Molecular typing of enteroviruses and parechoviruses in acute flaccid paralysis patients in Iran in 2019.

Enteroviruses (EVs) and parechoviruses (PeVs) are among the viral pathogens that can cause acute flaccid paralysis (AFP). There is not sufficient information about direct detection of EVs and PeVs in AFP patients in Iran. The aim of this study was to conduct a one-year study for direct detection and molecular typing of EVs and PeVs from stool samples of AFP patients in Iran. One hundred stool samples from polio-negative AFP patients who were referred to the Iran National Polio Laboratory were randomly chosen and analyzed during 2019. A one-step TaqMan probe-based real-time RT-PCR assay targeting the 5'-untranslated region (5' -UTR) was used to screen for EVs and PeVs. All positive samples were genotyped by direct sequencing, targeting the VP1 region of the genome. In total, twelve (12%) and four (4%) stool samples from polio-negative AFP children were positive for EVs and PeVs, respectively. Sequence analysis revealed the presence of echovirus 2 (E2), echovirus 13 (E13), echovirus 25 (E25), echovirus 30 (E30), coxsackievirus A2 (CVA2), coxsackievirus A9 (CVA9), coxsackievirus A16 (CVA16), human enterovirus A76 (HEV-A76), and human parechovirus 1 (HPeV1) in children with AFP-like symptoms. Phylogenetic analysis showed that E2 strains clustered together with the strains circulating in the Netherlands during 2014, whereas the PeV strains belonged to different lineages. This study demonstrates that different EV types are associated with AFP cases in Iran. However, the frequency of association of PeVs with AFP cases appears to be low.

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.

Vaccine-Derived Polioviruses and Children with Primary Immunodeficiency, Iran, 1995-2014.

Widespread use of oral poliovirus vaccine has led to an ≈99.9% decrease in global incidence of poliomyelitis (from ≈350,000 cases in 1988 to 74 cases in 2015) and eradication of wild-type poliovirus serotypes 2 and 3. However, patients with primary immunodeficiency might shed vaccine-derived polioviruses (VDPVs) for an extended period, which could pose a major threat to polio eradication programs. Since 1995, sixteen VDPV populations have been isolated from 14 patients with immunodeficiency in Iran. For these patients, vaccine-associated paralysis, mostly in >1 extremity, was the first manifestation of primary immunodeficiency. Seven patients with humoral immunodeficiency cleared VDPV infection more frequently than did 6 patients with combined immunodeficiencies. Our results raise questions about manifestations of VDPVs in immunodeficient patients and the role of cellular immunity against enterovirus infections. On the basis of an association between VDPVs and immunodeficiency, we advocate screening of patients with primary immunodeficiency for shedding of polioviruses.

Environmental Surveillance of Poliovirus and Non-polio Enteroviruses in Iran, 2017-2023: First Report of Imported Wild Poliovirus Type 1 Since 2000.

In Iran, which is at high risk of the Wild Poliovirus (WPV) and Vaccine-Derived Poliovirus (VDPV) importation due to its neighborhood with two polio endemic countries, Pakistan and Afghanistan, Environmental Surveillance (ES) was established in November 2017. Sistan-Balouchestan province was chosen for the ES due to its vicinity with Pakistan and Afghanistan. Five sewage collection sites in 4 cities (Zahedan, Zabol, Chabahar and Konarak) were selected in the high-risk areas. Since the establishment of ES in November 2017 till the end of 2023, 364 sewage specimens were collected and analyzed. The ES detected polioviruses which have the highest significance for polio eradication program, that is, Wild Poliovirus type 1 (WPV1) and Poliovirus type 2 (PV2). In April and May 2019, three of 364 (0.8%) sewage specimens from Konarak were positive for imported WPV1. According to phylogenetic analysis, they were highly related to WPV1 circulating in Karachi (Sindh province) in Pakistan. PV2 was also detected in 5.7% (21/364) of the sewage specimens, most of which proved to be imported from the neighboring countries. Of 21 isolated PV2s, 7 were VDPV2, of which 5 proved to be imported from the neighboring countries as there was VDPV2 circulating in Pakistan at the time of sampling, and 2 were ambiguous VDPVs (aVDPV) with unknown source. According to the findings of this study, as long as WPV1 and VDPV2 outbreaks are detected in Iran's neighboring countries, there is a definite need for continuation and expansion of the environmental surveillance.

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.

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.

First detection of enterovirus 71 from an acute flaccid paralysis case with residual paralysis in Iran.

In an attempt to determine the types of non-polio enteroviruses (NPEVs) in acute flaccid paralysis (AFP) cases in Iran, we detected enterovirus 71 (EV71) in an AFP case with residual paralysis for the first time. Cell culture detected no enteroviruses, while RT-PCR and subsequent sequencing revealed that the specimen was positive for EV71. EV71 is the causative agent of a variety of diseases from hand, foot and mouth disease to severe neurological complications and is now considered as an important cause of childhood acute flaccid paralysis.

Novel BTK mutation presenting with vaccine-associated paralytic poliomyelitis.

Oral polio vaccine (OPV) has been used safely and efficiently for more than 40 years in preventive medicine. Vaccine-associated paralytic poliomyelitis (VAPP) is a rare adverse event of OPV due to reversion of the vaccine strain virus to a neurovirulent strain. VAPP can occur in healthy recipients or their close contacts. However, persons with primary humoral immunodeficiencies are at a much higher risk. X-linked agammaglobulinemia (XLA) is a prototypic humoral deficiency caused by mutations in the Bruton's tyrosine kinase (BTK) gene. In addition to susceptibility to bacterial infections, patients with XLA are especially prone to enteroviruses. Here, we describe the occurrence of VAPP in a 15-month old Iranian boy. The child had received four doses of OPV, administered at birth, 2, 4, and 6 months of age. The patient's infectious history was unremarkable. Laboratory evaluation revealed low levels of immunoglobulin G and CD19(+) B cells of less than 1% of the lymphocyte population. A novel insertion (c.685_686insTTAC) in the SH3 domain of the BTK gene was detected as the underlying cause. Immunodeficient recipients of OPV can excrete poliovirus vaccine strains for a long period and are at risk of developing flaccid paralysis. They could also serve as a source of reverted virulent poliovirus to be reintroduced into the general population. This patient presented for the first time with VAPP, without any history of other major infections in 15 months. This suggests that a negative history for recurrent infections does not exclude the presence of a primary defect in the immune system.

Prevalence of mucosal types of human papillomavirus in skin lesions in north part of Iran.

Human papillomaviruses (HPVs) consist of more than 100 types and are known to be associated with numerous malignant tumors, including carcinomas of the mucosal and cutaneous epithelium. Non-melanoma skin cancer (NMSC) is the most frequently occurring malignancy worldwide in the Caucasian population. Some studies have shown that NMSC biopsy specimens harbor cutaneous as well as mucosal human papillomavirus, suggesting that mucosal types may play a role in development and progression of the tumor in skin. To investigate the presence of mucosal HPV types in skin lesions, we performed a retrospective study in which 288 paraffin embedded biopsies from benign and malignant skin lesions (NMSC) were collected. Using nested PCR with MY09/11 and GP5+/6+ primers mucosal HPVs were detected in 25.7% of malignant specimens, but just in 0.7% of benign lesions. Direct sequencing revealed HPV18 as the most frequent type, which was found in 75% of HPV-positive specimens. HPV16 and HPV56 were also detected, 22.3 and 2.7%, respectively. These findings suggest that, high-risk mucosal HPV types recently identified as significant risk factors for cervical cancer, may also represent a risk factor for non-melanoma skin cancer.

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.