The Molecular Basis for Antigenic Drift of Human A/H2N2 Influenza Viruses.

Influenza A/H2N2 viruses caused a pandemic in 1957 and continued to circulate in humans until 1968. The antigenic evolution of A/H2N2 viruses over time and the amino acid substitutions responsible for this antigenic evolution are not known. Here, the antigenic diversity of a representative set of human A/H2N2 viruses isolated between 1957 and 1968 was characterized. The antigenic change of influenza A/H2N2 viruses during the 12 years that this virus circulated was modest. Two amino acid substitutions, T128D and N139K, located in the head domain of the H2 hemagglutinin (HA) molecule, were identified as important determinants of antigenic change during A/H2N2 virus evolution. The rate of A/H2N2 virus antigenic evolution during the 12-year period after introduction in humans was half that of A/H3N2 viruses, despite similar rates of genetic change.IMPORTANCE While influenza A viruses of subtype H2N2 were at the origin of the Asian influenza pandemic, little is known about the antigenic changes that occurred during the twelve years of circulation in humans, the role of preexisting immunity, and the evolutionary rates of the virus. In this study, the antigenic map derived from hemagglutination inhibition (HI) titers of cell-cultured virus isolates and ferret postinfection sera displayed a directional evolution of viruses away from earlier isolates. Furthermore, individual mutations in close proximity to the receptor-binding site of the HA molecule determined the antigenic reactivity, confirming that individual amino acid substitutions in A/H2N2 viruses can confer major antigenic changes. This study adds to our understanding of virus evolution with respect to antigenic variability, rates of virus evolution, and potential escape mutants of A/H2N2.

A compensatory mutagenesis study of a conserved hairpin in the M gene segment of influenza A virus shows its role in virus replication.

RNA structures are increasingly recognized to be of importance during influenza A virus replication. Here, we investigated a predicted conserved hairpin in the M gene segment (nt 967-994) within the region of the vRNA 5' packaging signal. The existence of this RNA structure and its possible role in virus replication was investigated using a compensatory mutagenesis approach. Mutations were introduced in the hairpin stem, based on natural variation. Virus replication properties were studied for the mutant viruses with disrupted and restored RNA structures. Viruses with structure-disrupting mutations had lower virus titers and a significantly reduced median plaque size when compared with the wild-type (WT) virus, while viruses with structure restoring-mutations replicated comparable to WT. Moreover, virus replication was also reduced when mutations were introduced in the hairpin loop, suggesting its involvement in RNA interactions. Northern blot and FACS experiments were performed to study differences in RNA levels as well as production of M1 and M2 proteins, expressed via alternative splicing. Stem-disruptive mutants caused lower vRNA and M2 mRNA levels and reduced M2 protein production at early time-points. When the RNA structure was restored, vRNA, M2 mRNA and M2 protein levels were increased, demonstrating a compensatory effect. Thus, this study provides evidence for functional importance of the predicted M RNA structure and suggests its role in splicing regulation.

Intravenously injected Newcastle disease virus in non-human primates is safe to use for oncolytic virotherapy.

Newcastle disease virus (NDV) is an avian paramyxovirus with oncolytic potential. Detailed preclinical information regarding the safety of oncolytic NDV is scarce. In this study, we evaluated the toxicity, biodistribution and shedding of intravenously injected oncolytic NDVs in non-human primates (Macaca fascicularis). Two animals were injected with escalating doses of a non-recombinant vaccine strain, a recombinant lentogenic strain or a recombinant mesogenic strain. To study transmission, naive animals were co-housed with the injected animals. Injection with NDV did not lead to severe illness in the animals or abnormalities in hematologic or biochemistry measurements. Injected animals shed low amounts of virus, but this did not lead to seroconversion of the contact animals. Postmortem evaluation demonstrated no pathological changes or evidence of virus replication. This study demonstrates that NDV generated in embryonated chicken eggs is safe for intravenous administration to non-human primates. In addition, our study confirmed results from a previous report that naïve primate and human sera are able to neutralize egg-generated NDV. We discuss the implications of these results for our study and the use of NDV for virotherapy.

Middle East Respiratory Syndrome coronavirus (MERS-CoV) serology in major livestock species in an affected region in Jordan, June to September 2013.

Between June and September 2013, sera from 11 dromedary camels, 150 goats, 126 sheep and 91 cows were collected in Jordan, where the first human Middle-East respiratory syndrome (MERS) cluster appeared in 2012. All sera were tested for MERS-coronavirus (MERS-CoV) specific antibodies by protein microarray with confirmation by virus neutralisation. Neutralising antibodies were found in all camel sera while sera from goats and cattle tested negative. Although six sheep sera reacted with MERS-CoV antigen, neutralising antibodies were not detected.

Detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction.

We present two real-time reverse-transcription polymerase chain reaction assays for a novel human coronavirus (CoV), targeting regions upstream of the E gene (upE) or within open reading frame (ORF)1b, respectively. Sensitivity for upE is 3.4 copies per reaction (95% confidence interval (CI): 2.5­6.9 copies) or 291 copies/mL of sample. No cross-reactivity was observed with coronaviruses OC43, NL63, 229E, SARS-CoV, nor with 92 clinical specimens containing common human respiratory viruses. We recommend using upE for screening and ORF1b for confirmation.

Repository of Eurasian influenza A virus hemagglutinin and neuraminidase reverse genetics vectors and recombinant viruses.

Reverse genetics can be used to produce recombinant influenza A viruses containing virtually every desired combination of hemagglutinin (HA) and neuraminidase (NA) genes using the virus backbone of choice. Here, a repository of plasmids and recombinant viruses representing all contemporary Eurasian HA and NA subtypes, H1-H16 and N1-N9, was established. HA and NA genes were selected based on sequence analyses of influenza virus genes available from public databases. Prototype Eurasian HA and NA genes were cloned in bidirectional reverse genetics plasmids. Recombinant viruses based on the virus backbone of A/PR/8/34, and containing a variety of HA and NA genes were produced in 293T cells. Virus stocks were produced in MDCK cells and embryonated chicken eggs. These plasmids and viruses may be useful for numerous purposes, including influenza virus research projects, vaccination studies, and to serve as reference reagents in diagnostic settings.

Evaluation of a rapid molecular algorithm for detection of pandemic influenza A (H1N1) 2009 virus and screening for a key oseltamivir resistance (H275Y) substitution in neuraminidase.

BACKGROUND: Rapid and specific molecular tests for identification of the recently identified pandemic influenza A/H1N1 2009 virus as well as rapid molecular tests to identify antiviral resistant strains are urgently needed. OBJECTIVES: We have evaluated the performance of two novel reverse transcriptase polymerase chain reactions (RT-PCRs) targeting specifically hemagglutinin and neuraminidase of pandemic influenza A/H1N1 virus in combination with a conserved matrix PCR. In addition, we investigated the performance of a novel discrimination RT-PCR for detection of the H275Y resistance mutation in the neuraminidase gene. STUDY DESIGN: Clinical performance of both subtype specific RT-PCR assays was evaluated through analysis of 684 throat swaps collected from individuals meeting the WHO case definition for the novel pandemic influenza virus. Analytical performance was analyzed through testing of 10-fold serial dilutions of RNA derived from the first Dutch sequenced and cultured confirmed case of novel pandemic influenza infection. Specificity and discriminative capacities of the H275Y discrimination assay were performed by testing wild type and recombinant H275Y pandemic influenza. RESULTS: 121 throat swaps collected from April 2009 to July 2009 were positive by at least two out of three RT-PCRs, and negative for the seasonal H3/H1 subtype specific RT-PCR assays. 117 of these were tested positive for all three (Ct-values from 15.1 to 36.8). No oseltamivir resistance was detected. CONCLUSIONS: We present a sensitive and specific approach for detection of pandemic influenza A/H1N1 2009 and a rapid RT-PCR assay detecting a primary oseltamivir resistance mutation which can be incorporated easily into clinical virology algorithms.

Influenza A virus surveillance in wild birds in Northern Europe in 1999 and 2000.

Using reverse transcription/polymerase chain reaction (RT-PCR), we have screened more than 8500 wild birds in Northern Europe in 1999 and 2000 for the presence of influenza A virus. Although our primary focus was on ducks, geese, and shorebirds, we have also tested thousands of samples from other bird species. Approximately 1% of our samples were positive for influenza A virus by RT-PCR, and from half of these we were able to isolate influenza A virus in embryonated chicken eggs. A wide variety of isolates was obtained representing hemagglutinin (HA) subtypes 1 through 7, 10, 11, 13, an unidentifiable HA, and neuraminidase (NA) subtypes 1 through 8.

A primate model to study the pathogenesis of influenza A (H5N1) virus infection.

Cynomolgus macaques (Macaca fascicularis) infected with influenza virus A/HongKong/156/97 (H5N1) developed acute respiratory distress syndrome (ARDS) with fever. Reverse transcriptase/polymerase chain reaction (RT/PCR) and virus isolation showed that the respiratory tract is the major target of the virus. The main lesion observed upon necropsy, performed 4 or 7 days postinfection, was a necrotizing bronchointerstitial pneumonia, similar to that found in primary influenza pneumonia in human beings. By immunohistochemistry, influenza virus antigen proved to be limited to pulmonary tissue and tonsils. The data indicate that ARDS and multiple organ dysfunction syndrome (MODS), observed in both humans and monkeys infected with this virus, are caused by diffuse alveolar damage from virus replication in the lungs alone.

Antigenic and molecular heterogeneity in recent swine influenza A(H1N1) virus isolates with possible implications for vaccination policy.

In order to explore the occurrence of antigenic drift in swine influenza A(H1N1) viruses and the match between epidemic and vaccine strains, 26 virus isolates from outbreaks of respiratory disease among finishing pigs in the Netherlands in the 1995/1996 season and reference strains from earlier outbreaks were examined using serological and molecular methods. In contrast to swine H3N2 viruses, no significant antigenic drift was observed in swine H1N1 viruses isolated from the late 1980s up to 1996 inclusive. However, a marked antigenic and genetic heterogeneity in haemagglutination inhibition tests and nucleotide sequence analyses was detected among the 26 recent swine H1N1 virus strains. Interestingly, the observed antigenic and molecular variants were not randomly distributed over the farms. This finding indicates independent introductions of different swine H1N1 virus variants at the various farms of the study and points to a marked difference between the epidemiologies of human and swine influenza viruses. The observed heterogeneity may hamper the control of swine influenza by vaccination and indicates that the efficacy of current swine influenza vaccines requires re-evaluation and that the antigenic reactivity of swine influenza viruses should be monitored on a regular basis.

Pathogenesis of influenza A (H5N1) virus infection in a primate model.

Cynomolgus macaques (Macaca fascicularis) infected with influenza virus A/Hong Kong/156/97 (H5N1) developed acute respiratory distress syndrome and fever associated with a necrotizing interstitial pneumonia. Reverse transcription PCR, virus isolation, and immunohistochemistry showed that the respiratory tract is the major target of the virus.

Antigenic and genetic characterization of swine influenza A (H1N1) viruses isolated from pneumonia patients in The Netherlands.

It is generally believed that pigs can serve as an intermediate host for the transmission of avian influenza viruses to humans or as mixing vessels for the generation of avian-human reassortant viruses. Here we describe the antigenic and genetic characterization of two influenza A (H1N1) viruses, which were isolated in The Netherlands from two patients who suffered from pneumonia. Both viruses proved to be antigenically and genetically similar to avian-like swine influenza A (H1N1) viruses which currently circulate in European pigs. It is concluded that European swine H1N1 viruses can infect humans directly, causing serious disease without the need for any reassortment event.

Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene.

The recently raised awareness of the threat of a new influenza pandemic has stimulated interest in the detection of influenza A viruses in human as well as animal secretions. Virus isolation alone is unsatisfactory for this purpose because of its inherent limited sensitivity and the lack of host cells that are universally permissive to all influenza A viruses. Previously described PCR methods are more sensitive but are targeted predominantly at virus strains currently circulating in humans, since the sequences of the primer sets display considerable numbers of mismatches to the sequences of animal influenza A viruses. Therefore, a new set of primers, based on highly conserved regions of the matrix gene, was designed for single-tube reverse transcription-PCR for the detection of influenza A viruses from multiple species. This PCR proved to be fully reactive with a panel of 25 genetically diverse virus isolates that were obtained from birds, humans, pigs, horses, and seals and that included all known subtypes of influenza A virus. It was not reactive with the 11 other RNA viruses tested. Comparative tests with throat swab samples from humans and fecal and cloacal swab samples from birds confirmed that the new PCR is faster and up to 100-fold more sensitive than classical virus isolation procedures.

Antigenic drift in the influenza A virus (H3N2) nucleoprotein and escape from recognition by cytotoxic T lymphocytes.

Viruses exploit different strategies to escape immune surveillance, including the introduction of mutations in cytotoxic T-lymphocyte (CTL) epitopes. The sequence of these epitopes is critical for their binding to major histocompatibility complex (MHC) class I molecules and recognition by specific CTLs, both of which interactions may be lost by mutation. Sequence analysis of the nucleoprotein gene of influenza A viruses (H3N2) isolated in The Netherlands from 1989 to 1999 revealed two independent amino acid mutations at the anchor residue of the HLA-B27-specific CTL epitope SRYWAIRTR (383 to 391). A R384K mutation was found in influenza A viruses isolated during the influenza season 1989-1990 but not in subsequent seasons. In the influenza season 1993-1994, a novel mutation in the same CTL epitope at the same position was introduced. This R384G mutation proved to be conserved in all influenza A viruses isolated from 1993 onwards. Both mutations R384K and R384G abrogated MHC class I presentation and allowed escape from recognition by specific CTLs.

Influenza B virus in seals.

Influenza B virus is a human pathogen whose origin and possible reservoir in nature are not known. An influenza B virus was isolated from a naturally infected harbor seal (Phoca vitulina) and was found to be infectious to seal kidney cells in vitro. Sequence analyses and serology indicated that influenza virus B/Seal/Netherlands/1/99 is closely related to strains that circulated in humans 4 to 5 years earlier. Retrospective analyses of sera collected from 971 seals showed a prevalence of antibodies to influenza B virus in 2% of the animals after 1995 and in none before 1995. This animal reservoir, harboring influenza B viruses that have circulated in the past, may pose a direct threat to humans.

Chlamydia pneumoniae and Mycoplasma pneumoniae in children with acute respiratory infection in general practices in The Netherlands.

In this retrospective study Chlamydia pneumoniae and Mycoplasma pneumoniae infections were detected by polymerase chain reaction (PCR) in samples (n = 457) from children presenting with acute respiratory infection to general practitioners during 1992-97. Samples were collected in autumn and winter, and from 1994 onwards in spring and summer also. Overall, C. pneumoniae and M. pneumoniae were detected in throat or nasal samples by PCR in 3.1% and 2.4% of the cases, respectively. The proportion of both C. pneumoniae and M. pneumoniae infections varied between 0% and 6.9% over the years studied, whereas seasonal proportions varied from 1.8 to 9.1% and 1.2 to 4.5%, respectively. For both microorganisms the lowest proportion was detected during winter and the highest in summer. C. pneumoniae could already be detected by PCR in patients under 4 y of age, an observation not made in sero-epidemiological studies. In conclusion, both C. pneumoniae and M. pneumoniae infections play a minor role in children presenting with acute respiratory infection.

Low prevalence of Chlamydia pneumoniae and Mycoplasma pneumoniae among patients with symptoms of respiratory tract infections in Dutch general practices.

Acute respiratory disease is one of the most common reasons to consult a general practitioner. A substantial part of these diseases cannot be explained by an infection with a virus or a common pathogenic bacterium. To study this diagnostic deficit, the prevalence of Chlamydia pneumoniae and Mycoplasma pneumoniae infections was determined in two groups of patients consulting a general practitioner. DNA of C. pneumoniae and M. pneumoniae was detected by a polymerase chain reaction (PCR) in nose/throat swabs from six (1.1%), and from seven (1.3%) patients, respectively, of 557 patients consulting a general practitioner for complaints suggestive for a virus infection during the 1994/1995 respiratory infections season. Two patients remained C. pneumoniae PCR-positive for at least 4 weeks. All others were negative within 3 weeks. Double infections of C. pneumoniae and influenza virus (3/6), and of M. pneumoniae and respiratory syncytial virus (1/7) or rhinovirus (1/7) were diagnosed. During the 1992/1993 season, attempts to isolate C. pneumoniae in cell culture or to detect C. pneumoniae DNA by PCR using throat swabs were all negative for 80 patients with a sore throat, although serological data suggested a C. pneumoniae infection in 13 (16%) patients. A specimen from another patient of this group was M. pneumoniae PCR-positive and the corresponding serum specimens showed a persistent high antibody titre. In summary, the prevalence of acute C. pneumoniae and M. pneumoniae infections was less than 2% in patients consulting a general practitioner.

Antigenic drift in swine influenza H3 haemagglutinins with implications for vaccination policy.

In order to explore the occurrence of antigenic drift in swine influenza A(H3N2) virus, we examined virus strains from outbreaks of respiratory disease among finishing pigs in the Netherlands in 1996 and 1997 and from earlier outbreaks. In contrast to swine H3N2 strains from the 1980s, the recent isolates did not show significant cross-reactivity with human influenza A(H3N2) viruses from 1972-1975 in haemagglutination inhibition tests. These new strains form a separate branch in the phylogenetic trec of the HA1 parts of HA. We conclude that recently there has been considerable antigenic drift within the swine H3N2 viruses in the Netherlands and Belgium and recommend replacement of the A/Port Chalmers/1/73 (H3N2) strain in the current vaccine by a more recent swine H3N2 isolate.

Improved detection of rhinoviruses in clinical samples by using a newly developed nested reverse transcription-PCR assay.

This paper describes the development and evaluation of a new nested reverse transcription (RT)-PCR for the detection of rhinovirus in clinical samples. The nucleotide sequences of the 5' noncoding regions of 39 rhinoviruses were determined in order to map the most conserved subregions. We designed a set of rhinovirus-specific primers and probes directed to these subregions and developed a new nested RT-PCR. The new assay includes an optimal RNA extraction method and amplicon identification with probe hybridization to discriminate between rhinoviruses and the closely related enteroviruses. It proved to be highly sensitive and specific. When tested on a dilution series of cultured viruses, the new PCR protocol scored positive at 10- to 100-fold-higher dilutions than a previously used nested RT-PCR. When tested on a collection of clinical samples obtained from 1,070 acute respiratory disease patients who had consulted their general practitioners, the new assay demonstrated a rhinovirus in 24% of the specimens, including all culture-positive samples, whereas the previously used PCR assay or virus culture detected a rhinovirus in only 3.5 to 6% of the samples. This new assay should help determine the disease burden associated with rhinovirus infections.

Molecular characterization of a wild poliovirus type 3 epidemic in The Netherlands (1992 and 1993).

An outbreak of poliomyelitis due to wild poliovirus type 3 (PV3) occurred in an unvaccinated community in The Netherlands between September 1992 and February 1993. The outbreak involved 71 patients. The aim of this study was to characterize the virus at the molecular level and to analyze the molecular evolution of the epidemic virus. Molecular analysis was carried out by sequencing the VP1/2A junction region (150 nucleotides) of 50 PV3 strains isolated in association with this outbreak and the entire VP1 gene of 14 strains. In addition, the sequence of the VP1/2A junction region of strains from geographical regions endemic for PV3 (Egypt, India, and Central Asia) was analyzed and compared with the nucleotide sequence of the epidemic strain from The Netherlands. The earliest isolate was obtained from river water sampled 3 weeks before diagnosis of the first poliomyelitis patient and was found by VP1/2A sequence analysis to be genetically identical to the strain isolated from the first patient. Sequence divergence among the strains from the epidemic in The Netherlands was less than 2%. The closest genetic similarity (97.3%) was found with an Indian isolate (New Delhi, December 1991), indicating the likely source of the virus. A more than 99% sequence similarity was found in the VP1/2A region. Finally, the sequence information was used to design primers for the specific and highly sensitive molecular detection of PV3 strains during the epidemic.