Several Government documents, survey reports and unpublished program documents were studied and online searches were made to find literature on EPI Pakistan. World Health Organization (WHO), United Nations Children’s Fund (UNICEF) and other websites were also explored. The EPI program official database was also analyzed for this study. SPSS 16 and Microsoft Excel 7 were used for the statistical analysis, tabulation and compiling of collected data.

OPV III immunization in all provinces of Pakistan

A “fully immunized child” is one who has received at least 1 dose of Bacilli Calmette-Guérin (BCG) vaccine, 3 doses of oral polio vaccine (OPV), DPT3 and measles1 vaccine. EPI programs target is to immunize children of 0-11 months against seven EPI target diseases. According to EPI surveys 2001, Khyber Pakhtunkhwa was the best performing province with 89% immunization (OPV III) in fighting against polio. The lowest immunization results were in Baluchistan and Gilgit Baldistan with 52% and 34.6% immunization respectively. The EPI surveys are regularly conducted in Pakistan by the Ministry of the Health in collaboration with WHO and other organizations that are active in fight against polio. The main aim of the surveys is to get data about the immunization, to identify the regions that are at the risk so that the special consideration should be given to these areas in planning the programs, in future.

The vaccination campaigns conducted from 2002-2004 shows approximately the same results as in previous exercise in 2001. Azad Jammu and Kashmir (AJK) and KPK were the best performing provinces with 88% and 74% immunization and Baluchistan and Gilgit Baldistan having the lowest immunization 54%, 58% respectively. The main reason for the lowest immunization in Baluchistan and Gilgit Baldistan are the poor infrastructure, low education rate, lack of political well and highly diverse and inaccessible population. In 2005, over all reported immunization was 65%, the immunization campaign highly affected due to severe earthquake in the northern area of the Pakistan, due to which a huge population migrated to the other areas of the country. The surveys conducted during 2006 to 2010 in all the province of Pakistan shows that vaccine immunization has been greatly improved in door-to-door immunization campaigns.

This study is based on EPI (Expanded Program on Immunization) immunization surveys and surveillance of polio, its challenges in immunization and the way forward to overcome these challenges.

Methods

Several Government documents, survey reports and unpublished program documents were studied and online search was made to find information on EPI Pakistan. SPSS 16 and Microsoft Excel 2007 were used for the statistical analysis.

Results
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Immunization against polio is higher in urban areas as compared to rural areas. Marked variation in vaccination has been observed in different provinces of Pakistan in the last decade. Secondly 10-20% of the children who have received their first dose of trivalent polio vaccine were deprived of their 2nd and 3rd dose because of poor performance of EPI and Lack of information about immunization.

Conclusion

In spite of numerous successes, such as the addition of new vaccines and raising immunization to over 100% in some areas, EPI is still struggling to reach its polio eradication goals. Inadequate service delivery, lack of information about immunization and limited number of vaccinators were found to be the key reason for poor performance of immunization and for large number of cases reported each year due to the deficiency of second and third booster dose.
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Introduction

Many epidemics are caused by poliovirus in the last three centuries. About 100,000 new polio cases are reported each year worldwide. Europe and North America were the targets of Epidemic poliomyelitis in 1890s. Now a day most of these cases occur in Asia and Africa. German physician Jakob Heine recognized Poliomyelitis as a distinct condition for the first time in 1840. Poliovirus was identified by Austrian physicians Karl Landsteiner and E. Popper in 1908. The three serotypes 1, 2, and 3 infect cells via a specific receptor, PVR (polio virus receptors): CD-155. These receptors are only present in human cell that is why humans are the only reservoirs of this virus. The serological relationship is present between serotype 1 and serotype 2. This is conferred by significant protection against type 2 by the antibodies which were produced against serotype 1. Immunologically Type 2 is broad and it is the first serotype to disappear during vaccination campaigns. Albert Sabin and Jonas Salk developed effective vaccines against Polio virus in the early 1960s with different approaches. The Sabin oral vaccine which contains live attenuated polio virus is superior to the Salk inactivated vaccine in two ways, firstly it is easily administered; secondly it provides a long-lasting immunization. Pakistan use trivalent OPV that contain all the three serotypes in attenuated form.

While many PVs have been described in humans and other mammalian species, the number of known PVs infecting non-mammalian species is limited. The described MsPV1 genome represents the first PV isolated from a snake. It contains the characteristic ORFs E6, E7, E1, E2, L1 and L2, a large non-coding region between L1 and E6 as well as a small non-coding region between E2 and L2. The size of the viral genome (7048 bp) is comparable with the two turtle PV genomes (CcPV1; 7020 bp and CmPV1; 6953 bp), which are all relatively small. However, while the latter two viruses have short versions of E1, E2, L2 and E6 ORFs as well as the NCR, the small size of MsPV1 is primarily due to comparably short E2 and L2 ORFs and also comparably short NCRs.

The genomic sequences of the PVs from three bird and two turtle species have previously been published. According to our and other’s phylogenetic analysis, they cluster together. However, our newly discovered PV genome isolated from the snake did not at all cluster with the five other sauropsid PVs (CcPV1, CmPV1, FcPV1, FlPV1 and PePV1). Upon pairwise alignment of individual ORFs, MsPV1 shares much higher percentage of sequence identities with mammalian PVs than with any of the known sauropsid PVs. This finding raises interesting questions in the context of PV evolution. While co-evolution with the host has been suggested and demonstrated to play a role in PV evolution it has also been shown that PV evolution is probably a complex matter and other mechanism such as crossing of species barriers and adaptive radiation have to be considered as well.

Turtles as putative representatives of the Anapsida and snakes as representatives of the Diapsida are phylogenetically distinct with a common ancestor dating back more than 200 million years. However, the three PVs isolated from birds (FcPV1, FlPV1 and PePV1), which also belong to the Diapsida, are phylogenetically much closer to the turtle PVs (CcPV1, CmPV1) than to MsPV1. As the snake PV appears closer to mammalian PVs, one explanation could be that it belongs to a lineage of PVs, which existed already in Amniota species, that lived before the split of Synapsida (mammals and mammal-like reptiles) and Sauropsida. The five other sauropsid PVs could under these circumstances go back to a second distinct ancestral lineage. However, an alternative explanation could be that some ancestor of this virus had been able to cross species barriers between the Sauropsida and the Synapsida, even long after their separation.

The nucleotide sequence of cloned DNA and of precipitated RCA product was determined (Microsynth) on both strands by cycle sequencing using an ABI 377 sequencer (Applied Biosystems). The primary sequences were assembled using Contigexpress software (Vector NTI Informax).

Analyses of the cloned sequence confirmed that papillomavirus DNA had actually been detected. The amplified genome consists of 7048 bp and has a GC content of 41%. ORFs putatively encoding E6, E7, E1, E2, E4, L2 and L1 but no E5 were identified. Deduced amino acid sequences of the putative proteins revealed a degenerate ATP-dependent helicase motif GQPNTGKS in E1, two putative metal-binding motifs in E6, one such motif in E7, and one pRb binding domain. The deduced amino acid sequences of both structural proteins L1 and L2 are predicted to harbour a basic tail at their C termini.

A non coding region (NCR1) between the stop-codon of the L1 ORF and the start-codon of the E6 ORF was 473 nt in length. A second non coding region (NCR2) of 178 nt was between the stop-codon of the E2 and the start-codon of the L2 ORF.

In addition, papillomavirus-specific DNA motifs were identified in the readily determined genome sequence. Four putative consensus sequences for E2 binding (ACCN5-7GGT) were detected; two of these were located in the NCR1 (positions 6919-6931 and 32-43) and two were located within the predicted L1 ORF (positions 5936-5948 and 5990-6000). Within the NCR1, a putative origin of DNA replication was identified, consisting of two E2-binding regions flanking a region with 54% A/T content. Two polyadenylation consensus sequences (AATAAA) were predicted, one within the NCR1 (position 6729-6734) and the other within the NCR2 (position 3816-3821).

In order to possibly allocate the novel PV in the evolutionary context, phylogeny based on the aligned E1-E2-L2-L1 sequences was determined. Sequences of fifty PVs, representing all presently classified genera and MsPV1 were included in these analyses. While all other sauropsid PVs clustered together, MsPV1 was located far from any of them. Interestingly, MsPV1 was found in relative proximity to the PV (TmPV1) of a marine mammal, the manatee (sea cow).

The members of the family Papillomaviridae are non-enveloped, icosahedral viral particles with a diameter of 50 to 55 nm and a small (~8 kbp), circular, double stranded DNA genome, which is transcribed into one single direction. The family may be divided into at least 29 genera with a vast number of species, types, subtypes, and variants of papillomaviruses (PVs), based on nucleotide identities of the L1 open reading frames (ORFs). Due to the high genetic diversity and the host range it is anticipated, that the family Papillomaviridae has a long evolutionary history, but details remain yet vague.
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DNA of PVs can be detected in a wide range of vertebrate species, thus far including humans, various other mammals, birds, and turtles. PVs have been found to play a role in several human diseases of the skin and mucous membranes. The DNA of PVs can be amplified from samples of clinical asymptomatic individuals, but more importantly their influence on the development of certain benign and malignant disorders was demonstrated repeatedly.

The sequences of three PVs from birds, namely the Chaffinch (Fringgilla coelebs), the Yellownecked Francolin (Francolinus leucoscepus) and the Timneh African gray parrot (Psittacus erithacus timneh), have been determined. Various data suggest the existence of sauropsid-specific PVs in association with papillomas in lizards, snakes, crocodiles and turtles. Sequences of the entire genome from two PVs of turtles were determined recently from the Loggerhead turtle (Caretta caretta) and the Green seaturtle (Chelonia mydas). Upon phylogenetic analysis, these five sauropsid PVs cluster together and appear clearly distinct from the PVs infecting mammalian species.

Here, we report on the cloning, sequence determination and phylogenetic analysis of a PV-specific DNA from the Australian diamond python (Morelia spilota spilota).

Samples from a diamond python with small black papillated and pedunculated exophytic skin proliferations were taken and stored at -20°C until processing.

S1 genes of the newly strains contain mutations, insertions and deletions, resulting in different lengths of nucleotides. S1 genes of these strains were generated and confirmed from three time sequencing results, contained 1641, 1647, 1650, 1653, 1656, 1659 and 1662 nucleotides, amino acids sequences ranging from 547 (LC strain) to 554 (LC strain). The length differences indicated amino acid insertions and deletions exist among the different strains.

Most variations in the deduced amino acid sequences of Chinese IBVs were observed among residues 63-69, 211-212 and 354-358 (numbering was with reference to S1 sequence of the Mass41 strain).

The precursor protein of S glycoprotein is cleaved into amino-terminal S1 and S2 protein by the protease during viral maturation [9]. In this study, the most common cleavage recognition sites of S1 gene were RRF(S/L) RR (49/80) or HRRRR (28/80) in the China field strains. The exceptional ones included CQ8 (RRTGR), HY52 (RRSKR), and HY2 (RRSKR). The cleavage sites of these two strains containing amino acids K, T, and G, were novel motifs compared to the reference strains, and quite different with the other isolates of the cleavage site.

Phylogenetic analysis of the isolated strains

A phylogenetic tree was constructed from the nucleotides sequences of the S1 glycoprotein genes. The 80 isolates IBV strains were clustered into five distinct genetic groups or genotypes which were considerably heterogeneous, including A2-type (49 newly isolated strains), 4/91-type (9 newly isolated strains), HN08-type (20 newly isolated strains), Gray-type and M41-type. The newly isolated strains mainly belonged to A2-type, 4/91-type and HN08-type branch. The phylogenetic relationship of strains at different times and geographical regions displayed complexity and diversity.

Strains isolated from Hubei, Zhejiang, Jiangsu, Guangdong, Guangxi and Fujian province mainly belonged to the A2 branch, also including other seven published IBV strains from China (QXIBV, CK/CH/LJL/07II, CK/CH/LJS/07IV, CK/CH/LSD/08-12, IBVSX4, LZ05 and LZ07). The isolated strains of Hainan province and a few isolated strains from Guangdong and Fujian province belonged to the HN08 branch, included PSH050513 and CK/CH/LCQ/08II. Group Gray-type was correlative with the American strain (Gray), included other two classical American strains (ARK99 and Holte), one Japanese strain (JP9758), and the exceptional field strain (CQ08). Most of the current vaccine strains (H120, H52, Ma5, M41, W93, 4/91 and 28/86) were belonged to the M41 branch, which including one field strains (NJ). However, the current pandemic strains were mostly 4/91-type, A2-type (QXIBV-type) and HN08-type, indicating that the field IBVs co-circulating in chicken flocks in China were evolutionarily distant from the known vaccine strains.

The previous study by other researchers has been revealed that the variation in S1 sequences was closely confirmed relative to the emergence of novel strains, and S1 gene sequence was a good predictor of challenge of immunity in chickens. This study was conducted to identify the IBV strains that have escaped immune defenses conferred by vaccination in China. The genetic characterization of recent IBV field isolates in China was performed by sequencing the whole S1 genes, sequence alignment and phylogenetic analysis compared with other reference strains.

Results
Eighty IBV strains isolated during 2008-2009 in China

From unhealthy birds suspected of IBV infection in the vaccinated chicken flocks from Guangdong, Guangxi, Fujian, Hainan, Jiangsu, Zhejiang, Chongqing, Hubei, Sichuan and Jiangxi province of China, 80 filed IBV strains were isolated during 2008-2009. The isolation rates in the two years were season-dependent to some extent, 30 strains were isolated in October, while only seven strains were isolated in summer (from June to August). The ages of flocks at the time of the outbreak varied between 4 and 69 days. Most of the strains were isolated from the chickens between 10 to 30 days of age.

After three passage propagation, IBVs of all isolates induced peripheric lesions and growth retardation of embryo at 72 h post-inoculation. Since the fourth day post-inoculation, most of the chicks were listless and huddled together, showed ruffled feathers. The results of virus recovery in chicks indicated 87.5% (70/80) isolates caused serious kidney lesions, which were presented with swollen specked kidney and distended ureters filled with uric acid were nephropathogenic type, and the other ten isolates in the study caused respiratory system signs, which were consistent with the clinical record of each strain.

Homologies among S1 nucleotide and deduced amino acid sequences

The obtained strains were characterized phylogenetically by nucleotide sequence analysis of the hyper-variable S1 gene of IBV. The nucleotide and amino acid sequence similarities between the eighty IB strains were ranging from 75.4% (strain CQ8 and HY) to 100% (strain PT1 and PT3) and 73.9% to 100%, respectively. Compared to the 28 reference strains published in the GenBank, the identity of the nucleotide and amino acid sequence among the 108 isolates (including the 80 isolates in this study plus the 28 reference strains) were 75.1 to 99.8% and 73.1 to 99.8%, respectively, indicating low homology and high variation among the isolated and reference strains.

Methods

Virus and cells

The primary isolate, ANDV strain CHI-7913 was propagated in the epithelial Vero-E6 cell line (ATCC CRL 1586). Titrated supernatants of these cells were used to infect, at a MOI of 1 for 2 h, human iDCs derived from peripheral blood monocytes (PBMC), as previously described. In these experiments, UV (λ: 250 nm; 15 min)-irradiated ANDV was used as the negative control. Four days post-DC infection, ANDV N-protein was detected by indirect immunofluorescence (IFA) using a well characterized anti-ANDV N monoclonal antibody (MAb). Total RNA was extracted using the High Pure viral nucleic acid kit (Roche Molecular Biochemicals, Mannheim, Germany) following the manufacture’s protocol and 1 μl of total RNA was amplified in a one step RT-PCR (SuperScript III One-Step RT-PCR with Platinum Taq, Invitrogen) using primers that recognize the nucleocapsid coding region (forward primer: 5′ ACA CGA ACA ACA GCT CGT GAC ‘3 and reverse primer: 5′ AGG CTC AAG CCC TGT TGG ATC ‘3). To assess the viral infectivity, from ANDV-positive DCs, their supernatants were used to infect Vero-E6 cells.

Andes virus (ANDV), a rodent-borne Hantavirus, is the major etiological agent of Hantavirus cardiopulmonary syndrome (HCPS) in South America, which is mainly characterized by a vascular leakage with high rate of fatal outcomes for infected patients. Currently, neither specific therapy nor vaccines are available against this pathogen. ANDV infects both dendritic and epithelial cells, but in despite that the severity of the disease directly correlates with the viral RNA load, considerable evidence suggests that immune mechanisms rather than direct viral cytopathology are responsible for plasma leakage in HCPS. Here, we assessed the possible effect of soluble factors, induced in viral-activated DCs, on endothelial permeability. Activated immune cells, including DC, secrete gelatinolytic matrix metalloproteases (gMMP-2 and -9) that modulate the vascular permeability for their trafficking.

Methods

A clinical ANDES isolate was used to infect DC derived from primary PBMC. Maturation and pro-inflammatory phenotypes of ANDES-infected DC were assessed by studying the expression of receptors, cytokines and active gMMP-9, as well as some of their functional status. The ANDES-infected DC supernatants were assessed for their capacity to enhance a monolayer endothelial permeability using primary human vascular endothelial cells (HUVEC).

Results

Here, we show that in vitro primary DCs infected by a clinical isolate of ANDV shed virus RNA and proteins, suggesting a competent viral replication in these cells. Moreover, this infection induces an enhanced expression of soluble pro-inflammatory factors, including TNF-α and the active gMMP-9, as well as a decreased expression of anti-inflammatory cytokines, such as IL-10 and TGF-β. These viral activated cells are less sensitive to apoptosis. Moreover, supernatants from ANDV-infected DCs were able to indirectly enhance the permeability of a monolayer of primary HUVEC.

Conclusions

Primary human DCs, that are primarily targeted by hantaviruses can productively be infected by ANDV and subsequently induce direct effects favoring a proinflammatory phenotype of infected DCs. Finally, based on our observations, we hypothesize that soluble factors secreted in ANDV-infected DC supernatants, importantly contribute to the endothelial permeability enhancement that characterize the HCPS.

Background

Hantaviruses are rodent-born enveloped RNA-viruses belonging to Bunyaviridae family. Two major severe pathologies associated to Hantaviruses have been reported: hemorrhagic fever with renal syndrome (HFRS) in the Eurasia and Hantavirus cardiopulmonary syndrome (HCPS) in the Americas. HCPS is more frequently associated (40%) to fatal outcomes than HFRS (<1%). Andes Hantavirus (ANDV) is the major etiological agent of the HCPS in South America, syndrome characterized by the presence of high amounts of pulmonary fluids leading to an edema evolving to a cardiogenic shock that synergistically acts with hypovolemia due to capillary leakage resulting in an abrupt cardiopulmonary collapse. Although disease severity directly correlates with the viral RNA load [3], considerable evidence exists suggesting that immune mechanisms rather than direct viral cytopathology are indeed responsible for the massive vascular dysfunction and plasma leakage of HFRS and HCPS.

% of conversion = (mono- + di-acetylated forms)/(non acetylated + mono- + di-acetylated forms). Calculated with the above formula, the percentage of conversion is linear for values up to 50%. As expected, the X4-tropic virus (HXBc2 env) does not infect efficiently MDMs, while the dual tropic strain (89.6 env) displays the higher efficiency of infection in all the conditions tested. Our data demonstrate that, following HSV-2 infection, the ability of HIV-1 to superinfect target cells is overall enhanced. The effect is statistically significant (p < 0.05) when R5-tropic recombinant viral particles are examined, correlating this observation, at least partially, with the HSV-2 related increase in CCR5 expression levels on MDMs surface. Not surprisingly, the 89.6 dual tropic strain was extremely efficient in macrophage infection and the observed slight increase in HIV-1 entry upon HSV-2 infection was not statistically significant (p > 0.05).