Establishment and cryptic transmission of Zika virus in Brazil and the Americas.

Transmission of Zika virus (ZIKV) in the Americas was first confirmed in May 2015 in northeast Brazil. Brazil has had the highest number of reported ZIKV cases worldwide (more than 200,000 by 24 December 2016) and the most cases associated with microcephaly and other birth defects (2,366 confirmed by 31 December 2016). Since the initial detection of ZIKV in Brazil, more than 45 countries in the Americas have reported local ZIKV transmission, with 24 of these reporting severe ZIKV-associated disease. However, the origin and epidemic history of ZIKV in Brazil and the Americas remain poorly understood, despite the value of this information for interpreting observed trends in reported microcephaly. Here we address this issue by generating 54 complete or partial ZIKV genomes, mostly from Brazil, and reporting data generated by a mobile genomics laboratory that travelled across northeast Brazil in 2016. One sequence represents the earliest confirmed ZIKV infection in Brazil. Analyses of viral genomes with ecological and epidemiological data yield an estimate that ZIKV was present in northeast Brazil by February 2014 and is likely to have disseminated from there, nationally and internationally, before the first detection of ZIKV in the Americas. Estimated dates for the international spread of ZIKV from Brazil indicate the duration of pre-detection cryptic transmission in recipient regions. The role of northeast Brazil in the establishment of ZIKV in the Americas is further supported by geographic analysis of ZIKV transmission potential and by estimates of the basic reproduction number of the virus.

Standardization of Nucleic Acid Tests: the Approach of the World Health Organization.

The first World Health Organization (WHO) international standards (ISs) for nucleic acid amplification techniques were established two decades ago, with the initial focus on blood screening for three major viral targets, i.e., hepatitis C virus, hepatitis B virus, and human immunodeficiency virus 1. These reference materials have subsequently found utility in the diagnosis and monitoring of a wide range of infectious diseases in clinical microbiology laboratories worldwide. WHO collaborating centers develop ISs and coordinate international studies for their evaluation. The WHO Expert Committee on Biological Standardization is responsible for the endorsement of new standardization projects and the establishment of new and replacement ISs. Potencies of ISs are defined in international units (IU); the reporting in IU for assays calibrated with an IS (or secondary standards traceable to the IS) facilitates comparability of results for different assays and determination of assay parameters such as analytical sensitivities.

Unusual serological response to hepatitis E virus in plasma donors consistent with re-infection.

Hepatitis E virus (HEV)-positive plasma donations, identified by a plasma mini-pool screening approach, were analysed using serological methods for the presence of anti-HEV IgM and IgG. Avidity testing was performed on the IgG-reactive donations. Anti-HEV IgG with high avidity was observed in two donors together with high viral loads, but with the absence of anti-HEV IgM. These data are suggestive of re-infection in a small proportion of plasma donors, which has not previously been reported.

Collaborative study to establish a World Health Organization International genotype panel for parvovirus B19 DNA nucleic acid amplification technology (NAT)-based assays.

BACKGROUND AND OBJECTIVES: The aim of the collaborative study was to evaluate a panel of plasma samples containing different genotypes of parvovirus B19 (B19V) for use in nucleic acid amplification technology (NAT)-based assays. MATERIALS AND METHODS: The panel of samples [Center for Biologics Evaluation and Research Parvovirus B19 Genotype Panel 1; National Institute for Biological Standards and Control (NIBSC) code number 09/110] comprises four different members, i.e. Member 1, Member 2, Member 3, and Member 4 (M1-M4); these represent genotypes 1, 2, 3a B19V, and a negative plasma control, respectively. Thirty-five laboratories from 13 different countries participated in the study. Participants assayed the panel members concurrently with the 2nd World Health Organization (WHO) International Standard for B19V DNA (NIBSC code 99/802) on four separate occasions. RESULTS: A total of 44 sets of data were returned, 34 from quantitative assays and 10 from qualitative assays. The majority of assays used were in-house and based on real-time PCR. The results showed that all three genotypes were detected consistently by the majority of participants, although a small number of assays detected genotypes 2 and 3 less efficiently, or not at all. Real-time stability studies have indicated that the panel of B19V samples is stable under normal conditions of storage, i.e. ≤-70°C. CONCLUSIONS: The four-member panel is intended for use in evaluating the ability of NAT assays to detect different B19V genotypes (M1-M3). Based on the results of the collaborative study, the panel was established as the 1st WHO International Reference Panel for parvovirus B19 genotypes.

World Health Organization collaborative study to calibrate the 3rd International Standard for Hepatitis C virus RNA nucleic acid amplification technology (NAT)-based assays.

BACKGROUND AND OBJECTIVES: A collaborative study was undertaken to evaluate a replacement World Health Organization International Standard for hepatitis C virus (HCV) RNA for nucleic acid amplification technology (NAT)-based assays. The candidate preparations were calibrated in International Units (IUs). MATERIALS AND METHODS: Three new candidate preparations were produced from a single bulk containing anti-HCV-negative, genotype 1a HCV RNA-positive plasma. Two samples were lyophilized (coded Sample 2 and Sample 3), whilst a third (Sample 4) contained liquid/frozen material. The samples were distributed together with the 2(nd) International Standard (Sample 1, NIBSC code 96/798) for evaluation by thirty-three laboratories, from fourteen countries. The panel of samples were assayed on four separate occasions. Stability studies were performed for the lyophilized samples by accelerated thermal degradation. RESULTS: Participants returned data from a wide range of commercial and in-house quantitative and qualitative assays. Twenty-five data sets were returned for quantitative assays and fourteen for qualitative assays. Excellent agreement was observed between laboratories and assay methods. The mean relative potencies of Samples 2-4 were 5·19, 5·41 and 5·70 log(10) IU/ml, respectively, when compared against the 2(nd) International Standard. Samples 2 and 3 demonstrated stability of a similar order to the previous standards. CONCLUSIONS: Based upon the results of the collaborative study, Sample 2 (code number 06/100) was established as the 3rd International Standard for HCV RNA with an assigned unitage of 5·19 log(10) IU/ml. Each vial contains the equivalent of 0·5 ml of material; each vial contains 4·89 log(10) IU of HCV RNA.

Collaborative study to establish a replacement World Health Organization International Standard for parvovirus B19 DNA nucleic acid amplification technology (NAT)-based assays.

BACKGROUND AND OBJECTIVES: The aim of the study was to replace the 1(st) World Health Organization International Standard for parvovirus B19 DNA for nucleic acid amplification technique (NAT)-based assays (code 99/800). Two lyophilized preparations (coded 99/800 and 99/802) had been evaluated in the original collaborative study. The present study re-evaluates these two preparations in terms of potency, stability and encapsidation of virus DNA. MATERIALS AND METHODS: The 1(st) International Standard (99/800) and 99/802 were re-coded as Samples 1 and 2, respectively. The samples were distributed to six laboratories and assayed on four separate occasions. Accelerated thermal degradation samples of the two preparations were examined after storage at 20 degrees C for 7 years. Nuclease treatment was used to investigate the encapsidation of virus DNA. RESULTS: Data were returned from a total of six different quantitative NAT-based assays. The results of the present study confirm those of the original, with no significant differences found in estimated international units (IU)/ml for the 1(st) International Standard (Sample 1 in this study) and the proposed replacement preparation, Sample 2 (99/802). Accelerated thermal degradation studies demonstrate that both samples are very stable, with no loss of potency after storage at 20 degrees C for 7 years. Both lyophilized preparations contained the majority of B19V DNA encapsidated in virions. CONCLUSIONS: On the basis of the data presented in this collaborative study, Sample 2 (code number 99/802) was established as the 2(nd) International Standard for parvovirus B19 DNA for NAT-based assays with a potency of 10(6) IU/ml (500 000 IU/vial).

A proficiency testing study to evaluate laboratory performance for the detection of different genotypes of parvovirus B19.

BACKGROUND AND OBJECTIVES: In Europe, it is a regulatory requirement that parvovirus B19 (B19V) DNA nucleic acid amplification technique-based testing is performed on plasma pools for certain classes of plasma-derived medicinal products. This proficiency testing study set out to examine the ability of public quality control laboratories and plasma fractionation organizations to detect different genotypes of B19V using nucleic acid amplification technique-based assays. MATERIALS AND METHODS: Laboratories were supplied with cloned DNAs representing the main genotypes of B19V. All samples were adjusted to equivalent copy number and were distributed as part of a routine external quality assessment programme investigating the evaluation of B19V containing plasma samples by these laboratories. The plasmid clones were distributed to 25 laboratories, representing 13 quality control laboratories and 12 manufacturers of plasma derivatives. The criteria for acceptable detection of the different genotypes of B19V DNA was based upon the maximum theoretical efficiency for polymerase chain reaction amplification. Proficient laboratories were deemed to be those reporting results within 1 log(10) dilution for each of the different virus genotypes. RESULTS: Data were returned by 23 of the participating laboratories. Some laboratories returned data for more than one type of assay and in total 27 data sets were analysed. Nine of the participating laboratories were able to successfully detect all the virus genotypes according to the applied criteria, all except one used in-house assays. CONCLUSIONS: The results of the study highlight that there are still discrepancies in the detection of a broad spectrum of B19V genotypes, with implications for batch release testing.

Frequency of contamination of coagulation factor concentrates with novel human parvovirus PARV4.

Human parvovirus, PARV4 was identified in a plasma sample from a patient presenting with symptoms resembling acute HIV infection. Further strains of PARV4 and those of a closely related variant virus, were identified in plasma pools used in the manufacture of blood derivatives. DNA sequence analysis of these strains demonstrated two distinct PARV4 genotypes. It has subsequently been proposed that transmission of PARV4 occurs by parenteral routes. To investigate the risk of contamination of plasma-derived coagulation factor concentrates, we analysed 169 lots for PARV4 DNA by polymerase chain reaction. Positive samples were confirmed by nucleotide sequence analysis and quantification of the viral load. Twenty-one lots, representing eight different products were administered until the beginning of the 1980s and were not virally inactivated. Two lots examined were used in 1997, and 146 lots representing 13 products had been administered between October 2000 and February 2003. PARV4 DNA was detected in 7(33%) of the formerly administered lots, in one lot used in 1997, and in 13(9%) recently used lots. PARV4 genotype 2 DNA was predominantly present in the older concentrates, whilst genotype 1 was found more frequently in recently used lots. In three lots, both PARV4 genotypes were detected. Viral loads ranged between <100 and 10(5.8) copies mL(-1) of product, with higher viral loads in the older concentrates. The results show that PARV4 contamination can be detected in an appreciable proportion of clotting factor concentrates. Further studies are needed to determine whether or not PARV4 contamination of coagulation factors causes harm to the product recipients.

An international collaborative study to establish the 2nd World Health Organization International Standard for hepatitis B virus DNA nucleic acid amplification technology-based assays.

BACKGROUND AND OBJECTIVES: The aim of this study was to replace the 1st World Health Organization International Standard for hepatitis B virus DNA for nucleic acid amplification technique (NAT)-based assays (code 97/746) with a new International Standard. Two lyophilized preparations freeze dried from the same bulk were evaluated in the original collaborative study (coded 97/746 and 97/750, and termed AA and BB, respectively, in the original study). This present study re-evaluates these two preparations in terms of potency and real-time stability. MATERIALS AND METHODS: The 1(st) International Standard (97/746) and the second lyophilized preparation (97/750) were coded Samples 1 and 2, respectively, in the present study. The samples were distributed to six laboratories and assayed on four separate occasions. Accelerated thermal degradation samples of the two preparations were examined after long-term storage at 4 degrees C and 20 degrees C for more than 51 months. RESULTS: Data were returned from a total of nine different NAT-based assays, five in qualitative format and four in quantitative format. The results of this study confirm the results of the original collaborative study, with no significant differences being found in estimated international units (IU)/ml or polymerase chain reaction-detectable units/ml for the 1(st) International Standard (Sample 1 in this study) and the proposed replacement preparation, Sample 2 (97/750). Real-time and accelerated degradation studies indicate that both samples are very stable. Storage of both preparations at 20 degrees C for more than 51 months resulted in no detectable degradation. CONCLUSIONS: On the basis of the data presented in this collaborative study, Sample 2 (code 97/750) was established as the 2nd International Standard for hepatitis B virus DNA for NAT-based assays with a potency of 10(6) IU/ml (500,000 IU/vial).

Expression and localization of inducible and endothelial nitric oxide synthase in the rat ovary. Effects of gonadotropin stimulation in vivo.

Nitric oxide is reportedly involved in the regulation of several ovarian processes, yet the isoforms of nitric oxide synthase (NOS) expressed in the ovary are unknown. Our purpose was to identify and localize NOS isoenzymes in the rat ovary and to examine++ if mRNA expression of NOS isoenzymes change after gonadotropin stimulation. Using reverse transcriptase-PCR, we demonstrated that inducible (iNOS) and endothelial (eNOS), but not neuronal, NOS mRNAs are expressed in the ovary. In a gonadotropin-stimulated rat model, unstimulated ovaries had the highest levels of iNOS mRNA as quantified by ribonuclease protection assay. After gonadotropin injection, iNOS mRNA declined to undetectable levels in ovaries containing ovulatory follicles before increasing slighty in ovaries containing copora lutea. In situ hybridization studies localized iNOS to granulosa cells of secondary follicles and small antral follicles. Western blots of unstimulated ovaries demonstrated iNOS protein. In contrast to iNOS, eNOS mRNA levels, determined by quantitative PCR, increased after gonadotropin stimulation and peaked in ovaries containing ovulatory follicles before declining in the luteal phase. eNOS protein was localized to blood vessels in the ovary by immunohistochemistry. We conclude that two isoforms of NOS are expressed in the ovary and the mRNA levels for these isozymes are differentially regulated.

Collaborative study for the establishment of the Ph. Eur. Hepatitis E virus RNA for NAT testing biological reference preparation batch 1.

A new European Pharmacopoeia (Ph. Eur.) biological reference preparation (BRP) had to be established further to the decision to include nucleic acid testing (NAT) for the detection of hepatitis E virus (HEV) RNA in the monograph Human plasma (pooled and treated for virus inactivation) (1646). To this purpose, an international collaborative study was launched in the framework of the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM) and the Commission of the European Union (EU). The study was run in conjunction with the establishment of the 1st World Health Organization (WHO) international reference panel (IRP) for hepatitis E virus RNA genotypes (8578/13). Twenty-three laboratories used in-house developed and commercially available assays to calibrate a lyophilised candidate BRP prepared from a HEV 3f strain positive human plasma against the 1st WHO International Standard (IS) for HEV RNA (6329/10). Results from quantitative and qualitative assays were in good agreement and were combined to calculate an assigned potency. Real-time stability studies indicated that the candidate BRP is very stable at lower temperatures and is thus suitable for long-term use. Based on these results, in February 2016, the Ph. Eur. Commission adopted the candidate material as the hepatitis E virus RNA for NAT testing BRP batch 1, with an assigned unitage of 2.1 × 104 IU/vial (4.32 log10 IU/vial).

African swine fever virus encodes a gene with extensive homology to type II DNA topoisomerases.

Nucleotide sequencing of a virulent African swine fever virus (ASFV) isolate (Malawi LIL20/1) identified an open reading frame of 1191 amino acid residues encoding a protein of 134.9 kDa. This gene mapped to the SalI i and j restriction endonuclease fragments of the ASFV genome. The predicted polypeptide was found to share 21.1% identity over a 1077 amino acid region with the human type II DNA topoisomerase. The sequence is compared to other type II DNA topoisomerases and the possible roles in ASFV replication are discussed.

African swine fever virus genome content and variability.

A 55 kilobase pair (kb) region from the right end of the virulent African swine fever virus isolate, Malawi LIL20/1, has been sequenced. The 68 major open reading frames (ORFs) encoded are generally closely spaced and read from both DNA strands across the complete sequence. Comparison of the amino acid sequences of predicted ORFs with sequence databases identified 15 ORFs which encode proteins that are similar to proteins of known function. Two ORFs are homologous to copies of multigene family 360 (MGF360) and one ORF is homologous to copies of multigene family 110 (MGF110). Both of these multigene families have been described previously.

Regulatory expectations of validation/qualification of adventitious virus assays.

The European Union (EU) guideline concerning the virus safety evaluation of biotechnological investigational medicinal products (CPMP/BWP/398498/2005) recently came into force. In the guideline it is stated that analytical procedures supporting the qualification of cell banking systems, starting materials, as well as testing of unprocessed bulks for the presence of adventitious viruses, should be supported by appropriate qualification/validation studies. The validation protocols should prospectively set claims for assay performance, which should be verified by the validation experiments and demonstrate that a particular procedure is suitable for its intended purpose. Assay parameters for adventitious virus testing are discussed, and examples of validation of qualitative and quantitative assays for the detection of blood-borne viruses in human plasma are considered.

Standardization of nucleic acid amplification technique (NAT)-based assays for different genotypes of parvovirus B19: a meeting summary.

An extraordinary meeting of the International Working Group on the Standardization of Genome Amplification Techniques for the safety testing of blood, tissues and organs for blood borne pathogens was held on 2 March 2007, at the National Institute for Biological Standards and Control. The aim of the meeting was to investigate ways to harmonize results obtained for the detection and quantification of different genotypes of parvovirus B19 (B19V) DNA by control laboratories and manufacturers of plasma derivatives. The meeting explored issues of B19V such as the classification of B19V strains, the prevalence and distribution of different genotypes, the clinical and biological significance of different genotypes, the detection of different genotypes in plasma-derived products, and their susceptibility to virus-inactivation procedures. At this meeting and through subsequent studies, high titre, high volume samples have been identified representing different genotypes of B19V, which will be evaluated by collaborative study to prepare reference panels for the purposes of assay validation.