Molecular characterization of non-polio enteroviruses isolated from children with acute flaccid paralysis in IRAN, 2015-2018.

In addition to polioviruses, non-polio enteroviruses (NPEVs) are frequently isolated from patients with acute flaccid paralysis (AFP) worldwide. In polio-free countries, there have been expectations that with disappearing wild poliovirus from the community, the rate of AFP would decrease, but the increasing number of AFP cases proved this notion to be wrong. There are speculations that NPEVs might be the cause of increasing AFP rate. The aim of this study was to investigate frequency, genetic diversity, circulation patterns of NPEVs isolated from AFP cases in Iran from 2015 to 2018. Fifty-three NPEVs were isolated from stool specimens of AFP cases during four years of AFP surveillance. Nested PCR and VP1 sequencing revealed 20 NPEV types in which Echovirus 3 (13.2%), Echovirus 6 (13.2%), Echovirus 7 (7.5%), Echovirus 13 (7.5%) and Echovirus 21 (7.5%) were the most frequent. Coxsackie B viruses were isolated for the first time in AFP cases in Iran. The phylogenetic analysis of Echovirus 3 and Echovirus 6 revealed that Iranian echovirus strains belonged to the same cluster, indicating these viruses have been circulating in Iran for a long time. Compared to global Echovirus 3 and Echovirus 6 references, Echovirus 3 and Echovirus 6 strains detected in this study were closely related to Indian and Malaysia strains, respectively. The results of this study demonstrated a wide variety of NPEV types in Iranian patients, some of which had not been reported in previous studies. Moreover, this study highlights the need for NPEV surveillance in AFP cases.

Enteroviruses and Adenoviruses in stool specimens of paralytic children- can they be the cause of paralysis?

BACKGROUND AND OBJECTIVES: Acute flaccid paralysis (AFP) is a complicated clinical syndrome with a wide range of potential etiologies. Several infectious agents including different virus families have been isolated from AFP cases. In most surveys, Non-polio Enteroviruses (NPEVs) have been detected as main infectious agents in AFP cases; however, there are also some reports about Adenovirus isolation in these patients. In this study, NPEVs and Adenoviruses in stool specimens of AFP cases with or without Residual Paralysis (RP) with negative results for poliovirus are investigated. MATERIALS AND METHODS: Nucleic acid extractions from 55 AFP cases were examined by nested PCR or semi-nested PCR with specific primers to identify NPEVs or Adenoviruses, respectively. VP1 (for Enteroviruses) and hexon (for Adenoviruses) gene amplification products were sequenced and compared with available sequences in the GenBank. RESULTS: From 55 fecal (37 RP+ and 18 RP-) specimens, 7 NPEVs (12.7%) (2 cases in RP+) and 7 Adenoviruses (12.7%) (4 cases in RP+) were identified. Echovirus types 3, 17 and 30, Coxsackie virus A8, and Enterovirus 80 were among NPEVs and Adenoviruses type 2 and 41 were also identified. CONCLUSION: Our finding shows that NPEVs and Adenoviruses may be isolated from the acute flaccid paralyses but there is no association between the residual paralyses and virus detection.

The Effect of Different microRNA Backbones on Artificial miRNA Expression and Knockdown Activity Against HIV-1 Replication.

BACKGROUND: Artificial microRNAs (miRNAs) are designed to develop an RNAi-based gene therapy. Recently, it has been suggested that the flanking sequences and terminal loop structure play a critical role in RNAi biogenesis and target recognition, but no extensive study regarding the different miRNA backbone for artificial miRNAs optimization has been conducted. OBJECTIVE: We tested three artificial miRNAs with human hsa-miR30a (common miRNA), hsa-miR150 (T cell specific miRNA), and hsa-miR122 (liver specific miRNA) backbones in HEK-293T and Jurkat cell lines. METHODS: Artificial miRNA processing and knockdown efficiency were analyzed by stem-loop RT-PCR, qRT-PCR, luciferase assay and target challenging. RESULTS: We identified strikingly different RNAi activities among these different artificial miRNAs. Our results demonstrated that expression and function of art-miR150 was more than art-miR30 and artmiR122 in both HEK-293T and Jurkat cell lines. Since the main difference in these artificial miRNAs was flanking sequences and terminal loop structure, the change between the expression and function of artificial miRNAs can be attributed to these structures. CONCLUSION: This study showed that expression of cell-specific artificial miRNA in target and nontarget cells is not different, but variation in flanking sequences and terminal loop can be involved in expression and function of artificial miRNAs. These results can be important for improving artificial miRNA design in RNAi-based gene therapy.

Efficient inhibition of human immunodeficiency virus replication using novel modified microRNA-30a targeting 3'-untranslated region transcripts.

RNA interference (RNAi)-based gene therapy is currently considered to be a combinatorial anti-human immunodeficiency virus-1 (HIV-1) therapy. Although artificial polycistronic microRNAs (miRs) can reduce HIV-1 escape mutant variants, this approach may increase the risk of side effects. The present study aimed to optimize the efficiency of anti-HIV RNAi gene therapy in order to reduce the cell toxicity induced by multi-short hairpin RNA expression. An artificial miR-30a-3'-untranslated region (miR-3-UTR) obtained from a single RNA polymerase II was used to simultaneously target all viral transcripts. The results of the present study demonstrated that HIV-1 replication was significantly inhibited in the cells with the miR-3-UTR construct, suggesting that miR-3'-UTR may serve as a promising tool for RNAi-based gene therapy in the treatment of HIV-1.

No evidence of mammary tumor virus env gene-like sequences among Iranian women with breast cancer.

OBJECTIVE: The mouse mammary tumor virus (MMTV) is the well-established etiological agent of mammary tumors in mice. A series of studies have implicated that a human murine mammary tumor virus-like virus occurs in human breast cancer, but it is unclear whether it has any causal role. METHODS: The aim of the present study was to investigate the presence of MMTV env gene-like sequences in a group of Iranian women with or without breast cancer. A total of 65 breast cancer and 65 noncancerous breast specimens from the Department of Pathology of Tabriz University in East Azerbaijan, Iran, were analyzed by nested PCR. RESULTS: All breast cancer and benign breast samples were negative for MMTV env gene-like DNA. CONCLUSION: These results indicate that the MMTV env gene-like virus may not play a significant role in the etiology of breast cancer among Iranian women.

No detection of 'high-risk' human papillomaviruses in a group of Iranian women with breast cancer.

The presence of viral DNA in breast cancer cells is controversial. However, some studies have revealed a possible role for the human papillomavirus in the pathogenesis of breast cancer. The aim of the present study was to investigate the presence of HPV-DNA in breast tissue in a group of Iranian women with and without breast cancer and identification of the detected HPV types. Paraffin-embedded specimens from 65 malignant breast cancer cases and 65 cases with benign breast lesions were investigated for presence of HPV-DNA by nested polymerase chain reaction. We found HPV-DNA in 22 (33.8%) of the breast cancer specimens. All non-cancerous specimens were negative. Low and high-risk HPV types, including HPV-6 (26.2%), HPV-16 (1.5%), HPV-35 (1.5%), HPV-52 (1.5%), and HPV-11 (1.5%) were detected in our study. HPV-6 was the most prevalent type in the breast cancer specimens. Although high-risk HPV types have been shown to have a major role in cervix cancer, there have been no data that support the same relevance for other types of malignancies. Furthermore, presence of low-risk HPV types in malignancies still is a matter of debate. The data presented in this study indicates a strong need for epidemiological studies correlating different HPV types in human breast cancer.

Construction of recombinant bacmid containing m2e-ctxb and producing the fusion protein in insect cell lines.

BACKGROUND: Sequence variations in glycoproteins of influenza virus surface impel us to design new candidate vaccines yearly. Ectodomain of influenza M2 protein is a surface and highly conserved protein. M2e in influenza vaccines may eliminate the need for changing vaccine formulation every year. OBJECTIVES: In this study, a recombinant baculovirus containing M2e and cholera toxin subunit B fusion gene was generated with transposition process to express in large amounts in insect cell lines. MATERIALS AND METHODS: M2e-ctxB fusion gene was created and cloned into pFastBac HT. The recombinant vector was transformed into DH10Bac cells to introduce the fusion gene into the bacmid DNA via a site-specific transposition process. The recombinant bacmid was then extracted from white colonies and further analyzed using PCR, DNA sequence analyzing, and indirect immunofluorescence assay. RESULTS: PCR and DNA sequence analyzing results showed that the fusion gene was constructed as a single open reading frame and was successfully inserted into bacmid DNA. Moreover, indirect immunofluorescence results showed that the fusion gene was successfully expressed. CONCLUSIONS: Baculovirus expression vector system is valuable to produce M2e based influenza vaccines due to its simple utilization and ease of target gene manipulation. The expressed protein in such systems can improve the evaluating process of new vaccination strategies.

Methanobrevibacter smithii archaeosomes-entrapped mzNL4-3 virus-like particles induce specific T helper 1-oriented cellular and humoral responses against HIV-1.

Despite numerous and tremendous achievements in the development and standardization of HIV vaccines, there are still lots of vague concepts in HIV vaccinology. Various approaches have been applied to design an efficient HIV vaccine. Due to their lack of replication ability and expression of native antigens at the same time virus-like particles, such as previously introduced mzNL4-3 HIV-1 VLPs are among the highlighted candidates in this field. On the other part, application of adjuvants is an inseparable strategy in the vaccine development researches. Archaeosomes are liposomal adjuvants with intensifiying features of T helper 1 and cytotoxic T-cells responses. Archaeosomes derived from Methanobrevibacter smithii has been shown to enhance MHC class I-dependent antigen presentation and hence, are to be advantageous in the development of vaccines against viral infections. Herein, we have studied efficiency of mzNL4-3 VLPs entrapped in M. smithii archaeosomes as an HIV-1 vaccine candidate to induce humoral and cellular responses in BALB/c mice. Analysis of total and subtype-specific anti-Env IgG antibody, as well as, cytokine secretion pattern revealed an efficient promotion of anti-HIV specific T helper 1 responses in immunized animals. This finding was evidenced by the significant dominance of IgG2a subtype in the sera and considerable secretion of IFN-γ by specifically induced splenocytes of mice immunized with VLP-containing archaeosomes (VLP+ Archaeosome). In addition, ELISpot assay verified these results and indicated the significantly higher frequency of IFN-γ secreting splenocytes in immunized models. The ratio of IFN-γ to IL-4 spot forming cells (SFCs) in the VLP+ Archaeosome immunized mice was also higher than that of the other groups immunized with either VLP-free archaeosomes or VLPs formulated with complete/incomplete Freund's adjuvants. These results propound M. smithii archaeosomes-entrapped mzNL4-3 VLPs as a promising immunogen which specifically induces and augments T-helper 1 oriented responses against HIV antigens.

Development of a stable insect cell line constitutively expressing rotavirus VP2.

An insect High-Five cell line was generated constitutively and stably expressing the VP2 protein of rotavirus RF strain leading to the formation of core-like particles. The integration of the VP2 gene was confirmed by PCR, Real-time PCR and Southern blot, and the protein expression was confirmed by Western blot and immunofluorescence microscopy. Integrity and self assembly of VP2 as core-like particles was demonstrated by electron microscopy. The High-Five cell system stably expressing rotavirus core-like particles can be applied to the study of viral protein structure and functions; it may be useful for vaccine development, gene therapy and drug delivery.

Epidemiology of cocirculating human rotaviruses in Iran.

Rotaviruses are a major cause of severe diarrhea in children <5 years of age worldwide. The rotavirus positivity rate in Iran was 15.3% to 67.6% in all patients <5 years of age hospitalized for acute gastroenteritis, which was significantly higher than recorded in outpatient departments. Moreover, the most commonly detected genotype was G1P[8], although in a single study a peak prevalence of the G4P[8] genotype was reported.

Molecular characterization analysis of the outer protein layer (VP7) from human rotavirus A genotype G1 isolate identified in Iran: implications for vaccine development.

The full open reading frame of the outer protein layer VP7 from an isolate of human rotavirus identified in 2010 in an Iranian child admitted to hospital with gastroenteritis was amplified from a clinical stool specimen and subjected to molecular characterization. Genetic and phylogenetic analyses indicated that the analyzed gene falls into the G1 genotype forming a sub-cluster with sequences recently identified in Iran and geographically distant countries. Such results were confirmed by protein sequence alignment, showing a highly conserved "G1-like?? amino acid sequence pattern within the known three main immunodominant regions. These results are extremely relevant in a perspective of vaccine development. Indeed, the present study confirms that the A group G1 genotype is the most prevalent Rotavirus circulating in Iran and supports the development of G1 genotype-based rotavirus vaccine for this country.

Human papillomavirus is associated with breast cancer in the north part of Iran.

We have analyzed the possible relevance of HPV infection for breast cancer risk among Iranian women from north part of Iran. Among women with breast cancer, 25.9% had positive test results for HPV DNA in breast tumor samples in contrast to 2.4% of women with noncancer status (P = 0.002). The infection of HPV has increased the risk of breast cancer (OR 14.247; 95% CI 1.558-130.284, P = 0.019). The high-risk HPV genotypes (types 16 and 18) in samples of breast cancer patients were the predominant types (53.34%). Other genotypes detected in breast cancer were HPV-6, HPV-11, HPV-15, HPV-23, and HPV-124, and one isolate could not be genotyped compared to HPV reference sequences. While the sole detected HPV in control specimens was HPV-124. Our study reveals that HPV infection and age are the risk factors in breast cancer development in the north part of Iran.

Molecular epidemiology of human herpesvirus 8 variants in Kaposi's sarcoma from Iranian patients.

Kaposi's sarcoma (KS) is a rare cancer in Iran and there is no epidemiological and molecular information about HHV-8 variants circulating among the Iranian population. In this study HHV-8 sequences have been analyzed in 43 cutaneous KS biopsies from Iranian patients mainly affected by classic KS. DNA samples were subjected to PCR amplification of HHV-8 ORF26, T0.7 and K1 followed by direct nucleotide sequencing and phylogenetic analysis. The analysis of ORF26 showed that 30 (69.8%) and 13 (30.2%) samples belonged to subtypes A/C and K, respectively. In general, the clustering of HHV-8 T0.7 variants paralleled that of ORF26. Genotyping of K1 sequences showed that the majority of samples (39 out of 41) fall into the large C clade with only 2 belonging to the A clade. In conclusion, HHV-8 variants identified among classic Iranian KS are largely related to Eurasian genotypes previously identified in KS from Mediterranean, Middle East, and East Asian regions.

Seroprevalence of Human herpesvirus 8 (HHV-8) and incidence of Kaposi's sarcoma in Iran.

Seroepidemiological surveys show that the prevalence of human herpesvirus 8 (HHV-8) infection mostly varies in various geographical areas and reflects the local incidence of classic and endemic KS, being widespread in sub-Saharan Africa and Mediterranean countries and uncommon in the USA and Northern Europe. In the Middle East only few populations, such as Ashkenazi and Sephardic groups in Israel, have been adequately evaluated for HHV-8 seroprevalence. Among Iranian population a striking higher seroprevalence of HHV8 has been reported among haemodialysis (16.9%), renal transplant recipients (25%) and HIV (45.7%) patients compared to blood donors (2%). Kaposi's sarcoma (KS) is the rarest cancer in Iran, with an annual age-standardized incidence varying from 0.10 to 0.17 per 100,000 in males and from 0.06 to 0.08 per 100,000 in females. KS, however, is one of the most important malignancies in Iranian renal transplanted patients affecting up to 2.4% of organ recipients. The epidemiology of HHV8 and KS in Iran needs further evaluation. While the high prevalence of HHV-8 antibodies in HIV positive and haemodialysis individuals may be attributed to high-risk sexual behavior and polytransfusions, respectively, unknown determinants may be responsible for high seroprevalence of HHV8 and high incidence of KS in solid organ recipients. A global survey on HHV8 seroprevalence in Iran is mandatory to define co-factors associated with HHV8 infection and KS risk in the general Iranian population and in specific patient groups.

Vaccine-associated paralytic poliomyelitis in immunodeficient children, Iran, 1995-2008.

To determine the prevalence of vaccine-associated paralytic poliomyelitis (VAPP) in immunodeficient infants, we reviewed all documented cases caused by immunodeficiency-associated vaccine-derived polioviruses in Iran from 1995 through 2008. Changing to an inactivated polio vaccine vaccination schedule and introduction of screening of neonates for immunodeficiencies could reduce the risk for VAPP infection.

Molecular and genetic characteristics of hemagglutinin and neuraminidase in Iranian 2009 pandemic influenza A(H1N1) viruses.

Influenza virus infections cause severe illness worldwide. Vaccination reduces the morbidity and mortality of influenza. The efficacy of vaccines varies due to antigenic differences between the circulating influenza strains and the vaccine. Neuraminidase inhibitors are effective for prophylaxis and treatment of influenza infections, and the emergence of drug resistant mutants is an important challenge. Full-length nucleotide and deduced amino acid sequences of the hemagglutinin and neuraminidase genes of three 2009 pandemic influenza A/H1N1 isolates were compared with the vaccine strain and some strains from different countries. Phylogenetic analysis for hemagglutinin and neuraminidase showed they were related to their vaccine strain, with an average of 99.56 and 99.53% sequence identity, respectively. No genetic indication of resistance to neuraminidase inhibitors was found. Although genomic analysis of hemagglutinin and neuraminidase genes of Iranian strains in comparison to the corresponding vaccine strain revealed some mutations, none of these were identified in functionally important receptor-binding sites.

Environmental surveillance of Non-Polio Enteroviruses in Iran.

BACKGROUND: Enteroviruses can shed in feces for several weeks, so many excrete viruses can remain infectious for a long time in environment. Therefore, by detecting enteroviruses in environmental specimens and sewage, we can understand this virus circulation, the approximate ratio of contaminated persons in society and they are suitable indicators for environmental surveillance. METHODS: Since March 2006 to February 2007, 86 specimens from Sistan & Balouchestan, 63 specimens from Tehran and 48 samples from Fars sewage disposal systems and surface water were collected by Grab Sample method and tested for enteroviruses directly by using two concentration methods: Pellet and Two-phase. Then Non-Polio Enteroviruses (NPEV) were serotyped by microneutralization method. RESULTS: Enteroviruses were isolated from 49(56.98%) of specimens in Sistan & Baluchestan,38(60.32%) in Tehran and 11(22.92%) in Fars. Besides, the majority of Non-Polio Enteroviruses related to Non-typable Enteroviruses (N.T.E.V), E11 (31.52%), COX-B (27.58%), E7 (17.73%) and E4 (21.67%). CONCLUSION: Environmental surveillance has been used successfully in monitoring enteric virus circulation and assessing the extent or duration of epidemic non polioviruses in specific populations. The results of this research show the seasonal circulation of enteroviruses in different parts of Iran.

Isolation of a type 3 vaccine-derived poliovirus (VDPV) from an Iranian child with X-linked agammaglobulinemia.

Type 3 immunodeficiency-associated vaccine-derived polioviruses (iVDPVs) were isolated from a 15-month-old Iranian boy with acute flaccid paralysis (AFP) who was subsequently diagnosed with X-linked agammaglobulinemia (XLA). VP1 nucleotide sequences of the two isolates differed from Sabin 3 by 2.0% and 2.1% and from each other by 0.6%. Although the key determinant of attenuation and temperature sensitivity in the 5'-untranslated region (U(472)-->C) had reverted, a second capsid-region determinant (VP3:Phe(091)) was unchanged, but a presumptive suppressor (VP1:Ala(054)-->Val) was found. The isolates were Sabin 3/Sabin 1 recombinants, sharing a single recombination breakpoint in the 2C region. Although the two isolates were antigenically distinct from Sabin 3, only one amino acid replacement was found in the neutralizing antigenic sites (VP3:Ser(059)-->Asn in site 3). The patient was placed on intravenous immunoglobulin (IVIG) therapy within 9 days of onset of AFP, and iVDPV excretion ceased thereafter, but the patient remained severely paralyzed until his death approximately 11 months after paralysis. No secondary AFP cases were found, and none of the seven tested contacts of the patient were found to be infected with poliovirus.