REPISALUD (Instituto de Salud Carlos III)

We have applied the RNase A mismatch cleavage method to analyze genetic variability in RNA viruses by using influenza virus as a model system. Uniformly labeled RNA probes synthesized from a cloned hemagglutinin gene of a given viral strain were hybridized to RNA isolated from other strains of characterized or uncharacterized genetic composition. The RNA.RNA heteroduplexes containing a variable number of base mismatches were digested with RNase A, and the resistant products were analyzed by denaturing polyacrylamide gel electrophoresis. We show that many of these single base mismatches are cleaved by RNase A, generating unique and characteristic patterns of resistant RNA fragments specific for each of the different viral strains. Comparative analysis of the cleavage patterns allows a qualitative estimation of the genetic relatedness and evolution of field strains. We also show that cleavage by RNase A at single base mismatches can readily detect and localize point mutations present in monoclonal antibody-resistant variants. This method should have wide applications in the study of RNA viruses, not only for epidemiological analysis but also in some diagnostic problems, such as characterization of phenotypic mutants.This work was supported by National Institutes of Health Grant CA33021 awarded by the   Nationa l Cancer Institute to M.P. and by grants from the Comision Asesora de Investigacion Cientificay Tecnica (Grant 608/438) and Fondo de Investigaciones Sanitarias to J.O. and J.A.M.C.L.-G. was a recipient of a NATO short-term post doctoral fellow-ship while on leave from the Centro Nacional de Microbiologia, Majadahonda, Madri

Lopez-Galindez, Luis Cecilio

Lopez, Juan Antonio

Melero, Jose Antonio

Fuente, Luis de la

Martínez, Concepción

Ortin, Juan

Perucho, Manuel

Instituto de Salud Carlos III

English

Analysis of genetic variability and mapping of point mutations in influenza virus by the RNase A mismatch cleavage method

The immuno-double-diffusion (IDD) test was used to detect antibodies against the prevalent influenza A (H3N2) viruses in sera collected weekly in Madrid from 1972 to 1975. A total of over 14 000 sera were tested. The proportion of sera positive in the IDD test was found to reflect the level of recent influenza A infection of the population.S

Najera-Morrondo, Rafael

Perez-Breña, Pilar

Fernández, M V

Llacer Gil de Ramales, Alicia

Nájera, E

Schild, G C

Bulletin of the World Health Organization, 59 (1): 79-83 (1981)
The use of immuno-double-diffusion tests in
epidemiological studies of influenza in Spain
R. NAJERA,1 P. PEREZ-BRENA,2 M. V. FERNANDEZ,2 C. LOPEZ GALiNDEZ,3 A. LLACER,4
E. NAJERA,5 & G. C. SCHILD 6
The immuno-double-diffusion (IDD) test was used to detect antibodies against the
prevalent influenzaA (H3N2) viruses in sera collected weekly in Madridfrom 1972 to 1975. A
total of over 14 000 sera were tested. The proportion of sera positive in the IDD test was
found to reflect the level of recent influenza A infection of the population.
The methods available for the detection of anti-
bodies to influenza viruses include haemaggluti-
nation-inhibition (HI), neuraminidase-inhibition,
and complement-fixation tests. These tests are often
cumbersome because of the need to dilute the sera and
in the case of HI tests to remove non-specific
inhibitors. The immuno-double-diffusion (IDD) test
(2, 3, 5) has been employed for the detection of anti-
bodies to the antigenic components of influenza
viruses, and has been shown to be effective and
sensitive for the serodiagnosis of influenza infection
with human paired sera (3). Use of this test made it
possible to detect and identify antibody to the nucleo-
protein (NP), the neuraminidase (NA), and the
haemagglutinin (HA) antigens of influenza A virus in
convalescent serum samples. The antibodies to NP
and NA that were detected by IDD were apparently
short-lived (3) and thus their presence appeared to be
a useful indicator of recent influenza infection.
This paper describes studies to assess the value of
the IDD test in the serological surveillance of
influenza. Results obtained in Madrid by the routine
application of this method over four years are
described and related to information on the incidence
of clinical influenza in Spain obtained by the public
health services.
I Chief, Respiratory Viruses and Exanthems Department, Centro
Nacional de Microbiologia, Virologia e Immunologia Sanitarias,
Majadahonda, Madrid, Spain. Present address: Virus Diseases,
World Health Organization, 1211 Geneva 27, Switzerland.
2 Section Chief, Respiratory Viruses and Exanthems Department.
3Assistant, Respiratory Viruses and Exanthems Department.
4Section Chief, Epidemiology Department, Centro Nacional de
Microbiologia, Virologia e Immunologia Sanitarias, Majadahonda,
Madrid, Spain.
5 Chief, Epidemiology Department.
6 Head, Division of Viral Products, National Institute for
Biological Standards and Control, Holly Hill, London NW3 6RB,
England.
MATERIALS AND METHODS
Serum collection
Serum samples were obtained from a Madrid blood
bank (Instituto Nacional de Hematologia) which
serves the city and province of Madrid, an area about
8000 km2. From 1972 to 1975, approximately 100
serum samples were collected each week for IDD tests
from donors in the age range of 20-65 years.
Immuno-double-diffusion test
This test was performed in agar gel on a glass slide
or in a plastic immunoplate (Hyland Laboratories,
California, USA) as described by Schild et al. (3), the
antigen used being purified and concentrated
influenza virus disrupted by the addition of sarcosyl
detergent to a final concentration of 1 0o. There were
six peripheral wells and one central well containing the
antigen. The production of one or more visible
precipitin lines after staining the plate was taken as
evidence of positivity.
To evaluate the reproducibility and accuracy of our
results, 25 sera (15 IDD positive and 10 IDD negative)
were tested on 10 different occasions with different
batches of plates and antigens. Reference sera were
included in each set of tests.
The influenza A virus strains used, included the
following: A/Hong Kong/1/68 (H3N2), A/England/
42/72 (H3N2), A/Port Chalmers/1/73 (H3N2), and
A/Madrid/1492/74 (H3N2), a strain isolated in
Madrid in the winter of 1974 that is antigenically
similar to A/Scotland/840/74 (H3N2) (6).
Viruses were cultivated in the allantoic cavity of
10-day-old embryonated eggs and concentrated and
purified as described by Schild et al. (3). The final
4034 -79-
R. NAJERA ET AL.
virus preparations contained 4-5 x 106 haemagglu-
tinin units/ml, and 10-15 mg of protein/ml.
Epidemiological data
The number of reported cases of influenza (which is
a notifiable disease in Spain) as well as the number of
deaths from the three main respiratory disease
headings in list B of the ICD (1)-influenza, pneu-
monia and bronchopneumonia, and bronchitis,
emphysema, and asthma-were used as the basis for
evaluating the epidemiological impact of influenza.
The data on cases of influenza were obtained from the
existing influenza surveillance system for the city and
province of Madrid, covering a population of nearly
4 million.
RESULTS AND DISCUSSION
Fig. 1 shows the weekly influenza notifications for
Madrid from mid-1971 to mid-1975 compared with
the maximum and median numbers of influenza cases
for the previous five years-the "normal epidemic
area". The median values of the previous five years
were considered as the "non-epidemic" level. In
addition, the method of Serfling (4) for the analysis of
mortality data was applied in order to obtain a curve
for the non-epidemic level of influenza, calculating
expected values from a sinusoidal function, where the
constant and the amplitude are exponential functions
of time: (a) the length of cycle is one year (weekly
intervals of 2n/52); (b) the amplitude is equal to half
the range between the minimum and maximum values
in non-epidemic years, and (c) the phase angle is 3n/2
to fit the minimum value of the influenza year with the
-1 value of the sine function.
We considered that an epidemic level of influenza
had been reached when the weekly rate of increase of
cases was more than double that of the previous week
and the number of cases was above that represented by
the non-epidemic curve. This method, based only on
the number of notified cases, proved to be of value
and correlated well with mortality figures, but has the
disadvantage of not being etiologically based. Clini-
cally mild influenza outbreaks might be missed and
other respiratory infections may be counted as
influenza.
The results of IDD tests performed on the human
sera collected between 1972 and 1975 are shown in
Table 1. Most (93.90/) of the sera recorded as positive
gave only one precipitin line, which was identified, by
reference to a specific hyperimmune serum, as corre-
sponding to the line produced by antibody to influ-
enza A nucleoprotein antigen. A further 6% gave two
precipitin lines identified as corresponding to
antibodies to NP and NA. A small number of sera
(<0.20/) gave three precipitin lines, the third line
corresponding to antibody to HA. There was con-
siderable annual variation in the proportion of sera
positive by IDD tests.
Fig. 2 illustrates the proportions of sera found to be
seropositive in IDD tests, by 4-week periods from
1972 to 1975, and also the weekly incidence of influ-
enza in Madrid over the same period (log scale). There
was, in general, a good correlation between the
periods of high incidence of influenza and periods
when a high proportion of sera were positive by the
IDD test.
The antibodies to influenza antigens detected by
IDD in individual sera were found to persist for only
2-3 months after natural infection and could be
detected as early as a week after the onset of illness in
virologically confirmed cases (R. Najera, unpublished
data, 1975). The proportion of sera positive in the
IDD test can therefore be regarded as a cumulative
index of recent infection.
Table 1. Results of IDD tests
IDD positive sera
One line Two lines Three lines
(NP) (NP + NA) (NP + NA + HA) Total
Year No. of sera
examined No. % No. % No. % No. %
1972 4 548 571 12.55 70 1.54 1 0.02 642 14.11
1973 3 865 255 6.60 17 0.44 0 0 272 7.04
1974 3 470 471 13.57 20 0.57 2 0.05 493 14.21
1975 3100 536 17.29 10 0.32 0 0 546 17.61
Total 14 983 1833 12.23 117 0.78 3 0.02 1953 13.07
80
SEROEPIDEMIOLOGY OF INFLUENZA 81
#0tl06 OHAhA~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-.
- _~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~r
IL4I
la~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~0
VW C
0~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~0
a)
_s 1 N~~~~~~~~~~~~~~~~~~~~~~~~~~~~~0
_~~~~~~~~~~~~~~~~~~~~~~~~~~~~*
O~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~,' OCCU0*U
(9L x : uo!Ielndod ) looM jed sasm eZUnulJUI O 'ON
R. NAJERA ET AL.
(0 x>: uoilelndod) laaM jed sesez ezuanl,ua 1o 'ON
m
0)
>, 2
_
> ,
0)
a
CM CN_
eJas OA!Xisod lo 9BelU83J8d
r._
0)
N
V
C
C
0
._
0
-o
4-
a)
C'
NN
C
0
C.
C
C
a)0) CC
0)
LL
-
._-
82
SEROEPIDEMIOLOGY OF INFLUENZA 83
High levels of seropositivity were observed during
the epidemics that commenced in January 1973 and
December 1974, as well as after the period of high
incidence of influenza which occurred in December
1973. This indicates that an increase in the number of
notified cases to twice the non-epidemic level is
accompanied by an increase in the proportion of sera
found positive by IDD tests. The highest level of sero-
positivity observed in December 1974 to January 1975
coincided with the occurrence of the highest number
of notified cases. It was concluded that the use of the
IDD test to monitor human sera may provide useful
information on the recent prevalence of influenza A
infection in the community. Nevertheless this
information should be regarded as complementary to
information provided by virus isolation studies and
serological diagnosis based on paired sera.
RtSUM
UTILISATION DES EPREUVES D'IMMUNODIFFUSION DOUBLE DANS DES ETUDES
EPIDEMIOLOGIQUES SUR LA GRIPPE EN ESPAGNE
Les epreuves d'immunodiffusion double (IDD) ont e
effectuees sur des echantillons de serum preleves toutes les
semaines dans une banque de sang de Madrid, de 1972 A
1975, et les proportions de serums positifs ont e comparees
au nombre de cas cliniques enregistres par le systeme de
surveillance de la grippe dans la meme zone. Les anticorps
detectes par l'IDD ne persistaient que deux A trois mois et
etaient donc consideres comme un indice cumulatif d'infec-
tion recente. Les resultats de l'etude ont montre qu'une
augmentation de deux fois, par rapport au niveau non epid&
mique, du nombre des cas de grippe signales s'accompagnait
d'un accroissement du niveau de la seropositivite. Bien que
les epreuves IDD puissent donc fournir des renseignements
utiles sur la prevalence de l'infection recente par le virus
grippal A, ces renseignements doivent etre consideres
comme des complements aux donnees provenant des etudes
par isolement du virus et du diagnostic serologique fonde sur
des paires de s6rums.
REFERENCES
1. International classification of diseases. 1965 Revision,
Vol. 1. Geneva, World Health Organization, 1967.
2. SCHILD, G. C. & PEREIRA, H. G. Characterization of
ribonucleoprotein and neuraminidase of influenza A
viruses by immunodiffusion. Journal of general
virology, 4: 355 (1969).
3. SCHILD, G. C. ET AL. Serological diagnosis of human
influenza infection by immunoprecipitin techniques.
Bulletin of the World Health Organization, 45: 465
(1971).
4. SERFLING, R. E. Methods for current statistical analysis
of excess pneumonia-influenza deaths. Public health
reports (Washington), 78: 494 (1963).
5. STYK, B. & HANA, L. Immunodiffusion studies on the
reaction of influenza virus with specific antibody and
non-specific serum beta inhibitor. Acta virologica, 10:
281 (1966).
6. Weekly epidemiological record, 3: 26 (1975).


The use of immuno-double-diffusion tests in epidemiological studies of influenza in Spain

Not a member yet

Analysis of genetic variability and mapping of point mutations in influenza virus by the RNase A mismatch cleavage method

The use of immuno-double-diffusion tests in epidemiological studies of influenza in Spain

10,310

16,912