First Human Cosavirus Detection From Cerebrospinal Fluid in Hospitalized Children With Aseptic Meningitis and Encephalitis in Iran.

OBJECTIVE: Human cosavirus (HCosV) is a newly recognized virus that seems to be partly related to nonpolio flaccid paralysis and acute gastroenteritis in pediatric patients. However, the relationship between HCosV and diseases in humans is unclear. To assess an investigation for the occurrence of HCosV among pediatric patients involved in meningitis and encephalitis, we implemented a real-time quantitative polymerase chain reaction assay for detection and quantification of HCosV in stool specimens. MATERIALS AND METHODS: In this study, a total of 160 cerebrospinal fluid samples from September 2019 to October 2020 were collected from presenting pediatric patients with meningitis and encephalitis in a Karaj hospital, Iran. After viral RNA extraction, the real-time quantitative polymerase chain reaction was performed to amplify the 5'Un-Translated Region region of the HCosV genome and viral load was analyzed. RESULTS: Of the 160 samples tested, the HCosV genomic RNA was detected in 2/160 (1.25%) of samples. The minimum viral load of HCosV was 3.5 × 103 copies/mL from 4 years male patient. The maximum viral load was determined to be 2.4 × 105 copies/mL in one sample obtained from 3.5 years female patient. CONCLUSIONS: This is the first documentation of HCosV detection in cerebrospinal fluid samples that better demonstrates relation of HCosV with neurologic diseases including meningitis and encephalitis. Also, these results indicate that HCosV has been circulating among Iranian pediatric patients.

High viral load detection of human Cosavirus in Iranian pediatric patients with acute gastroenteritis.

AIM: The present study implemented an RT-qPCR assay for the detection and quantification of human cosavirus in stool specimens from pediatric patients involved in acute gastroenteritis. BACKGROUND: Human cosavirus is a newly recognized virus that seems to be partly related to acute gastroenteritis in pediatric patients. However, the relationship between human cosavirus and diseases in humans is unclear. METHODS: From January 2018 to December 2019, a total of 160 stool samples were collected from pediatric patients presenting with acute gastroenteritis in a hospital in Karaj, Iran. After viral RNA extraction, RT-qPCR was performed to amplify the 5'UTR region of the human cosavirus genome and viral load was analyzed. RESULTS: The human cosavirus genomic RNA was detected in 4/160 (2.5%) stool samples tested. The maximum viral load was determined to be 4.6×106 copies/ml in one sample obtained from a 4-year-old patient. CONCLUSION: The human cosavirus as a new member of the Picornaviridae family was illustrated in fecal samples from pediatric patients with acute gastroenteritis in Iran. This is the first documentation of human cosavirus circulation in Iranian children.

A Real-Time RT-PCR Assay for Genotyping of Rotavirus

Background: Human rotavirus (HRV) is the causative agent of severe gastroenteritis in children and responsible for two million hospitalizations and more than a half-million deaths annually. Sequence characteristics of the gene segments encoding the VP7 and VP4 proteins are used for the genotype classification of rotavirus. A wide variety of molecular methods are available, mainly based on reverse transcription PCR for rapid, specific and sensitive genotyping of rotaviruses. This study describes an alternative real-time PCR assay for genotyping of rotavirus. Methods: The samples of stools studied in this research have been collected from patients referred to Children's Medical Centers, Tehran, Iran. Rotavirus detection and genotyping were performed using the RT-PCR and semi-nested RT-PCR, respectively. Samples were then genotyped with a new real-time PCR. Results: The real-time PCR was able to genotype all positive samples with a mean Ct of 28.2. Besides, a concordance rate of 100% was detected between real-time PCR and semi-nested RT-PCR. Conclusion: In this study, the genotyping of rotavirus with real-time PCR showed that this method can provide several favorable features, including high sensitivity and specificity, and a wide dynamic range for rotavirus genotyping.

High Frequency of Aichivirus in Children With Acute Gastroenteritis in Iran.

BACKGROUND: Initially, detection and isolation of Aichivirus as a new member of Picornaviridae family was documented in Japan. Aichivirus species belongs to genus Kobuvirus, including 3 genotypes A, B and C. In previous studies, it has been suggested that Aichivirus infect humans by fecal-oral route. To establish an investigation for the occurrence of Aichivirus among pediatric patients involved to acute gastroenteritis, we developed a reverse transcription quantitative polymerase chain reaction assay for detection and quantification of Aichivirus in stool specimens. MATERIAL AND METHODS: In this study, a total of 160 stool samples from September 2018 to May 2019 were collected from pediatric patients presenting with acute gastroenteritis in Karaj hospital, Iran. After viral RNA extraction, the reverse transcription quantitative polymerase chain reaction was performed to amplify the 3CD junction region of Aichivirus genome and viral load was assessed. Aichivirus genomic RNA was detected in 13/160 (8.1%) of stool samples. The highest Aichivirus detection rate was in December (30.7%). The maximum viral load was determined to be 3.9 × 10 copies/g in one sample obtained from a 1-month-old patient. The co-infection of Aichivirus with salivirus and saffold virus was also assessed by reverse transcription quantitative polymerase chain reaction, among which frequent mixed infections by 2 or more viruses were identified. CONCLUSIONS: This is the first documentation of Aichivirus detection in stool samples that demonstrates Aichivirus has been circulating among Iranian pediatric patients.

First Occurrence of Saffold Virus in Sewage and River Water Samples in Karaj, Iran.

Saffold virus as a newly discovered virus, which seems to be related to acute gastroenteritis as with other enteric viruses and to human airway diseases in children belongs to Cardiovirus genus in picornaviridae family with 11 genotypes. Saffold virus initially was detected in America from infant stool sample. Saffold virus has also been detected in environmental water samples. Until now, two reports have demonstrated that sewage water sources are contaminated with Saffold viruses. Molecular detection of Saffold virus mostly depended on reverse transcription PCR methods and RT-qPCR, which had targeted 5'UTR region of the viral genome. The present study aims to evaluate the molecular detection and quantity of Saffold virus in sewage water and river water specimens by RT-qPCR assay in Karaj, Iran. Fifty samples collected from environmental waters containing treated and untreated sewage water and river water samples were included in this study. After viral RNA extraction, the Real-time PCR was developed to amplify the 5'UTR sequence of Saffold virus genome and viral load was assessed. Out of the 50 samples tested (consisting 28 river water samples and 22 sewage water samples), the Saffold virus genomic RNA was identified in 10/28 (35.7%) of river water samples and in 4/12 (33.3%) of treated and 4/10 (40%) of untreated sewage samples. The maximum viral load was 6.8 × 106 copies/l in untreated sewage water sample in December, and the lower viral load was 1.2 × 106 copies/l related to treated sewage water taken in October. Our results for the first time indicate that Saffold virus has apparently been circulating among Iranian peoples. Also, the viral prevalence of Saffold virus in each of the three sets of tested samples was within moderate to high in range.

Optimization of RT-qPCR for Detection of Aichi Virus in Sewage and River Water Samples in Karaj, Iran.

BACKGROUND: Aichi virus (AiV) is an emerging virus, which belongs to Kobuvirus genus of the Picornaviridae family. AiV was recently determined as an etiologic agent of gastroenteritis in susceptible humans. After shedding of virus particles from affected people, AiV particles can contaminate water sources. Then, infection with this virus occurs in humans by the fecal-oral route after exposure with contaminated waters. Thus far, some research around the world demonstrated that different kinds of water sources including river water, ground water and treated or untreated sewage water have contamination with AiVs. Molecular detection of AiV has been mostly depended on reverse transcription polymerase chain reaction (RT-PCR) methods, which targeted 3CD junction region of the virus genome. METHODS: The present study aims to assess the molecular detection of AiVs in treated and untreated sewage water and river water specimens by the development of reverse transcription-quantitative PCR (RT-qPCR) assay for all AiV genotypes. RESULTS: Out of 50 samples tested (consisting of 28 river water samples and 22 sewage water samples), the AiV genomic RNA was identified in 15/28 (~50%) river water samples and in 14/22 (~70%) sewage samples. CONCLUSION: Our results, for the first time, indicate that AiVs have been circulating in Iran.

Occurrence of Salivirus in Sewage and River Water Samples in Karaj, Iran.

Salivirus is a newly discovered virus which seems to be related to acute gastroenteritis in children. Salivirus may infect susceptible children by fecal-oral route after exposure to contaminated water. The present study aims to evaluate the occurrence and quantity of Salivirus in treated and untreated sewage water and river water samples collected in the city of Karaj, Iran by reverse transcription-quantitative PCR assay. A total of 50 samples were collected from environmental waters containing 22 treated and untreated sewage water in volume of 1 l and 28 river water samples in volume of 5 l were included in this study. After viral RNA extraction, the Real-time PCR was performed to amplify the 5'UTR sequence of Salivirus genome and viral load was assessed. Out of the 50 samples tested, the Salivirus genomic RNA was identified in 5/12 (41.6%) of treated and 3/10 (30%) of untreated sewage samples and in 8/28 (28.5%) of river water samples. The maximum viral load was 4.8 × 106 copies/l in treated sewage water sample in September and the lower viral load was 4 × 105 copies/l related to treated sewage water taken in December. This is the first report of Salivirus occurrence in the environmental waters in Iran. The viral prevalence of Salivirus in each of the three sets of tested samples was within low to moderate in range.

A comparative approach between heterologous prime-boost vaccination strategy and DNA vaccinations for rabies.

BACKGROUND AND AIM: Rabies is a widespread neurological zoonotic disease causing significant mortality rates, especially in developing countries. Although a vaccine for rabies is available, its production and scheduling are costly in such countries. Advances in recombinant DNA technology have made it a good candidate for an affordable vaccine. Among the proteins of rabies virus, the Glycoprotein (RVG) has been the major target for new vaccine development which plays the principal role in providing complete protection against RV challenge. The aim of this study is to produce recombinant RVG which could be a DNA vaccine candidate and to evaluate the efficiency of this construct in a prime-boost vaccination regimen, compared to commercial vaccine. METHODS: Cloning to pcDNA3.1(+) and expression of rabies virus glycoprotein gene in BSR cell  line were performed followed by SDS-PAGE and Western blot analysis of the expressed glycoprotein. The resulting genetic construct was used as a DNA vaccine by injecting 80 µg of the plasmid to MNRI mice twice. Prime-Boost vaccination strategy was performed using 80 µg plasmid construct as prime dose and the second dose of an inactivated rabies virus vaccine. Production of rabies virus neutralizing antibody (RVNA) titers of the serum samples were determined by RFFIT. RESULTS: In comparisons between heterologous prime-boost vaccination strategy and DNA vaccinations, the potency of group D that received Prime-Boost vaccine with the second dose of pcDNA3.1(+)-Gp was enhanced significantly compared to the group C which had received pcDNA3.1(+)-Gp as first injection. CONCLUSION: In this study, RVGP expressing construct was used in a comparative approach between Prime-Boost vaccination strategy and DNA vaccination and compared with the standard method of rabies vaccination. It was concluded that this strategy could lead to induction of acceptable humoral immunity.

Construction of an eGFP Expression Plasmid under Control of T7 Promoter and IRES Sequence for Assay of T7 RNA Polymerase Activity in Mammalian Cell Lines.

BACKGROUND: Recently, the use of T7 RNA polymerase instead of other viral and cellular promoters is increasing due to high efficacy of transcription in the cell cytoplasm by this polymerase. In order to translate the transcripts produced by T7 RNA polymerase in mammalian cell lines, it is necessary to include Internal Ribosome Entry Site (IRES) sequences. In addition, if sequence of poly A signal would be included after interested gene, the rate of expression could be increased in the cells. METHODS: For expression of eGFP in HEK-293 and T7-BHK cells by T7 RNA polymerase, the sequence of eGFP as well as IRES sequences upstream of eGFP gene and poly A signal were inserted into a pUC57 plasmid. On the other hand, gene of T7 RNA polymerase was cloned into modified pIRES2-EGFP plasmid. Then, the constructed plasmids were transfected into HEK-293 cells. T7-BHK cell was used for control of T7 RNA polymerase activity. RESULTS: Our results showed that using T7 RNA polymerase for expression of foreign genes in mammalian cell lines is highly efficient. CONCLUSION: Highly efficient eGFP expression in HEK-293 cells showed that T7 RNA polymerase could be used for cytoplasmic RNA transcription such as production of anti-cancer proteins and oncolytic viral genomic RNA by reverse genetics.

HCV NS3 Blocking Effect on IFN Induced ISGs Like Viperin and IL28 With and Without NS4A.

BACKGROUND: Hepatitis C virus (HCV) is able to down-regulate innate immune response. It is important to know the immune pathways that this virus interacts with. HCV non-structural protein 3 (NS3) plays an important role in this viral feature. HCV NS3 protein could affect the expression of antiviral protein such as viperin, and interleukin 28whichare important proteins in antiviral response. OBJECTIVES: HCV has developed different mechanisms to maintain a persistent infection, especially by disrupting type I interferon response and subsequent suppression of expression of Interferon stimulatory genes (ISGs). Viperin, a member of ISGs, is considered as a host antiviral protein, which interferes with viral replication. Since it is a good target for some viruses to evade host responses, it is interesting to study if HCV has evolved a mechanism to interfere with this member of ISGs. MATERIALS AND METHODS: We evaluated the impact of NS3, NS3/4A and a mutated nonfunctional NS3 on ISGs expression such as viperin and IL-28 after the induction of IFN signaling Jak-STAT pathway using IFN-. RESULTS: NS3 protein disrupted the expressions of viperin gene and IL-28, an inducer for the expression of ISGs and viperin itself. By comparing the roles of NS3 and NS3/4A protease activities in suppressing the innate immune responses, we also showed that NS3 (without NS4A) has the ability to down-regulate ISGs expression, similar to that of NS3/4A. CONCLUSIONS: ISGs expression is impeded by NS3 protease activity and its interaction with Jak-STAT pathway proteins. In addition, the NS3/4A substrates spectrum seems to be similar to those of NS3.