The mouse mammary tumor virus (MMTV) Pr77Gag polypeptide is an essential retroviral structural protein without which infectious viral particles cannot be formed. This process requires specific recognition and packaging of dimerized genomic RNA (gRNA) by Gag during virus assembly. Most of the previous work on retroviral assembly has used either the nucleocapsid portion of Gag, or other truncated Gag derivatives—not the natural substrate for virus assembly. In order to understand the molecular mechanism of MMTV gRNA packaging process, we expressed and purified full-length recombinant Pr77Gag-His6-tag fusion protein from soluble fractions of bacterial cultures. We show that the purified Pr77Gag-His6-tag protein retained the ability to assemble virus-like particles (VLPs) in vitro with morphologically similar immature intracellular particles. The recombinant proteins (with and without His6-tag) could both be expressed in prokaryotic and eukaryotic cells and had the ability to form VLPs in vivo. Most importantly, the recombinant Pr77Gag-His6-tag fusion proteins capable of making VLPs in eukaryotic cells were competent for packaging sub-genomic MMTV RNAs. The successful expression and purification of a biologically active, full-length MMTV Pr77Gag should lay down the foundation towards performing RNA–protein interaction(s), especially for structure-function studies and towards understanding molecular intricacies during MMTV gRNA packaging and assembly processes.

MPMV precursor polypeptide Pr78Gag orchestrates assembly and packaging of genomic RNA (gRNA) into virus particles. Therefore, we have expressed recombinant full-length Pr78Gag either with or without His6-tag in bacterial as well as eukaryotic cultures and purified the recombinant protein from soluble fractions of the bacterial cultures. The recombinant Pr78Gag protein has the intrinsic ability to assemble in vitro to form virus like particles (VLPs). Consistent with this observation, the recombinant protein could form VLPs in both prokaryotes and eukaryotes. VLPs formed in eukaryotic cells by recombinant Pr78Gag with or without His6-tag can encapsidate MPMV transfer vector RNA, suggesting that the inclusion of the His6-tag to the full-length Pr78Gag did not interfere with its expression or biological function. This study demonstrates the expression and purification of a biologically active, recombinant Pr78Gag, which should pave the way to study RNA-protein interactions involved in the MPMV gRNA packaging process.

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ObjectivesThe prevalence of hMPV infections in Sri Lanka has not been reported and here we report a case series of hMPV infection in children less than 5 years. Patients with ARTI were included from Teaching Hospital, Anuradhapura from March 2013 to August 2014. Indirect fluorescence assay was performed on nasopharyngeal aspirates for the identification of respiratory viruses [respiratory syncytial virus (RSV), parainfluenza virus 1, 2 and 3, influenza A and B and hMPV]. Moreover, reverse transcriptase-polymerase chain reaction was done to further confirm the hMPV infection.ResultsIn this case series, hMPV infection showed a range of respiratory symptoms from common cold to life threatening lower respiratory tract infections with varying severity. In some cases, the clinical presentation of hMPV infection was similar to the ARTI caused by RSV. hMPV co-infections with of RSV have also been seen in some cases of ARTI. A child delivered through cesarean section and birth order > 3 has an Odds ratio of 3.5 and 4.3 (95% CI) for developing co-infection with RSV compared to hMPV mono-infections. Lack of diagnostic facilities to identify the viral aetiology has contributed to the use of antibiotics indicating the need for establishing viral diagnostic facilities in the country.Electronic supplementary materialThe online version of this article (10.1186/s13104-018-3239-3) contains supplementary material, which is available to authorized users.

Hepatitis B virus (HBV) infection is a major public health concern in many Southeast Asian, South Asian and African countries. HBV infection is transmitted sexually or by other parenteral routes. HBV causes an acute viral hepatitis or chronic infection, largely based on the age at which the infection is acquired. In the majority of infected infants, HBV causes a chronic protracted infection for decades without symptoms. After several decades, the infection might flare up to give rise to an active hepatitis followed by sero-conversion to anti-HBs. However, the vast majority of patients with chronic HBV (CHB) infection will have chronic liver disease. Some with CHB infection develop a primary liver cancer, hepatocellular carcinoma (HCC) later in life. Approximately 25 percent of chronically infected individuals die prematurely of cirrhosis or HCC. The prevalence of HBV infection in Sri Lanka is estimated to be less than 2%. Sri Lanka is therefore considered a country of low endemicity. This prevalence rate of HBV in Sri Lanka is very different to that in many other South Asian countries such as India and Bangladesh. Diagnosis of HBV infection in Sri Lanka is carried out in many private and state laboratories using immunochromatography based rapid assays or ELISAs for HBV surface antigen (HBsAg) detection. Those with latter stages of CHB are treated with currently available nucleotide / nucleoside analogues in some private sector hospitals where testing facilities for virus load during different stages of treatment is available. Sri Lanka has a policy of immunizing healthcare workers (HCW) who are at risk of acquiring HBV occupationally. Sri Lanka included HBV immunization in the Expanded Program of Immunization (EPI) with effect from 2003. In all HBsAg immunization programmes for HCW, testing for the protective anti-HBs response is mandatory in those who receive a complete course of immunization in order to ensure immunity in responders and to re-vaccinate non-responders.

The feline immunodeficiency virus (FIV) full-length Pr50Gag precursor is a key player in the assembly of new viral particles. It is also a critical component of the efficient selection and packaging of two copies of genomic RNA (gRNA) into the newly formed virus particles from a wide pool of cellular and spliced viral RNA. To understand the molecular mechanisms involved during FIV gRNA packaging, we expressed the His6-tagged and untagged recombinant FIV Pr50Gag protein both in eukaryotic and prokaryotic cells. The recombinant Pr50Gag-His6-tag fusion protein was purified from soluble fractions of prokaryotic cultures using immobilized metal affinity chromatography (IMAC). This purified protein was able to assemble in vitro into virus-like particles (VLPs), indicating that it preserved its ability to oligomerize/multimerize. Furthermore, VLPs formed in eukaryotic cells by the FIV full-length Pr50Gag both in the presence and absence of His6-tag could package FIV sub-genomic RNA to similar levels, suggesting that the biological activity of the recombinant full-length Pr50Gag fusion protein was retained in the presence of His6-tag at the carboxy terminus. Successful expression and purification of a biologically active, recombinant full-length Pr50Gag-His6-tag fusion protein will allow study of the intricate RNA-protein interactions involved during FIV gRNA encapsidation.

The present study was carried out to identify the hepatitis B virus (HBV) genotypes in six patients attending the surgical clinic who were positive for hepatitis B surface antigen. DNA was extracted from the serum of patients and subjected to a modified nested PCR to detect a 585 bp region within the S gene of the HBV genome. Positive PCR products were purified and sequenced via a cycle sequencing method. The sequence data were analyzed with reference sequences in the HepSEQ database to identify the particular HBV genotype of the samples. Nested PCR for the S gene of the HBV genome was positive in 2 out of 6 samples. The genotyping and sequence analysis of the PCR products showed HBV genotype A with a homology of 98% to the reference sequences in the HepSeq database.

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