Genotype replacement of the human parainfluenza virus type 2 in Croatia between 2011 and 2017 - the role of neutralising antibodies.

Previously we reported on the HPIV2 genotype distribution in Croatia 2011-2014. Here we expand this period up to 2017 and confirm that G1a genotype has replaced G3 genotype from the period 2011-2014. Our hypothesis was that the G1a-to-G3 genotype replacement is an antibody-driven event. A cross-neutralisation with anti-HPIV2 sera specific for either G1a or G3 genotype revealed the presence of genotype-specific antigenic determinants. By the profound, in silico analyses three potential B cell epitopic regions were identified in the hemagglutinin neuraminidase (regions 314-361 and 474-490) and fusion protein (region 440-484). The region identified in the fusion protein does not show any unique site between the G1a and G3 isolates, five differentially glycosylated sites in the G1a and G3 genotype isolates were identified in epitopic regions of hemagglutinin neuraminidase. All positively selected codons were found to be located either in the region 314-316 or in the region 474-490 what indicates a strong positive selection in this region and reveals that these regions are susceptible to evolutionary pressure possibly caused by antibodies what gives a strong verification to our hypothesis that neutralising antibodies are a key determinant in the inherently complex adaptive evolution of HPIV2 in the region.

Low concentration of PDGF-AB shows synergism with IFN-α in induction of IFN-ß and -γ in MRC5 fibroblasts.

Platelet-derived growth factor (PDGF) is a potent mediator of fibroblast proliferation and chemotaxis. Also it has been reported as a strong suppressor of interferon (IFN) expression. IFN-α has opposite effect on fibroblast function and IFN induction. Here is our early report on the effect of low concentration of PDGF-AB alone or in combination with IFN-α on IFN mRNA production in MRC5 fibroblasts. MRC5 cells incubated with IFN-α or PDGF-AB, alone or in combination, produced significant induction of IFN-α, -ß and -γ mRNA in comparison with untreated cells. The induction was dose-dependent, with higher effect in cells treated with lower concentrations of PDGF-AB. Also, low concentration of PDGF-AB showed synergism with IFN-α in IFN-ß and -γ induction. Results presented here open new possibilities in multi-cytokine therapy and expand previous results on PDGF activity.

Native human interferon-α is a strong inductor of endogenous cytokines involved in the suppression of procollagen type I.

Native human interferon-α (nHuIFN-α) has a stronger reductive effect on procollagen type I mRNA expression than recombinant human interferon-α (rHuIFN-α). It is partially due to the additive activity of interleukin-1ß (IL-1ß), which is present in small concentrations in nHuIFN-α. Here, we show that the reductive effect is also the result of the endogenous cytokines induced by the activity of nHuIFN-α. In the culture of MRC5 fibroblasts, we have further found that nHuIFN-α induces endogenous interferons in higher amounts than rHuIFN-α, measured with PCR. That is more pronounced when interferon-γ (IFN-γ) is measured. This result puts a new light on IFN-γ activity in the nHuIFN-α treatment because its role was neglected due to the loss of its activity during the nHuIFN-α preparation process. The findings lead to the conclusion that endogenous cytokines play a significant role in the nHuIFN-α -mediated reduction of procollagen type I mRNA and are therefore an important factor in potential therapeutic usage.

Antigenic differences between vaccine and circulating wild-type mumps viruses decreases neutralization capacity of vaccine-induced antibodies.

A recent resurgence of mumps in doubly vaccinated cohorts has been observed, identifying genotype G as the current predominant genotype. In this study, the neutralization efficacy of guinea pig sera immunized with three vaccine viruses: L-Zagreb, Urabe AM9 and JL5, was tested against seven mumps viruses: three vaccine strains and four wild-type strains (two of genotype G, one of genotype C, one of genotype D) isolated during 1998-2011. All sera neutralized all viruses although at different levels. The neutralization efficiency of sera decreases several fold by temporal order of virus isolation. Therefore, we concluded that gradual evolution of mumps viruses, rather than belonging to a certain genotype, results in an antigenic divergence from the vaccine strains that decrease the neutralization capacity of vaccine-induced antibodies. Moreover, the amino-acid sequence alignment revealed three new potentially relevant regions for escape from neutralization, i.e. 113-130, 375-403 and 440-443.

Spotlight on measles 2010: excretion of vaccine strain measles virus in urine and pharyngeal secretions of a child with vaccine associated febrile rash illness, Croatia, March 2010.

We describe excretion of measles vaccine strain Schwarz in a child who developed a febrile rash illness eight days after primary immunisation against measles, mumps and rubella. Throat swabs and urine specimens were collected on the fifth and sixth day of illness, respectively. Genotyping demonstrated measles vaccine strain Schwarz (genotype A). If measles and rubella were not under enhanced surveillance in Croatia, the case would have been either misreported as rubella or not recognised at all.

Detection of hepatitis C virus RNA in alpha interferon derived from in vitro culture of leukocytes of human origin.

In order to assure the virological safety of blood products, in addition to serological testing of individual donations and virus inactivation steps undertaken during manufacture, routine PCR testing for HCV RNA of starting materials (plasma, cells), intermediates or final product is necessary. The aim of this study was to determine the rate of HCV RNA positive batches of human native leukocyte interferon during large-scale production. Our findings indicate the presence of HCV RNA in 6.1% batches despite acidification of intermediates in order to inactivate Sendai virus.

Incidence of hepatitis C virus RNA in anti-HCV negative plasma pools in Croatia.

The risks of transmitting viral infection by blood and products derived from plasma have long been known and still remain an area of concern. Blood banks and transfusion centres are faced with the imminent introduction of nucleic acid amplification testing (NAT) of plasma pools as used by the plasma industry. In this paper, we show a part of our results of a validation study of an in-house method for routine polymerase chain reaction (PCR) screening for hepatitis C virus (HCV) RNA in plasma pools and the results of testing 2,718 anti-HCV negative plasma pools for the presence of HCV RNA. The European Committee for Proprietary Medical Products (CPMP) recommended that from 1 July 1999, only batches derived from plasma pools tested and found non-reactive for HCV RNA, using validated test methods of suitable sensitivity and specificity, should be batch released by authorities. The quality and efficiency of NAT detection of HCV RNA is among others influenced by the efficacy of RNA isolation, the primer selection and the use of control samples. Using modern molecular biology techniques (sensitive and specific in-house amplification methods for detection of HCV RNA and automated sequencing), we analysed samples of plasma pools from different Croatian transfusion centres. By detection of HCV RNA in an NIBSC working reagent (genotype 3) and a Pelispy HCV RNA run control (genotype 1) we determined a high reproducibility and sensitivity (below 100 International Units (IU)/ml) for our in-house method. By direct sequencing PCR cDNAs we proved the specificity of the test system and the possibility of determining the HCV genotype when the method was used for PCR screening of HCV RNA in single donations. Of 2,718 anti-HCV negative plasma pools we have found that 2.1$ were HCV RNA positive. Results of our investigation confirm the necessity of testing HCV RNA in plasma pools to further increase the safety of human plasma-derived drugs.

Comparative study of the effects of peptidoglycan monomer and structurally related adamantyltripeptides on humoral immune response to ovalbumin in the mouse.

Peptidoglycan monomer, GlcNAc-MurNAc-L-Ala-D-isoGln-mesoDAP(omega NH2)-D-Ala-D-Ala (PGM) originating from Brevibacterium divaricatum and synthetic adamantyltripeptides, diastereoisomers of D,L-(adamant-2-yl)-Gly-L-Ala-D-isoGln (AdTP1 and AdTP2) exhibit immunomodulating activity. An experimental model in the mouse has been established with suboptimal amounts of ovalbumin (OVA) as the immunogen, and parallel testing of adjuvant activity of these three immunomodulators was carried out in Balb/c, C57B16 or CBA mice. Tested compounds (100 or 200 micrograms/mouse) mixed with OVA in saline (50 micrograms/mouse) were administered s.c. Anti-OVA was assayed by ELISA in the sera of mice taken 7 days after the boosters (given on days 14 and 28). The treatment with PGM and one of the diastereoisomers, AdTP2, resulted in significantly higher increase in anti-OVA IgG levels (stimulation index up to 46) with respect to controls and groups treated with AdTP1. The effect of AdTP2 treatment was comparable to that of PGM in most experiments after the first booster, but after the second booster PGM exhibited markedly better effect. PGM and AdTP2 also induced markedly higher levels of IgG1 and IgG2 anti-OVA subclasses than detected in controls and AdTP1 treated mice, indicating that these two immunomodulators might upregulate both Th1-like and Th2-like immune responses.

Introduction of the gene amplification technique to decrease the risk of hepatitis C virus transmission by plasma products.

The viral safety of plasma-derived products with respect to hepatitis C virus (HCV) is assured by selection of donors, screening of individual donations for antibodies to HCV and the incorporation of effective viral inactivation-removal steps into manufacturing processes. As antibody screening of single donations is not sufficient to completely eliminate HCV RNA positive plasmas from plasma pools, testing for HCV RNA by gene amplification techniques may be necessary to identify positive donations. Using modern molecular biology techniques, we developed a specific, sensitive and reproducible method for routine PCR screening for HCV RNA in plasma pools.