Sequence diversity of Jeryl Lynn strain of mumps virus: quantitative mutant analysis for vaccine quality control

The Jeryl Lynn strain of mumps vaccine live (MVL) was developed in 1966 by Merck Co. and has been widely used in the U.S. and other countries since the early 1970s. Partial sequencing has recently shown that the vaccine contains a mixture of two substrains with substantially different nucleotide sequences. We have determined the complete genomic sequences of both substrains and identified 414 nucleotide differences (2.69%), leading to 87 amino acid substitutions (1.67%). We used this information to develop methods for quantification of the substrain components in vaccine samples based on PCR and restriction enzyme cleavage and oligonucleotide microarray hybridization and monitored their dynamics in viral populations propagated in different conditions. Passaging Jeryl Lynn strain in Vero or CEF cell cultures resulted in rapid selection of the major component JL1, while growth in embryonated chicken eggs (ECE) favored accumulation of the minor component JL2. Based on the findings presented here, it is proposed that the substrain composition of Jeryl Lynn vaccine can be monitored as a part of its quality control to ensure consistency of the vaccine.     

Molecular characterisation of varicella-zoster virus strains in Germany and differentiation from the Oka vaccine strain

With the introduction of varicella vaccination, surveillance of varicella-zoster virus (VZV) strains occurring in cases of chickenpox or zoster should be considered. Differentiating Oka vaccine strain from wild-type VZV can be achieved only using molecular genotyping. In the present study, the VZV genotype was examined in 53 VZV strains isolated from patients with varicella or zoster and in 73 samples from skin eruptions, cerebrospinal fluid, and throat swabs obtained from patients with VZV infections in Germany. The polymerase chain reaction and restriction fragment length polymorphisms analysis using DNA fragments of the open reading frames 38, 54, 62, and the R5 repeat region were used. Whereas all VZV isolates could be typed, direct genotyping of viral DNA in patients' samples was achieved in 63 of 73 cases (86.3%). The dominant genotype of VZV found in 88.8% of 116 patients had the wild-type pattern PstI(+) BglI(-) R5A followed by the wild-genotype PstI(+) BglI(+) R5A in 6.0%, the wild-genotype PstI(+) BglI(-) R5B in 3.4%, the wild-genotype PstI(+) BglI(-) R5C and the Oka vaccine genotype PstI(-) BglI(+) R5B in 0.9% of patients each. BglI(-) wild-types were found in 90.7% of patients with zoster and in 9.3% of patients with varicella. By contrast, the BglI(+) wild-type was diagnosed in five patients with varicella and in two patients with zoster. In conclusion, VZV strains found in Germany are similar to strains circulating in the United States and the United Kingdom. VZV wild-type strains containing a BglI restriction site in ORF 54 as well as Oka vaccine strains can rarely be detected.     

Comparative analysis of hepatitis B virus genotype a molecular evolution in patients infected with HBV and in patients co-infected with HBV and HIV

HIV infection has a significant impact on the natural progression of liver disease caused by infection with hepatitis B virus (HBV), but its role in the molecular evolution of HBV is unknown. It is difficult to study the molecular evolution of HBV longitudinally considering its genomic complexity, which implies the analysis of paired samples. This study aimed to analyze the difference in the evolutionary dynamics of HBV among patients with HIV and uninfected individuals. In this study, 17 patients infected chronically with HBV were recruited, 9 of them were co-infected with HIV. Patients were HBe antigen-positive and infected with HBV genotype A. Paired plasma samples were collected from each patient 3 years apart, and they were compared subsequently to each other. The HBV phylogenetic inference among isolates from patients infected with HBV and co-infected with HBV and HIV tends to cluster separately. Likewise, when comparing the HBV evolutionary rate and genetic distances, values were higher in the former in both preC/C and S genomic regions. Intra-host analyses of HBV isolates revealed high diversity and complexity of quasispecies among patients infected with HBV exhibiting high numbers of viral variants and genetic distance. In summary, after studying the HBV molecular evolution among isolates ascribed to genotype A at inter- and intra-host levels, HBV exhibited low quasispecies complexity and diversity as well as low evolutionary rates in the presence of HIV co-infection, suggesting that the co-infection may have an impact on the HBV molecular evolution most likely from the weakened cellular immune response.     

Antigenic and molecular analysis of influenza A (H3N2) virus strains isolated from a localised influenza outbreak in South Africa in 2003

A severe acute institutional influenza outbreak occurred in a police residential college in Pretoria amongst new recruits and staff members at the end of May 2003. The outbreak was characterised by marked illness which affected a total of 648 students, 26 of whom were admitted to hospital. Symptoms included pyrexia, severe headache, and myalgia. The attack rate per dormitory building ranged from 20 to 47%, with an overall attack rate of 34%. Throat swabs and bronchoalveolar lavage specimens were sent to the National Institute for Communicable Diseases (NICD) from 20 patients. All were positive for influenza A by multiplex PCR and/or indirect immunofluorescence, and were further identified as subtype H3N2. Additional specimens from sporadic influenza cases in Johannesburg and surrounding areas were collected through the NICD active viral surveillance programme for respiratory viral testing and were also positive for influenza A H3N2 viruses. Viruses isolated from patients from both the institutional outbreak as well as from sporadic cases were analysed both antigenically and at the molecular level to determine the characteristics of the influenza strain responsible for the epidemic. The results showed clearly that the outbreak was caused by the introduction in 2003 into South Africa of the novel A/Fujian/411/02-like H3N2 influenza strain, which is antigenically distinct from the A/Panama/2007/99 vaccine strain. The rapid spread of these variant viruses to the southern hemisphere indicates that the H3N2 component of the influenza vaccine needs to be updated for the 2004 southern hemisphere winter.     

Measles virus strains circulating in Ethiopia in 1998-1999: molecular characterisation using oral fluid samples and identification of a new genotype

A measles outbreak in December 1998 in Bedelle (vaccine coverage <40%) and two sporadic cases in Addis Ababa, Ethiopia, were investigated. Paired serum and oral fluid samples were collected 2-8 days after the onset of symptoms. A total of 53 of 55 outbreak cases and both sporadic cases were positive for serum measles virus-specific IgM. Oral fluid measles-specific IgM was positive in 71% of cases collected up to 5 days after onset and in 90% collected at 6-8 days. By contrast, 100% of oral fluid samples were positive for measles virus RNA by RT-PCR, suggesting that early collection of samples favoured the detection of measles virus RNA by RT-PCR. The measles virus strain in the outbreak was identified as genotype D4. One strain from a sporadic case was also genotype D4; the strain from the other sporadic case was assigned to clade D but was distinct. The degree of divergence from recognised clade D strains suggested that, together with three strains from the United Kingdom, it represents an additional genotype of clade D (GenBank accession numbers AF280800-280807).