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.

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.

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.

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.

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.

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.

Relationship between lung cancer and human papillomavirus in north of Iran, Mazandaran province.

Lung cancer is a major health problem and the leading cause of cancer deaths in the world. The pathogenesis of lung cancer is complex, and is believed to be due to the interaction between environmental and genetic factors. Various evidences show that HPV might be involved in bronchial carcinogenesis. In this study, 141 lung cancer patients and 92 non-cancer control subjects were enrolled to examine whether HPV DNA existed in lung tumor and normal tissues in Mazandaran, north part of Iran by nested PCR. Our data showed that 33 of 129 lung tumors had HPV DNA compared with 8 of 90 non-cancer control subjects (25.6% vs. 9.0%, P=0.002). The infection of HPV had an OR of 3.48 (95% CI 1.522-7.958; P=0.002). Meanwhile infection of high risk HPV types (16 and 18) had a significantly high OR of lung cancer incidence as 8.00 (95% CI 1.425-44.920; P=0.021) compared with 4.423 (95% CI 2.407-8.126; P0.0001) of smoking status. This result suggests that HPV infection is associated with lung cancer development in Mazandaran, Iran.

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.

An increased lung cancer risk associated with codon 72 polymorphism in the TP53 gene and human papillomavirus infection in Mazandaran province, Iran.

The TP53 gene has a polymorphism in exon 4 at codon 72 that presents the arginine or proline genotype. The association of TP53 codon 72 polymorphism with lung cancer risk has been studied by several groups, although with inconsistent results. Our previous study showed that the human papillomavirus (HPV) infection is associated with the development of lung cancer in Mazandaran, north part of Iran (cases=25.6% versus controls=9.0%, P=0.002). The frequency of TP53 codon 72 polymorphism was studied in a north part Iranian group of 92 healthy controls and 141 lung cancer patients. The allelic distribution of the three genotypes (ArgArg, ArgPro, ProPro) in healthy normal controls was 46.1, 32.6 and 21.3%, respectively, which differs from that of lung cancer patients showing genotype frequency as 42.6, 49.6 and 7.8%. A relation between the presence of the Arg allele and lung cancer risk was observed. Our study reveals that Arg allele, active smoking and HPV infection are the important risk factors in lung cancer development in the north part of Iran, Mazandaran province.

Vaccine-associated paralytic poliomyelitis in a patient with MHC class II deficiency.

Vaccine-associated paralytic poliomyelitis (VAPP) is a rare complication of oral polio vaccine. We describe a fatal case of VAPP in an 8-month-old boy with Major Histocompatibility Class II deficiency. The isolated poliovirus was a Sabin type 2-type 1 recombinant that showed 1.4% VP1 divergence from Sabin type 2.

Detection of enteroviruses by reverse-transcriptase polymerase chain reaction in cell culture negative stool specimens of patients with acute flaccid paralysis.

The aim of this study was the detection of a 114 base pairs amplicon in 5' non-translated region of enterovirus genome in stool specimens of patients with acute flaccid paralysis (AFP) which were negative on cell culture. One hundred and twenty stool specimens were collected from AFP cases and tested with cell culture (RD, L20B and Hep2 cell lines). RT-PCR was carried out for the specimens with negative cell culture result. A 10% raise in enterovirus detection was observed with RT-PCR. This increased sensitivity can improve the detection of enterovirus serotypes which grow poorly in cell culture, and can thus alter significantly the medical care of patients with acute flaccid paralysis.

Evaluation of immunity against rubella in Iranian after mass campaign for measles-rubella vaccination on December 2003.

BACKGROUND: Prevention of congenital rubella syndrome (CRS) is the main goal of rubella immunization. The World Health Organization (WHO) recommends that countries undertaking measles elimination should take the opportunity to eliminate rubella as well through use of measles-rubella (MR) or measles-mumps-rubella (MMR) vaccine in their childhood immunization programs and campaigns. On December 2003, during a mass campaign for measles-rubella vaccination in Iran, more than 33 million doses of vaccine were administered to 5- to 25-year-old males and females. METHODS: In this study, immunoglobulin (Ig)G avidity assay was used to evaluate the rubella vaccine produced immunity among susceptible populations. RESULTS: Findings indicated that 61.9% of vaccinees (1202 out of 1940) were immune against rubella before vaccination, and 38.1% (738 out of 1940) were susceptible to rubella before vaccination. Our results also showed that 98% of the susceptible group (723 out of 738) acquired immunity against rubella after vaccination, and 2% of them (15 out of 738) did not acquire vaccine-induced immunity to rubella. CONCLUSION: Findings revealed that mass vaccination in December 2003 provided appropriate immune coverage among vaccinees. Also, rubella vaccine induced favorable immunity in a part of the childbearing age female (15-25 years) population, which could prevent congenital rubella syndrome among those females.

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.

Assessment of IgM enzyme immunoassay and IgG avidity assay for distinguishing between primary and secondary immune response to rubella vaccine.

The primary test for the laboratory confirmation of rubella is IgM serology. It is important to distinguish IgM reactivity caused by primary infection from that caused by reinfection or persistence, especially in pregnant women; as termination of pregnancy is considered when primary rubella is diagnosed during the first trimester. In this study, the performance of rubella IgM enzyme immunoassay (IgM-EIA) and rubella IgG avidity assay were compared using well-defined panels of sera from persons vaccinated against rubella and commercial rubella IgM and IgG enzyme immunoassay kits (Dade Behring, Marburg, Germany). The sensitivity and specificity of rubella IgM-EIA were found to be 77.4 and 97.9%, respectively, while the results for rubella IgG avidity assay were 100 and 100%. IgG avidity assay showed higher positive and negative predictive values than the IgM-EIA (100 and 100% compare to 96.9 and 82.9%). In conclusion, the rubella IgG avidity assay is more sensitive and specific than IgM-EIA for differential detection of primary rubella infection from rubella reinfection.