Natural circulation of human enterovirus in Maputo city, Mozambique

The human enteroviruses (HEV) are responsible for a large diversity of infections affecting humans. Most infections are asymptomatic, but these viruses can cause a wide spectrum of diseases, including severe cases involving the central nervous system. The aim of this study was to isolate and identify human enteroviruses in natural circulation in children less than 15 years of age admitted at the Mavalane General Hospital in Maputo City, Mozambique. In this study, 178 stool samples were processed, obtained during November 2011 to February 2012. Samples were inoculated onto cell culture and the isolates were identified as enterovirus by conventional RT-PCR in the 5’ non-coding region followed by partial VP1 sequence. Twenty-six (26) out of the 45 cell-culture positive samples were constituted by Enterovirus (14.6% of the total 178 samples). EV-29 was the serotype most prevalent. The results show the importance of maintaining the cell line Hep2C in the diagnosis and Enterovirus circulating in the Maputo city, Mozambique.


INTRODUCTION
The human enteroviruses (HEV) belong to the genus Enterovirus and Picornaviridae family.These agents infect millions of people worldwide each year, resulting in a wide variety of clinical conditions ranging from unapparent infection, undifferentiated fevers, common cold to serious diseases such as aseptic meningitis, hand-foot-mouth disease, acute hemorrhagic conjunctivitis, myocarditis, encephalitis and paralytic poliomyelitis (Pallansch and Roos, 2001).Children are more susceptible to infection and transmission occurs either by the fecal-oral or respiratory tract.The virus can be excreted in the feces for several weeks (Pallansch and Roos, 2001).
There are more than 80 species of HEV divided into four group, designated HEV-A to D based on the genetic characteristics of the VP1 capsid gene (Oberste et al., 2006;Knowles et al., 2011).In general, the 5' and 3' noncoding regions (NCRs) are highly conserved, and the most variable region of the genome lies within the genes encoding the capsid proteins exposed on the virus surface, VP1, VP2, VP3, and VP4 (Hogle et al., 1985;Racaniello, 1988;Oxman, 1999).

Ethics statement
National Bioethics Committee on Health of Mozambique approved the study with the reference: 280/CNBS/11.All study participants were under 15 years old and parents or guardians authorized written consent for their participation by signing an informed consent form where each party stayed with a copy.

Patients and specimens
A cross-sectional study involves enrolling a total of 178 stool specimens from children under 15 years old.Clinical specimens were collected from pediatric patients admitted in general hospital in Maputo City, Mozambique in the period from November 2011 to February 2012, with at least three or more of these signs and symptoms: fever, vomiting/nausea, difficulty breathing, cough, diarrhea, headaches joint pain.Patient information's were recorded on a standard questionnaire including demographic and clinical details.Specimens were transported in dry ice to the Enterovirus Laboratory, Oswaldo Cruz Institute/Fundação Oswaldo Cruz/Rio de Janeiro, Brazil.

Virus isolation
Fecal suspensions were treated with chloroform as previously described (WHO, 2004).Each specimen extract (0.2 ml) was inoculated in duplicate tubes of the three cell culture lineages, RD, L20B and Hep2C, as recommended (WHO, 2004).The tubes were incubated at 37°C and examined daily for evidence of cytophatic effect (CPE) and these were accompanied by negative control.Positive cultures were harvested and kept frozen (-20°C) until further analysis.

Molecular characterization and Enterovirus genotyping
Isolates showing characteristic enterovirus CPE were subject to in vitro amplification by RT-PCR using enterovirus primers as previously described (Dos Santos et al., 2012).The Pan-EV pair of primers (PanEVF and PanEVR) used in one-step conventional RT-PCR, are broader reactive for the human enterovirus genus targeting conserved nucleotide sequences at the 5'-NCR of the EV genomes.Briefly 1.0 μl of sample was added to 3.2 μl reaction mixture A (containing 2.5 μl of 10× PCR buffer and 0.7 μl of reverse primer at 50 pmol/μl).The reaction mixture was first incubated at 95°C for 5 min and then chilled in ice for 5 min.The reaction mixture B containing 0.5 μl of each dNTP´s 10 mM, 0.15 μl of RNASin at 5 U/μl, 0.7 μl of foward primer, 0.2 μl of transcriptase reverse at 20 U/μl, 0.2 μl of Taq DNA polymerase at 5 U/μl, 19.05 μl of water were added to mixture A. The tubes were placed in thermo cycler (Veriti TM 96-well Thermal Cycler) and cycled as follows: 42°C for 30 min, 30 cycles at 95°C for 45 s, 55°C for 45 s and 72°C for 45 s, and these were accompanied by negative and positive control of the reaction, respectively.
All positive samples in Pan-EV were subjected to Pan-PV to test for the presence of polioviruses, as previously described (WHO, 2004).All isolates was identify by partial VP1 sequence (Oberste et al., 1999;Oberste et al., 2003).After edition, sequences were compared with those located at GenBank using the Blast Program 2.2.27 for confirmation of the viral identity and species determination (Altschul et al. 1990).
A phylogenetic three was constructed based on MEGA 4.0.2 program (Kumar et al., 2001) using the Neighbor-Joining reconstruction method (Saitou and Nei, 1987).The genetic distances were estimated to using Kimura-Two parameter model (Kimura, 1980).The robustness of the branches in the phylogenetic tree was statistically evaluated by 1,000 bootstrap replicates (Felsenstein, 1985).All samples negative by RT-PCR PAN EV were subjected to PCR to search Adenovirus members because in cell line Hep2C showed a highly suggestive adenovirus cytopathic effect for virus isolation in cell culture.The Adenoviruses were screened through a PCR reaction using specific primers previous described (Xu et al., 2000).

RESULTS
A total of 45 (25.3%) samples had viral isolation in RD, L20B or Hep2C cell lines although it was not homogenous.The EV was identified in 14.6% of the samples (26/178).Molecular typing based on partial VP1 sequence showed a wide diversity of human enterovirus with 16 types detected (Table 1).The HEV-B was the most prevalent.Echovirus 29 (E29) serotype was presented in 5/26 (19.2%) of the EV samples, followed by EC99 in 3/26 (11.5%).The male and female ratio for enterovirus positivity was 1.8:1.Among the 26 participants who had enterovirus positive samples, 18 were from the age group of 0-24 months.The reported signs and symptoms (fever and vomiting/nausea, coughing) of participants show that the clinical manifestations were similar and occurred irrespective of the enterovirus group, HEV-A, B or C, detected (data not shown).
Two samples (32 and 163) were identified Poliovirus sabin like.The complete sequence of the gene encoding the protein VP1 of the two samples identified as Poliovirus Sabin like was performed to detect mutations that may be associated with reversion to virulence of the vaccine strains poliovirus.The sample 32 was identified as Poliovirus Sabin like 2 while sample 163 Poliovirus Sabin like 3 with 99.7% identity with the vaccine strain.Both samples were collected from children at one month of life, the first of which had record of having received polio vaccine and the second had no history of having received the vaccine.
Adenovirus PCR reaction and run electrophoretic run gave a fragment of 134 pb in 20 of the 23 samples.The samples positive for adenovirus was 11.2% (20/178)  (data not shown) and only two samples (75 and 128) showed co-infection between enterovirus and adenovirus and both the species HEV-B.

DISCUSSION
This is the first study that reports the natural circulation of Enterovirus in Mozambique.The isolation in cell culture shows a low permissive cell line RD for members of the species EV-C and confirms the interest of maintaining the cell line Hep2C in the diagnosis for monitoring the circulation of Enterovirus as evidenced by Bessaud and colleagues (2012).
The results of the molecular identification demonstrate the existence of serotypes; the species HEV-B (54%), and within this specie, E29 was most identified.In fact, the HEV-B was the most identified enterovirus in previous studies conducted in Taiwan and Central African Republic (Lo et al., 2010;Bessaud et al., 2012).
A higher frequency of enterovirus isolation in male children was observed although this finding was consistent with the larger number of male participants (1.8:1).Enterovirus infection has been reported more frequently in males compared to females (Moore and Morens, 1984;Gondo et al., 1995;Dechkum et al., 1998).
The phylogenetic analysis of the two samples Poliovirus Sabin like had 99.7% nucleotide identity with strain Poliovirus Sabin 2 and 3 isolated by Rezapkin and colleagues (2002).However, for the sample 32, although the participant had received dose of trivalent oral polio vaccine, this fact can be explained by more efficient replication of live attenuated poliovirus type 2 in the gastrointestinal tract, among serotypes that comprise the vaccine (Modlin, 1995).
In sample 163, according to the information collected during the survey, the participant did not receive the vaccine poliomyelitis.This event is predictable in countries or areas where the trivalent oral polio vaccine is used, which is one of the advantages of oral vaccination by enabling the expansion of vaccine virus in other subjects and the environment, conferring protection by inducing production of antibodies not only serum but also an intestinal resistance and speed with which people vaccinated develop a long immunity (Melnick, 1996;da Silva et al., 2005).
However these findings reinforce the necessity of epidemiological surveillance of poliovirus in Mozambique.In 2011 there was two cases of virus circulation derived polio vaccine (cVDPV) serotype 1 in the region center of the country although the present study was conducted in the southern region of Mozambique (Conceição et al., 2011;MISAU, 2012).
The overall period of study and sample collection was short (November, 2011to February, 2012), which did not allow a seasonal analysis, which would be important in future studies.
Most (70%) of the enterovirus cases were detected in children up to 24 months of age.In fact, these age groups (0-24 months/old) are subject to different susceptibility, disease severity and prognosis of infections and the peak of enterovirus infections occur in the first two years of age (Pallansch and Roos, 2001).
In summary, this finding shows the importance of maintaining the cell line Hep2C in the diagnosis and existence of serotypes of human enteroviruses in natural circulation in Maputo city.
(s)agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Molecular epidemiology of enteroviruses (EVs) is poorly known in Southern Africa, particularly in Mozambique.Therefore, the aim was to isolate and identify human enteroviruses from stool samples of children less than 15 years of age receiving medical attention in Mavalane General Hospital in Maputo city, Mozambique and to understand the natural circulation of HEV.

Table 1 .
Enterovirus serotypes identified, and socio-demographic data of children younger than 15 years old.