Nucleotide Sequence Homology Research Articles

Identification, Characterization, and Homology Analysis of a Novel Strain of the Crimean-Congo Hemorrhagic Fever Virus from Yunnan, China.

Wildlife serve as potential microbial reservoirs, accounting for approximately 70% of emerging infectious diseases. Crimean-Congo hemorrhagic fever virus (CCHFV), which causes Crimean-Congo hemorrhagic fever (CCHF) in humans, is a highly pathogenic tick-borne virus prevalent in several parts of Asia, Africa, and Europe with high case fatality rates. Several CCHFV cases have been reported in Asia, the Middle East, Africa, and Southern and Eastern Europe. The disease is endemic in several parts of western China, particularly Xinjiang. Ticks of the genus Hyalomma have been identified as a principal vector and reservoir for CCHFV, although other tick species may also have a crucial role in maintaining CCHFV in endemic regions. On infection, CCHF begins as a nonspecific febrile illness that can progress to severe hemorrhagic manifestations with a higher case fatality due to the unavailability of vaccines or other therapeutic agents. In this study, we collected tissue samples from a wild dead Chinese serow (Capricornis milneedwardsii) and three Naemorhedus griseuses from Deqin County, Tibetan Autonomous Prefecture, Yunnan, China, to investigate for contagious viruses that could be transmitted to humans. We identified a novel CCHFV strain, YNDQL-415G, in the liver tissue of a dead C. milneedwardsii. We performed nucleotide and amino acid sequence homology on the full-length viral genome. The results revealed significant homology between the viral S segment to that of the Africa1 strain, while the M and L segments showed similarity with the Asia CCHFV strain, indicating potential gene reassortment in the YNDQL-415G strain. The genetic characterization of a novel CCHFV strain from a dead C. milneedwardsii raises concerns about the possibility of a new zoonotic infection. A regular survey program is recommended to track the distribution of wild animals as well as the viruses they may transmit to humans and other domestic mammals in the region.

Brain invasion of bovine coronavirus: molecular analysis of bovine coronavirus infection in calves with severe pneumonia and neurological signs.

Although the role of bovine coronavirus (BCoV) in calf diarrhea and respiratory disorders is well documented, its contribution to neurological diseases is unclear. This study conducted virological investigations of calves showing diarrhea and respiratory and neurological signs. An outbreak of diarrhea, respiratory, and neurological disorders occurred among the 12 calves in July 2022 in Istanbul, Türkiye. Two of these calves exhibited neurological signs and died a few days after the appearance of symptoms. One of these calves was necropsied and analyzed using molecular and histopathological tests. BCoV RNA was detected in the brain, lung, spleen, liver, and intestine of the calf that had neurological signs by real-time reverse transcription polymerase chain reaction. Immunostaining was also observed in the intestine and brain. A 622 bp S1 gene product was noted on gel electrophoresis only in the brain. Phylogenetic analysis indicated that the BCoV detected in this study had a high proximity to the BCoV strain GIb with 99.19% nucleotide sequence homology to the strains detected in Poland, Israel, Türkiye, and France. No distinct genetic lineages were observed when the brain isolate was compared with the respiratory and enteric strains reported to GenBank. In addition, the highest identity (98,72%) was obtained with the HECV 4408 and L07748 strains of human coronaviruses. The strain detected in a calf brain belongs to the GIb-European lineage and shares high sequence homology with BCoV strains detected in Europe and Israel. In addition, the similarity between the human coronaviruses (4408 and L07748) raises questions about the zoonotic potential of the strains detected in this study.

The Prevalence and Molecular Characterization of Bovine Babesia Species and the First Report of B. bovis from Kashmir Himalayas.

Bovine babesiosis, a global disease, has not been studied so far in Kashmir valley, which is having temperate type of climate as compared to rest of India having tropical to sub-tropical climate, so we felt the need to investigate it. To diagnose the babesiosis in clinically suspected cattle (n=450), peripheral blood film examination and PCR tests using generic and species-specific primers targeting Babesia/Theleria genera and B. bigemina, B. bovis as well as B. divergens, respectively were conducted. Four PCR products were sequenced and subjected to BLASTn analysis. Ticks were collected from the clinically suspected animals and identified as per the standard morphological keys. The prevalence of babesiosis among suspected cattle in central Kashmir by peripheral blood film examination and PCR technique was 11.11% and 33.62%, respectively. The 18S rRNA gene of Isolate B1 of Babesia spp. showed 99.0 to 100% nucleotide sequence homology with 18S rRNA gene of different isolates of B. bigemina registered in the GenBank, while as 18S rRNA gene of Isolate Z showed 98.5 to 99.2% and 93.1 to 93.9% nucleotide sequence homology with 18S rRNA gene of different isolates of Babesia spp. and B. bigemina, respectively, registered in the GenBank. Rhipicephalus spp. and Haemaphysalis spp. were the two major tick genera identified in the present study. Bovine Babesiosis in Kashmir is attributed to B. bovis, B. bigemina and some other Babesia spp. or strains which needs further investigation. To our knowledge, this is the first report of Babesia bovis from northern India in cattle.

Improvement of the Genome Editing Tools Based on 5FC/5FU Counter Selection in Clostridium acetobutylicum.

Several genetic tools have been developed for genome engineering in Clostridium acetobutylicum utilizing 5-fluorouracil (5FU) or 5-fluorocytosine (5FC) resistance as a selection method. In our group, a method based on the integration, by single crossing over, of a suicide plasmid (pCat-upp) followed by selection for the second crossing over using a counter-selectable marker (the upp gene and 5FU resistance) was recently developed for genome editing in C. acetobutylicum. This method allows genome modification without leaving any marker or scar in a strain of C. acetobutylicum that is ∆upp. Unfortunately, 5FU has strong mutagenic properties, inducing mutations in the strain's genome. After numerous applications of the pCat-upp/5FU system for genome modification in C. acetobutylicum, the CAB1060 mutant strain became entirely resistant to 5FU in the presence of the upp gene, resulting in failure when selecting on 5FU for the second crossing over. It was found that the potential repressor of the pyrimidine operon, PyrR, was mutated at position A115, leading to the 5FU resistance of the strain. To fix this problem, we created a corrective replicative plasmid expressing the pyrR gene, which was shown to restore the 5FU sensitivity of the strain. Furthermore, in order to avoid the occurrence of the problem observed with the CAB1060 strain, a preventive suicide plasmid, pCat-upp-pyrR*, was also developed, featuring the introduction of a synthetic codon-optimized pyrR gene, which was referred to as pyrR* with low nucleotide sequence homology to pyrR. Finally, to minimize the mutagenic effect of 5FU, we also improved the pCat-upp/5FU system by reducing the concentration of 5FU from 1 mM to 5 µM using a defined synthetic medium. The optimized system/conditions were used to successfully replace the ldh gene by the sadh-hydG operon to convert acetone into isopropanol.

Detection and genetic evolution analysis of porcine parvovirus type 7 (PPV7) in Fujian Province

Porcine parvovirus (PPV) is an important pathogen causing reproductive disorders in sows, with clinical symptoms including stillbirth, mummified fetuses, embryonic dysplasia and death, and sow infertility. Porcine parvovirus 7 (PPV7) is a recently discovered type of PPV and its widespread distribution and rapid evolution has caused huge economic losses in the pig industry. To investigate the molecular epidemiology of PPV7 in Fujian Province, China, we collected 491 blood samples and 72 tissue samples from diseased pigs in large-scale pig farms across selected areas of Fujian Province from 2019 to 2022. PPV7 infection was determined using real-time quantitative PCR, and positive samples underwent whole-genome amplification, sequencing, and subsequent homology, phylogenetic, and recombination analyses. The PPV7 positive detection rate was 25.73% (145/563) in Fujian Province, among which the positive rate of blood and tissue samples was 26.47% (130/491) and 20.83% (15/72), respectively. The nucleotide sequence homology among the 29 PPV7 whole-genome sequences obtained in this study was 90.0%–97.2%, whereas that with 128 reference strains from China and other countries was 88.9%–98.1%. Six strains had partial nucleotide deletions or insertions. Phylogenetic analysis based on the whole-genome sequences classified the 29 PPV7 strains and 128 reference strains into eight subtypes (PPV7a–PPV7h), and PPV7h was the predominant subtype in Fujian Province. Recombination analysis revealed evidence of inferred recombination events in the genomes of four strains. This study provides significant insights into the molecular characteristics of PPV7 in Fujian Province and serves as a crucial foundation for further advancements in PPV7 prevention and control strategies.

Variants in the mitochondrial genome sequence of Oryctes rhinoceros (Coleoptera: Scarabaeidae) infected with Oryctes rhinoceros nudivirus in oil palm and coconut plantations

The CRB (coconut rhinoceros beetle) haplotype was classified into CRB-S and CRB-G, based on the presence of single nucleotide polymorphisms (SNPs) in the mitochondrial cox1 gene. Mitochondrial genomes (mitogenomes) are the most widely used genetic resources for molecular evolution, phylogenetics, and population genetics in relation to insects. This study presents the mitogenome CRB-G and CRB-S which were collected in Johor, Malaysia. The mitogenome of CRB-G collected from oil palm plantations in 2020 and 2021, and wild coconut palms in 2021 was 15,315 bp, 15,475 bp, and 17,275 bp, respectively. The CRB-S was discovered in coconut and oil palms in 2021, and its mitogenome was 15,484 bp and 17,142 bp, respectively. All the mitogenomes have 37 genes with more than 99% nucleotide sequence homology, except the CRB-G haplotype collected from oil palm in 2021 with 89.24% nucleotide sequence homology. The mitogenome of Johor CRBs was variable in the natural population due to its elevated mutation rate. Substitutions and indels in cox1, cox2, nad2 and atp6 genes were able to distinguish the Johor CRBs into two haplotypes. The mitogenome data generated in the present study may provide baseline information to study the infection and relationship between the two haplotypes of Johor CRB and OrNV in the field. This study is the first report on the mitogenomes of mixed haplotypes of CRB in the field.

Филогенетический анализ дерматофитов, выделенных от мелких домашних животных

Dermatophytoses are diseases of skin and its accessory structures that are widely spread worldwide. They are most commonly caused by fungi of the genera Micro sporum and Trichophyton. The identification of the agent’s species has a great epidemiological significance and is essential for effective therapy. The aim of the study is the identification and phylogenetic analysis of dermatophytes isolated from dogs and cats in the Republic of Kazakhstan and the Russian Federation by means of molecular techniques. The fungal isolate species were confirmed by sequencing using two rDNA internal transcribed spacer (ITS) primer pairs, and this allowed for their deposition to the GenBank database. Based on the sequencing results, Microsporum canis (12 strains) and Trichophyton benhamiae (2 strains) were identified. The nucleotide sequences were analysed, and phylogenetic trees were constructed, taking into account the results of the dermatophyte identification using two primer pairs. The constructed phylogenetic trees reflecting the relationships of dermatophytes showed that, irrespective of the primer pairs used, the Microsporum and Trichophyton pathogens are in all cases reliably assigned to different clades. The analysis of ITS4F/ITS5R sequence fragment structures enabled the establish- ment of genetic relatedness between the Trichophyton benhamiae strains first isolated from cats in Russia and the Russian strain recovered from a guinea pig. The comparative analysis of the genomes of the Microsporum and Trichophyton fungi and reference strains revealed a relatively low level of intraspecies polymor- phism and point mutations of the sequences. The data analysis demonstrated a high percentage of nucleotide sequence homology, and this allows using the primers for PCR tests intended for dermatophytosis diagnosis in cats and dogs.

Molecular Identification of Planococcus citri (Hemiptera: Pseudococcidae)

Planococcus citri is an important phytophagous pest in different agroecosystems. The species is very difficult to distinguish by morphological features. The goal of this research was to present basic data on the molecular data to confirm P. citri. The primers used in the molecular identification of P. citri consisted of mitochondrial cytochrome c oxidase subunit 1 (COI), ribosomal small subunit RNA (18S rDNA) gene, and internal transcribed spacer 2 (ITS2) gene. The 1% (w/v) agarose gel showed the presence of successful amplification of the COI, 18S rDNA, and ITS2 genes indicated with positive PCR products. The Internal transcribed spacer (ITS2), 18S ribosomal and Cytochrome c oxidase I (COI) mitochondrial genes used in this study confirmed the identity of mealybugs as P. citri. The 18S ribosomal gene showed 98% nucleotide sequence homology to the P. citri USA isolate while the COI gene and ITS2 gene showed less than 95% nucleotide sequence homology to the P. citri USA isolate and P. citri Israel isolate, respectively. At present, the nucleotide sequence data of mealybugs is still lacking; consequently, mealybug species must be correctly identified for the proper management of this pest.

Molecular characterization and phylogenetic analysis of pseudorabies virus isolated from pigs in Ukraine

The article presents the results of a molecular genetic study of two isolates of the Pseudorabies virus that were isolated from pigs in Ukraine. Bioinformatic analysis of the gE gene fragment of Aujeszky's disease virus (Pseudorabies virus) isolates was carried out in order to determine the phylogenetic relationships and homology of nucleotide sequences. Fragments of the Aujeszky disease virus genome corresponding to the C-terminal region of the gE gene were selected for sequencing and further analysis. As a result of the conducted studies, it was demonstrated that the nucleotide sequences of the analyzed samples differ from each other by the presence of ACG insert in the tandem repeats region. Comparison of the studied sequences with the sequences of strains/isolates of the Aujeszky's disease virus found in Europe and Asia, presented in the GenBank database, indicates that such an insert is characteristic for the Min-A and HNJZ strains (position 1487 in the gE gene) isolated in Asia. Analysis of the homology of nucleotide sequences showed that the sequence of the gE gene fragment of sample No. 1 is 100% identical to the sequences of strains 89V87 and 00V72 isolated in Belgium. The homology of the nucleotide sequence of the gE gene fragment of sample No. 3 with strains 89V87 and 00V72 was 99.13%. In order to clarify the analyzed samples belonging to a particular genogroup (genetic cluster), a phylogenetic dendrogram was constructed. This demonstrates the phylogenetic relationships between strains/isolates of the Aujeszky's disease virus. It was found that the analyzed samples belong to the genetic cluster uniting European strains/isolates, and the studied isolates are most genetically close to strains 89V87 and 00V72.

Isolation and Identification of New Soil Strains with Phosphate-Solubilizing and Exopolysaccharide-Producing Abilities in the Yellow River Wetland Nature Reserve of Luoyang City, China

The establishment of the Yellow River wetland nature reserves improves the local soil structure and fertility through the long-term succession of microorganisms. However, little is known about which indigenous microbial resources can accelerate the process of soil improvement and ecology restoration. To fill this gap, exopolysaccharides-producing bacteria and phosphate-solubilizing bacteria were isolated from soil samples of the wetland nature reserve with higher soil organic matter, available phosphorus, and available nitrogen content. 16S rRNA nucleotide sequence homology analysis and physiological-biochemical assay showed that the strain PD12 with the highest phosphate solubilization activity and higher EPS production was identified as Klebsiella variicola, and other high yield EPS-producing strains (EPS12, EPS15, EPS18, and EPS19) were identified as Pseudomonas migulae, Pseudomonas frederiksbergensis, Aeromonas media, and Pseudomonas vancouverensis, respectively. These results provided new potential microbial resources for the research and development of biofertilizers and added new insights into accelerating the restoration of physical, chemical, and biological properties of soil in the Yellow River basin.

DNA barcoding detects resurrected taxon Giuris laglaizei (Sauvage 1880) in Sulawesi, Indonesia: Bolano Sau Lake payangka phylogeny, phenotypic characters and implications for Giuris spp. conservation

Background: The freshwater ichthyofauna of Wallacea is diverse and understudied. A baseline survey of Bolano Sau Lake in Parigi Moutong District, Central Sulawesi Province, Indonesia in 2019 found an eleotrid goby (local name payangka) with characters conforming to the genus Giuris, long considered monophyletic as G. margaritacea/G. margaritaceus but recently found to comprise at least eight species. This study focused on the molecular (DNA barcoding) identification and phenotypic characters of the payangka. Methods: Payangka samples were collected from August to December 2019 in collaboration with local fishermen, weighed and measured, and preserved in 75% ethanol. Length, weight, sex (n=111) and 17 morphometric characters/six meristic counts (n=42) were recorded. DNA barcoding was performed on a fin clipping preserved in 96% ethanol. Homologous nucleotide sequences were obtained from public (GenBank and BOLD) databases, analysis conducted in MEGA X, and phylogenetic trees edited in the Interactive Tree of Life (iToL). Results: Within the deeply divided Giuris clade, the payangka sequence resolved into a sub-clade identified as Giuris laglaizei (Sauvage 1880), a recently resurrected taxon, based on a sequence provided by Philippe Keith. The length-weight relationship (L = 0.0087∙W3.162) indicated mildly allometric positive growth. Size distribution differed significantly between male and female fish with significantly larger mean size of males (13.56 cm) than females (11.62 cm). The meristic formula was: D VI-I,8 A I,8 P 13 V I,5 C15. Phylogenetic analysis indicated four Giuris species in wetlands around Tomini Bay and five in Sulawesi. Conclusions: This first record of G. laglaizei in Indonesia advances knowledge of Wallacean and Indo-Pacific Gobiiformes biogeography and highlights the need for a revision of the conservation status of the taxa currently grouped under Giuris margaritacea/G. margaritaceus in the IUCN Red List and FishBase databases. The data will inform biodiversity and fisheries management at local and regional levels.

Analisis Nukleotida dan Homologi Sekuens Fragmen Gen p72 (B646L) Virus African Swine Fever Virus (ASF) Asal Kota Kupang

African Swine Fever (ASF) is an important infectious disease in pigs caused by African Swine Fever Virus (ASFV). Despite not being zoonotic, this disease has the potential to severely affect the socioeconomic conditions in the impacted regions. The majority of pig farmers in Indonesia, particularly those in Kupang City, that raise pigs in backyards or on a small scale, experience the impact of ASF's effects. Early in 2020, the ASF cases were confirmed in Timor Island, including the Kupang City area in Nusa Tenggara Timur (NTT) Province. The molecular information on ASFV in this area is still limited. In order to determine the homology and nucleotide analysis using BLAST NCBI, the ASFV p72 (B646L) gene fragment sequence from Kupang City was compared to ASFV p72 (B646L) gene segments from other parts of Indonesia and several other Asian countries. The results of nucleotide analysis and sequence homology of the original ASFV p72 (B646L) gene fragment from Kupang City showed a high level of homology to the ASFV p72 (B646L) gene fragment from West Java, North Sumatra, and several Asian countries. The findings from this study indicate that the source of ASF viral transmission across different regions may be comparable. Therefore, to prevent the dissemination of ASF, strict biosecurity measures must be implemented along with monitoring of animal and product transportation.

SARS-CoV-2 Omicron Variant Genomic Sequences and Their Epidemiological Correlates Regarding the End of the Pandemic: In Silico Analysis.

Emergence of the new SARS-CoV-2 variant B.1.1.529 worried health policy makers worldwide due to a large number of mutations in its genomic sequence, especially in the spike protein region. The World Health Organization (WHO) designated this variant as a global variant of concern (VOC), which was named "Omicron." Following Omicron's emergence, a surge of new COVID-19 cases was reported globally, primarily in South Africa. The aim of this study was to understand whether Omicron had an epidemiological advantage over existing variants. We performed an in silico analysis of the complete genomic sequences of Omicron available on the Global Initiative on Sharing Avian Influenza Data (GISAID) database to analyze the functional impact of the mutations present in this variant on virus-host interactions in terms of viral transmissibility, virulence/lethality, and immune escape. In addition, we performed a correlation analysis of the relative proportion of the genomic sequences of specific SARS-CoV-2 variants (in the period from October 1 to November 29, 2021) with matched epidemiological data (new COVID-19 cases and deaths) from South Africa. Compared with the current list of global VOCs/variants of interest (VOIs), as per the WHO, Omicron bears more sequence variation, specifically in the spike protein and host receptor-binding motif (RBM). Omicron showed the closest nucleotide and protein sequence homology with the Alpha variant for the complete sequence and the RBM. The mutations were found to be primarily condensed in the spike region (n=28-48) of the virus. Further mutational analysis showed enrichment for the mutations decreasing binding affinity to angiotensin-converting enzyme 2 receptor and receptor-binding domain protein expression, and for increasing the propensity of immune escape. An inverse correlation of Omicron with the Delta variant was noted (r=-0.99, P<.001; 95% CI -0.99 to -0.97) in the sequences reported from South Africa postemergence of the new variant, subsequently showing a decrease. There was a steep rise in new COVID-19 cases in parallel with the increase in the proportion of Omicron isolates since the report of the first case (74%-100%). By contrast, the incidence of new deaths did not increase (r=-0.04, P>.05; 95% CI -0.52 to 0.58). In silico analysis of viral genomic sequences suggests that the Omicron variant has more remarkable immune-escape ability than existing VOCs/VOIs, including Delta, but reduced virulence/lethality than other reported variants. The higher power for immune escape for Omicron was a likely reason for the resurgence in COVID-19 cases and its rapid rise as the globally dominant strain. Being more infectious but less lethal than the existing variants, Omicron could have plausibly led to widespread unnoticed new, repeated, and vaccine breakthrough infections, raising the population-level immunity barrier against the emergence of new lethal variants. The Omicron variant could have thus paved the way for the end of the pandemic.

The role of merA gene of mercury-resistant Escherichia coli from Kahayan River, Central Kalimantan, Indonesia in emerging antibiotic resistance

Abstract. Martani NS, Notobroto HB, Wasito EB, Jabal AR. 2022. The role of merA Gene of Mercury-resistant Escherichia coli from Kahayan River, Central Kalimantan, Indonesia in emerging antibiotic resistance. Biodiversitas 23: 6629-6634. This study collected nine water samples from the upstream, middle, and downstream parts of the Kahayan river, which was heavily contaminated with mercury (Hg). The water was sampled following the collection method of surface water sample (SNI 6989.57-2008). Escherichia coli has been found in the four samples out of nine samples collected. The merA gene of bacteria was identified at 1695 base pairs (bp), bla at 199 bp, tet at 494 bp, cat at 623 bp, and gyrA at 577 bp. The merA gene from S1 from the PCR results was identified at 219 bp. However, the merA gene was not amplified with a primer measuring 1695 bp. Then, sequencing was performed, and the results indicated a 48.551% homology with E. coli Tn5075 nucleotide sequences. On examination of the cat gene, all samples are positive, or target-specific bands are observed (i.e., samples 1, 5, 6, and 7). Following sequencing, 623 bp were found to have nucleotide sequences that were 100% identical to E. coli. In all samples, the gyrA gene is either positive or has a band that corresponds to the target. Meanwhile, the results of nucleotide sequence homology of 99.826% were identical to E. coli at 586 bp. No bla gene or tet gene was identified. Sample 1 plate 1, 3, 4, 5, and sample 5 plate 3 exhibited intermediate results for ampicillin antibiotics, while the remaining results are sensitive to the tested antibiotics. Therefore, the mercury-resistant merA gene found in E. coli from the Kahayan river in Central Kalimantan has not been conclusively linked to antibiotic resistance.

Occurrence of Viruses and Viroids in Chrysanthemum Plants (Dendranthema morifolium) Cultivated in Yesan-gun, Chungcheongnam-do in Korea

Chrysanthemum plants are one of the most economically important plants in South Korea. Both virus and viroid can cause diseases and economic damage to the plants. In this study, we investigated the detection of seven viruses and two viroids in 350 chrysanthemum plants cultivated in Yesan-gun, Chungcheongnam-do. Two viruses, chrysanthemum virus B (CVB) and tomato aspermy virus (TAV), and two viroids, chrysanthemum chlorotic mottle viroid (CChMVd) and chrysanthemum stunt viroid (CSVd), were detected in this study. The two viruses were detected in six samples and one sample, respectively. The two viroids were detected in 97 samples and 21 samples, respectively. The nucleotide sequences of the CVB-CN-Y, TAV-CN-Y, CChMVd-CN-Y, and CSVd-CN-Y obtained in this study showed 83.7–86.9%, 99.2–100.0%, 94.4–99.5%, and 95.7–99.7% identity, respectively, compared to their other strains/isolates. The CVB-CN-Y and TAV-CN-Y showed the greatest nucleotide sequence homology to CVB-GS1 and three TAV isolates (TAV-V, TAV-P, and TAV-ChJ), respectively. The CChMVd-CN-Y and CSVd-CN-Y showed the greatest nucleotide sequence homology to CChMVd-Horst and four CSVd isolates (Au1.1, K4pop, Sagae, and Tochigi), respectively. This study is the report on the infection rate of viruses and viroids in chrysanthemum plants cultivated in Yesan-gun in 2021.

Variations in the NSP4 gene of the type 2 porcine reproductive and respiratory syndrome virus isolated in China from 1996 to 2021.

Porcine reproductive and respiratory syndrome virus (PRRSV) has continuously mutated since its first isolation in China in 1996, leading to difficulties in infection prevention and control. Infections caused by PRRSV-2 strains are the main epidemic strains in China, as determined by phylogenetic analysis. In this study, we focused on the prevalence and genetic variations of the non-structural protein 4 (NSP4) from PRRSV-2 over the past 20years in China. The fundamental biological properties of the NSP4 were predicted, and an analysis and comparison of NSP4 homology at the nucleotide and amino acid levels was conducted using 123 PRRSV-2 strains. The predicted molecular weight of the NSP4 protein was determined to be 21.1kDa, and it was predicted to be a stable hydrophobic protein that lacks a signal peptide. NSP4 from different strains exhibited a high degree of amino acid (85.8-100%) and nucleotide sequence homology (81.0-100%). Multiple amino acid substitutions were identified in NSP4 among 15 representative PRRSV-2 strains. Phylogenetic analysis showed that the lineage 8 and 1 strains, the most prevalent strains in China, were indifferent clades with a long genetic distance. This analysis will help fully elucidate the parameters of the PRRSV NSP4 epidemic in China to lay a foundation for adequate understanding of the function of NSP4. Genetic information results from the accumulation of conserved and non-conserved sequences. The high conservation of the NSP4 gene determines the most basic life traits and functions of PRRSV. Analyzing the spatial structure of NSP4 protein and studying the genetic evolution of NSP4 not only provide the theoretical basis for how NSP4 participates in the regulation of the innate response of the host but also provide a target for genetic manipulation and a reasonable target molecule and structure for new drug molecules.

Molecular Characterization of HN1304M, a Cat Que Virus Isolated from Midges in China.

The Cat Que orthobunyavirus has been found in mosquitoes, birds, pigs, and humans, suggesting its wide range of hosts and potential public health implications. During arbovirus surveillance in 2013, the HN1304M virus was isolated from naturally occurring Culicoides biting midges in Hunan Province, southern China. The virus was cytopathic to BHK-21 cells and showed stable passage, but was not cytopathic to C6/36 cells. Determination and analysis of the viral genome sequence revealed that HN1304M is an RNA virus with three gene segments, namely, L, M, and S. The nucleotide and amino acid sequence homologies of HN1304M to Cat Que viruses in the Manzanilla species complex were 90.3–99.4%, and 95–100%, respectively, while the homologies to other viruses in this species complex were 74–86.6% and 78.1–96.1%, respectively. A phylogenetic analysis of the viral genes revealed that HN1304M formed an evolutionary branch with other Cat Que viruses isolated from mosquitoes, pigs, birds, and humans, which was completely independent of the other viruses in this complex. The fact that the Cat Que virus was isolated from Culicoides suggests that biting midges may participate in the natural circulation of Cat Que viruses.

Nephritis-Associated Plasmin Receptor (NAPlr): An Essential Inducer of C3-Dominant Glomerular Injury and a Potential Key Diagnostic Biomarker of Infection-Related Glomerulonephritis (IRGN).

Nephritis-associated plasmin receptor (NAPlr) was originally isolated from the cytoplasmic fraction of group A Streptococci, and was found to be the same molecule as streptococcal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and plasmin receptor (Plr) on the basis of nucleotide and amino acid sequence homology. Its main functions include GAPDH activity, plasmin-binding capacity, and direct activation of the complement alternative pathway (A-P). Plasmin trapped by deposited NAPlr triggers the degradation of extracellular matrix proteins, such as glomerular basement membranes and mesangial matrix, and the accumulation of macrophages and neutrophils, leading to the induction of plasmin-related endocapillary glomerular inflammation. Deposited NAPlr at glomerular endocapillary site directly activates the complement A-P, and the endocapillary release of complement-related anaphylatoxins, C3a and C5a, amplify the in situ endocapillary glomerular inflammation. Subsequently, circulating and in situ-formed immune complexes participate in the glomerular injury resulting in NAPlr-mediated glomerulonephritis. The disease framework of infection-related glomerulonephritis (IRGN) has been further expanded. GAPDH of various bacteria other than Streptococci have been found to react with anti-NAPlr antibodies and to possess plasmin-binding activities, allowing glomerular NAPlr and plasmin activity to be utilized as key biomarkers of IRGN.

Identification and primary distribution of Citrus viroid V in citrus in Punjab, Pakistan.

Citrus plants are prone to infection by different viroids which deteriorate their vigor and production. Citrus viroid V (CVd-V) is among the six citrus viroids, belongs to genus Apscaviroid (family Pospiviroidae) which induces symptoms of mild necrotic lesions on branches and cracks on trunk portion. A survey was conducted to evaluate the prevalence of CVd-V in core and non-core citrus cultivated areas of Punjab, Pakistan. A total of 154 samples from different citrus cultivars were tested for CVd-V infection by RT-PCR. The results revealed 66.66% disease incidence of CVd-V. Citrus cultivars Palestinia Sweet lime, Roy Ruby, Olinda Valencia, Kaghzi lime, and Dancy were identified as new citrus hosts of CVd-V for the first time from Pakistan. The viroid infection was confirmed by biological indexing on indicator host Etrog citron. The reported primers used for the detection of CVd-V did not amplify, rather showed non-specific amplification, which led to the designing of new primers. Whereas, new back-to-back designed primers (CVd-V AF1/CVd-V AR1) detected CVd-V successfully and obtained an expected amplified product of CVd-V with 294bp. Sequencing analysis confirmed the new host of CVd-V showing 98-100% nucleotide sequence homology with those reported previously from other countries while 100% sequence homology to the isolates reported from Pakistan. Based on phylogenetic analysis using all CVd-V sequences in GenBank, two main CVd-V groups (I and II) were identified, and newly identified isolates during this study fall in the group I. The study revealed that there are some changes in the nucleotide sequences of CVd-V which made difficult for their detection using reported primers. All isolates of Pakistan showed high sequence homology with other isolates of CVd-V from Iran and USA whereas; the isolates from China, Japan, Tunisia, and Africa are distantly related. It is evident that CVd-V is spreading in all citrus cultivars in Pakistan.

Genetic variation of sequences of partial mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 1 (pnad1) and pnad5 genes of Toxascaris leonina isolates from foxes and dogs in Jiuquan City of Gansu Province

To investigate the genetic variations of Toxascaris leonina isolates from different hosts in Jiuquan City, Gansu Province. The mitochondrial sequences of partial mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 1 (pnad1) and pnad5 of eleven T. leonina isolates from domestic dogs, foxes and pet dogs in Jiuquan City, Gansu Province, were amplified using PCR, and the amplification product was sequenced. The genetic variations of pnad1 and pnad5 genes in T. leonina isolates were analyzed. The sequences of T. leonina pnad1 and pnad5 genes measured 530 bp and 550 bp in size, respectively. The nucleotide sequence homology was 99.4% to 100.0% for T. leonina pnad1 gene and 99.5% to 99.8% for T. leonina pnad5 gene, and the sequences of T. leonina pnad1 and pnad5 genes shared 99.2% to 99.9% and 99.1% to 99.9% with corresponding sequences of known T. leonina isolates. In addition, there were 19 and 24 polymorphic sites detected in the sequences of T. leonina pnad1 and pnad5 genes, with 10 and 9 haplotypes, haplotype diversity of 0.982 and 0.964 and nucleotide diversity of 0.039 4 and 0.034 2, respectively. Phylogenetic analysis based on pnad1 and pnad5 gene sequences showed that the eleven T. leonina isolates and known T. leonina isolates were clustered into the same branch with a random distribution, which were close to the branch where Toxocara canis was clustered, and far from the branch where other Ascaris species were clustered. There is a minor genetic variation in pnad1 and pnad5 genes of T. leonina isolates from different hosts in Jiuquan City, Gansu Province, and the pnad1 gene is more suitable as a molecular marker than pnad5 gene for analysis of genetic variations in T. leonina.