NS1 Gene Research Articles

Molecular detection and quantification of bluetongue virus in small ruminant and Culicoides vectors in Gujarat, India

The present study was carried out to detect bluetongue virus (BTV) in Culicoides vectors and the biological samples of small ruminants with the use of PCR based molecular methods. The vectors were collected using the UV LED CDC traps, placed near animal sheds and then identified by morphological and PCR based methods. Primers specific to genus Culicoides and species Culicoides oxystoma were used in PCR reaction. The end point RT-PCR protocols were used for tentative and confirmatory diagnosis of BTV and its two serotypes viz. BTV-1 and BTV-16, whereas, the real time RT-PCR was used for confirmation of BTV and quantization of viral RNA load. Out of a total of 132 blood samples and 55 tissue samples viz., spleen, liver, kidney, thymus and heart blood of aborted fetuses and 09 pooled samples of Culicoides, total 21 (6.93%) samples i.e., 11 blood samples, 08 spleens and 02 pooled Culicoides samples were detected positive for BTV by NS1 gene specific RT-PCR. In nested PCR, 8 additional samples were found positive for BTV. Among these 29 positive samples, 6 were collected from spleens of aborted fetuses which was indicative of transplacental transmission of BTV among small ruminants. All the positive samples were of either of BTV-1 and BTV-16 serotypes as confirmed by serotype specific PCR. In real time PCR, plasmid vector based positive control was prepared, which confirmed the 21 samples to be BTV positive, whereas, quantization of viral RNA, showed that out of all clinical samples tested, highest and lowest RNA copy numbers i.e. 3566.41 and 81.79 were found in blood sample and spleen of sheep, respectively.

SCREENING OF POTATO SELECTIVE AND COLLECTING MATERIAL FOR RESISTANCE TO PVM AND PVS USING DNA MARKERS

The article presents the research results on the assessment of potato samples that were created in the laboratory of potato genetics and varieties that are cultivated in the collection nursery of RUE «Scientific and Practical Center of the NAS of Belarus for
 Potato, Vegetable and Fruit Growing», for resistance to PVM and PVS, and detection of resistance sources to these pathogens with the use of DNA markers. Based on the research results, a number of stable forms was found with the following markers: SC878885 to the Gm gene, GP250510, UBS8221079 to the Rm gene and markers SCG17321 and UBC811660 to the NS gene, which can be used in potato selection for virus resistance.

Growth characteristics of experimental live influenza vaccine strains with modified NP and NS genes

BACKGROUND: Vaccination is the most effective means of fighting influenza epidemics, but the immunogenicity of licensed influenza vaccines is not always satisfactory. One of the ways to increase the immunogenicity of an attenuated live influenza vaccine is to shorten the open reading frame of the NS1 protein, a modulator of innate antiviral immunity. In addition, the T-cell response to vaccination can be optimized by including the NP gene from the epidemic parental virus into the genome of vaccine strains.
 MATERIALS AND METHODS: The open reading frame of the NS1 protein of the master donor virus A/Leningrad/134/17/57 was truncated to 126 amino acids by site-directed mutagenesis. The HA, NA, and NP genes of the model virus A/Anhui/1/2013 (H7N9) were cloned into the pCIPolISapIT vector. The rescue of recombinant influenza viruses was performed by transfection of Vero cells with a desired set of plasmids. The growth properties of the recombinant viruses were determined in embryonated chicken eggs incubated at different temperatures, as well as in the tissues of the respiratory tract of mice (nasal turbinates, lungs).
 RESULTS: Experimental live influenza vaccine strains of subtype H7N9 with genome compositions 6:2 and 5:3 and carrying a full-length or truncated NS1 gene were actively replicated in eggs under optimal conditions, while maintaining the temperature-sensitive and cold-adapted phenotypes characteristic of classical live influenza vaccine strains. All viruses lacked the ability to grow in the lungs of C57BL/6J mice, which confirms the attenuated phenotype of the viruses. In the nasal passages of mice, only viruses with the full-length NS1 gene replicated, while viruses expressing the truncated NS1 protein were not detected in the respiratory tract of animals.
 CONCLUSIONS: The results indicate that modification of the NS1 gene of the vaccine virus and the inclusion of wild-type NP gene in its genome does not affect its growth characteristics in eggs. A decrease in the activity of viral replication in the upper respiratory tract of mice with a shortening of the NS1 open reading frame indicates an increase in the attenuating properties of modified vaccines, which opens up prospects for the use of new vaccines in children under three years of age.

Generation and in vitro characterization of engineered cold-adapted influenza A strains with modified NS gene

BACKGROUND: The high variability of influenza strains and the emergence of new variants of viruses lead to the need for constant updating of the composition of influenza vaccines. One of the options for solving this problem is the development of vaccines with enhanced cross-protection against a wide range of influenza strains. Genetically engineered preparations based on live influenza vaccine can be used for targeted stimulation of the cellular immune response. It has been experimentally established that CTL epitopes inserted into the NS gene of the live influenza vaccine strain cause the activation of lymphocytes and the formation of a pool of resident memory T-cells in the lungs of model animals. It is optimal to use experimentally confirmed immunogenic regions for insertion.
 AIM: The aim of this study was to rescue a panel of experimental cold-adapted live attenuated influenza vaccine strains with a modified NS gene using A/Leningrad/134/17/57 backbone and recent influenza strains of H1N1, H3N2 and H7N9 subtypes, and evaluate their properties in vitro.
 MATERIALS AND METHODS: A cassette encoding immunogenic, conserved among a wide range of influenza strains T-cell epitopes of the influenza virus PB1 protein restricted by common HLA-allotypes was inserted into the gene encoding the NS1 protein. The modified NS gene was cloned into the pCIPolISapIT influenza virus reverse genetics vector. Chimeric influenza viruses were rescued by transfection of Vero cells by electroporation using a standard 8-plasmid system. The growth characteristics of viruses were assessed in developing chicken embryos.
 Results: Three strains were successfully obtained based on the live influenza vaccine master donor virus A/Leningrad/ 134/17/57 with a modified NS gene and influenza viruses of the H1N1, H3N2, H7N9 subtypes. Thus, modification of NS gene by insertion of immunogenic PB1 epitopes did not affect the viability and replicative activity of the rescued chimeric live influenza vaccine strains, regardless of the composition of the surface proteins. The strains replicated well at an optimal temperature, had temperature-sensitive phenotype and were able to grow at low temperature.
 CONCLUSIONS: The strains will be further studied as candidates for influenza prophylaxis as an experimental universal influenza vaccine.

Molecular and Phylogenetic Characterisation of a Highly Divergent Novel Parvovirus (Psittaciform Chaphamaparvovirus 2) in Australian Neophema Parrots.

Parvoviruses under the genus Chaphamaparvovirus (subfamily Hamaparvovirinae) are highly divergent and have recently been identified in many animals. However, the detection and characterisation of parvoviruses in psittacine birds are limited. Therefore, this study reports a novel parvovirus, tentatively named psittaciform chaphamaparvovirus 2 (PsChPV-2) under the genus Chaphamaparvovirus, which was identified in Australian Neophema birds. The PsChPV-2 genome is 4371 bp in length and encompasses four predicted open-reading frames, including two major genes, a nonstructural replicase gene (NS1), and a structural capsid gene (VP1). The NS1 and VP1 genes showed the closest amino acid identities of 56.2% and 47.7%, respectively, with a recently sequenced psittaciform chaphamaparvovirus 1 from a rainbow lorikeet (Trichoglossus moluccanus). Subsequent phylogenetic analyses exhibited that the novel PsChPV-2 is most closely related to other chaphamaparvoviruses of avian origin and has the greatest sequence identity with PsChPV-1 (60.6%). Further systematic investigation is warranted to explore the diversity with many avian-associated parvoviruses likely to be discovered.

Influenza virus vector iNS1 expressing bovine papillomavirus 1 (BPV1) antigens efficiently induces tumour regression in equine sarcoid patients.

Bovine papillomaviruses types 1 and 2 (BPV1, BPV2) commonly induce skin tumours termed sarcoids in horses and other equids. Sarcoids seriously compromise the health and welfare of affected individuals due to their propensity to resist treatment and reoccur in a more severe form. We have developed influenza (Flu) A and B virus vectors that harbour a truncated NS1 gene (iNS) assuring interferon induction and co-express shuffled BPV1 E6 and E7 antigens for sarcoid immunotherapy. In a safety trial involving 12 healthy horses, intradermal administration of iNSA/E6E7equ and iNSB/E6E7equ was well tolerated, with the only transient side effect being mild fever in four horses. Repeated screening of secretions and faeces by RT-PCR and plaque assay revealed no virus shedding, thus also confirming biological safety. In a patient trial involving 29 horses bearing BPV1-induced single or multiple sarcoids, at least one lesion per horse was intratumourally injected and then boosted with iNSA/E6E7equ and/or iNSB/E6E7equ. The treatment induced a systemic antitumour response as reflected by the synchronous regression of injected and non-injected lesions. Irrespective of vaccination schemes, complete tumour regression was achieved in 10/29 horses. In 10/29 horses, regression is still ongoing (May 2021). Intriguingly, scrapings collected from former tumour sites in two patients tested negative by BPV1 PCR. Nine severely affected individuals with a history of unsuccessful therapeutic attempts did not (6/29) or only transiently (3/29) respond to the treatment. INSA/E6E7equ and iNSB/E6E7equ proved safe and effective in significantly reducing the tumour burden even in severe cases.

Genetic characterization of atypical porcine pestivirus (APPV) in China and the successful isolation of a novel APPV strain within genotype 2

Atypical porcine pestivirus (APPV) is one major causative agent of congenital tremor (CT) type A-II. In this study, the geographical distribution and genetic characteristics of APPV strains in China were established. To date, APPV has been detected in at least ten provinces/regions of China. Genetic variability analysis showed that NS3 genes were highly conserved among Chinese APPV strains, while NS5A-, Npro-, and Erns-encoding genes were highly variable. Phylogenetic analysis revealed that all of the reference strains could be classified into 3 genotypes (1–3) and 7 subgenotypes (1.1–1.7). The Chinese APPV strains were assigned to all 3 genotypes and to 5 subgenotypes (1.2 and 1.4–1.7) of genotype 1. Amino acid insertions/deletions in nonstructural proteins, including NS3, NS5A, and NS5B, could only be found in Chinese APPV strains. Among the three envelope glycosylation proteins (Erns, E1, and E2), Erns was the most heavily glycosylated protein, and the N-glycosylation sites of E2 in different APPV strains showed apparent correlations with genetic types. Furthermore, a novel APPV strain named China/HeN01/2018 belonging to genotype 2 was identified in Henan Province. It was then isolated and successfully propagated in embryonic porcine kidney epithelial cells (SPEV cells). This study provides updated information to better understand the divergence of APPV strains in China. This first successful isolation of a Chinese APPV strain provides a research foundation for future studies.

Genetic Characterization and Pathogenesis of Three Novel Reassortant H5N2 Viruses in South Korea, 2018.

The outbreaks of H5N2 avian influenza viruses have occasionally caused the death of thousands of birds in poultry farms. Surveillance during the 2018 winter season in South Korea revealed three H5N2 isolates in feces samples collected from wild birds (KNU18-28: A/Wild duck/South Korea/KNU18-28/2018, KNU18-86: A/Bean Goose/South Korea/KNU18-86/2018, and KNU18-93: A/Wild duck/South Korea/KNU18-93/2018). Phylogenetic tree analysis revealed that these viruses arose from reassortment events among various virus subtypes circulating in South Korea and other countries in the East Asia–Australasian Flyway. The NS gene of the KNU18-28 and KNU18-86 isolates was closely related to that of China’s H10N3 strain, whereas the KNU18-93 strain originated from the H12N2 strain in Japan, showing two different reassortment events and different from a low pathogenic H5N3 (KNU18-91) virus which was isolated at the same day and same place with KNU18-86 and KNU18-93. These H5N2 isolates were characterized as low pathogenic avian influenza viruses. However, many amino acid changes in eight gene segments were identified to enhance polymerase activity and increase adaptation and virulence in mice and mammals. Experiments reveal that viral replication in MDCK cells was quite high after 12 hpi, showing the ability to replicate in mouse lungs. The hematoxylin and eosin-stained (H&E) lung sections indicated different degrees of pathogenicity of the three H5N2 isolates in mice compared with that of the control H1N1 strain. The continuing circulation of these H5N2 viruses may represent a potential threat to mammals and humans. Our findings highlight the need for intensive surveillance of avian influenza virus circulation in South Korea to prevent the risks posed by these reassortment viruses to animal and public health.

Construction of an Influenza D Virus with an Eight-Segmented Genome.

Influenza D virus (IDV) may cause the bovine respiratory disease complex, which is the most common and costly disease affecting the cattle industry. Previously, we revealed that eight segments could be actively packaged in its single virion, suggesting that IDV with the seven-segmented genome shows an agnostic genome packaging mechanism. Herein, we engineered an eight-segmented recombinant IDV in which the NS1 or NS2 genes were separated from NS segment into independent segments (NS1 or NS2 segments, respectively), leading to monocistronic translation of each NS protein. We constructed two plasmids: one for the viral RNA (vRNA)-synthesis of the NS1 segment with a silent mutation at the splicing acceptor site, which controls NS2 transcription in the NS segment; and another for the RNA synthesis of the NS2 segment, with deletion of the intron in the NS segment. These plasmids and six other vRNA-synthesis plasmids were used to fabricate an infectious eight-segmented IDV via reverse genetics. This system enables analysis of the functions of NS1 or NS2. We tested the requirement of the N-terminal overlapping region (NOR) in these proteins for viral infectivity. We rescued a virus with NOR-deleted NS2 protein, which displayed a growth rate equivalent to that of the eight-segmented virus with intact NS2. Thus, the NOR may not influence viral growth. In contrast, a virus with NOR-deleted NS1 protein could not be rescued. These results indicate that the eight-segmented rescue system of IDV may provide an alternative method to analyze viral proteins at the molecular level.

Emergence and Characterization of a Novel Reassortant Canine Influenza Virus Isolated from Cats.

Cats are susceptible to a wide range of influenza A viruses (IAV). Furthermore, cats can serve as an intermediate host, and transfer avian influenza virus (AIV) H7N2 to a veterinarian. In this report, a novel reassortant influenza virus, designated A/feline/Jiangsu/HWT/2017 (H3N2), and abbreviated as FIV-HWT-2017, was isolated from nasal swab of a symptomatic cat in Jiangsu province, China. Sequence analysis indicated that, whilst the other seven genes were most similar to the avian-origin canine influenza viruses (CIV H3N2) isolated in China, the NS gene was more closely related to the circulating human influenza virus (H3N2) in the region. Therefore, FIV-HWT-2017 is a reassortant virus. In addition, some mutations were identified, and they were similar to a distinctive CIV H3N2 clade. Whether these cats were infected with the reassortant virus was unknown, however, this random isolation of a reassortant virus indicated that domestic or stray cats were “mixing vessel” for IAV cannot be ruled out. An enhanced surveillance for novel influenza virus should include pet and stray cats.

Nuclear-localized human respiratory syncytial virus NS1 protein modulates host gene transcription.

SUMMARYHuman respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in the pediatric, elderly, and immunocompromised individuals. RSV non-structural protein NS1 is a known cytosolic immune antagonist, but how NS1 modulates host responses remains poorly defined. Here, we observe NS1 partitioning into the nucleus of RSV-infected cells, including the human airway epithelium. Nuclear NS1 coimmunoprecipitates with Mediator complex and is chromatin associated. Chromatin-immunoprecipitation demonstrates enrichment of NS1 that overlaps Mediator and transcription factor binding within the promoters and enhancers of differentially expressed genes during RSV infection. Mutation of the NS1 C-terminal helix reduces NS1 impact on host gene expression. These data suggest that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription. Our study identifies another layer of regulation by virally encoded proteins that shapes host response and impacts immunity to RSV.

Identification and molecular characterization of H9N2 viruses carrying multiple mammalian adaptation markers in resident birds in central-western wetlands in India.

We report here a targeted risk-based study to investigate the presence of influenza A viruses at the migratory-wild-domestic bird interface across the major wetlands of central India's Maharashtra state during the winter migration season. The H9N2 viruses have been isolated and confirmed in 3.86% (33/854) of the fecal samples of resident birds. To investigate the genetic pools of H9N2 circulating in resident birds, we sequenced two isolates of H9N2 from distant wetlands. Sequence and phylogenetic analyses have shown that these viruses are triple reassortants, with HA, NA, NP, and M genes belonging to G1 sub-lineage (A/quail/Hong Kong/G1/1997), PB2, PB1, and NS genes originating from the prototype Eurasian lineage (A/mallard/France/090360/2009) and PA gene deriving from Y439/Korean-like (A/duck/Hong Kong/Y439/97) sub-lineage. It was confirmed not only that four of their gene segments had a high genetic association with the zoonotic H9N2 virus, A/Human/India/TCM2581/2019, but also that they had many molecular markers associated with mammalian adaptation and enhanced virulence in mammals including the unique multiple basic amino acids, KSKR↓GLF at the HA cleavage site, and analog N-and O-glycosylation patterns on HA with that of the zoonotic H9N2 virus. Furthermore, future experiments would be to characterize these isolates biologically to address the public health concern. Importantly, due to the identification of these viruses at a strategic geographical location in India (a major stop-over point in the Central Asian flyway), these novel viruses also pose a possible threat to be exported to other regions via migratory/resident birds. Consequently, systematic investigation and active monitoring are a prerequisite for identifying and preventing the spread of viruses of zoonotic potential by enforcing strict biosecurity measures.

First Clinical Case of Equine Parvovirus-Hepatitis-Related Theiler's Disease in Asia.

Equine parvovirus-hepatitis (EqPV-H) is a newly identified etiologic agent of Theiler’s disease (TD). We present a case of EqPV-H-related fulminant hepatitis in a 14-year-old thoroughbred mare in Korea. The mare had acute hepatopathy and gastrointestinal symptoms, with abnormal liver-related blood parameters. The horse was born in the USA and imported to Korea in 2017, with no history of administration of equine biological products after entry into Korea. The horse was diagnosed with EqPV-H-associated hepatitis after abdominal ultrasonography, laparotomy, and nested polymerase chain reaction (PCR) and in situ hybridization (ISH) assays. The serum, nasal swab, oral swab, and liver biopsy were positive for EqPV-H according to the PCR assay. Genetic analysis of the partial NS1 gene of EqPV-H showed a unique nucleotide substitution, distinct from that in previously deposited strains. EqPV-H DNA was found not only in hepatocytes but also in bile duct epithelium and Kupffer cells, particularly via ISH. To the best of our knowledge, this is the first case of EqPV-H-associated TD in Asia, providing the first clinical evidence for viral shedding from the mouth and nose, and identification of EqPV-H in the liver. This study contributes to a better understanding of the pathological features of EqPV-H-associated TD.

Assessment of Aleutian mink disease virus (AMDV) prevalence in feral American mink in Iceland. Case study of a pending epizootiological concern in Europe.

BackgroundRecurring escapes or deliberate releases and subsequent infiltration or establishment of feral populations by individuals from fur farms have been commonly noted since the beginning of fur industry expansion. Once animals have invaded ecosystems adjacent to source farms escapees can change the demography of the feral populations through hybridization, outbreeding depression, competition and spreading of various pathogens which can decimate wild populations. In our study, we aimed to assess spread of Aleutian mink disease virus (AMDV) in the feral population of American mink (Neovison vison) in Iceland. The additional objective was to elucidate whether basic morpho-anatomical parameters (i.e., Fulton’s condition factor or spleen to body weight ratio) might be used as a preliminary indicator of AMDV infection.MethodsAmerican mink (n = 164) were captured by professional hunters in 8 regions of Iceland. The detection of AMDV in the spleen of male and female individuals was based on PCR amplification of an NS1 gene fragment.ResultsWe confirmed AMDV presence in 23.8% (n = 39) of collected samples with no significant difference in infection rate between males and females. Additionally, we revealed that the prevalence of virus in the feral population was higher closer to fur farms. However, the countrywide prevalence and direction of AMDV distribution needs to be further investigated. Comparison of condition indices in non-infected and infected animals showed significant deterioration of body and spleen parameters in the latter group. Therefore, the application of basic measurements of the American mink may be used to evaluate the health status of individuals in terms of pathogen infection.ConclusionsThe study shed a new light on prevalence and distribution of AMDV in the feral population of American mink in Iceland and the results might be successfully applied to develop models to infer dynamics of various pathogens, even those latently transmitted by disease-free animals.

First identification and genomic characterization of equine hepacivirus subtype 2 in China.

Equine hepacivirus (EqHV) is a newly discovered hepatitis C virus-like virus that can infect equines. EqHV strains circulating worldwide have been classified into subtypes 1-3. In previous studies, we detected the presence of EqHV strains of subtype 1 and 3 in China. To determine whether EqHV strains of subtype 2 are prevalent in China, serum samples were collected from 133 racehorses in Guangdong province in 2021 and were tested for EqHV RNA by RT-PCR, and the positive rate was 9% (12/133). Sequencing of the NS3 gene revealed that one field strain (GD2021) had a high degree of genetic similarity to EqHV strains of subtype 2. Subsequent genome sequencing and analysis demonstrated that strain GD2021 belongs to subtype 2. The present study enriches our knowledge about the genetic diversity of EqHV in China.

Participation of interferon type I during canine parvovirus infection.

Canine parvovirus (CPV) is a single-stranded DNA virus that causes severe and fatal gastrointestinal diseases in dogs. CPV has developed several strategies to evade innate immune response mediated by type I interferons (IFN-I) to achieve a successful infection. The aim of this work was to evaluate the capability of CVP-2c to evade the IFN-I mediated response in infected cells. To establish the role of this response, the gene expression of interferon β (IFNβ), IFIT1, IFIT3, MAVS, and STING were estimated in MDCK cells infected with CPV-2c. Viral replication and gene expression was evaluated by quantitative PCR, also, a treatment with IFN-I (interferon omega) was included to confirm the role of IFN-I during CPV infection. The results revealed that CPV-2c infection stimulates the expression of IFNβ moderately, in these cells. Due to low IFNβ induction, the IFIT1 and IFIT3 expression were also low, and therefore CPV-2c was able to replicate in these cells. However, when the cells were treated with exogenous IFN-I, the IFNβ expression was higher, leading to an increased gene expression of IFIT1 and IFIT3, responsible for antiviral control. The overexpression of these proteins reduced the expression of NS1 and VP2 viral genes and hence viral replication. MAVS and STING expression on infected cells showed a mild increase compared to IFNβ, suggesting that the viral infection could partially modify its expression. All results obtained in this study showed that during CPV-2c infection in MDCK cells, the IFNβ expression was altered since this cytokine is one of the most critical factors for the control and inhibition of viral replication.

A SYBR Green I-based real-time polymerase chain reaction assay for detection and quantification of canine bufavirus

Canine bufavirus (CBuV) was first discovered in puppies in Italy in 2016, and subsequent studies have reported its possible relationship with acute enteritis. Currently, there is no specific and quantitative detection method for CBuV. This study examined the conserved NS1 gene and used a pair of specific primers to establish a direct SYBR Green I-based real-time quantitative polymerase chain reaction (qPCR) method for the detection and quantification of CBuV. In the sensitivity experiment, the detection limit of SYBR Green I-based real-time qPCR was 4.676 × 101 copies/μL and that of conventional PCR (cPCR) was 4.676 × 103 copies/μL. Furthermore, the qPCR method did not detect other viruses in dogs, indicating good specificity. The intra-assay coefficient of variation was 0.07–0.55% and the inter-assay coefficient of variation was 0.03–0.11%, indicating good repeatability. In clinical sample testing, the detection rate of qPCR was 5.0% (6/120), higher than that of cPCR (2.5%, 3/120). In addition, the samples that tested CBuV-positive in this experiment were all from dogs with acute enteritis. In summary, the SYBR Green I-based qPCR method established in this study has good sensitivity, specificity, and reproducibility for clinical sample detection and can also assist in future research on CBuV.

Non-primate hepacivirus transmission and prevalence: Novel findings of virus circulation in horses and dogs in Morocco.

Non-primate hepacivirus (NPHV) is a homolog of hepatitis C virus and has been isolated from dogs and horses. Data on NPHV prevalence and distribution are not complete, and there is a particular lack of reports from the African continent. The present study represents the first investigation of NPHV prevalence in horses and dogs in North Africa. Blood was collected from 172 horses and 36 dogs at different locations in Morocco, and screened for NPHV RNA using nested PCR targeting 5'UTR and NS3 regions and analyzed for anti-NPHV NS3 antibody using a Gaussia luciferase immunoprecipitation system-to determine seroprevalence. Eight sequences of the NS3 region isolated from positive serum samples were targeted for phylogenetic analysis. Horses and dogs showed respective NPHV RNA positivity rates of 10.5% and 5.5%, and seroprevalences of 65.7% and 8.33%. Juvenile horses appeared more susceptible to infection, with a 23.5% NHPV RNA positivity rate. Seropositivity was more extensive in mares than stallions (77.14% vs. 46.27%, p<0.0001). Phylogenetically, that NPHV NS3 genes isolated from horses and dog are clustered together. The NPHV strains we detected showed no correlation with geographic location within Morocco. In conclusion, Moroccan horses showed much evidence of previous and/or current NPHV infection, with young age and female sex as noted potential risk factors. Interestingly, NPHV is circulating in dogs as well as horses, suggesting that it has crossed species barriers and that horses and dogs are potential vectors by which an ancestor to hepatitis C virus was transmitted into human populations.

Effect of siRNA targeting dengue virus genes on replication of dengue virus: an in vitro experimental study.

Dengue is a notorious viral infection, which affects a large segment of world populations in absence of vaccines and anti-viral treatment. The current study evaluates role of effective siRNA in dengue virus replication. Eight siRNA were synthesized against five different genes (Capsid, CprM, NS1, NS3 and NS5) of all serotypes of dengue virus. All serotype of DV were transfected with all synthesized siRNA in vitro, using BHK-21 cell lines. Culture fluid from test and control was tested by Real time PCR for CT value comparison in siRNA treated cell line (test) and untreated cell line (controls). Percent knockdown (%KD) was calculated by ∆∆CT methods to know the difference in test and control CT value. It was found that siRNA targeted against capsid gene worked best and showed inhibition of all four DV serotypes. DV-1, DV-2, DV-3 and DV-4 showed 93.8%, 99.3%, 87.5% and 93.8% knock down (%KD) respectively by siRNA targeted against capsid gene. Additionally, Si2 (target CprM gene 60-899) and Si 6 (target NS1 gene 3007-3025) were also showing inhibition of replication. Most serotypes of DV (with few exceptions) were not inhibited by siRNA targeted against NS-1, NS-3, and NS-5 genes. Animal studies using siRNAs are warranted to establish their therapeutic role.

In vitro Inhibition of Border Disease Virus Replication With Lentivirus-Mediated shRNAs.

Background: Border disease is believed to be one of the most important diseases in the animal husbandry industry, which has not yet been eradicated in Iran. The development of approaches based on the application of interfering RNA (RNAi) for antiviral therapy has attracted a great deal of attention over the recent years. The present research was conducted to design, construct, and apply shRNA against the NS3 gene of BDV to evaluate the prevention of BDV proliferation in the cell culture system. For this purpose, the suitable oligonucleotide sequence of NS3 gene coding was selected utilizing BDV- X818 strain. Afterwards, using shRNA design software, shRNA molecules were designed and synthesized. These shRNAs were cloned into the desired vectors and were finally transfected in HEK293T cells employing the third generation of lentiviral packaging system. Subsequently, these shRNA expressing lentiviruses were transduced to the MDBK cell line to challenge to border virus. In order to evaluate the efficacy of shRNAs, the viral infectious titer and RNA copy number were calculated with TCID50 and Real-time RT-PCR tests, respectively.Results: The results revealed that shRNAs 1, 2, and 3 decreased viral RNA by more than 90% compared to the control groups. BDV titer noticeably decreased after the challenge with shRNAs 1, 2, and 3 from ~88% up to 99% in comparison with the control groups.Conclusions: Overall, it could be concluded that RNAi may be considered as a strong treatment proposal against viruses, such as BDV.