prevalence of hepatitis B and D viruses among HIV-infected residents of South Vietnam.Materials and methods. The study material was represented by 316 blood serum samples collected from HIV-infected residents of the Socialist Republic of Vietnam taking antiretroviral therapy. The subjects were examined for the presence of HBV markers with a qualitative detection of HBsAg, HBs IgG, HBcore IgG, anti-HDV, DNA HBV, and RNA HDV. HBV and HDV complete genomes nucleotide sequences were obtained for 23 samples from HIV+HBV+HDV co-infected patients. Amplification and subsequent sequencing of HBV and HDV were performed using nested PCR with pair’s overlapping primers jointly flanking the complete HBV and HDV genomes, respectively.Results. Serological markers of HBV and HDV were presented in the following ratios: HBsAg — 9.17%, anti-HBs Ig G — 10.44%, anti-HBcore Ig G — 42.08%, total anti-HDV — 9.81%. HBV DNA was detected in 32.58% of cases, including 23.41% of HBsAg-negative individuals. HDV RNA was detected in 24.13% of HBsAg-positive individuals and 21.62% of HBsAg-negative, which amounted to 22.33% of HBV-positive individuals and 7.27% of the total group, respectively. In phylogenetic analysis, HBV subgenotype B4 (60.89%) prevailed among HIV-infected patients compared to C1 (21.73%), B2 (8.7%), C2 (4.34%) and C5 (4.34%). Phylogenetic analysis of HDV nucleotide sequences showed the prevalence of HDV genotype 1 (78.26%) compared to genotype 2 (21.74%). The hepatitis Delta virus prevalence in patients with HIV+HBV coinfection, and the prevalence of seronegative HDV in patients with OBI indicate the need to use PCR in hepatitis highly endemic regions for hepatitis B and hepatitis D screening of the general population and especially those at-risk groups.

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.

​Molecular Characterization and Phylogenetic Analysis of Canine Parvovirus Type 2 (CPV-2) in the Aizawl Region of Mizoram Reveals Circulation of CPV-2c Antigenic Variant: A Hospital based Study

Background: The objective of the present study was to confirm the presence of CPV-2 in dogs with acute gastroenteritis and to characterize the antigenic variants circulating in the Aizawl region of Mizoram, India. Methods: A hospital based epidemiological study was carried out from April, 2019 to January, 2020. Faecal samples were collected from the dogs with gastroenteritis and screened for CPV-2 infection by polymerase chain reaction (PCR). The distribution of positive cases according to various epidemiological factors was studied. Partial sequencing of VP2 gene was performed for the analysis of CPV-2 variants. Result: A total of 96 faecal samples were collected during the study period out of which 70 (72.91%) were found positive for CPV-2 infection. The distribution of positive cases was highest in young dogs of the age group between 3-6 months (47.14%), cross-bred (48.57%) and unvaccinated dogs (64.28%). The percentage of CPV-2 infection was relatively higher in male (54.28%) as compared to female dogs (45.71%). The analysis of nucleotide sequences from amplified products and phylogenetic analysis revealed presence of CPV-2a (1/5, 20.0%) and CPV-2c (4/5, 80.0%). Furthermore, sequence analysis of large number of samples is required to ensure the prevalent antigenic variant of CPV-2 linked to the infection.

Occurrence of cucurbit yellow stunting disorder virus causing yellowing disease of cucurbits in India

Cucurbitaceous crops are major vegetable group cultivated widely in India. Leaves of cucumber, bitter gourd and watermelon were observed with virus like yellowing symptoms of interveinal chlorosis. DAS-ELISA and RT-PCR assay were performed to identify the infecting virus. Nucleotide sequence analysis confirmed the association of Cucurbit yellow stunting disorder virus (CYSDV), a Crinivirus belonging to family Closteroviridae. This study documented the prevalence of CYSDV on cucurbitaceous crops for the first time from India.

Molecular investigation of occult hepatitis B virus infection in a reference center in Northern Brazil

The goal of this study was to investigate the prevalence of occult HBV infection in a reference center for the Northern Brazil from 2005 to 2015 and to identify mutations associated with occult hepatitis B. Molecular analysis was performed on 110 serum samples in which anti-HBc was the only positive serological marker. Regions of the HBV genome were amplified by polymerase chain reaction to detect HBV DNA. A prevalence of 4.1% (793/18,889) for anti-HBc alone was identified. Molecular analysis revealed a prevalence of occult HBV infection of 0.04%. HBV DNA detected were identified in individuals who underwent hemodialysis, infected with the hepatitis C virus and from area of high endemicity for HBV. Direct DNA nucleotide sequencing and phylogenetic analysis identified that genotypes A and D and mutations E164D, I195M, P217L and P120S were associated with occult HBV infection in the S gene. This study contributed with epidemiological and molecular information on Northern Brazil samples with a suggestive profile of occult HBV infection in addition to reinforcing the importance of molecular diagnosis in this type of infection.

Zng1 is a GTP-dependent zinc transferase needed for activation of methionine aminopeptidase.

The evolution of zinc (Zn) as a protein cofactor altered the functional landscape of biology, but dependency on Zn also created an Achilles' heel, necessitating adaptive mechanisms to ensure Zn availability to proteins. A debated strategy is whether metallochaperones exist to prioritize essential Zn-dependent proteins. Here, we present evidence for a conserved family of putative metal transferases in human and fungi, which interact with Zn-dependent methionine aminopeptidase type I (MetAP1/Map1p/Fma1). Deletion of the putative metal transferase in Saccharomyces cerevisiae (ZNG1; formerly YNR029c) leads to defective Map1p function and a Zn-deficiency growth defect. Invitro, Zng1p can transfer Zn2+ or Co2+ to apo-Map1p, but unlike characterized copper chaperones, transfer is dependent on GTP hydrolysis. Proteomics reveal mis-regulation of the Zap1p transcription factor regulon because of loss of ZNG1 and Map1p activity, suggesting that Zng1p is required to avoid a compounding effect of Map1p dysfunction on survival during Zn limitation.

STUDY OF THE 5,8S-ITS2 RIBOSOMAL DNA REGIONS IN THREE SPECIES OF THE GENUS METASTRONGYLUS (NEMATODA: METASTRONGYLIDAE)

Nematodes of the genus Metastrongylus (Nematoda: Metastrongylidae) have adapted to parasitizing the bronchi and bronchioles of the wild and domestic pig. Metastrongyles are widely spread in wild pig populations throughout the world and occur in a mixed form under natural infection conditions. The development cycle of all three types of the pathogens proceeds in the same way involving intermediate hosts, various types of earthworms. It is difficult to identify the species of these nematodes only by morphological characters. The study used nematodes Metastrongylus elongatus, M. pudendotectus, and M. salmi isolated from the lungs of wild pigs in the Tashkent Region of Uzbekistan. DNA was isolated using the Insect DNA Purification kit (India). Comparative analysis of the pairwise nucleotide sequence showed that these three species differed from each other, and the found relationships were similar to those previously described based on the morphology. Phylogenetic trees were built using maximum likelihood methods and it was proved that all the studied species turned out to have high bootstrap support and had found a separate place in the cladogram. The analysis of the study results using rDNA and morphological characters allowed us to determine the relationships of the considered taxa.

Comparative study on molecular epidemiology of measles H1 outbreak and sporadic cases in Shandong Province, 2013\u20132019

BackgroundMeasles caused by measles virus (MeV) is a highly contagious viral disease which has also been associated with complications including pneumonia, myocarditis, encephalitis, and subacute sclerosing panencephalitis. The current study isolated 33 strains belonging to 2 groups, outbreak and sporadic strains, in 13 cities of Shandong province, China from 2013 to 2019. Comparison of genetic characterization among 15 outbreak strains and 18 sporadic strains was performed by analyzing nucleotide sequences of the C-terminal region of N protein gene (N-450).ResultsAll 33 stains belonged to genotype H1. The outbreak strains and sporadic strains distributed crossly in phylogenetic tree. Sequences alignment revealed some interesting G to A transversion which changed the amino acids on genomic sites 1317, 1422, and 1543. The nucleotide and amino acid similarities among outbreak isolates were 98–100% (0–10 nucleotide variations) and 97.7–100%, respectively; They were 97.3–100% and 96.6–100%, respectively for sporadic isolates. Evolutionary genetics analysis revealed that the mean evolution rates of outbreak and sporadic isolates were 1.26 N 10− 3 and 1.48 N 10− 3 substitutions per site per year separately, which were similar with corresponding data before 2012. Local transmission analysis suggested that there were three transmission chains in this study, two of them originated from Japan. Outbreak cases and sporadic cases emerged alternatively and were reciprocal causation on the transmission chains.ConclusionsOur study investigated the phylogeny and evolutional genetics of MeV during a 7-year surveillance, and compared epidemic and genetic characteristics of outbreak strains and sporadic strains. These results underscore the importance of evolutionary study alongside with sporadic cases in discovering and tracing possible outbreaks, especially in the stage of measles elimination.